John MOORE, Plaintiff and Appellant, v. The REGENTS OF the UNIVERSITY OF CALIFORNIA; David W. Golde, M.D.; Shirley G. Quan; Genetics Institute, Inc.; Sandoz Ltd., Sandoz United States, Inc., Sandoz Pharmaceutical Corporation aka Sandoz, Inc., et al., Defendants and Respondents.
This appeal raises fundamental questions concerning a patient's right to the control of his or her own body, and whether the commercial exploitation of a patient's cells by medical care providers, without the patient's consent, gives rise to an action for damages. This appears to be a case of first impression.
In 1976, plaintiff and appellant sought medical treatment at the Medical Center of the University of California, Los Angeles (UCLA),2 for a condition known as hairy-cell leukemia. He was seen by Dr. David W. Golde, who confirmed the diagnosis. As a necessary part of the treatment for this disease, plaintiff's spleen was removed at UCLA in October of 1976.
Without plaintiff's knowledge or consent, Dr. Golde and Shirley G. Quan, a UCLA employee, determined that plaintiff's cells were unique. Through the science of genetic engineering, these defendants developed a cell-line from plaintiff's cells which is capable of producing pharmaceutical products of enormous therapeutic and commercial value.3 The Regents, Golde and Quan patented the cell-line along with methods of producing many products therefrom. In addition, these defendants entered into a series of commercial agreements for rights to the cell-line and its products with Sandoz Pharmaceuticals Corporation (Sandoz) and Genetics Institute, Inc. (Genetics). The market potential of products from plaintiff's cell-line was predicted to be approximately three billion dollars by 1990. Hundreds of thousands of dollars have already been paid under these agreements to the developers. Without informing plaintiff, and in pursuit of their research efforts, Golde and UCLA continued to monitor him and take tissue samples from him for almost seven years following the removal of his spleen.
THE THIRD AMENDED COMPLAINT
On September 11, 1984, plaintiff instituted a lawsuit based on these events. He eventually filed a third amended complaint for damages and declaratory relief which is the subject of this appeal. The complaint names Golde, Quan, the Regents, Sandoz, and Genetics, as defendants, alleging causes of action for: (1) conversion; (2) lack of informed consent; (3) breach of fiduciary duty; (4) fraud and deceit; (5) unjust enrichment; (6) quasi-contract; (7) breach of implied covenant of good faith and fair dealing; (8) intentional infliction of emotional distress; (9) negligent misrepresentation; (10) interference with prospective advantageous economic relationships; (11) slander of title; (12) accounting; and (13) declaratory relief.
A. ALLEGATIONS OF THE CONVERSION CAUSE OF ACTION
The first cause of action for conversion includes allegations which form the foundation for most of the other causes of action. Plaintiff alleges the following:
Plaintiff's blood and blood components are unique, possessing properties with a potential for significant breakthroughs in medical research and the treatment of disease. His “blood and bodily substances” (by which term plaintiff means all bodily tissue, including cells, spleen, blood and genetic material) have commercial value.
“8. Defendants, based upon their experience and familiarity with developments in scientific fields relevant to such matters, were aware that certain blood products and blood components were of great value in a number of commercial and scientific efforts, and that a steady and abundant natural source of these substances in a human being would be highly desirable.
“9. In addition, defendants, were aware that certain characteristics and properties of the blood and blood components of certain rare patients could prove to be highly useful in defendants' efforts to locate, isolate and cultivate large quantities of these valuable substances.
“10. Defendants were also aware that direct and exclusive access to such a patient would provide them with a steady and natural source of these unique living substances and properties, and would provide defendants with competitive, commercial, and scientific advantages.”
In the course of treating plaintiff, defendants drew blood and bodily substances from him. Defendants confirmed that plaintiff had hairy-cell leukemia, and planned to remove his spleen as part of the treatment.
The complaint further alleges:
“14. Prior to the surgical removal of plaintiff's spleen, Defendant David W. Golde, M.D., Defendant Shirley G. Quan, and other defendants, without advising plaintiff or obtaining his consent, formed the intent and made arrangements to obtain portions of his spleen following its removal from plaintiff in connection with their desire to have regular and continuous access to, and possession of, plaintiff's unique and rare Blood and Bodily Substances.
“15. Plaintiff was never informed by the defendants of the research and commercial value of his Blood and Bodily Substances, including his spleen, prior to its surgical removal, nor was he ever informed of defendants' prior formed intent to obtain from the operating room or the Surgical Pathology Department immediately after said surgery a portion or portions of his spleen and to remove said portion or portions to an entirely separate research unit where defendants, without plaintiff's knowledge or consent, would store, study, culture, grow and otherwise use them to establish cell-lines, cultures, conditioned media and other by-products having the aforementioned valuable, unique and rare characteristics possessed by his Blood and Bodily Substances, all of which activities did not have, and were not intended to have, any relation to plaintiff's medical and health care.”
Additionally, before the splenectomy on October 20, 1976:
“17. ․ Defendant Golde provided written instructions to members of the research staff of Defendant, The Regents of the University of California, informing them of the unique characteristics of plaintiff's condition and Blood and Bodily Substances, and instructing them to study and characterize the nature of his unique cells, Blood and Bodily Substances, utilizing a portion of plaintiff's spleen. At no time did Defendant Golde, nor Defendant Quan, nor any of the other defendants or researchers involved in this action ever inform plaintiff of their intentions in this regard, nor did they request, nor did they ever receive any permission or consent from plaintiff to perform these activities on plaintiff's spleen tissue or the unique Blood and Bodily Substances contained therein.” 4
After the splenectomy, a portion of plaintiff's spleen was taken to a separate research unit at UCLA where it was stored and used for research. In furtherance of the research and commercial activities of defendants, plaintiff's spleen tissue was used to establish cell-lines with unique characteristics, capable of producing valuable pharmaceutical products. These activities were unrelated to the treatment of plaintiff.
Plaintiff alleges that had he known what was taking place, he would not have consented to the splenectomy for these research and commercial activities; would have insisted on participating in control of the use of his blood and bodily substances; would not have permitted these materials to be used by defendants solely for their independent research, commercial activity, and economic benefit; would have considered treatment at another medical facility where his wishes would have been carried out; and would have sought participation in the economic benefit.
At defendants' direction, plaintiff returned from the State of Washington to UCLA many times from November of 1976 through September of 1983. Defendants represented that these visits were necessary for plaintiff's well-being, and at each visit, blood and bodily substances were withdrawn (including blood, blood serum, skin, bone marrow aspirate, and sperm). All of the blood and bodily substances removed from plaintiff on these visits furthered defendants' purpose of establishing a cell-line for commercial exploitation. Defendants did not inform plaintiff that these withdrawals served the purpose of defendants' independent research and commercial activity, rather than his medical treatment. Defendants misrepresented the nature of the research to plaintiff by advising plaintiff that there was no commercial or financial value in his tissues.
During the time plaintiff was under defendants' care, defendants engaged in a number of activities aimed at commercial exploitation of the cell-line, without disclosing these activities to plaintiff and without his knowledge. These activities included the following: Golde and Quan actively asserted the unique and valuable properties of plaintiff's cell-line and its capabilities; defendants provided commercial firms with samples of the cell-line and products therefrom; defendants applied for patents for plaintiff's cell-line and the products derived therefrom, with Golde and Quan listed as the inventors; Golde engaged in negotiations and entered into contracts with firms to commercially develop and scientifically investigate the cell-line; specifically, Golde entered into contracts in 1981, 1982 and 1983 with Genetics Institute, Sandoz Ltd., Sandoz United States, Inc., and Sandoz Pharmaceutical Corporation to collaborate on commercial exploitation of plaintiff's cell-line and products therefrom; pursuant to these contracts Genetics gave Golde 75,000 shares of stock at a nominal price, and paid the Regents and Golde $330,000 over three years, while Sandoz paid Golde and the Regents $110,000; defendants renamed the cell-line from “Moore” to “RLC” to avoid detection by plaintiff; Golde and Quan devoted a large percentage of their time on activities related to plaintiff's cell-line and products therefrom; the patents and research leading to them gave defendants a significant competitive advantage over other commercial entities, and defendants have predicted a potential market of over three billion dollars by the year 1990 for a whole range of products derived exclusively from plaintiff's blood and bodily substances and from cells and cell-lines extracted from him.
During one of plaintiff's visits to UCLA, on April 11, 1983, defendants extracted tissue from plaintiff to further their research. At that time, defendants presented plaintiff with a consent form to allow them to engage in research. Plaintiff signed the consent without knowing the defendants' true purpose. He would not have consented had he been aware of defendants' true research and commercial activities. On September 20, 1983, plaintiff again consented to withdrawals, but on this occasion expressly denied defendants any rights to his cell-line. The consent form of September 20, 1983, was attached as an exhibit to the complaint. (See Appendix B to this opinion.) Though plaintiff did not know, nor was he informed, of defendants' activities regarding his cell-line, he refused to consent to further activity. In spite of this express lack of consent, defendants continued their commercial exploitation of the cell-line.
Plaintiff alleged that defendants had a fiduciary relationship to him because they represented that they were acting in his best interest. They had a duty to fully disclose both their activities and relevant facts to him, and to neither obtain an interest adverse to his, nor unfairly profit from him.
Golde and Quan obtained a patent to the cell-line and products obtained from it on March 20, 1984.5 This patent was assigned to the Regents. The subject of the patent was the Mo cell-line derived from plaintiff's tissues and nine products derived from the cell-line.6 Defendants are in the process of developing additional patent applications from plaintiff's cell-line. Plaintiff was never told of this patent activity which defendants concealed from him.
“60. Plaintiff alleges that his Blood and Bodily Substances, including but not limited to his blood, blood components, by-products, bodily tissues, lymphokines, genetic material, genetic sequences, conditioned medium, spleen, sperm, bone marrow aspirate, and other substances derived from his Blood and Bodily Substances, are his tangible personal property, and the activities of the defendants as set forth herein constitute a substantial interference with plaintiff's possession or right thereto, as well as defendants' wrongful exercise of dominion over plaintiff's personal property rights in his Blood and Bodily Substances.”
According to the complaint, plaintiff's cell-line and the products from it were not developed through any extraordinary scientific methodology invented or employed by defendants, nor created by defendants. The cell-line and products were obtained exclusively through live specimens and samples of plaintiff's blood and bodily substances. Plaintiff alleged damages, including lost profits, and punitive damages.
B. RULING ON REGENTS' DEMURRERS
The Regents, Golde and Quan jointly demurred to all the causes of action in the third amended complaint. These demurrers and the ruling thereon will be referred to as the Regents' demurrers.
After hearing, the trial court sustained the Regents' demurrers to the first cause of action for conversion, and held that since the first cause of action was incorporated into the remaining causes of action, those causes of action were defective for the same reasons. The demurrers to those remaining causes of action were sustained on that limited basis. The court articulated the following reasons for sustaining the demurrers to the first cause of action:
1. Plaintiff failed to “specifically allege that when he first went to UCLA he did not know or have reason to know that tissue removed during the care and treatment of patients by that facility might be used for medical or scientific study as well as for diagnosis and treatment of the patient.”
2. Plaintiff failed to specifically allege that at neither the time defendants withdrew blood and tissue on October 5, 1976, nor at the removal of his spleen a few weeks later, defendants (1) “knew such substances had commercial or potential commercial value as a product (as a raw substance or as a component of other substances)” and (2) “had formed the intent to commercially exploit such substances.”
3. Plaintiff failed to “specifically allege whether he consented to the removal of his spleen for therapeutic purposes.” The court noted that plaintiff did not allege whether the removal was unrelated to therapeutic purposes, but merely that he consented to removal.
4. Plaintiff failed to attach a copy of either the consent to the splenectomy in October of 1976, or the consent of September 20, 1983, as exhibits to the complaint.
Since the court did not conclude that no cause of action could be stated on these facts, the demurrers were sustained with leave to amend. Following a hearing to clarify how the ruling on the first cause of action affected the twelve other causes of action, however, plaintiff declined to file a fourth amended complaint, and allowed the court to dismiss the complaint as to the Regents, Golde and Quan pursuant to Code of Civil Procedure section 581, subdivision (c) (now § 581, subd. (f)(2)).
C. RULING ON GENETICS' DEMURRERS
Some months later, the demurrers of Genetics and Sandoz were heard by another judge. These demurrers and the ruling thereon will be referred to as the Genetics' demurrers. The judge sustained these demurrers without leave to amend for the following reasons:
1. That there was no recognized cause of action for the claim being made by plaintiff, and the court did not intend to create a new cause of action.
2. The court adopted the ruling on the Regents' demurrers, summarized above. The court believed it important that the plaintiff allege, if it were true, that the splenectomy had no medical purpose.
3. The court believed that Genetics and Sandoz were much removed from UCLA and its personnel, that the allegations of agency were conclusory, and that “[t]hose of ratification presuppose the existence of the fundamental right contended for.”
4. The court also held that the demurrers for lack of specificity, relating to the pleading of misrepresentations, were well taken.
The court declined to grant leave to amend because an appeal was already pending from the Regents' demurrers “that may resolve the central issues.” The court ordered dismissal as to Genetics and Sandoz.
ISSUES ON APPEAL
Plaintiff contends that the trial courts erred in sustaining the demurrers. He argues that the grounds given by the trial courts for sustaining the demurrers to the conversion cause of action were not well-taken, and that neither court should have sustained the demurrers to the twelve remaining causes of action based on the conclusion that the first cause of action for conversion was imperfectly pled. Finally, plaintiff invites this court “to fashion a new remedy to prevent the breach of duties and deceptions alleged in the Complaint from being rewarded with commercial profit” if we are not satisfied that the novel facts of this action fit traditional classifications.
We conclude that the demurrers to the first cause of action were improperly sustained because the plaintiff has adequately stated a cause of action for conversion. We also conclude that the matter should be remanded to the trial court in light of this ruling for decision of the remaining causes of action, which have never been expressly ruled upon.
DISCUSSION: SUFFICIENCY OF THE CONVERSION CAUSE OF ACTIONA. THE ELEMENTS OF CONVERSION
In deciding whether plaintiff can state a cause of action for conversion on the facts set forth in his complaint, it is necessary to review the requirements of conversion.
Conversion is “a distinct act of dominion wrongfully exerted over another's personal property in denial of or inconsistent with his title or rights therein, ․ without the owner's consent and without lawful justification.” (18 Am.Jur.2d, Conversion, § 1, pp. 145–146; fns. omitted.) It is “ ‘an act of wilful interference with a chattel, done without lawful justification, by which any person entitled thereto is deprived of use and possession.’ ” (De Vries v. Brumback (1960) 53 Cal.2d 643, 647, 2 Cal.Rptr. 764, 349 P.2d 532, quoting Prosser on Torts (2d ed. 1955) § 15, p. 66; see also Civ.Code, § 1712.)
For conversion, a plaintiff need only allege: “(1) plaintiffs' ownership or right to possession of the property at the time of the conversion; (2) defendants' conversion by a wrongful act or disposition of plaintiffs' property rights; and (3) damages. [Citation.]” (Baldwin v. Marina City Properties, Inc. (1978) 79 Cal.App.3d 393, 410, 145 Cal.Rptr. 406.)
Conversion is a strict liability tort. (City of Los Angeles v. Superior Court (1978) 85 Cal.App.3d 143, 149, 149 Cal.Rptr. 320.) “ ‘The foundation for the action of conversion rests neither in the knowledge nor the intent of the defendant. It rests upon the unwarranted interference by defendant with the dominion over the property of the plaintiff from which injury to the latter results. Therefore, neither good nor bad faith, neither care nor negligence, neither knowledge nor ignorance, are of the gist of the action. “The plaintiff's right of redress no longer depends upon his showing, in any way, that the defendant did the act in question from wrongful motives, or generally speaking, even intentionally; and hence the want of such motives, or of intention, is no defense. Nor, indeed, is negligence any necessary part of the case. Here, then, is a class of cases in which the tort consists in the breach of what may be called an absolute duty; the act itself (in some cases it must have caused damage) is unlawful and redressible as a tort.” [Citation.]’ ” (Byer v. Canadian Bank of Commerce (1937) 8 Cal.2d 297, 300, 65 P.2d 67, quoting 1 Bigelow on Torts, p. 6; Henderson v. Security Nat. Bank (1977) 72 Cal.App.3d 764, 770–771, 140 Cal.Rptr. 388.)
In a leading case on the subject, Poggi v. Scott (1914) 167 Cal. 372, 139 P. 815, the defendant bought a building in which plaintiff had been a subtenant, using a basement space to store numerous barrels of a “sound wine, seven years old.” (Id., at p. 373, 139 P. 815.) The defendant, unaware that the barrels contained wine, thought they were junk and sold them. He was held liable in spite of his lack of knowledge or intent. “The foundation for the action of conversion rests neither in the knowledge nor the intent of the defendant. It rests upon the unwarranted interference by defendant with the dominion over the property of the plaintiff from which injury to the latter results.” (Id., at p. 375, 139 P. 815; emphasis added.)
B. TISSUE OF A LIVING PERSON AS A FORM OF TANGIBLE PERSONAL PROPERTY
The complaint alleges that plaintiff's tissues, including his spleen, blood, and the cell-line derived from his cells “are his tangible personal property.” This is the crux of plaintiff's case for conversion.
As the ensuing analysis will establish, we have concluded that plaintiff's allegation of a property right in his own tissue is sufficient as a matter of law. We have been cited to no legal authority, public policy, nor universally known facts of biological science concerning the particular tissues referred to in this pleading (Evid.Code, § 451) which compel a conclusion that this plaintiff cannot have a sufficient legal interest in his own bodily tissues amounting to personal property. Absent plaintiff's consent to defendants' disposition of the tissues, or lawful justification, such as abandonment,7 the complaint adequately pleads all the elements of a cause of action for conversion.
We have approached this issue with caution. The evolution of civilization from slavery to freedom, from regarding people as chattels to recognition of the individual dignity of each person, necessitates prudence in attributing the qualities of property to human tissue. There is, however, a dramatic difference between having property rights in one's own body and being the property of another. To our knowledge, no public policy has ever been articulated, nor is there any statutory authority, against a property interest in one's own body. We are not called on to determine whether use of human tissue or body parts ought to be “gift based” or subject to a “free market.” That question of policy must be determined by the Legislature. In the instant case, the cell-line has already been commercialized by defendants. We are presented a fait accompli, leaving only the question of who shares in the proceeds.
Until recently, the physical human body, as distinguished from the mental and spiritual, was believed to have little value, other than as a source of labor. In recent history, we have seen the human body assume astonishing aspects of value. Taking the facts of this case, for instance, we are told that John Moore's mere cells could become the foundation of a multi-billion dollar industry from which patent holders could reap fortunes. For better or worse, we have irretrievably entered an age that requires examination of our understanding of the legal rights and relationships in the human body and the human cell.
“Genetic engineering promises a revolution more far-reaching than that wrought by the computer, one that may bring to a close the industrial revolution that has been the major shaper of our society for three hundred years․ [I]t is impossible to exaggerate the potential of genetic engineering for good and, if misused, for evil.” (Sylvester and Klotz, The Gene Age (1983) pp. 1–2.)
Since it is not possible to foresee all of the implications of our decision, judicial determinations will be necessary on a case-by-case basis. A scientific revolution of this magnitude may also compel legislative intervention.
In our evaluation of the law of property, we consider the definition of the word “property” and cases and statutes involving such issues as the right of dominion over one's own body; disposition of bodies after death; cornea transplants from deceased persons; and medical experimentation on live human subjects. We find nothing which negates, and much which supports, the conclusion that plaintiff had a property interest in his genetic material.
“As a matter of legal definition, ‘property’ refers not to a particular material object but to the right and interest or domination rightfully obtained over such object, with the unrestricted right to its use, enjoyment and disposition. In other words, [in] its strict legal sense ‘property’ signifies that dominion or indefinite right of user, control, and disposition which one may lawfully exercise over particular things or objects; thus ‘property’ is nothing more than a collection of rights.” (63A Am.Jur.2d, Property, § 1, p. 228; fns. omitted.)
The definitions of property are not restrictive and exclusive. “In legal usage, the word ‘property’ is a term of broad and extensive application, and it is also a term of large import, with the very broadest and most extensive signification.” (73 C.J.S., Property, § 4, pp. 163–164.)
Civil Code section 654 defines property as follows: “The ownership of a thing is the right of one or more persons to possess and use it to the exclusion of others. In this Code, the thing of which there may be ownership is called property.” Civil Code section 14, subdivision (3) provides: “The words ‘personal property’ include money, goods, chattels, things in action, and evidences of debt․” Although the definition of section 14 seems narrower than section 654, use of the word “include” indicates that the section is plainly not intended to be an exhaustive list. These sections have been interpreted to mean that “the word ‘property’ is a generic term which includes anything subject to ownership [citation]․” (Canfield v. Security First Nat. Bk. (1935) 8 Cal.App.2d 277, 284, 48 P.2d 133.)
Property is “ ‘every species of right and interest capable of being enjoyed as such upon which it is practicable to place a money value.’ ” (Yuba River Power Co. v. Nevada Irr. Dist. (1929) 207 Cal. 521, 523, 279 P. 128, quoting 22 R.C.L., p. 43, sec. 10.)
Plaintiff's spleen, which contained certain cells, was something over which plaintiff enjoyed the unrestricted right to use, control and disposition.
The rights of dominion over one's own body, and the interests one has therein, are recognized in many cases. These rights and interests are so akin to property interests that it would be a subterfuge to call them something else.
Venner v. State (1976) 30 Md.App. 599, 354 A.2d 483, involved determination of whether police illegally seized narcotics filled balloons found with defendant's feces in a hospital bedpan. The court dealt with the question of whether the balloons were abandoned. In discussing this point, the court noted: “It could not be said that a person has no property right in wastes or other materials which were once a part of or contained within his body, but which normally are discarded after their separation from the body. It is not unknown for a person to assert a continuing right of ownership, dominion, or control, for good reason or for no reason, over such things as excrement, fluid waste, secretions, hair, fingernails, toenails, blood, and organs or other parts of the body, whether their separation from the body is intentional, accidental, or merely the result of normal body functions.” (Id., 354 A.2d at p. 498, fn. omitted.)
Although the subject matter here does not equate with or have the dignity of the human cell, the legal significance of this reference is the court's statement that it cannot be said that a person has no property right in materials which were once part of his body.
In Bouvia v. Superior Court (1986) 179 Cal.App.3d 1127, 225 Cal.Rptr. 297, the court confirmed the long established rule that: “ ‘[A] person of adult years and in sound mind has the right, in the exercise of control over his own body, to determine whether or not to submit to lawful medical treatment.’ [Citation.] It follows that such a patient has the right to refuse any medical treatment, even that which may save or prolong her life. [Citations.]” (Id., at p. 1137, 225 Cal.Rptr. 297; emphasis in original.) The court reiterated Judge Cardozo's statement in Schloendorff v. Society of New York Hospital (1914) 211 N.Y. 125, 105 N.E. 92, 93, that: “ ‘Every human being of adult years and sound mind has a right to determine what shall be done with his own body․’ ” (Bouvia v. Superior Court, supra, 179 Cal.App.3d at p. 1139, 225 Cal.Rptr. 297.) This control of one's own body is certainly an incident of interest in property.
Some of the briefs suggest that the law which has developed with respect to dead bodies is analogous to the instant case. We do not completely agree. The “care of dead human bodies and the disposition of them by burial or otherwise have such a relation to the public health that they may well be regulated by law. [Citation.]” (22 Am.Jur.2d, Dead Bodies, § 1, p. 9, fn. 2.) Significant limitations imposed by law on the disposition of a body after death reflect public health concerns, rather than a legislative policy against a property interest in a living body. We see no inconsistency between the cases dealing with dead bodies and our conclusion.
In O'Donnell v. Slack (1899) 123 Cal. 285, 55 P. 906, the court summarized existing authorities by saying that although a decedent's next of kin might not have full proprietary ownership of the body, there exist some “property rights in the body which will be protected, and for a violation of which [the next of kin] are entitled to indemnification.” (Id., at p. 289, 55 P. 906; emphasis added.)
In Enos v. Snyder (1900) 131 Cal. 68, 63 P. 170, a decedent's surviving estranged wife and daughter demanded possession of decedent's body, for the purpose of burial, from the person with whom the decedent had been living at the time of his death. The decedent had expressed in his will the desire that he be buried in accordance with the wishes of the person with whom he had lived. The issue before the court was whether the next of kin had a right to the possession of decedent's body notwithstanding his wishes. The court held that, absent statute, “there is no property in a dead body, that it is not part of the estate of the deceased person, and that a man cannot by will dispose of that which after his death will be his corpse.” (Id., at p. 69, 63 P. 170.)
Over the years, the legal position set forth by Enos has been clarified. The present state of the law regarding a dead body is reflected in Cohen v. Groman Mortuary, Inc. (1964) 231 Cal.App.2d 1, 41 Cal.Rptr. 481. In Cohen, the court noted that there is “a quasi property right to its possession ․ for the limited purpose of determining who shall have its custody for burial. [Citations.]” (Id., at p. 4, 41 Cal.Rptr. 481.) The court pointed to the duty to bury the corpse and preserve the remains as a legal right the courts have recognized. (Cohen v. Groman Mortuary, Inc., supra, 231 Cal.App.2d at pp. 4–5, 41 Cal.Rptr. 481.) Thus, even though full property rights are not recognize in a dead body, a limited property interest has been found. (See Health & Saf. Code, § 7100, concerning the right to control disposition of remains, and § 7150, et seq., the Uniform Anatomical Gift Act.) 8
This property interest is also reflected in statutes and cases concerning the use of the corneas of a deceased person and statutes regarding medical experimentation on human subjects.9
The law that has emerged in the area of cornea transplants recognizes that body parts are not free for the taking, but are subject to the heir's right to dispose of them. (See Tillman v. Detroit Receiving Hosp. (1984) 138 Mich.App. 683, 360 N.W.2d 275, 277; Georgia Lions Eye Bank, Inc. v. Lavant (1985) 255 Ga. 60, 335 S.E.2d 127.)
The Protection of Human Subjects in Medical Experimentation Act, adopted in 1978, expresses a strong public policy that medical experimentation on human subjects “shall be undertaken with due respect to the preciousness of human life and the right of individuals to determine what is done to their own bodies.” (Health and Saf. Code, § 24171; emphasis added.) The statutory scheme is designed “to provide minimum statutory protection for the citizens of this state with regard to human experimentation․” (Health & Saf. Code, § 24171; emphasis added.) The principal device for this protection is the adoption of an “experimental subject's bill of rights,” which includes full advisement of the experiment and informed consent. (See Health & Saf. Code, §§ 24172 to 24175.) 10
The essence of a property interest—the ultimate right of control—therefore exists with regard to one's own human body.
Even though the rights and interests one has over one's own body may be subject to important limitations because of public health concerns, the absence of unlimited or unrestricted dominion and control does not negate the existence of a property right for the purpose of a conversion action. (See Corey v. Struve (1915) 170 Cal. 170, 173, 149 P. 48) (disapproved on another point Maben v. Rankin (1961) 55 Cal.2d 139, 144, 10 Cal.Rptr. 353, 358 P.2d 681.)
Defendants' position that plaintiff cannot own his tissue, but that they can, is fraught with irony. Apparently, defendants see nothing abnormal in their exclusive control of plaintiff's excised spleen, nor in their patenting of a living organism derived therefrom. We cannot reconcile defendant's assertion of what appears to be their property interest in removed tissue and the resulting cell-line with their contention that the source of the material has no rights therein.
Defendants also claim that plaintiff cannot have an interest in the cell-line derived from his tissues since he is only the source of the thing studied. “Thus, the cells involved have no inherent value until a cell line is derived and created to produce useful products. Plaintiff tries to obscure this unassailable fact by creating the impression his cells themselves have miraculous curative powers․” (Emphasis in original.)
Defendants contend that plaintiff has no property right in the knowledge gained or the new things made in the course of the study of his cells. This is an inaccurate characterization of this case. Plaintiff's complaint does not allege a conversion of ideas gained from study of his cells, but conversion of the cells, and their progeny, themselves. Plaintiff was not simply the object of study to increase knowledge. His spleen and cells were taken by defendants to their laboratory, where they extracted his genetic material, placed it in a growth medium, and, by cell division, created an “immortal” cell-line—one that could forever produce products of enormous commercial value to defendants.11 Human cells were essential to the creation of the cell-line. (See sources cited in Note, Toward the Right of Commerciality: Recognizing Property Rights in the Commercial Value of Human Tissue (1986) 34 UCLA L.Rev. 207, 209, fns. 6–9 (hereinafter Toward the Right of Commerciality.)) The complaint alleges that defendants exploited plaintiff's cells, not just the knowledge gained from them. Without these small indispensable pieces of plaintiff, there could have been no three billion dollar cell-line.
The fact that defendants' skill and efforts modified the tissue and enhanced its value does not negate the existence of a conversion. Any questions as to defendants' alteration of plaintiff's tissue go to what damages, if any, he might be entitled if successful at trial.12
Defendants further contend that plaintiff's spleen could not be converted because, being diseased, it was a thing of no value. The allegations in the complaint, however, indicate the opposite is true. While removal of plaintiff's spleen may have been necessary to the treatment of his disease, the cells it contained became the foundation of a multi-billion dollar industry. The extraordinary lengths to which defendants went to obtain a specimen for their work shows their belief in its value. This belief has proven correct. The fact the spleen was at one time of little utility to plaintiff does not mean its value was so minimal it could not be converted.13
Defendants' argument that the DNA from plaintiff's cells is not a part of him over which he has the ultimate power of disposition during his life is also untenable.
In Motschenbacher v. R.J. Reynolds Tobacco Company (9th Cir.1974) 498 F.2d 821, the court found that, although California courts had not yet spoken on the subject, they would “afford legal protection to an individual's proprietary interest in his own identity.” (Id., at p. 825.) The court went on: “We need not decide whether they would do so under the rubric of ‘privacy,’ ‘property,’ or ‘publicity’; we only determine that they would recognize such an interest and protect it.” (Id., at pp. 825–826, fns. omitted; see also Midler v. Ford Motor Co. (9th Cir.1988) 849 F.2d 460.)
In Lugosi v. Universal Pictures (1979) 25 Cal.3d 813, 160 Cal.Rptr. 323, 603 P.2d 425, the Supreme Court avoided determining whether the interest of Bela Lugosi in his name, face, likeness and Count Dracula persona, in connection with any kind of business was “property.” Turning to Prosser, the court found the dispute “pointless.” “ ‘Once protected by the law, [the right of a person to the use of his name and likeness] ․ is a right of value upon which plaintiff can capitalize by selling licenses.’ ” (Id., at p. 819, 160 Cal.Rptr. 323, 603 P.2d 425, emphasis omitted, quoting Prosser, Law of Torts (4th ed. 1971) p. 807.) The court found the right was personal to Lugosi and did not survive him.
Plaintiff's cells and genes are a part of his person. Putting aside the effect of environment, “[a]n individual's genotype contains all of the genetic instructions essential for human development, growth, and reproduction․ [¶] All human traits, including weight, strength, height, sex, skin color, hair texture, fingerprint pattern, blood type, intelligence and aspects of personality (for example, temperament), are ultimately determined by the information encoded in the DNA.” (Gordon Edlin, Genetic Principles—Human and Social Consequences (1984) pp. 406–407.) If the courts have found a sufficient proprietary interest in one's persona, how could one not have a right in one's own genetic material, something far more profoundly the essence of one's human uniqueness than a name or a face?
A patient must have the ultimate power to control what becomes of his or her tissues. To hold otherwise would open the door to a massive invasion of human privacy and dignity in the name of medical progress.
In oral argument, the Regents expressed concern over the implication of our decision on medical research. They contended that unencumbered access to human tissue for research is essential to progress and public health. They argued that these sources must remain unencumbered, and medical researchers be free to both combine these materials with tissue taken from others, and dispose of the tissues, without answering to the person from whom the tissue was taken.14
The Regents cite no statutory or case law which authorizes such an invasion of fundamental rights. We have no reason to believe that medical research will suffer by requiring the consent of a donor of the tissue before it can be appropriated. Absent lawful authority, medical researchers are no more free to impose their priorities over the unconsented use of cells than any intruder on any other property.
We are told that if plaintiff is permitted to have decision making authority and a financial interest in the cell-line, he would then have the unlimited power to inhibit medical research that could potentially benefit humanity. He could conceivably go from institution to institution seeking the highest bid, and if dissatisfied, “would claim the right simply to prohibit the research entirely.”
We concede that, if informed, a patient might refuse to participate in a research program. We would give the patient that right. As to defendants' concern that a patient might seek the greatest economic gain for his participation, this argument is unpersuasive because it fails to explain why defendants, who patented plaintiff's cell-line and are benefiting financially from it, are any more to be trusted with these momentous decisions than the person whose cells are being used. It has been suggested by writers that biotechnology is no longer a purely research oriented field in which the primary incentives are academic or for the betterment of humanity. Biological materials no longer pass freely to all scientists. As here, the rush to patent for exclusive use is rampant. The links being established between academics and industry to profitize biological specimens are a subject of great concern.15 If this science has become science for profit, then we fail to see any justification for excluding the patient from participation in those profits.
It is also argued that by giving patients a financial interest in their tissues, donations could be inhibited, costs increased and people of modest means driven out of the health care market. To the extent that unacceptable consequences, which can now only be the subject of speculation, do follow, legislative solutions are possible and likely. The court's role in this instance is not to provide solutions to all possible social concerns, but to resolve the dispute presented as to individual rights and interests.
C. ISSUE OF ABANDONMENT OF TISSUE IN SURGERY
Defendants argue that even if plaintiff's spleen is personal property, its surgical removal was an abandonment by him of a diseased organ. They assert that he cannot, therefore, bring an action for conversion.
The essential element of abandonment is the intent to abandon. The owner of the property abandoned must be “ ‘entirely indifferent as to what may become of it or as to who may thereafter possess it.’ ” (Martin v. Cassidy (1957) 149 Cal.App.2d 106, 110, 307 P.2d 981, quoting 1 Cal.Jur.2d, Abandonment, § 2, p. 2.)
“It may be said that abandonment is made up of two elements, act and intent, and the intent must be gathered from all the facts and circumstances of the case. [Citations.]” (Peal v. Gulf Red Cedar Co. (1936) 15 Cal.App.2d 196, 199, 59 P.2d 183.)
The question whether the plaintiff abandoned his spleen, or any of the other tissues taken by the defendants, is plainly a question of fact as to what his intent was at the time. A demurrer does not reach such questions. A consent to removal of a diseased organ, or the taking of blood or other bodily tissues, does not necessarily imply an intent to abandon such organ, blood or tissue. The only fact alleged in the complaint on the subject is that the spleen was surgically removed, and that, had plaintiff known of defendants' intentions regarding the spleen, he would not have consented to its removal at UCLA. While it may be true that many people under such circumstance would be entirely indifferent to the disposition of removed tissue, we cannot assume plaintiff shared this state of mind. Nothing in the complaint indicates that plaintiff had an intent to abandon his spleen, and we do not find that, as a matter of law, anyone who consents to surgery abandons all removed tissue to the first person to claim it. Certainly, in the example of an unconscious patient, the concept of abandonment becomes ridiculous.
In California, absent evidence of a contrary intent or agreement, the reasonable expectation of a patient regarding tissue removed in the course of surgery would be that it may be examined by medical personnel for treatment purposes, and then promptly and permanently disposed of by internment or incineration in compliance with Health and Safety Code section 7054.4.16 Simply consenting to surgery under such circumstances hardly shows indifference to what may become of a removed organ or who may assert possession of it. Any use to which there was no consent, or which is not within the accepted understanding of the patient, is a conversion. It cannot be seriously asserted that a patient abandons a severed organ to the first person who takes it, nor can it be presumed that the patient is indifferent to whatever use might be made of it.
An inference of abandonment is particularly inappropriate when it comes to the use undertaken by defendants involving recombinant DNA technology. Almost from the beginning, this technology has incited intense moral, religious and ethical concerns.17 There are many patients whose religious beliefs would be deeply violated by use of their cells in recombinant DNA experiments without their consent, and who, on being informed, would hardly be disinterested in the fate of their removed tissue.
D. ISSUE OF THE SCOPE OF CONSENT TO TISSUE REMOVAL
Defendants next contend that there was no conversion since the plaintiff consented to the removal of the spleen and other tissues.
The complaint is clear that plaintiff consented to the splenectomy and some of the other removals of tissue and blood. It is also clear that he did not consent to the commercial exploitation and research on his tissue which were not directly related to his treatment. He repeatedly alleges that the defendants did not inform him of the use they were making of his spleen or other tissues.
The consent to the splenectomy is not attached to the complaint and there is no basis for concluding on the facts before us that this consent contained a specific authorization for either research unrelated to treatment or commercial exploitation. If it should develop that plaintiff did expressly consent to what took place, or if such consent can be implied from some other facts, plaintiff will have no basis for a claim for conversion. We conclude, however, that simple consent to surgery does not imply a consent to medical research on the patient's tissues unrelated to treatment nor to commercial exploitation of the patient's tissues.
The complaint also alleges that on April 11, 1983, when plaintiff returned to UCLA to have blood and tissue samples taken, he signed a consent form. The complaint indicates that plaintiff consented to research on the tissue samples taken on that occasion. There is nothing that indicates he consented to commercial exploitation of his tissues. Nor was this consent form in any way retroactive. The defendants had already filed their second Mo cell-line patent (Appendix A) on January 6, 1983, three months before the consent for research was obtained in April of 1983. The consent to research on this blood sample, therefore, could have no effect on plaintiff's claim that his tissues were converted to produce the Mo cell-line.18
Defendants also seemingly assert that Health and Safety Code section 7054.4 gives them the right to engage in unlimited scientific use of a patient's bodily parts without the patient's consent. (See fn. 16 above.) We find nothing in this section authorizing indiscriminate research on human organs, let alone their commercial exploitation without the consent of the living patient. There is nothing in the history of these laws to indicate that the Legislature intended that patients relinquish all rights to excised tissue or that such tissue may be commercially exploited by medical researchers. On the facts before us, plaintiff's claim of conversion is not barred by his consent.
E. SPECIFIC RULINGS ON FIRST CAUSE OF ACTION
We now address the sufficiency of the specific grounds given by the trial court in sustaining the Regent's demurrers.
1. PLEADING PLAINTIFF'S KNOWLEDGE OF RESEARCH USES
In sustaining the Regents' demurrers, the court ruled that plaintiff must allege that he neither knew nor had reason to know that his tissue might be used for medical or scientific study as well as for his diagnosis and treatment. The court that ruled on the Genetics demurrers concurred in this requirement, and added that plaintiff must also allege that there was no medical purpose in the splenectomy.
Defendants do not dispute that the demurrers should not have been sustained on this ground. Whether plaintiff knew or had reason to believe that defendants might use the spleen for purposes other than treatment is not germane to a conversion cause of action; any use of plaintiff's tissues required his consent. It is no defense to claim that plaintiff should have known his spleen might be expropriated by medical science. If he did not consent, its taking was conversion. If a trier of fact should find that plaintiff did indeed consent to any or all of the uses defendants made of his body parts, then, to that extent, his claims will be defeated.
2. PLEADING DEFENDANTS' KNOWLEDGE OF COMMERCIAL VALUE
In sustaining the Regents' demurrers, the trial court concluded that it was necessary for the plaintiff to allege that the defendants knew of the commercial value of his cells and intended to exploit them before the first taking of blood and tissue occurred on October 5, 1976, three weeks before the splenectomy.
Plaintiff's complaint alleges that on October 5, 1976, defendants took blood, bone marrow aspirate and other bodily substances from plaintiff. Plaintiff did not include the allegations required by the court's ruling, and we find such allegations unnecessary to state a cause of action for conversion. The intent of the defendants is not relevant for conversion since, as noted supra, conversion is a species of absolute liability. (See Byer v. Canadian Bank of Commerce, supra, 8 Cal.2d at p. 300, 65 P.2d 67.)
3. PLEADING LACK OF THERAPEUTIC PURPOSE
In sustaining the Regents' demurrers, the court also criticized plaintiff's failure to allege whether or not he consented to the removal of his spleen for therapeutic purposes. The court implied that if it was necessary to remove the spleen to save the plaintiff's life, no conversion occurred.
The plaintiff's reasons for agreeing to removal of his spleen are of no consequence to this cause of action. If the defendants used the spleen, or part of it, for a purpose beyond plaintiff's consent, then there has been a conversion. The essence of the complaint is not the removal of the spleen, blood, or other tissues, but the defendants' alleged subsequent use of them.
4. ATTACHMENT OF CONSENTS
The court also found that the plaintiff should have attached both a copy of the consent to the splenectomy in October of 1976, and the September 20, 1983 consent mentioned in the complaint (Appendix B here).
The record on appeal from the Superior Court contains the consent form dated September 20, 1983, as an attachment to the third amended complaint. The splenectomy consent is not attached as an exhibit and is not referred to as an exhibit in the complaint. The consent of September 20, 1983, therefore seems to have been temporarily misplaced in the trial court's file.
We have not been directed to any authority by any party which requires that a document be attached to a complaint. Indeed, the contrary appears to be the rule. (See 4 Witkin, Cal. Procedure (3d ed. 1985) Pleading, § 381, p. 431.) A document may be pled in haec verba or by legal effect. The pleading here, by legal effect, was perfectly acceptable.
In sum, none of the grounds given by the trial court for sustaining the Regent's demurrers were appropriate.
DISCUSSION: PROCEDURAL ISSUES ON APPEALA. LACK OF LEAVE TO AMEND—GENETICS' DEMURRERS
As noted above, both trial courts sustained demurrers to the entire complaint consisting of thirteen causes of action. The demurrers by Genetics and Sandoz were sustained without leave to amend. The Regents' demurrers were sustained with leave, but the plaintiff elected to stand on his complaint.
The order sustaining the demurrers of Genetics and Sandoz must be reversed for failure to grant leave to amend.
It is well settled that the “trial court abuses its discretion in sustaining a demurrer without leave to amend, if there is a reasonable possibility that a defect in the complaint can be cured by amendment or if the pleading can be liberally construed to state a cause of action. [Citations.]” (Hooper v. Deukmejian (1981) 122 Cal.App.3d 987, 994, 176 Cal.Rptr. 569; emphasis in original.) The trial court should have allowed an amendment regarding those allegations which it found defectively pled.19
The trial court's order was based on the conclusion that no cause of action could be stated on the facts of the case under any legal theory. As we have pointed out in Part IV of this opinion, a conversion cause of action was adequately pleaded. The court's ruling on the Genetics and Sandoz demurrers made certain additional rulings concerning the adequacy of allegations of agency, and in respect to the fourth cause of action for misrepresentation. We briefly discuss these issues.
The court below concluded that Genetics and Sandoz “are much removed from the medical treatment given to plaintiff by the hospital and its personnel. The allegations of agency are highly conclusionary. Those of ratification presuppose the existence of the fundamental right contended for.” 20 In regard to the misrepresentation cause of action, the judge ruled: “the specific grounds of demurrer, and in particular the lack of specific pleading as to who made which misrepresentations, and when and where, their authority to bind these defendants and like technical matters are for the most part well taken.”
The court, however, went on to say: “Normally the court would consider granting leave to amend; it does not do so here since there already is an appeal pending that may resolve the central issues.”
Contrary to defendants' contention, the plaintiff did not agree to the court's decision to deny leave to amend. The record reflects that counsel for the plaintiff, although willing to submit on the court's tentative ruling sustaining without leave, added: “Obviously, plaintiff would prefer an opportunity to amend his pleadings. In light of the recent discovery, they may have been able to give us more specific allegations regarding the conduct of Genetics Institute.”
The judge explained that his reason for not granting leave to amend was that all the defendants were in the same position in regard to the central issue to be reviewed by the appellate court, whether an action of any kind could be maintained by plaintiff under these novel facts. He said: “If you were to say to me now I want leave to amend because these defendants [Genetics and Sandoz] are different than yours [Golde, Quan and the Regents], I might consider that. But I view these defendants as being in an a fortiori position to the other defendants, really. If Judge Deering's ruling [on the Regents' demurrers] is affirmed, then I think the result will automatically follow as to these defendants.”
The foregoing was not an offer by the court to give plaintiff another opportunity to amend. It was an expression of the court's reasons for not doing so. The only amendment, in the court's view, that might warrant leave to amend would be one which placed Genetics and Sandoz in a position different from the other defendants on the central issue of the case. Since plaintiff could not offer such an amendment because all defendants indeed are in the same position in respect to the central issues of the case, the court did not grant leave to amend.
At the hearing on the demurrers, all parties were aware of the appeal pending from the Regents' demurrers, which they believed would resolve the central issues in both actions. The parties understood that placement of the Genetics demurrers in the same posture would facilitate appellate review, but this did not amount to a stipulation by the plaintiff that the court's order sustaining the demurrers without leave was proper, particularly as to the specific rulings on agency and misrepresentation.
Since we hold that the conversion cause of action was properly pleaded, we remand the Genetics demurrers to the trial court for rehearing on the specific defects alleged in the remaining causes of action.
B. SCOPE OF REVIEW AFTER VOLUNTARY DISMISSAL—REGENTS' DEMURRERS
As to the Regents' demurrers, the court allowed leave to amend which the plaintiff declined to do. Defendants point to the ruling in Gonzales v. State of California (1977) 68 Cal.App.3d 621, 635, 137 Cal.Rptr. 681: “where the lower court sustains the defendant's demurrer with leave to amend and the plaintiff declines to do so, electing to stand on his complaint, the judgment of dismissal must be affirmed if the complaint is objectionable on any ground raised by the demurrer. [Citations.]” Defendants ask this court to uphold the trial court if we find the complaint is objectionable on any ground.
One example of an issue that this contention encompasses is the Regents' argument that the complaint fails to allege facts sufficient to overcome the governmental tort immunity established under Government Code section 818.8. This section gives immunity to public entities for misrepresentations by their employees. This was one of the grounds of the demurrer that was not expressly addressed in the trial court's decision. Were this court to agree with defendants' reasoning, we would have to affirm the dismissal of the entire complaint if we found any basis to support the trial court's ruling on the demurrer even though it was not addressed by the trial court. There may well be parts of the pleading that are defective, with the consequence, under defendants' theory, that the entire complaint would fail.
This result would be incongruous since it would eliminate an action against the Regents, Golde and Quan, even though the trial court's ruling on the demurrers was erroneous. At the same time, such a holding would continue proceedings against two other defendants, Genetics and Sandoz, because the plaintiff in their demurrer did not choose to stand on his pleading. This result was not contemplated by the courts and the parties below. Instead, the parties sought a clear ruling by the trial courts, which could then be promptly reviewed by an appellate court.
Under these circumstances, we elect to follow the procedure outlined in Collins v. Marvel Land Co. (1970) 13 Cal.App.3d 34, 44–45, 91 Cal.Rptr. 291. In that case, demurrers were sustained with leave to amend on all four counts in the pleading. On plaintiff's failure to amend, the action was dismissed. The court of appeal held that the demurrers to three of the four causes of action were erroneously sustained, and that “the judgment should be reversed with directions to overrule those demurrers. The reversal reinstates the status quo [ante] which existed prior to the judgment thus nullified [citation], including leave to plaintiffs to amend which was not terminally cut off until entry of the judgment of dismissal. Thus, subject to the trial court fixing the period of time, plaintiffs in the milieu of this case should be given the opportunity to amend their third alleged cause of action, the demurrer to which we find to have been properly interposed.” (Id., at pp. 44–45, 91 Cal.Rptr. 291.)
The court was aware of the rule “that if plaintiff fails to take advantage of the leave to amend granted by the trial court and the demurrer is found to have been properly sustained, the appellate court will not reverse a judgment of dismissal even if plaintiff could possible amend his complaint. [Citations.] However, we have not found the rule applied in a situation such as here presented where it could be applied only to one count or cause of action of a multi-count complaint, and where the ruling sustained the demurrers is reversed on three other counts. In a sense our disposition is a grant of a new trial [citation] and there is no question but that parties may be permitted to amend their pleadings upon a new trial. A judgment on this single count or cause of action found to be defective could not be entered until final disposition of the other causes of action, because this case involves one overall dispute between two identical sets of parties in each count or cause. [Citations.]” (Id., at p. 45, 91 Cal.Rptr. 291.)
In view of Collins, we do not resolve the Regents' demurrers beyond the first cause of action, and remand the action to the trial court to review the remaining causes of action, including the potential for amendment, in light of our decision.
C. FAILURE TO DISCUSS ISSUES IN APPELLATE BRIEFS
Defendants contend that plaintiff abandoned his fifth through twelfth causes of action since he failed to discuss them in his opening brief. We note that, absent a general reference in the reply brief that he stood by all his causes of action, plaintiff did not reply to this claim by the defendants. It is the appellant's obligation to point out the errors committed below. The reviewing court “will notice only those assignments pointed out in the brief of an appellant, all others are deemed to have been waived or abandoned.” (Title G. & T. Co. v. Fraternal Finance Co. (1934) 220 Cal. 362, 363, 30 P.2d 515.) The rule is not mandatory, but was developed for the convenience of the court. (9 Witkin, Cal. Procedure (3d ed. 1985) Appeal, §§ 479–480, pp. 469–472.) Because of our decision to allow the parties and the court to reexamine this pleading in light of our ruling on the primary issue in the case, we decline to apply the rule of Title in the instant case.
D. EFFECT OF DISMISSAL ON SCOPE OF COMPLAINT
Defendants lastly contend that one of the effects of plaintiff's refusal to amend following the trial court's ruling on the Regents' demurrers is that he has thereby voluntarily dismissed his claims based on any taking of his tissue after the splenectomy. Defendants assert that the trial judge's order was directed only to inadequacies in pleading the events up to the removal of the spleen. They contend that for the order to be a final judgment, plaintiff would have had to voluntarily drop all claims that the trial judge did not decide since there cannot be an appealable judgment that decides less than all issues in a case. (See IFS Industries, Inc. v. Stephens (1984) 159 Cal.App.3d 740, 756, 205 Cal.Rptr. 915.) We disagree.
The trial judge ruled on what he regarded as the fundamental defects in the pleading, and, from those defects, concluded that the entire balance of the complaint was defective. Although the trial judge did not specifically mention post-splenectomy conversion, his order was directed to the portion of the first cause of action where those allegations are raised. The order, therefore, encompassed the conversion of plaintiff's blood and tissue samples in the years following the splenectomy.
The judgments of dismissal are reversed with directions to the trial court to take proceedings consistent with the views set forth in the foregoing opinion.
Appellant to recover costs.
APPENDIX A. THE MO CELL–LINE PATENT
Saxon et al., Annals of Internal Medicine, (1978), 58:323-326.
Weisbart et al., Clin. Immunology & Immunopathology, (1979), 14:441-448.
Weisbart et al., J. Lab. Clin. Med., (1979), 93:622-626.
Lusis et al., In Viva and In Vitro Erythropoiesis, 1980, pp. 97-106.
Golde et al., Blood, (1978), 51:1068-1071.
Golde et al., PNAS, USA, (1980), 77:593-596.
Golde et al., Annals of Internal Medicine, (1980), 92:650-662.
Primary Examiner—Howard E. Schain
Attorney, Agent, or Firm—Bertram I. Rowland
Human T-lymphoblast cell line, Proteinaceous products produced therefrom, messenger RNA and DNA expressing the proteinaceous products. A human T-lymphoblast celi line (Mo) maintained as a continuous culture constitutively produces proteins, including immune interferon, neutrophil migration inhibition factor, granulocyte-macrophage colony-stimulating activity and erythroid-potentiating activity, as well as other proteins produced by T-cells.
22 claims, No Drawings
UNIQUE T-LYMPHOCYTE LINE AND PRODUCTSDERIVED THEREFROM
The invention described herein was made in the course of or under a grant from the United States Public Health Service.
CROSS-REFERENCE TO RELATED
This application is a continuation of pending prior application Ser. No. 229,900, filed Jan. 30, 1981. Now abandoned.
Field of the Invention
Cell regulation is mediated by a wide variety of polypeptides. Historically, few of these polypeptides are produced in sufficient amount to be isolated and characterized. Even in those situations where a particular polypeptide was capable of isolation and characterization, the number of amino acids constituting the polypeptide normally precluded synthesis by conventional peptide bond formation in commercially useful amounts.
In the last few years a number of discoveries have occurred which at the present time promise opportunities for the detection, isolation, and production in commercially useful amounts of naturally occurring proteins, which fulfill a wide variety of cell regulatory functions.
The ability to insert a gene into a replicating vector, such as a plasmid or phage, and transform a microorganism with the resulting hybrid has introduced new techniques for the production of macromolecular polypeptides. These techniques not only afford the opportunity to obtain polypeptides in abundance, but allow for study of the polypeptides and use of the polypeptides in regulating cell functions in vitro and in vivo.
At the present time, in order to be able to produce a polypeptide of interest by “genetic engineering,” there are basically three methods which may be employed. Once the amino acid sequence of DNA sequence is known, for relatively small polypeptides, the sequence can be synthesized. See for example, Wu, R. (1978) Ann. Rev. of Biochem. 47, 607. Alternatively, one can excise the gene either directly, where the chromosome has been mapped and there are available restriction sites adjacent the gene, or indirectly by genomic cloning. Thirdly, one can isolate the messenger RNA and employing reverse transcriptase produce cDNA from which dsDNA may be obtained.
Because of the cumbersome nature and difficulties associated with synthesis and the existence of introns present in chromosomal DNA, the messenger RNA is frequently the desired route where genetic engineering is involved.
In each cell, there is continuously produced a large number of different messenger RNA molecules. Therefore, means must be provided for isolating the messenger RNA of interest from the other messenger RNA molecules. Where a messenger RNA of interest is normally produced in only small amounts as compared to the total amount of messenger RNA, it is frequently desirable, if not necessary, to obtain cells which enhance the amount of messenger RNA of interest present in the cell.
As an alternative to genetic engineering, the ability to culture tumor cells in vitro offers an opportunity for the production of a wide variety of polypeptides. Where the tumor cells do not regulate the production of one or more polypeptides of interest, the tumor cells will constitutively produce these polypeptides. By isolating specific tumor cells and establishing a culture, which can be expanded and maintained for long periods of time, one can directly produce the polypeptides of interest from a “normal” host cell. In this manner, one avoids the need to isolate the gene of interest and perform the numerous steps involved with successful genetic engineering. In addition, where modification of the polypeptide naturally occurs, such as glycosylation, and the modification affects the activity of the polypeptide, it will be observable to employ the native host as the polypeptide source.
Brief Description of the Prior Art
The existence of the Mo line was first described in a series of articles by Golde, et al., Blood 52:1068-1072, 1978; Saxon et al. Ann. Intrn. Med. 88:323-326, 1978 and Saxon et al., J. Immunol. 120:778-782, 1978. At no time has the Mo cell line been available to other than the investigators involved with its initial discovery and only the conditioned medium from the cell line has been made available to a limited number of investigators for collaborative work with the original discoverers of the Mo cell line. The following additional articles have been published concerning the Mo cell line: Cline and Golde, Nature 277:177-181, 1979; Minden et al. Blood 54:186-195,1979; Weisbart et al. J. Lab. Clin. Med. 93:622-626, 1979; Weisbart et al. Clin. Immunol. Immunopathol. 14:441-448, 1979; Golde et al. Proc. Natl. Acad. Sci. USA 77:593-596, 1980; In, Biochemical Characterization of Lymphokines, edited by A. L. de Weck, Academic Press, New York, 1980 pages 221-225; Lusis and Golde, In In Vivo and In Vitro Erythropoiesis: The Friend System, edited by G. B. Rossi, Elsevier/North-Holland Biomedical Press, Amsterdam, 1980, pages 97-106; and Quan et al., J. Histochem. Cytochem. 28:434-440, 1980.
SUMMARY OF THE INVENTION
A cell line (Mo) has been established with spleen cells from a patient with a T-cell variant of hairy-cell leukemia. The cells have been shown to be capable of continuous culture for an indefinite period of time, while maintaining the properties of T-lymphoblasts. The cells constitutively produce a wide variety of proteins, including growth factors, such as colony stimulating factor, useful for the growth of granulocyte-macrophage colonies in vitro and erythroid-potentiating activity, which is capable of potentiating the formation of both large and small human erythroid colonies in vitro; human immune interferon, neutrophil migration-inhibition activity, as well as other polypeptides produced by T-lymphoblast cells many of which are secreted and isolatable from the medium.
The cells provide a continuous source of the above proteins, as naturally modified which can be isolated by conventional ways. In addition, due to the constitutive production of the proteins, the cells provide either directly or indirectly, a source of the genes for the proteins of interest, which by conventional genetic engineering techniques, can be introduced into microorganisms for continuous large scale production of the proteins.
DESCRIPTION OF THE SPECIFIC
A novel cell line was established, referred to as the Mo cell line. The cell line was established with spleen cells from a patient with a T-cell variant of hairy-cell leukemia. The cells have been shown to be capable of growing in continuous culture for an indefinite period while maintaining the properties of T-lymphoblasts. More than 60% of the cells rosette with sheep erythrocytes and carry the tartrate-resistant isozyme five of acid phosphatase, characteristic of hairy-cell leukemia. No evidence of immunoglobulin synthesis was observed. The Mo cells are not infected with Epstein-Barr virus and are lysed by antithymocyte globulin in the presence of complement. The cells respond to the mitogen phytohemagglutinin by increased incorporation of [3 H] thymidine.
The Mo cell line is conveniently cultured in alpha medium containing 20% fetal calf serum and 104 M alpha-thioglycerol. While the cell line can also be cultured in serum-free medium, it grows considerably more slowly.
The Mo cell line is unique in liberating many of the products known to be elaborated by lectin- or antigen- stimulated normal T cells. The Mo cell line constitutively produces a wide variety of proteins having different physiological activity in isolatable amounts. Thus, the Mo cell line provides a method for production of a large number of polypeptides of physiologic interest. In addition, because of the constitutive production of these polypeptides, the Mo cell line also offers the messenger RNAs for these polypeptides in relatively large amounts compared to the total amount of messenger RNA present. By employing conventional techniques, the messenger RNAs for the desired polypeptides may be separated from the mass of messenger RNA and used for production of cDNA.
In describing the various products produced by the Mo cell line, the direct production of the polypeptides by the Mo cell line and the isolation of the polypeptides will first be described. This will then be followed by a description of the production of the polypeptides employing hybrid DNA techniques. EPA
The first polypeptide of interest is referred to as erythroid-potentiating activity (EPA). This polypeptide enhances the proliferation of human erythroid progenitors in vitro. The polypeptide is an acidic glycoprotein of molecular weight about 45 kdal. The EPA activity in Mo-conditioned medium is bound by DEAE-Sephadex at pH 7.4 and by use of a linear NaCl gradient is eluted from the resin as a single broad peak between 0.15 and 0.25 M NaCl. When subjected to preparative isoelectric focusing in granulated gel, the bulk of the EPA is found in the very acid portions of the gel corresponding to pI 3.5-4.8. The EPA is bound by concanavalin A-Sepharose and most of the activity can be recovered from the lectin by elution with 0.2 M methyl-alpha-D-mannoside. The EPA is heat stable as evidenced by its retaining substantially all of its activity after heating a Mo-conditioned medium in a boiling water bath for 10 min.
By conventional techniques the EPA can be concentrated and substantially freed of other protienaceous components of the Mo-conditioned medium. Concentrates having a specific activity of at least 25,000 units/mg protein, usually at least 50,000 units/mg protein, are readily obtained by gel exclusion chromatography usually preceded by other techniques e.g. chromatography and electrophoresis. (The unit activity will be defined in the experimental section.)
The EPA in the presence of 0.5 units/ml erythropoietin is active at a concentration less than 0.1 nM, usually less than about 0.01 nM. Stimulation is obtained with both early (14 day BFU-E) and late (7-day CFU-E) human erythroid progenitors.
The subject EPA is important in early events in erythropoiesis. The EPA potentiates the formation of both large and small human erythroid colonies in vitro.
The EPA can find use in enhancing the development of erythrocytes in blood. Other uses for EPA are the production of erythrocytes in vitro, use of EPA in evaluating the ability of stem cells to form erythrocytes, treatment of human host having a deficiency of EPA, and the like.
The next polypeptide of interest is colony stimulating factor (CSF) which provides in vitro proliferation and differentiation of granulocyte-macrophage progenitors.
The CSF obtained from the Mo cell line is heat stable, being stable at temperatures up to about 70 ° C. and about half the activity is recovered after 5 mins. of incubation at 90 ° C. in 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.2, containing 0.1% bovine serum albumin. The molecular weight is about 34 kdal for the glycosylated polypeptide. The presence of sialic acid is indicated, based on changes in isoelectric focusing after neuraminidase treatment. The removal of sialic acid did not affect the activity of the polypeptide. The pI is between pH 4.0 and 5.2. With ion-exchange chromatography, elution from DEAE-Sephadex (equili-brated with 0.02 M Tris, pH 7.4) is between 0.12 and 0.22 M NaCl from serum-free medium, while between 0.18 M and 0.22 M NaCl from serum-containing conditioned medium. The CSF is not inactivated by mercapto ethanol in concentrations as high as 0.1 M in the absence of denaturants, such as 8 M urea.
By conventional purification techniques, such as combinations of affinity chromatography and gel exclusion chromatography, CSF can be obtained in concentrates having a specific activity of greater than 0.5 units/mg, usually greater than 3.5 units/mg protein and preferably greater than 10 units/mg protein. (Units will be defined in the experimental section.) The CSF from the Mo-cell conditioned medium is more active than leukocyte CSF in stimulating granulocyte-macrophage colonies and relatively inert in stimulating mouse granulocyte-macrophage colonies from bone marrow cells.
The CSF can be used for assaying stem cells for the ability to form granulcytes and macrophage, to grow granulocytes and macrophage in vitro, and to enhance the formation of granulocytes and macrophage in a host which is deficient in CSF.
The next protein of interest is neutrophil migration inhibition factor (NIF-T). The neutrophil migration inhibition factor is a heat stable 35-45 kdal glycoprotein resistant to diisopropyl fluorophosphate and reversibly inhibites neutrophil migration.
Type 2 immune interferon
In addition to the other proteins, immune interferon, Type 2 is observed. The presence of interferon was established by assays employing human fibroblasts and vesicular stomatitis virus in a classical plaque-reduction assay for interferon. The fact that it is immune interferon Type 2 was established by its instability at pH2; instability upon heating, abolishment of the activity by exposure to an antibody to Type 2 interferon; the absence of augmentation of interferon activity by exposure to usual Type 1 inducers, including poly-IC and Sendai virus; and induction of interferon formation by exposure to phytohemagglutinin.
The Mo-cell line interferon is substantially free of other interferons. The Type 2 interferon may be produced by stimulation with lectins to concentrations of greater than about 4000 units/ml, even greater than about 10,000 units per ml. (For units/ml, see Stewart et al., (1977) Proc. Natl. Acad. Sci. USA 74, 4200-4204.)
Interferon purification can be achieved using control-pore-glass beads with ethylene glycol stepwise elution (Wiranoska-Stewart et al., In, Biochemical Characterization of Lymphokines, supra). Further purification can be achieved by dialysis and applying the residue to a poly-U-Sepharose column with sequential chromatographic purification. Specific activities of at least about 106 units/mg protein are achieved.
In addition to the other indicated protein factors, activity was observed for macrophage-activating factor, a factor that stimulates colony formation by human leukemic cells in vitro and a factor that stimulates megakaryocyte growth in vitro.
The Mo cell line can be used for providing the conditioned medium having all of the above factors. As to each of the factors, the proteins can be separated by electrophoresis, chromatography, including affinity, adsorption, and gel exclusion chromatography, gradient density separations, radial immunodiffusion, ultrafiltration, or other conventional techniques which allow for isolation of a protein based on its structure, charge, molecular weight, shape or combinations thereof.
The conditioned medium prepared from the Mo cell line can be used directly, where one or more of the factors may be destroyed or retained, where such factor does not interfere with the purpose of the conditioned medium. For example, heat labile factors may be removed by heating at 90 °C. for 5 min, while retaining heat stable factor activities.
The colony stimulating factor can be concentrated from the Mo medium to a specific activity of at least 3.5 x 106 colonies per 105 Ficoll-Hypaque-separated human bone marrow cells plated per milligram protein. (Golde and Cline, J. Clin. Invest. 51:2981-2983, 1972). The erythroid-potentiating activity has been concentrated to 51,000 units/mg protein, wherein one unit is the amount required to stimulate human peripheral blood burst forming unit-erythrocyte (BFU-E) by 50% in the presence of 0.5 units/ml human urinary erythropoitein.
The production of the various factors can be used to produce the proteins employing hybrid DNA technology. The individual factors can be isolated as described above and in the experimental portion and a portion of the molecule sequenced to determine the amino acid sequence. A sequence of from 15 to 20 amino acids will usually suffice. Based on the observed sequence, one can translate the amino acid sequence back to an RNA or DNA sequence which would express the particular amino acid sequence. The 45 to 60 base sequence can then be used as a probe to isolate the messenger RNA which is translated to the desired protein. The Mo cells can be lysed and the RNA separated from the DNA. The poly-(A) enriched RNA may then be separated on an oligo dT or -dU column. This will separate the messenger RNA from other RNA which is present.
One can further separate the messenger RNA by electrophoresis and employing the Southern technique, label the probe with32 P, whereby the probe will hybridize solely to the messenger of interest. Once one has identified the messenger RNA of interest, one can employ preparative electrophoresis and by excising the band identified as having the RNA of interest from the electrophoretic gel, a useful amount of the messenger RNA can be obtained.
Employing common procedures, the gene for the particular protein factor may now be prepared. cDNA may be prepared from the messenger RNA employing reverse transcriptase. The cDNA may then be used as a template for the production of dsDNA which expresses the desired protein. The dsDNA may be further modified by adding control signals such as a promoter, RNA polymerase stop signal, ribosomal start and stop signals or other control signals, as appropriate. These signals can be added by ligation, employing oligomers, by joining the dsDNA to a vector, where the signals are present in the proper reference frame with the dsDNA, by addition of one or more mono- or dinucleotides employing the proper ligase, or other well known conditions.
Conveniently, one can add to the blunt ends of the dsDNA an oligomer having a blunt end and a staggered end, where the staggered end upon binding to a complementary end of a vector provides a restriction site.
The gene as modified may be joined to an appropriate vector for transformation or transfection of bacteria or yeast for replication of the hybrid DNA and expression of the desired gene. The transformant cells are cloned and cultivated under conditions where the desired protein is expressed. When the protein is not excreted, the cells are harvested and lysed in accordance with conventional procedures, and the protein isolated in a conventional manner. Where the protein is excreted, the protein may be extracted from the spent nutrient medium.
The Mo cell line can be employed to produce conditioned media. The Mo cell line can be grown in an appropriate nutrient medium, whereby the growth regulating factors and lymphokines excreted into the medium provide a conditioned medium for the growth of other cells. That is, the conditioned medium can be used for the proliferation and differentiation of stem cells, so as to produce progenitors of erythrocytes and granulocyte-macrophage. As already indicated, the conditioned medium can also be used as a source of the individual proteins serving as the growth factors and lymphokines.
The Mo-cell line is normally grown in a conventional nutrient medium e.g. alpha medium containing fetal calf serum, normally about 20%, and a small amount of a mercaptan, generally about 104 M alpha-thioglycerol. It can also be grown in a serum-free medium, where the cells grow more slowly.
A 33-year old white man experienced fatigue, mild abdominal discomfort, and easy bruising during a period of 2 years. On presentation to a physician the patient had massive splenomegaly and mild depatomegaly without associated lymphadenopathy. Although bone marrow could not be aspirated, a bone-marrow biopsy showed diffuse replacement with neoplastic cells typical of hairy-cell leukemia. Hemoglobin count was 8 g/dl, packed cell volume 24%, platelet count 45 000/micro l, leukocyte count 2900/micro l with 26% polymorphonuclear leukocytes. About 20% of the peripheral blood leukocytes showed morphologic characteristics of hairy cells. Serum protein electorphoresis, immunoelectrophoresis, and quantitative serum immunoglobulins were normal. The patient was referred to UCLA Medical Center. Bone-marrow and peripheral blood hairy cells had abundant tartrate-resistant acid phosphatase. In October 1976 the patient underwent splenectomy. The spleen weighed about 6 kg and histopathologic examination showed diffuse infiltration with tartrate-resistant acid-phosphatase-containing hairy cells. Transmission electron microscopy revealed the neoplastic cells to have ultrastructure typical of hairy cells, and several characteristic “ribosome-lamellar complexes” were identified. Liver biopsy showed similar abnormal cells in the portal triads. Post-operatively, absolute platelet and granulocyte counts returned to normal.
Mo cells grow in suspension culture and exhibit the morphology of relatively mature T-lymphoblasts. They are lysed by antithymocyte serum in the presence of complement, do not produce immunoglobulin, and over 60% of the cells rosette with sheep erythrocytes. They contain the tartrate-resistant isozyme 5 of acid phosphatase, a marker for hairy-cell leukemia, and are not infected with Epstein-Barr virus. The line has now been cultured for over three years and 100 passages. It is generally cultured in alpha medium (Flow Laboratories, Inglewood, CA) containing 20% fetal calf serum (screened lot) and 104 M alpha-thioglycerol (Calbiochem, San Diego, CA). It can also be cultured in serum-free medium, where it grows considerably more slowly. The Mo cells are responsive to phytohemagglutinin as judged by increased incorporation of radioactive thymidine and increased production of certain lymphokines, including CSF. The cells do not contain measurable terminal deoxynucleotidyl transferase.
Culture of erythroid progenitors
CFU-E and BFU-E were cultured in methylcellulose (Dow Chemical, Medland, MI) using normal human bone marrow or peripheral blood, as described in Golde et al. (1977) Science, 196, 1112-1113 and Bersch and Golde, (1978) in In Vitro Aspects of Erythropoiesis, eds. Murphy et al., Springer-Verlag, New York, pp. 252-253. Human bone marrow CFU-E, consisting of clusters of 8 or more hemoglobinized cells, were enumerated after 7-8 days. Human bone-marrow and peripheral blood BFU-E, consisting of erythroid colonies of 50 or more cells were counted after 14 days. In contrast to some other investigators the formation of a small number of CFU-E and BFU-E colonies was observed in the absence of added erythropoietin. This apparently results from the presence of low levels of endogenous erythropoietin in fetal calf serum or associated with the target cells. Mouse CFU-E and BFU-E were assayed as described in Bersch and Golde, ibid. The serum-free methylcellulose technique for the culture of erythroid cells was adopted from the procedure of Guilbert and Iscove, (1976) Nature, 263 594-595. The culture medium contained 1% bovine serum albumin (Sigma, St. Louis, MO), transferrin (350 micro g/ml), ferric chloride (1.6 X 10 6 M), sodium selenite (107 M), insulin (1 ng/ml) and human growth hormone (200 ng/ml). Human urinary erythropoietin was obtained from the Division of Blood Diseases and Resources, National Heart, Lung, and Blood Institute and had an activity of about 44 units/mg protein. Friend erythroleukemia cells (GM-86, clone 745) were cultured in methylcellulose with alpha medium and 0.5% bovine serum albumin. Colonies of 8 or more cells were enumerated after 72 hours.
Culture of granulocyte-macrophage progenitors
Ficoll-Hypaque separated, light density nonadherent cells were prepared from bone marrow and cultured as previously described. Colonies containing 40 or more cells were enumerated after 11-14 days.
Ultrogel AcA 44 was from LKB and concanavalin A-Sepharose and DEAE-Sephadex were from Pharmacia. Concanavalin A-Sepharose chromatography was performed using serum-free Mo-conditioned medium in 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4, and specifically bound glycoproteins were eluted using 0.2 M methyl-alpha-D-mannoside.
Protease inactivation of EPA
Mo-conditioned medium was incubated in 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4, with 0.3 mg/ml pronase (Calbiochem) or a buffer control for 1 hour at 37 ° C. Pronase activity was then destroyed by immersing samples in a boiling water bath for 5 minutes.
Colony stimulating factor (CSF) activity was assayed using a two-layer agar technique. (Golde, D.W. and Cline, M.J., J. Clin. Invest. 51:2981-2983, 1972). The target cells were normally 1 X 105 Ficoll-Hypaque-separated, light-density bone marrow cells obtained from normal volunteers. Colonies (containing 40 or more cells) were scored after 11 days. One unit of activity is defined as the amount stimulating one colony in 105 separated human bone marrow cells plated. Cellular morphology in the colonies was examined either by staining individual colonies picked from cultures with Wright's stain or by staining entire culture dishes for a lipase activity. (Willcox M.B., et al., J. Histochem. Cytochem. 24:979-983, 1976) The latter method was particularly useful for distinguishing granulocyte and macrophage colonies.
Small erythroid colonies (CFU-E) and large erythroid colonies (BFU-E) were grown in methycellulose using normal human bone marrow and peripheral blood. (Golde, D.W. et al. Proc. Natl. Acad. Sci. USA 77:593-596, 1980).
Comparative Studies Utilizing CSFs from Placental-
and Leukocyte-conditioned Medium (CM)
Placental CM was prepared as described in Nicola, N.A. et al., Leukemia Res. 2:313-322, 1978. Using 50 micro l of the resulting CM, about 70 colonies were obtained with the clonogenic human bone marrow CFU-G,M assay described; approximately the same number of colonies was stimulated by a peripheral leukocyte underlayer. Phytohemagglutinin-stimulated leukocyte-conditioned medium was prepared by culturing Ficoll-Hypaque-separated peripheral blood mono-nuclear cells (3 X 106 cell/ml) in alpha medium containing 20% fetal calf serum and phytohemagglutinin (Wellcome Research Laboratories)(Price, G. B. et al., Biochem. J. 148:209-217, 1975). Using 50 micro l of the resulting CM, about 60 colonies were obtained with the clonogenic human bone marrow CFU-G,M assay.
The CSFs from placental CM, phytohemagglutinin-stimulated leukocyte CM, and Mo CM (serum containing) were then partially purified by gel exclusion chromatography to remove any inhibitors of granulocyte-macrophage colony formation. The CM from each source was first concentrated about five-fold by ultrafiltration(Amicon apparatus equipped with a PM 10 membrane) and then chromatographed on an Ultrogel AcA 44 column equilibrated with 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4. The major peak of CSF activity from each source eluted in a volume corresponding to molecular weight between 30,000 and 40,000. The fractions containing the bulk of activity were pooled and utilized for comparative studies of CSFs from these sources. The degree of purification of CSF from each source was about 20-fold.
Purification of Mo CSF
Serum-free Mo CM (1.8 liter) was concentrated by ultra-filtration (using an Amicon apparatus equipped with a PM10 membrane) to 8 ml. The solution was then heated at 57 ° C. for 30 min and the resulting precipitate removed by centrifugation (9,000 RPM, 15 min, using a Sorvall HB-4 rotor). The clear supernatant solution was applied to a 1.6X78-cm column of Ultrogel AcA 44 (LKB) equilibrated with 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4, containing 0.01% polyethylene glycol (average molecular weight 6,000). Fractions of 3.8 ml were collected at a rate of 7.2 ml/hr and assayed for CSF activity. The major contaminating protein in serum-free Mo CM was serum albumin, which accounts for the protein peak eluting at molecular weight about 68,000. Presumably, the albumin was associated with Mo cells upon transfer from serum-containing to serum-free medium. Peak fractions (24-29) were pooled, dialyzed against 0.02 M Tris, pH 7.4, containing 0.01% polyethylene glycol, and applied to a 0.9X5-cm column of DEAE-Sephadex (Pharmacia) equilibrated with the same buffer. The column was washed with several volumes of equilibration buffer and then developed with a linear NaCl gradient (from 0 to 0.4 M NaCl in equilibration buffer) with a total volume of 90 ml. Fractions of 3.6 ml were collected at a rate of 5.4 ml/hr and those with peak activity, eluting between about 0.12 and 0.20 M NaCl (total volume, 18 ml) were pooled. The pooled preparation was applied directly to a 0.9X1.0-cm column of concanavalin-A-Sepharose (Pharmacia) equilibrated with 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4, containing 0.01% polyethylene glycol. The column was washed with equilibration buffer (6 ml) and developed with equilibration buffer containing 0.2 M methyl-alpha-D-mannoside at the rate of about 2 ml/hr. Essentially all of the CSF activity bound to the lectin column and the bulk of the activity was eluted in the first two column volumes after the application of methyl-alpha-D-mannoside. Unless otherwise stated, this was the source of CSF for the biological and physical studies employing purified Mo CSF.
Polyacrylamide gel electrophoresis of the purified CSF in a native gel system (Clarke, J. T., Ann. N.Y. Acad. Sci. 121:428-436, 1964) gave multiple, poorly resolved protein bands, possibly resulting from the extensive charge heterogeneity of the CSF (see below). CSF activity was associated with several protein bands and was poorly resolved. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Laemmli, U.K., Nature (London) 227:680-685, 1970) yielded two major protein bands, corresponding to molecular weights of about 33,000 and 18,000. CSF activity was not recovered from gel slices after extracting for 24-48 hr with buffers. Thus, it was concluded that the CSF is not homogeneous, probably constituting 40% or less of the preparation. However, the relatively high specific activity of CSF in the preparation suggests that CSF is probably not a minor protein species in the preparation.
Flat-bed isoelectric focusing in granulated gel was performed using an LKB apparatus as suggested by the manufacturer. The CSF from serum-free Mo Cm was concentrated by absorption to 1/50 volume of calcium phosphate gel (BioRad Laboratories, Richmond, CA). The CSF was eluted by incubating the gel with 2 volumes of 0.05 M sodium phosphate, pH 6.0. The eluate was dialyzed against 0.02 M Tris-HCl, pH 7.4, and incorporated into a gel slurrY (100 ml total volume) containing 2.5 ml pH 4-6 ampholines, 2.5 ml pH 3.5-10 ampholines and 5 g Ultrodex-granulated gel (LKB). The mixture was dried to the proper consistency and subjected to flat-bed isoelectric focusing using 1,000 V (maximum 8 watts) for 16 hr at 5 ° C. The gel was subdivided and each fraction was extracted with 10 ml 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4 containing 0.01% polyethylene glycol (average molecular weight 6,000). The fractions were dialyzed against extraction buffer and assayed.
Isoelectric focusing in polyacrylamide gel was performed as previously described (Lusis, A. J. and Pargen, K., J. Biol. Chem., 253:7336-7345, 1978) with the following modifications: Focusing was for 12 hr at 3 ° C. and 200 V. Gels contained 1.8% pH 4-6 ampholines (LKB) and 1.8% pH 3.5-10 ampholines. The anode reservoir contained 0.02 M sodium hydroxide and the cathode reservoir contained 0.02 M phosphoric acid.
Isolation of Mo CSF
When cultured in alpha medium containing 15-20% fetal calf serum the Mo cells continuously elaborated a CSF that stimulated the formation of granulocyte-macrophage colonies in vitro. The specific activity of the conditioned medium (CM) prepared in the presence of serum was about 700 units/mg protein (using target human bone marrow cells), roughly similar to the specific activities observed using several other sources of human CSF, such as placental CM, phytohemagglutinin-stimulated leukocyte CM, and lung CM. The Mo cells continued to release CSF when transferred into serum-free alpha medium, although their growth was slowed considerably. After 7 days of incubation in serum-free culture at a density of about 5 X 106 cells/ml, the medium had a specific activity in the range of 3,000 to 20,000 units/mg protein.
Because of the higher specific activity of serum-free Mo-CM, it was used as the starting material for purification experiments. A variety of fractionation methods were first surveyed using crude CM and partially purified preparations of Mo CSF. On the basis of yield and separation achieved using these methods, a purification protocol for Mo CSF was devised.
The yield of greater than 100% during the first steps of the purification is probably due to the removal of inhibitors of colony formation present in the crude CM. The final product had a specific activity of about 3.5 X 106 units/mg protein, representing about a thousand-fold purification with an overall yield of 31%. This is the highest specific activity reported for a human CSF (using human CFU-G,M). However, as judged by electrophoresis, the CSF in this preparation was not more than about 40% pure.
The responsiveness of human bone marrow cells to various concentrations of Mo CSF was determined. Curves relating the number and size of the colonies observed in soft-agar cultures to the concentrations of CSF were sigmoidal. High doses of the crude conditioned medium resulted in inhibition of colony fornation; this probably results from the presence of inhibitors, since the inhibition was not observed with partially purified preparations.
Crude and purified Mo CSF stimulated the formation of both granulocyte and macrophage colonies. Normally, macrophage colonies predominated slightly. The ratio of colony types was not greatly dependent upon the concentration of CSF used. The fraction of macrophage colonies stimulated by Mo CSF was significantly greater than for CSFs from human PHA-stimulated leukocyte CM and human placental CM.
Crude Mo CM gave little or no stimulation of macrophage-granulocyte colonies using mouse bone marrow cells, while partially purified preparations of Mo CSF showed very weak stimulation. The relative stimulation of mouse, as compared to human, target cells was considerably less for Mo-CSF than for CSFs examined from other human sources.
The CSF activity in crude Mo CM was stable indefinitely when stored frozen at -20 ° C. and for weeks when stored at 4 ° C. However, highly purified preparations of the CSF were relatively labile when stored either frozen or at 4 ° C., with the bulk of the activity being lost within days. The addition of protein (0.1% bovine serum albumin) or polyethylene glycol (0.01%) stabilized CSF preparations, and polyethylene glycol was routinely included in buffers during the latter stages of the purification of CSF. The Mo CSF was stable over a wide pH range; most of the activity was recovered after incubating the CSF in buffers between pH 3 and pH 10 at 37 ° C. for 1 hr or at 4 ° C. overnight. It was also quite stable in the presence of denaturing agents; for example, the bulk of the activity could be recovered after treating CSF for 15 hr at room temperature with either 8 M urea or 6 M guanidinc hydrochloride (in 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4).
In contrast to CSFs from placental CM and stimulated leukocyte CM, the Mo CSF was stable at temperatures up to about 70 ° C. Even at temperatures as high as 90 ° C., about half the CSF activity was recovered after 5 min of incubation. Tests were carried out in 0.02 M sodium phosphate, 0.15 M NaCl (pH 7.2), 0.1% bovine serum albumin.
Since certain CSFs are inactivated in the presence of sulfhydryl compounds, the effect of mercaptoethanol on the activity of Mo CSF was tested. In the absence of denaturing agents, exposure to concentrations of mercaptoethanol as high as 0.1 M had little effect on CSF activity. However, in the presence of 8 M urea, treatment with mercaptoethanol (1 mM) resulted in greater than 90% loss of activity.
When subjected to gel exclusion chromatography, the Mo-CSF activity eluted as a single major peak, in a volume corresponding to an apparent molecular weight of about 34,000. The molecular weight of the CSF did not change during the course of purification, although the crude conditioned medium contained variable amounts of a higher molecular weight form of CSF. This latter activity had an apparent molecular weight about 50,000 and generally comprised less than 20% of the total activity.
Isoelectric point and charge heterogeneity
Using isoelectric focusing in granulated gel, the Mo CSF from serum-free CM focused between pH 4.0 and pH 5.2. The CSF from serum-containing CM, on the other hand, was slightly more acidic, focusing between about pH 4.0 and pH 4.6. A similar charge difference was noted using ion-exchange chromatography; for example, the bulk of the activity from serum-containing medium eluted from DEAE-Sephadex (equilibrated with 0.02 M Tris, pH 7.4) between 0.18 and 0.22 M CaCl 2, while CSF from serum-free medium eluted over a wider range of salt concentrations, between about 0.12 amd 0.22 M NaCl. In contrast to the variation in charge, the CSFs from serum-containing and serum-free Mo CM were not discernibly different in size (as judged by gel exclusion chromatography) or heat stability.
Experiments involving treatment with neuraminidase suggested that the charge heterogeneity of the CSF is due in part to the presence of varying amounts of sialic acid. When CSF preparations were subjected to isoelectric focusing in adjacent wells of polyacrylamide gels, neuraminidase treatment was found to shift the charge of the CSF toward a more basic pH. This effect was also observed using ion exchange chromatography.
The Mo CSF appears to be glycosylated, since it was bound by the lectin concanavalin-A and was eluted using methyl-alpha-D-mannoside. Moreover, neuraminidase treatment altered the charge, but not the activity of Mo CSF, suggesting the presence of sialic acid.
Relationship to Other Hematopoietins Present in
The Mo T-lymphocyte cells liberate into the medium several lymphokines in addition to the CSF. These include an activity that stimulates human erythroid colony formation in vitro and an activity that inhibits the migration of neutrophils. To test whether these activities resulted from distinct factors, fractions obtained after chromatographic separations of Mo CM were tested for each activity. Using gel exclusion chromatography, the CSF eluted in a volume corresponding to apparent molecular weight 34,000 and was clearly separable from the activities potentiating erythroid colony formation and inhibiting neutrophil migration, both of which eluted in a volume corresponding to molecular weight about 45,000. Moreover, the CSF was less heat stable than the latter activities. The purified CSF had little or no erythroid-potentiating activity as judged by the stimulation of human CFU-E or BFU-E in vitro; in fact, the addition of purified CSF to human erythroid cultures (BFU-E) decreased colony formation slightly.
The Mo cells elaborate and release into the medium a variety of products characteristic of activated T lymphocytes.
PRODUCTS OF Mo LINE
1. Colony-stimulating factor (CSF)
2. Erythroid-potentiating activity (EPA)
3. Immune interferon (type II)
4. Neutrophil migration-inhibitory factor (NIF-T)
5. T-cell growth factor (TCGF, interleukin II)
6. Macrophage-activating factor (MAF)
7. Factor stimulating fibroblast growth
8. Factor stimulating human pluripotent hematopoietic stem cell (may be same as #2)
9. Factor stimulating human leukemic cells in vitro (may be same as #1 or #2)
Among these is erythrocyte potentiating activity (EPA), which stimulates the formation of colony forming units-erythrocytes (CFU-E) and burst forming units-erythrocytes (BFU-E) colonies in cultures of human hematopoietic tissues. The purified EPA was diluted to a concentration of about 1 micro g/ml using 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4 (PBS buffer), containing 0.1% bovine serum albumin (Sigma, recrystallized). Erythroid colonies were grown using a methylcellulose technique (Golde, D.W. et al., Proc. Natl. Acad. Sci. USA, 77:593-596, 1980). Cultures contained 0.5 units/ml human urinary erythropoietin (specific activity 44 untis/mg protein), 0.8% methylcellulose (Dow), alpha medium (Flow), 30% fetal calf serum, 0.1 mM alpha-thioglycerol, and penicillin and streptomycin. Human bone marrow CFU-E containing 8 or more hemoglobinized cells were counted after 7 days of incubation and human peripheral blood BFU-E containing 50 or more cells were counted after 14 days.
Optimum stimulation of erythroid colony formation was observed at about 20-50 micro l Mo-conditioned medium (CM) per 1-ml culture dish. At higher concentrations a decrease in colony formation was observed due to the presence of inhibitory activities. The inhibition was partially removed by heat treatment and was less pronounced at low erythropoietin concentrations. Colony formation was augmented by Mo EPA at all erythropoietin concentrations tested (up to 2 units/ml).
During fractionation experiments EPA was normally assayed at a suboptimal erythropoietin concentration (0.1 unit/ml) or in the absence of added erythropoietin. In contrast to some other investigators, slight human erythroid colony formation was observed in the absence of added erythropoietin. This probably results from erythropoietin bound to target cells or present in fetal calf serum. One unit of EPA is arbitrarily defined as the amount required to stimulate human peripheral blood BFU-E by 50% in the presence of 0.5 units/ml human urinary erythropoietin.
The specific activity of EPA in serum-free Mo CM was much higher than that in serum-containing CM, and therefore, it was utilized for purification experiments. For example, after 7 days of serum-free culture at a density of 5 X 106 cells/ml, serum-free Mo CM had a specific activity of about 200 units/mg protein as compared with about 3 units/mg protein for serum-containing CM prepared under similar conditions.
The EPA in serum-free Mo CM was first partially purified and concentrated by adsorption to and elution from calcium phosphate gel. Calcium phosphate gel (36 ml) was added to 1.8 liter serum-free Mo CM containing about 0.1 mg/ml protein. After 1 hr incubation, the gel was collected by centrifugation, and the EPA was eluted by incubating the gel with two volumes of 0.05 M sodium phosphate, pH 6.0. The eluate was dialyzed against 0.02 M Tris, pH 7.4, and incorporated into a gel slurry (100 ml total volume) containing 2.5 ml pH 4-6 ampholines (LKB), 2.5 ml pH 3.5-10 ampholines, and 5 g Ultrodex ® granulated gel (LKB). The mixture was subjected to flat-bed isoelectric focusing as suggested by LKB (8 watts constant power, 5 ° C., 16 hr). The gel was then subdivided and fractions were extracted with PBS buffer containing 0.01% polyethylene glycol. The fractions were dialyzed against PBS buffer and assayed for EPA protein or G,M-CSF. Fractions 22-31, containing the highest specific activity of EPA, were pooled and used for further purification experiments. The eluate was then dialized and subjected to flat-bed preparative isoelectric focusing.
EPA was present primarily in acidic fractions, ranging in pH from 3.5 to 4.8. In addition, a distinct peak of activity focusing at about pH 6.5 was observed in several separate experiments. Fractions with highest EPA specific activity, ranging from about pH 3.5 to 4.6, were pooled and concentrated. The concentrate was subjected to gel exclusion chromatography using Ultrogel AcA 44. Pooled fractions of EPA from preparative isoelectric focusing were concentrated to 7 ml (using an Amicon apparatus equipped with a PM-10 membrane) and applied to a 1.6X78-cm column of Ultrogel AcA 44 (LKB) equilibrated with PBS. The column was developed with PBS at a rate of 7.2 ml/hr and fractions of 3.6 ml were collected and assayed for EPA and protein. Fractions with peak specific activity were pooled for biological and physical studies. The large peak eluting at a volume corresponding to molecular weight 68,000 probably represents serum albumin which remained associated with Mo cells upon transfer from serum-containing to serum-free medium. Peak EPA activity eluted in fractions corresponding to a molecular weight of about 40,000. Fractions with highest specific activity were pooled and used for subsequent biological and physical characterization of EPA. The final product had specific activity of about 51,000 units/mg protein, representing about a 250-fold purification with an overall yield of about 20%. The activity measurements reflect the removal of inhibitors of erythroid colony formation present in Mo CM as well as the recovery of EPA. The purification scheme is summarized in Table III.
The potent inhibitors of erythroid colony formation present in the crude Mo CM were largely or completely removed by the purification procedure. The purified EPA significantly stimulated erythroid colony formation in nanogram quantities per 1-ml plat, although it was not homogeneous. Assuming a molecular weight of 45,000 for EPA, we calculate that the EPA stimulates erythroid colony formation below concentrations of 0.1 nM.
The partially purified EPA stimulated both early (14-day BFU-E) and late (7-day CFU-E) human erythroid progenitors, suggesting that the two activities reside in a single factor. This possibility is supported by the observation that both activities are unusually heat stable.
Previous studies suggested that the G,M-CSF and EPA present in Mo CM are distinct since they differ in heat stability, charge and molecular weight. Our present findings support this conclusion since the partially purified EPA contained little or no CSF activity. Moreover, a highly purified preparation of Mo CSF was found to inhibit, rather than stimulate, erythroid colony formation.
The partially purified EPA preparation retained an activity stimulating mixed erythroid-granulocyte-megakaryocyte colony formation in vitro. In addition, the activity stimulating mixed colonies was, like EPA, exceptionally heat stable. This raises the possibility that the activities stimulating BFU-E and the multipotent myeloid stem cell detected in vitro result from the same factor. Alternatively, the two activities may reside in separate but structurally similar factors, possibly members of a family of closely related, homologous proteins. The sources of activities stimulating formation of mixed myeloid colonies in vitro all contain activities stimulating committed erythroid progenitors as well.
Partially purified EPA, but not crude Mo CM, stimulates the clonal growth of mouse Friend erythroleukemia cells and of human K562 leukemia cells. Certain sublines of K562, including the one tested, exhibit properties characteristic of erythroleukemia cells.
The following is a summary of observed EPA properties. EPA appears to be glycoprotein in nature, since it binds to concanavalin A-Sepharose and is destroyed by treatment with proteases. It exhibits remarkable heat stability and is highly resistant to denaturing agents. When subjected to gel exclusion chromatography at neutral pH the bulk of the activity eluted in a peak of apparent molecular weight about 45,000, although a higher molecular weight shoulder was also observed. This shoulder was diminished after partial purification. Using isoelectric focusing, a broad major peak of EPA was observed in the acid regions of the gradient; in addition a second peak of activity focusing at about neutral pH was observed in four separate experiments. The origin of the size and charge heterogeneity is not clear. In view of the complexity of the clonal assay procedure, the presence of multiple modulator activities in Mo CM would not be surprising. Also, EPA could exhibit heterogeneity in the degree of modification (e.g., glycosylation or proteolytic cleavage) or form complexes with other constituents in the medium.
The neutrophil migration inhibition factor was isolated and characterized as follows.
Lymphocyte Culture Techniques
Mononuclear cells were isolated from heparinized human peripheral blood by Ficoll-Hypaque gradient centrifugation and washed with Hanks' buffered salt solution (BSS). Mononuclear cells were cultured at 3X106 /ml for 24 hr at 37 ° C. in the presence of 5% CO 2 in Medium 199 (Flow Laboratories, Anaheim, Calif.) with 20 mM Hepes buffer, penicillin, 100 units/ml, and streptomycin, 100 micro g/ml. Stimulated cultures contained concanavalin A (Con A), 25 micro g/ml. After 24 hr, the cell-free supernatants were harvested, and the nonstimulated culture supernatants were reconstituted with the same concentrations of Con A. All cultures were viable as determined by trypan blue exclusion. The T-lymphoblast cell lines 8402 (established from a patient with acute lymphoblastic leukemia) and Mo were cultured similarly, except that Con A was not used. Mo cells spontaneously produce a migration inhibition factor for neutrophils whereas other lymphoblast cell lines including 8402 do not. Moreover, 8402 could not be stimulated to produce such an activity in the presence of mitogen. Therefore, the 8402 cells were used as control for Mo cells in these studies. All culture supernatants were stored at -20 ° c. until assayed.
Microassay for Neutrophil Migration
The details of this assay have been reported (Weisbart, R.H. and Mickey, M.R., J. Immunol. Methods 16:269, 1972). Briefly, neutrophils were purified (97-99%) from the peripheral blood of healthy subjects by Ficol-Hypaque gradient centrifugation, and the red blood cells were removed by dextran sedimentation. Four microliters of a neutrophil suspension (50X106 /ml) were mixed with 4 micro l of cell culture supernatants and incubated for 30 min at 37 ° C. One-microliter aliquots of these neutrophil-supernatant suspensions were then distributed into quadruplicate 1.5-mm diameter wells cut in a 1% agarose gel that contained Medium 199 with 10 mM Hepes buffer and 10% horse serum. The wells were filled under mineral oil in 150-mm round culture dishes (Integrid, Falcon Plastics, Oxnard, Calif.) with 128 migrations on each dish. After the neutrophils migrated overnight, the cells were fixed with ethyl alcohol and the agarose gel was removed. The largest diameter of each migration was measured with an ocular micrometer, and the data were expressed as the percentage inhibition in the area of migration.
Heat Stability of Neutrophil Migration Inhibition
Control and active serum-free culture supernatants were heated at 60 °>>>>, 80 °, and 100 ° C. for 10 to 30 min and centrifuged at 2000 g for 10 min. The supernatant fractions were decanted and assayed for neutrophil migration inhibition activity.
Effect of Pronase on Neutrophil Migration Inhibition
Pronase was added to 0.5-ml aliquots of control and active cultur e supernatants (1 mg/ml final concentration) and incubated at 37 ° C. for 30 min. The supernatants were then heated to 80 ° C. for 15 min to inactivate the pronase. The supernatants were assayed for neutrophil migration inhibition activity.
Affinity of Con A Sepharose for Neutrophil Migration
Sepharose beads coated with Con A (Pharmacia, Piscataway, N.J.) were washed with Hanks' BSS, and 0.1 ml of packed beads were incubated at 37 ° C. for 30 min with 0.5 ml of control (8402) and active (Mo) lymphoblast culture supernatants with constant mixing. In addition, PBL were cultured with sepharose beads alone, and with Con A sepharose for 24 hr. The beads were recovered by centrifugation, washed with Hanks' BSS, and eluted at 24 °>>>> C. for 30 min with 0.5 ml of 10% alpha-methyl-D-glucoside in 0.1 M acetate buffer, pH 6.0, containing 1 M NaCl, 103 M CaCl 2, 10-3 M MgCl 2, and 103 M MnCl 2. The eluates were dialyzed against Hanks' BSS. Aliquots of original supernatants and the dialyzed eluates were assayed for neutrophil migration inhibition activity.
Isolation of Neutrophil Migration Inhibition Activity by Electrophoresis in Polyacrylamide Gradient Slab Gels
Neutrophil migration inhibition activity was concentrated from culture supernatants by treatment with diethylaminoethyl (DEAE) Sephacel (Pharmacia, Piscataway, N.J.). Batch method preparation was used with sterilized beads and buffers. Ten milliliters of PBL or lymphoblast culture supernatants was dialyzed against 0.1 M trischloride, pH 8.0, and incubated at 24 ° C. for 30 min with 2 ml of packed beads. The beads were eluted in 1 ml of 0.3 M NaCl. The eluates were dialyzed against distilled water, lyophylized, and reconstituted with 20 micro l of Hanks' BSS. In preliminary experiments substantial migration inhibition activity was demonstrated in these preparations. Fivemicroliter aliquots of DEAE Sephacel purified and concentrated material were applied to preformed polyacrylamide gradient slab gels (PAA 4/30 Pharmacia, Piscataway, N.J.) and electrophoresed for 4 hr at 300 V in 1% trisborate buffer, pH 8.1. Control and test supernatants were electrophoresed simultaneously on the same gel, and corresponding fraction of each were compared for biological activity. Hen egg albumin (45,000 daltons) and bovine serum albumin (67,000 daltons) markers were electrophoresed on the opposite half of the gel. Horse serum was electrophoresed to provide column markers. Purified hemoglobin A (Gelman, Ann Arbor, Mich.) was electrophoresed on both halves of the slab gel. The gel was sectioned and the half of the gel containing the standards was stained with amido black and destained by electrophoresis. The two halves of the gel were then aligned by superimposition of the hemoglobin bands. Portions of the gel containing the control and test samples were sectioned and eluted by electrophoresis (140 V for 16 hr) into 50-micro l dialysis chambers formed in Lucite. The eluates were dialyzed against Hanks' BSS and tested for neutrophil migration inhibition activity.
Treatment of Culture Supernatants with Diisopropyl
The culture supernatants (Mo T, pH 8.9, and peripheral blood lymphocytes, pH 7.2) were treated with DFP to provide final concentrations of 103, 104, and 105 M DFP. Ten microliters of the appropriate dilution of a 5% stock solution of DFP in isopropyl alcohol was incubated with 0.5 ml of the control and active culture supernatants for 30 min at 37° C. DFP-Treated supernatants were assayed before and after dialysis against Hanks' BSS.
In order to test the efficacy of this procedure in destroying serine esterase activity thought to be present in the culture supernatants, 0.25 ml samples of both active supernatant (Mo T and PBL) were incubated as described above following addition of 2.5 micro g bovine trypsin in 25 micro l of 1.2 103 M Tris HCl containing 5X105 M CaCl 2. Two control samples containing trypsin but no DFP were incubated similarly. Trypsin activity in all samples was determined at the start and end of the experiment by incubating a 10-micro l aliquot with a mixture of 0.49 ml of 0.1 M Tris HCl, pH 8.2, containing 2.5X103 M CaCl 2,Triton X-100(1:4000,and 0.4 ml of an aqueous solution of carbobenzoxyglycylglycylarginyl-2-naphthylamide (90 mg/100ml) in a water bath at 25 ° C. for 15 min. The reaction was terminated by addition of 0.1 ml 1 M citrate buffer, pH 4.5, and fluorescence measured at lambda EX 345 nm, lambda EM 415 nm (uncorrected) (Penderknecht, H. et al., Clin. Chim. Acta 73:369, 1976).
Reversibility of Neutrophil Migration Inhibition
Supernatants from control and Con A stimulated peripheral blood lymphocyte cultures and the lymphoblast cells 8402 and Mo were preincubated for 30 min at 37 ° C. with purified human neutrophils. The cells were then washed with Hanks' BSS, resuspended in Medium 199 with 10% horse serum, and incubated at 24 ° C. Aliquots of the neutrophils were removed at 2, 4, and 20 hr, washed with Hanks' BSS, and the cell concentration was adjusted to 25X10 6 /ml viable cells. One-microliter aliquots of cells were dispensed in agarose wells and the migrations were assessed as previously described.
Heat Stability of Neutrophil Migration Inhibition
The results of heating serum-free supernatants containing neutrophil migration inhibition activity from peripheral blood lymphocytes and the T-lymphoblast cell line, Mo, are shown in Table IV.
Similar heat stabilities were demonstrated for the inhibition activities from PBL and Mo cells, with full stability at 60 ° and 80 ° C. for 30 min. Neutrophil migration inhibition activity was stable even after boiling for 5 min. The activity was destroyed, however, after boiling for longer periods of time.
Effect of Pronase on Neutrophil Migration Inhibition
Before treatment with pronase, serum-free supernatants from Mo cells and Con A induced PBL showed 56 ± 6% and 42 ± 4% inhibition in the area of neutrophil migration, respectively, compared to supernatants from 8402 cells and nonstimulated PBL. After treatment with pronase, the Mo and PBL activities were abolished (-4 ± 6% and 4 ± 4% inhibition in the area of migration). Control supernatants containing pronase that were heated to 80 ° C. for 15 min did not inhibit neutrophil migration in comparison to supernatants that did not contain pronase. Supernatants heated to 80 ° C. did not lose migration inhibition activity as shown above.
Affinity of Con A Sepharose for Neutrophil Migration
All of the neutrophil migration inhibition activity in supernatants from Mo cells was removed by Con A Sepharose with 37 ± 4% inhibition in the area of migration before and 0 ± 11% after incubation with Con A Sepharose. Full activity was recovered after elution with alpha-methyl-D-glucoside (45 ± 7% inhibition). The dialyzed eluates of Con A Sepharose cultured with PBL gave 41 ± 5% inhibition in the area of migration of neutrophils compared to the eluates of Sepharose beads without Con A similarly cultured with PBL.
Isolation of Neutrophil Migration Inhibition Activity
by Electrophoresis in Polyacrylamide Gradient SlabGels
The biological activities identified in eluates of the various fractions are shown in Table V.
Migration inhibition activity from PBL was localized to the pre 45,000 dalton-region with no substantial activity identified elsewhere on the gel. Activity was obtained from the Mo supernatants in the 45,000-dalton region as well as the region corresponding to 35,000 to 45,000 daltons.
Effect of Treatment of Culture Supernatants with
Diisopropyl Fluorophosphate DFP)
The effect of DFP on neutrophil migration inhibition activity from supernatants of PBL, Mo cells, and a partially purified preparation from Mo isolated by polyacrylamide gel electrophoresis were determined. There was no effect on the migration inhibition activity at 10-3, 10-4, or 10-5 M DFP. The efficacy of DFP in destroying serine esterase activity was demonstrated by adding trypsin to an aliquot of the supernatants tested, and assaying for the presence of trypsin in the presence and absence of DFP. In the samples containing trypsin and DFP (10-3 M), more that 99% of trypsin activity was destroyed; in the samples containing trypsin but no DFP, only 4% of trypsin activity was lost. These results demonstrate the complete removal of serine esterase by the above treatment with DFP.
Reversibility of Neutrophil Migration Inhibition
Neutrophils that were incubated with activated supernatants, washed, and cultured for 2 hr, showed maximum inhibition of migration. If the neutrophils exposed to activated supernatants were washed and left at 24 ° C. for 20 hr, rewashed, and migrated, the inhibition activity was lost. Neutrophils treated in this way could still be inhibited by activated supernatants. When reincubated with active supernatants for 30 min and migrated, the neutrophils were inhibited in their migration 39 ± 6%. Neutrophils were viable at 24 hr as evidenced by their ability to migrate and respond to activated supernatants.
The immune interferon is produced as follows in a conditioned medium. Single cell suspensions in minimum essential medium supplemented with non-essential amino acids (F-15) and 10% FCS are seeded into 16 mm wells at a density such that at 37 ° C., confluence is achieved in about 20 hrs. post-plating. After achieving confluence, the medium is removed, the cell cultures washed twice, and phorbol esters in about 1-100 micro g/ml with phytohemeagglutinin in about 1-100 micro g/ml are added. After 15 hrs. at 37 ° C., the cells are separated from the supernatant, and the interferon activity of the medium determined. Human virus susceptible cells of single cell suspension in appropriate nutrient medium are seeded into 16 mm wells at a density such that at 37 °, confluence is achieved within about 20 hrs. post-plating. After achieving confluence, the medium is removed, the cultures washed twice, and an appropriate virus at an input multiplicity of infection (MOI) equal to 7 added in a nutrient medium containing varying amounts of the Mo conditioned medium. The virus is absorbed at 37 ° C. for 30 mins. At the end of this period and again 90 min. post infection, the monolayers are washed twice and one ml of fresh nutrient medium added. After 15 hrs. at 37 ° C., the cultures are harvested and the yield of the virus determined. In accordance with the above test, the Type 2 immune interferon produced by the Mo cell line is effective in reducing the amount of virus produced, as compared to control samples where no Mo conditioned medium is present.
In addition to obtaining the individual factors by cultivation of the Mo cell line, isolation of the conditioned medium, and separation and purification of the desired protein factor, the individual factors can be produced by hybrid DNA technology.
The Mo cells are homogenized and cytoplasmic RNA purified from a postnuclear supernatant by Mg2 precipitation. (Palmiter, R. (1974) Biochemistry 13, 3603-3615). The precipitate is extracted with phenol and chloroform and enriched for poly(A)-containing RNA by oligo (dT)-cellulose chromatography. (Avir, H. and Leder, P., (1972) Proc. Nat. Acad. Sci. USA 69, 1408-1412.
The RNA is washed with 0.01 M Tris HCl (pH 7.5)-0.5 M KCl with 100A 260 units of RNA dissolved in the above buffer applied to a 2 ml column previously washed with buffer. After eluting non-absorbed material, the absorbed material was eluted by washing initially with 0.01 M Tris-HCl (pH 7.5)-0.1 M KCl, followed by 0.01 M Tris-HCl(pH 7.5). The eluted material was precipitated in 2% potassum acetate (pH 5.5) with two volumes of ethanol, and the mixture allowed to stand overnight at -20 ° C. The precipitate was dissolved in a solution containing 10 mM Tris-HCl/1 mM EDTA (pH 7.4) and disaggregated by heating at 80 ° for 1 hr.
The desired mRNA is isolated as follows, as described in Alwine et al., Proc. Nat. Acad. Sci USA (1977) 12, 5350-5354. See also U.S. Pat. No. 4,139,346 and Reiser et al. Biochem. Biophys. Res. Comm., 85, 1104 (1978).
The mRNA is separated by electrophoresis on a horizontal slab gel (23X14X0.4 cm) containing 1.5% agarose and 4 mM methyl mercuric hydroxide. Samples in water are mixed with an equal volume of starting buffer [1XE buffer/10% glycerol/bromphenol blue] (for E buffer, see Bailey and Davidson (1976) Anal. Biochem. 70, 75-85) and made 10 mM in methyl mercuric hydroxide.
DBM paper is prepared as described in Alwine et al., supra. After electrophoresis, a strip of DBM paper saturated with 50 mM sodium borate buffer (pH 8.0) is placed on the gel with Plexiglas strips positioned to prevent the DBM-paper from contacting the two layers of Whatman 3 MM paper saturated with the borate buffer underlying the gel. The DBM-paper is then repetitively covered with two layers of dry 3 MM paper, several layers of paper towels and a Plexiglas weight and the buffer allowed to soak through before each change.
The position of the desired mRNA is determined using a probe prepared as follows. From the amino acid sequence of the desired factor a DNA sequence is synthesized as described in European Patent No. 0001931. The DNA sequence will be from about thirty to sixty bases. By employing derivatized bases, codons are prepared which are then coupled with other codons to produce oligodeoxyribonucleotides of from 9 to 18 bases. The oligodeoxynucleotides are then combined to prepare the DNA probe. The probe is then labeled with32 P employing [alpha-32 P] - ATP and polynucleotide kinase.
Hybridization of the RNA with the DNA probes is performed in accordance with the method of Denhardt (1966) Biochem. Biophys. Res. Comm. 23, 641-652. The paper strips containing transferred RNA are treated for at least four hours at 42 ° C. with hybridization buffer (50% formamide/0.75 M NaCl/75 mM sodium citrate containing 0.02% wt/vol each of bovine serum albumin, ficoll and polyvinylpyrrolidone, 1.0-2.5 mg of sonicated denatured calf thymus DNA/ml and 1% wt/vol glycine. Hybridizations are performed by placing the paper strips, hybridization buffer without glycine (50-100 micro l/cm2 of paper surface area), and the single-stranded32 P labeled probe (5X104 cpm/cm2 of paper surface area; 5X107 cpm/micro g) into plastic boiling bags from which the excess air is removed prior to sealing by heat. After laying the bags horizontally and gently rocking for 36 hr. at 42 ° C., the paper is washed at 42 ° C. for at least 4 hrs. with at least 6 changes of a 50% formamide/0.75 M NaCl/75 mM sodium citrate solution. After blotting to remove excess solution, the paper strips are covered with Saran ® wrap and visualized autoradiographically with Kodak XF-5 X-ray film. The band to which the probe hybridized is excised from the electrophoretic gel to provide the desired mRNA.
The mRNA is used to make cDNA as follows. The reaction is carried out in 100 micro l using approximately 5 micro g mRNA, 45 units avian myeloblastosis virus reverse transcriptase and dCTP labeled to 4 Ci/mmole. In 30 min, 1.5 micro g cDNA is synthesized. The reaction is terminated by adding EDTA (to 10 mM), followed by extraction with phenol, then ether and then passing the mixture over a Sephadex G-50 fine column in 10 mM Tris (pH 7.4), 2 mM EDTA, 10 mM NaCl (TEN). The void volume is collected, precipitated with ethanol, centrifuged and the RNA hydrolyzed with NaOH. The cDNA is isolated and used as a template for dsDNA.
The formation of dsDNA is achieved by combining in 100 micro l 1.1 micro g cDNA, 10 units DNA polymerase I and 200 micro M of each deoxynucleotide triphosphate with dCTP adjusted to 30 Ci/mmole and the reaction allowed to proceed for 10 min. at 42 ° C. The reaction is stopped and and extracted as above before being passed over a Sephadex G-50 fine column in TEN containing only 0.1 mM EDTA. Column fractions containing the ds cDNA were pooled avoiding the inclusion of unincorporated deoxynucleoside triphosphates and the mixture adjusted to 30 mM sodium acetate (pH 4.5), 3 mM ZnCl 2, 10 micro g of native and 10 micro g denatured salmon sperm DNA/ml.
Purified A. oryzae single strand specific nuclease S1 is added to a final concentration of 5 units/ml (1 unit = amount required to completely degrade 10 micro g denatured DNA in the presence of 10 micro g native DNA in a 2 hr. incubation at 37 ° in above described S1 buffer). After 1 hr. at 37 ° C. the digestion is stopped by ice-cooling and adding SDS to a final concentration of 1%. After adding about 10 micro g of E. coli tRNA, the mixture is extracted at r.t. with an equal volume of chloroform and the organic phase re-extracted with S1 buffer. After precipitation by the addition of 2.5 volumes ethanol at 20 ° C. overnight, terminal addition of dCTP to the ds cDNA by terminal deoxynucleotidyl transferase was performed with 1 micro g ds cDNA in 500 micro l containing 140 mM cacodylic acid, 30 mM Tris base, 110 mM KOH (final pH 7.6), 0.1 mM dithiothreitol, sufficient dCTP to provide about 103 pM 3' termini/micro l and 0.5 micro l (2.3X105 units/ml) of the transferase. After sufficient time to add about 30 dC residues per 3' terminus (10 min) at 37 ° C., the reaction is cooled, extracted, desalted and precipitated as described above. The dC-tailed ds cDNA is then preparatively electrophoresed on a 1.7% agarose gel in Tris-acetate-NaCl (Dugarczyk et al. J. Mol. Biol. 96, 171-184 (1975)), cut out of the gel and electrophoretically eluted into a dialysis bag (McDonnell et al. J. Mol. Biol. 110, 119-146 (1977). After centrifugation, the dC-tailed ds cDNA is redissolved in 10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA, 100 mM NaCl.
The ds cDNA is inserted into the PstI site of plasmid pBR322 by linearizing pBR322 DNA with PstI and adding approximately 15 dG residues per 3' end by the procedure described for dC-tailing, substituting dGTP for dCTP (See also, Roychoudbury et al. Nucleic Acids Res. 3, 101-116 (1976)). After extraction with phenol and ethanol precipitation the dG-tailed pBR322 is combined with the dC-tailed ds cDNA at a weight ratio of about 1:1 in 10 mM Tris (pH 8) and dialyzed against 0.1 M NaCl, 10 mM EDTA, 10 mM Tris (pH 8). After heating the mixture (4 ml) at 56 ° C. for 2 min, the mixture is heated at 42 °>>>> C. for 2 hrs.
The resulting plasmids are introduced into Chi1776 using a modification of the transfection procedure described by Errea et al. J. Mol. Bio. 96, 495-509 (1975). Into 100 ml of L broth supplemented with diaminopimelic acid (DAP 50 micro g/ml) and thymidine (4 micro g/ml), is innoculated 1 ml of an overnight bacterial culture and the bacterial culture grown until exponential phase at 35 ° C. and then harvested by centrifugation at 4 ° C. After washing the cells in 0.3 volume 10 mM NaCl, the cells are resuspended in 30 ml freshly prepared MCN buffer (70 mM MnCl 2, 40 mM NaOAc (pH 5.6) and 30 mM CaCl 2 ) and chilled on ice for 20 min. Cells are collected, resuspended in 1 ml MCN buffer and added in 200 micro l aliquots to 50 micro l DNA in TEN buffer (10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 50 mM NaCl). After chilling for 30 min. at 0 ° C., the mixtures are incubated at 27 °>>>> C. for 5 min., chilled again for 30 min. and 50 micro l aliquots plated onto Penassaybroth agar supplemented with DAP and thymidine. Plates containing transformants are incubated at 32 ° C. and colonies scored 2 to 3 days after plating.
Colonies are screened by the HART method (Patersen et al. Proc. Nat. Acad. Sci. USA 74, 4370-4374 (1977)) by digesting plasmid DNA isolated from the clones with PstI, precipitating with ethanol and dissolving directly into 20 micro l deionized formamide. After heating for 1 min. at 95 ° C., each sample is placed on ice. The samples are mixed with 1.5 micro g oligo dT-cellulose bound RNA, 10 mM PIPES (pH 6.4), 0.4 M NaCl and incubated for 2 hr. at 50 ° C. After diluting with 75 micro l H 2 O and ethanol and adding 10 micro g wheat-germ tRNA, the precipitate is washed with 70% ethanol, dissolved in H 2 O, and added to a wheat-germ cell-free translation mixture (Roberts et al. Proc. Natl. Acad. Sci. USA 70, 2330-2334 (1973)). After 3 hrs. at 23 ° C., the reaction mixture is treated with ribonuclease and analyzed for the presence of the desired protein by an immunoassay, if antibodies are available, or by a bioassay after purification by conventional techniques described previously.
The clone(s) shown to produce the desired protein may then be expanded and used for the production of the desired protein product.
It is evident from the above results, that a conditioned medium can be produced by cultivating Mo cells which can be used for a wide variety of growth and regulatory factors, such as erythroipoietic and colony stimulating factor, immune interferon, neturophil migration-inhibition factor, as well as others previously indicated. The conditioned medium can be used for directing cell differentiation to the growth of a particular type of cell, for protection of cells in vitro from destruction by viral invasion, and for the production of various proteins which may be isolated from the conditioned medium. In addition, the cells provide a source for messenger RNA as well as chromosomal DNA which can be used in hybrid DNA technology for the formation of plasmids for transformation microorganisms. The resulting transformed microorganisms can then be used for the production of the individual factors.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
What is claimed is:
1. A method for producing in isolatable amounts an excretory protein produced by a T-lymphocyte, said method comprising:
cultivating as a single cell suspension the Mo cell line in a nutrient medium, whereby said excretory proteins are produced and excreted into said nutrient medium.
2. A method according to claim 1, wherein at least one of said proteins is a member of the group consisting of colony stimulating factor, erythroid potentiating factor, neutrophil migration inhibition factor and immune interferon Type II.
3. A method according to claim 2, wherein said protein is immune interferon, Type II.
4. A method according to claim 2, wherein said protein is colony stimulating factor.
5. A method according to claim 2, wherein said protein is erythroid potentiating factor.
6. A method according to claim 2, wherein said protein is neutrophil migration inhibition factor.
7. A method according to claims 3, 4, 5, or 6, including the step of isolating and purifying said protein.
8. A method for cloning DNA including a gene capable of expressing a protein product produced by a T-lymphocyte,
said method comprising:
isolating messenger RNA from Mo cells;
separating the messenger RNA coding for expression for a predetermined polypeptide from other messenger RNA;
generating cDNA with reverse transcriptase from said messenger RNA;
preparing double-stranded DNA from said cDNA employing DNA polymerase;
inserting said double-stranded DNA into a vector;
transforming a host with said vector, whereby said double-stranded DNA is cloned.
9. A method according to claim 8, wherein said double-stranded DNA is inserted into said vector to provide expression of said double-stranded DNA; and
including the additional step of isolating protein expressed by said double-stranded DNA.
10. A method according to claim 9, wherein said protein is immune interferon, Type II.
11. A method according to claim 9, wherein said protein is erythroid potentiating factor.
12. A method according to claim 9, wherein said protein is colony stimulating factor.
13. A method according to claim 10, wherein said protein is neutrophil migration inhibition factor
14. A protein composition comprising erythroid potentiating factor in an amount of at least about 50,000 units/mg.
15. A protein composition comprising colony stimulating factor in an amount sufficient to provide at least about 10 units/mg.
16. A single cell suspension of the Mo cell line in a nutrient medium.
17. a single cell suspension according to claim 16, wherein said nutrient medium is serum free.
18. a single cell suspension according to claim 16, wherein said nutrient medium contains serum.
19. A genetic library comprising DNA fragments derived by restriction cleavage of DNA from the Mo cell line.
20. A method for stimulating the poliferation of human bone marrow cells, which comprises contacting said bone marrow cells an amount sufficient to cause proliferation of a composition according to claim 4.
21. A method for stimulating the proliferation of erythrocytes which comprises contacting human bone marrow cells in the presence of erythropoietin with a composition according to claim 5 in an amount sufficient to cause proliferation.
22. A method for inhibiting viral lysis of a human cell susceptible to viral lysis which comprises applying an inhibiting amount to a host human cell of a composition according to claim 3.
Reversing the trial court's judgment of dismissal following the sustaining of demurrers, the majority holds that a cause of action for conversion is stated by the allegations in plaintiff's third amended complaint that, with plaintiff's consent, defendants surgically removed his spleen, tissue, and blood, the majority reaching this conclusion because of the allegation plaintiff was not apprised prior thereto of the research and commercial uses that would be made of these bodily substances. Because the trial court sustained demurrers to the remaining causes of action by reason of the pleading having incorporated by reference the first cause of action for conversion into the remaining causes of action, the majority remands the entire matter to the trial court “for decision of the remaining causes of action, which have never been expressly ruled upon.” (Majority opn., ante, p. 503.)
Although the majority views this case as raising “fundamental questions concerning a patient's right to the control of his or her own body, and whether the commercial exploitation of a patient's cells by medical care providers, without the patient's consent, gives rise to an action for damages” (majority opn., ante, p. 498), the majority correctly recognizes that the “crux of plaintiff's case for conversion” is whether “plaintiff's tissues, including his spleen, blood, and the cell-line derived from his cells ‘are his tangible personal property.’ ” (Majority opn., ante, p. 504.)
WHETHER BLOOD SAMPLES, TISSUE, AND A DISEASED SPLEEN, SURGICALLY REMOVED FROM A PATIENT, CONSTITUTE THE REQUISITE ELEMENT OF “PROPERTY,” THE WRONGFUL DISPOSITION OF WHICH WOULD GIVE RISE TO A CAUSE OF ACTION FOR CONVERSION ON THE PART OF THE PATIENT AGAINST THE TREATING PHYSICIAN AND OTHERS
I part company with the majority in its conclusion that “plaintiff's allegation of a property right in his own tissue is sufficient as a matter of law” (majority opn., ante, p. 504), and that “the law of property” contains “nothing which negates ․ the conclusion” that tissue of living persons constitutes tangible personal property, the wrongful disposition of which can constitute conversion. (Majority opn., ante, pp. 504–505.)
It is plaintiff's burden to establish that his bodily substances constituted “personal property” at the time they allegedly were converted by defendants. “The elements of a conversion are: (1) plaintiff's ownership or right to possession in the property at the time of the conversion; (2) defendant's conversion by a wrongful act or disposition of plaintiff's property rights; and (3) damages. [Citation.] In this action [plaintiff ] had the burden of proving the existence of each of the above elements.” (Chartered Bank of London v. Chrysler Corp. (1981) 115 Cal.App.3d 755, 759–760, 171 Cal.Rptr. 748, emphasis added.)
In order to determine what constitutes “property” in the context of a suit for conversion, resort must be had to the provisions in the Civil Code setting forth a definition of that concept. The Legislature, in enacting the Code in its original form, still in effect today, provided in nineteenth-century prose that “the thing of which there may be ownership is called property,” stating further that “[t]he ownership of a thing is the right of one or more persons to possess and use it to the exclusion of others.” (Civ.Code, § 654.) 1 “Property is either: 1. Real or immovable; or, 2. Personal or movable.” (§ 657.) “Every kind of property that is not real is personal.” (§ 663.) “The words ‘personal property’ include money, goods, chattels, things in action, and evidences of debt.” (§ 14, subd. 3.)
“Goods” comprise “ ‘movables; household furniture; personal or movable estate; wares; merchandise; commodities bought and sold by merchants and traders.’ ” (In re Holmes (1921) 187 Cal. 640, 643, 203 P. 398. See also § 1802.1; Com. Code, § 2105.) “Chattels” comprise personal and movable property, as opposed to real property, and “may refer to animate as well as inanimate property.” (Black's Law Dict. (5th ed. 1979) p. 215, col. 1.) “Conversion has been defined as ‘an act of wilful interference with a chattel, done without lawful justification, by which any person entitled thereto is deprived of use and possession.’ [Citations.]” (de Vries v. Brumback (1960) 53 Cal.2d 643, 647, 2 Cal.Rptr. 764, 349 P.2d 532, emphasis added.)
Unlike the gizzards of domestic poultry (§ 655), the spleens of human beings do not come within the definition of “goods” or “chattels” any more than they fit the remaining definitions of personal property (“money,” “things in action,” and “evidences of debt”). (§ 14.)
The majority, seeking to avoid the constraints of the statutory definition of personal property, asserts that section 14 “is plainly not intended to be an exhaustive list” and that the provisions of the Civil Code “have been interpreted to mean that ‘the word “property” is a generic term which includes anything subject to ownership,’ ” citing Canfield v. Security First Nat. Bk. (1935) 8 Cal.App.2d 277, 284, 48 P.2d 133. (Majority opn., ante, p. 23.) Specific provisions such as section 14, however, qualify a general provision such as section 654. (See §§ 3509, 3534; Neumarkel v. Allard (1985) 163 Cal.App.3d 457, 463, 209 Cal.Rptr. 616.)
The majority also finds in the “rights of dominion over one's own body, and the interests one has therein,” something “so akin to property interests that it would be a subterfuge to call them something else.” (Majority opn., ante, p. 505.)
I find the foregoing assertions by the majority unpersuasive, as I do its reliance on case authority dealing with the harvesting of a beet crop,2 the right of a deceased motion picture actor during his lifetime to exploit his own name and likeness as Count Dracula,3 and the right of surviving family members to control the burial of a dead body.
A spleen is not “personal property” which the patient, to avoid sale by the hospital as unclaimed property, must claim within “a period of 180 days following the departure of the owner from the hospital.” (§ 1862.5.) It is instead the type of “recognizable anatomical part[ ]” or “human tissue [ ]” which is subject to “scientific use.” (Health & Saf. Code, § 7054.4.)
Treatment of plaintiff's spleen, body tissue, and blood as “property” is incompatible with other statutory provisions dealing with the acquisition and disposition of property. As provided by section 1000, “Property is acquired by: 1. Occupancy; 2. Accession; 3. Transfer; 4. Will; or, 5. Succession.” “Property of any kind may be transferred, except as otherwise provided․” (§ 1044.) “A will may dispose of ․ property” (Prob. Code, § 6101), which term is defined in the Probate Code as “anything that may be the subject of ownership and includes both real and personal property and any interest therein.” (Prob.Code, § 62.)
“An interest labelled ‘property’ normally may possess certain characteristics: it can be transferred to others; it can be devised and inherited; it can descend to heirs at law; it can be levied upon to satisfy a judgment; it comes under the jurisdiction of a bankruptcy court in a bankruptcy proceeding; [fn. omitted] it will be protected against invasion by the courts; it cannot be taken away without due process of law. [Fn. omitted.]” (First Victoria Nat. Bank v. United States (5th Cir.1980) 620 F.2d 1096, 1103–1104.)
The only direct support in the law cited by the majority opinion, reflecting its expansive definition of personal property encompassing human parts and human refuse, is Venner v. State (1976) 30 Md.App. 599, 354 A.2d 483, a case dealing with a seizure of human feces from a hospital bedpan by police officers in quest of contraband narcotics. Although the Maryland Court of Special Appeals observed that “[i]t is not unknown for a person to assert a continuing right of ownership, dominion, or control ․ over such things as excrement, [fn. omitted] fluid waste, secretions, hair, fingernails, toenails, blood, and organs or other parts of the body” (id. at p. 498), this dictum is a weak foundation upon which to construct a property right in a patient's surgically-removed body tissue and fluid. In any event, I am not prepared to extend the constitutionally sanctified right of property (U.S. Const., 5th & 14th Amends.; Cal. Const., art. I, §§ 1, 7) to the refuse found on the floor of the barbershop or nail salon, in the hospital bedpan, or in the operating room receptacle.
WHETHER, IN ANY EVENT, CONSENT TO A SPLENECTOMY AND THE TAKING OF BLOOD AND TISSUE SAMPLES AMOUNTS TO ABANDONMENT OF ANY PROPERTY INTEREST THE PATIENT MIGHT POSSESS IN THE REMOVED BODILY SUBSTANCES
Even if one accepts the majority's premise that the diseased 4 spleen and other bodily tissue and fluids removed from plaintiff constituted “property,” the allegations in the third amended complaint necessitate consideration of the issue whether plaintiff abandoned any property interest he possessed in such substances.
Plaintiff alleges, “Prior to surgical removal of his spleen, ․ plaintiff signed a written consent form authorizing ․ surgeons at the UCLA [University of California at Los Angeles] Medical Center, to perform a splenectomy upon plaintiff.” On several subsequent occasions he voluntarily agreed to the removal of blood samples.
The only suggestion that no abandonment took place is the allegation plaintiff's consent to the surgery and the taking of blood samples was not an unconditional relinquishment of whatever interest he possessed in such bodily tissue and fluids, because he did not assent expressly to research and commercial use of those substances, a matter considered in the following section of this dissenting opinion.
Aside from the alleged economic interest in their use asserted by plaintiff, a patient who consents to surgical removal of his bodily substances has no reasonable expectation as to their subsequent use other than an understanding that the licensed medical personnel involved in the removal and use of these substances will comply with applicable medical standards and legal constraints, such as the mandate that substances removed from his body “following conclusion of scientific use ․ be disposed of by interment, incineration, or any other method determined by the state department to protect the public health and safety.” (Health & Saf. Code, § 7054.4, emphasis added.)
WHETHER THE CONSENT OF A PATIENT TO SURGICAL PROCEDURES IS UNINFORMED UNLESS HE OR SHE IS ADVISED OF THE POTENTIAL RESEARCH AND COMMERCIAL USES OF SUBSTANCES REMOVED FROM THE PATIENT'S BODY
The majority concludes “that simple consent to surgery does not imply a consent to medical research on the patient's tissues unrelated to treatment nor to commercial exploitation of the patient's tissues” (majority opn., ante, p. 511), based on plaintiff's allegation in the third amended complaint that he would not have agreed to the removal of his diseased spleen and bodily fluids had he known of “defendants' true research and commercial intentions and plans in this regard, and the value of his Blood and Bodily Substances in those intentions and plans․”
Even apart from the above-quoted statutory provision recognizing “scientific use” of surgically removed “anatomical parts” and “human tissue,” and plaintiff's admitted consent on at least two occasions (April 11, 1983, and September 20, 1983) to “research” unrelated to his own medical treatment being conducted on blood samples taken from his body, there is nothing in logic or law to support the majority's action engrafting onto an apparently unqualified consent a proviso that the ensuing scientific use of the removed body tissue or fluid exclude research and commercial use.
The majority's holding invalidates the standard surgical consent form insofar as it would authorize research and commercial use of surgically removed bodily tissue and fluid. (See 5 Witkin, Summary of Cal. Law (9th ed. 1988) Torts, §§ 360–361, pp. 446–449.) The majority's opinion will require hospitals and surgeons to give prospective patients an expanded advisement concerning potential research and commercial use of their removed bodily tissue and fluid or otherwise risk foregoing such use or incurring potential liability. (In this regard, it should be noted that no perfect dichotomy exists between research and commercial use.)
To the extent the foregoing holding is applicable to “the practice or research of medicine in a manner not reasonably related to maintaining or improving the health of [the patient] or otherwise directly benefiting [the patient]” (Health & Saf. Code, § 24174), it is inconsistent with the Protection of Human Subjects in Medical Experimentation Act. (Health & Saf. Code, § 24170 et seq., enacted in 1978, after plaintiff's splenectomy but prior to the removal of bodily substances during some of his later visits to the UCLA Medical Center.)
In enacting this legislation, the California Legislature recognized “that medical experimentation on human subjects is vital for the benefit of mankind,” but declared that “such experimentation shall be undertaken with due respect to the preciousness of human life and the right of individuals to determine what is done to their own bodies.” (Health & Saf. Code, § 24171.) With these objectives in mind, the Act sets forth an “experimental subject's bill of rights,” which among other advisements requires informing the patient concerning the nature and purpose of the experiment and obtaining the patient's informed consent thereto. (Health & Saf. Code, §§ 24172–24175.)
Significantly, the advisement and consent required by the Act do not include any mention of the commercial use which might ensue from experimental research.5
THE PROBLEM OF DAMAGES UNDERLYING A CAUSE OF ACTION FOR CONVERSION OF BODILY SUBSTANCES
The inappropriateness of a conversion suit as a means of redressing the wrong allegedly suffered by plaintiff becomes even more apparent when considered in light of the measure of damages specified for such a cause of action. Under section 3336, “The detriment caused by the wrongful conversion of personal property is presumed to be: First—The value of the property at the time of the conversion, with the interest from that time, or, an amount sufficient to indemnify the party injured for the loss which is the natural, reasonable and proximate result of the wrongful act ․; and Second—A fair compensation for the time and money properly expended in pursuit of the property.” (Emphasis added. See Krueger v. Bank of America (1983) 145 Cal.App.3d 204, 215, 193 Cal.Rptr. 322.)
As previously noted, I cannot accept the majority's characterization of plaintiff's diseased spleen as “property,” a necessary element of a cause of action for conversion. But if the majority's premise is accepted, the question arises whether this “property” was worthless as a matter of law “at the time of the conversion” and whether there was any further “loss” as the “proximate result” of the purported conversion. (§ 3336.) It was only after defendants expended great effort, time, and skill that—in my opinion—plaintiff's spleen acquired any of the characteristics of property, at which time this diseased organ became transmuted from human waste into patentable blood cell-lines. Although it can be argued the blood extracted from plaintiff had a monetary value, its commercial worth is substantially restricted by provisions severely curtailing the sale of blood. (Health & Saf. Code, §§ 1606, 1626.)
Even if—as the majority views them—plaintiff's bodily substances are assumed to have constituted “property” at the time they were removed from plaintiff, their value at that time would be de minimis and plaintiff's potential damages for their conversion therefore negligible. Although the raw material from plaintiff's body may have been unique, it evolved into something of great value only through the unusual scientific expertise of defendants, like unformed clay or stone transformed by the hands of a master sculptor into a valuable work of art.
Plaintiff in his third amended complaint alleges that his post-surgical visits to UCLA Medical Center (but not the splenectomy) were “without plaintiff's knowledge or consent solely to further defendants' research and commercial activities and to promote defendants' economic, financial, and competitive benefit, and were not intended to, and did not provide, any assistance in the actual medical and health care plaintiff received from the defendants.” (Emphasis added.) Because plaintiff has made the foregoing allegation, it is appropriate to observe that surgery or blood extraction performed without (or beyond) the consent of the patient is a battery, and that a consent given without adequate disclosure is uninformed and therefore invalid. Accordingly, if properly and timely pleaded by a patient, allegations such as those made by plaintiff would afford the patient the remedy of seeking damages on a cause of action against medical personnel for the intentional tort of battery (which is not among the 13 causes of action pleaded by plaintiff). (See 5 Witkin, Summary of Cal. Law, supra, §§ 352, 357, 358, pp. 439–440, 443–445.) Additionally, the aforementioned Protection of Human Subjects in Medical Experimentation Act provides substantial civil and criminal liability for the conduct of a medical experiment without the patient's informed consent. (Health & Saf. Code, § 24176.) However, I reiterate that no legal authority (in that Act or elsewhere) requires that a patient be given an advisement of the potential commercial use of the substances removed from his or her body in order for the patient's consent to be an informed one.
POLICY CONSIDERATIONS MILITATING AGAINST JUDICIAL RECOGNITION OF A CAUSE OF ACTION FOR CONVERSION OF HUMAN BODILY SUBSTANCES
Even prior to the filing of the court's opinion in this appeal, it was recognized that “Moore promises to create valuable precedent in the area[ ] of the patient's property rights in his or her body․” (Hardiman, Toward the Right of Commerciality: Recognizing Property Rights in the Commercial Value of Human Tissue (1986) 34 UCLA L.Rev. 207, 213. See also Sherman, The Selling of Body Parts (1987) National L.J. 1.)
I am greatly concerned about the full implications of the majority's ruling that body fluids and parts constitute a form of property. There are those who believe “ ‘society will benefit ․ from a market in body parts'․ More body parts would be available for recipients, and donors would gain a means to make money.” “Some people are revolted by the thought of a woman selling her aborted fetus. Others think it is only fair.” (Sherman, The Selling of Body Parts, supra.)
Plaintiff himself admits, in his third amended complaint, that had he been aware of the commercial potential of his diseased spleen he “would have considered whether to avail himself of medical, surgical and health care services at other facilities and institutions, where his wishes in this regard would have been inquired into, respected, and carried out. [¶] ․ [P]laintiff would have sought to participate in the economic and financial benefit defendants ․ are likely to receive, as a result of their research and commercial activities․”
The absence of legislation regulating the trafficking in human body parts (except where transplantation is involved) raises the specter of thriving “used body parts” establishments emulating their automotive counterparts, but not subject to regulation comparable to that governing the latter trade. (Veh. Code, §§ 220, 11500; Bus. & Prof. Code, § 9884.10.)
I believe we are not authorized, and should not be inclined, to create new rights and remedies in an area which so clearly lies outside the bounds of the legislatively defined cause of action for conversion (§§ 14; 3336) and is so unsuited to judicial intervention.
The majority rationalizes the ramifications of its willingness to enter this uncharted area by observing, “To the extent that unacceptable consequences, which can now only be the subject of speculation, do follow, legislative solutions are possible and likely.” (Majority opn., ante, p. 509.) In my opinion, it is regrettable that the majority's expressed deference to the legislative process comes only after the fact. I would defer to the Legislature not only because I believe we must, but because that body has shown itself willing, able, and best-suited to regulate areas involving comparable competing interests.
As previously observed, the Legislature in 1978 enacted the Protection of Human Subjects in Medical Experimentation Act. (Health & Saf. Code, §§ 24170–24179.5.) Extensive provisions now prohibit the sale of human organs for the purpose of transplantation. (Pen.Code, § 367f, enacted in 1984.) The Uniform Anatomical Gift Act (enacted in 1970 as ch. 3.5 of the Health & Saf. Code) specifies that the “use of any human tissue donated ․ for the purpose of transplantation in the human body shall be construed for all purposes as a rendition of a service by each person participating therein and shall not be construed as a sale of such tissue.” (Health & Saf. Code, § 7155.6. See also §§ 1603.3; 1606; 1625, subd. (e); 1626 of that Code for comparable provisions dealing with the donation of blood and blood products and severely limiting the circumstances under which the “donor” of blood may receive compensation.) Only last year, Congress enacted the National Organ Transplant Act prohibiting the transfer in interstate commerce of any “human organ for valuable consideration for use in human transplantation.” (42 U.S.C.A. § 274(e).)
Under the Uniform Anatomical Gift Act, among the persons who may “become donees of gifts of bodies or parts thereof for the purposes stated” are “[a]ny hospital, surgeon, physician, or coroner, for medical or dental education, research, advancement of medical or dental science, therapy, or transplantation.” (Health & Saf. Code, § 7153.5, subd. (a).) Where the donation of organs from a dead body is involved, “custody” (not ownership) “of the remainder of the body vests in the surviving spouse, next of kin,” etc.6 (Health & Saf. Code, § 7155.5, subd. (a).)
The Legislature also has seen fit to provide that “Human whole blood, plasma, blood products, and blood derivatives, or any human body parts held in a bank for medical purposes, shall be exempt from taxation for any purpose.” (Rev. & Tax. Code, § 33, enacted in 1965.)
Various “arguments against recognition of property rights” in human bodily substances are listed in Hardiman, Toward the Right of Commerciality, supra, at pages 236–237. They warrant recitation, not because I feel we are in a position to adopt or reject these arguments, but because they illustrate the complexity of the issues before us and the superiority of the Legislature's fact-finding capabilities over our own in resolving these questions: “Although compelling arguments support recognizing property rights in human tissue, arguments against recognition may also be raised. These arguments include: the potential for adverse effects on organ donation for transplantation usage; the moral aversion to treating the body as a commodity; the effect of patient hold-outs and higher transaction costs on research and tissue availability; and the threat of improper motivation in the area of tissue acquisition․
“Several potentially persuasive arguments against property rights arise in the context of organ transplantation. Opponents of organ sales predict a variety of adverse effects should a market in human organs arise: decreases in the number of organs charitably donated; increases in the number of inferior organs; competitive bidding between patients for limited resources; financial pressure on the poor to sell their organs; and unacceptable risks of death for a pecuniary profit. [Fn. omitted.] Without question, any adverse effects of property rights on the organ donation system are of critical importance to the health of the populace. If these rights seriously interfere with the availability of life-saving transplantable organs, the cost of recognizing the patient's rights may be too high for society to bear. [Fn. omitted.]”
If a patient with unusual bodily substances is encouraged (as he or she will be, by today's decision) to shop around for the highest bid on the patient's spleen, tissue, blood, or other bodily substance, the quality of health care and medical research undoubtedly will suffer. By recognizing a property right in such substances, and expanding informed consent to include advisement of potential research and commercial use of the matter removed from the patient's body, the majority clearly establishes a right to bargain over body parts and share in the financial windfall that sometimes ensues from years of expensive medical research.
The right to be advised of research and commercial use of substances taken from one's body implies the right to withhold consent for such use. As one of many illustrations of the potential ramifications of the majority's ruling, we need consider only the current massive effort to eradicate the lethal AIDS virus. Any determination granting a patient the unilateral right to forbid the use of tissues and fluids, already taken from his or her body, in research designed to find a cure to a disease posing a grave threat to public health, should be left to the Legislature. It should also be noted that the problems of consent, and of “profit-sharing,” are compounded where specimens from several individuals (or perhaps several hundred or several thousand) are utilized in effecting a cure for a deadly disease.
Much of the New Jersey Supreme Court's characterization of “baby-bartering” in the celebrated case of Matter of Baby M. (1988) 109 N.J. 396, 537 A.2d 1227 is equally applicable to “body-part bargaining”: “The evils inherent in baby-bartering are loathsome for a myriad of reasons. The child is sold․ [¶] Baby-selling potentially results in the exploitation of all parties involved.” (Id. 537 A.2d at pp. 1241–1242.) “Whatever idealism may have motivated any of the participants, the profit motive predominates, permeates, and ultimately governs the transaction. The demand for children is great and the supply small․ The situation is ripe for the entry of the middleman who will bring some equilibrium into the market by increasing the supply through the use of money.” (Id. 537 A.2d at p. 1249.) “There are, in a civilized society, some things that money cannot buy․ There are, in short, values that society deems more important than granting to wealth whatever it can buy, be it labor, love, or life.” (Ibid.)
Like the court below, I conclude plaintiff has not pleaded any recognized cause of action for the claim asserted by him, and decline to expand the legislatively defined cause of action for conversion to encompass a situation never contemplated by the Legislature or the courts that have helped develop the law on this subject. Instead, I would leave to the Legislature's demonstrated competence in this field the task of adding, to the several comprehensive enactments in the area of body substance transfer, an informed regulatory scheme designed to balance the vital competing interests of the patient, the health care provider, the commercial research laboratory, and the public at large in the removal and transfer of human bodily substances for research and commercial use.
1. Because this is an appeal from the sustaining of demurrers, our review of the case is necessarily limited to an examination of “the face of the pleading” alone. (5 Witkin, Cal. Procedure (3d ed. 1985) Pleading, § 895, p. 334.) Accordingly, statements in this opinion referring to the facts of this case are based on the unproven allegations of the complaint.
2. The Regents of the University of California (Regents) are the legal entity that operates UCLA.
3. Cells are “the basic structure and functional units of all living organisms,” and carry within their nuclei an individual's unique genetic information in DNA (deoxyribonucleic acid) macro molecules. DNA cloning is “the production of billions of copies of a piece of DNA as part of a plasmid introduced into a microorganism,” such as Escherichia coli (E. coli) bacteria. (Gordon Edlin, Genetic Principles—Human and Social Consequences (1984) pp. 15, 29–30, 446–448, 452.) This technology is not new. It is based on the scientific break through in 1953. Technology emerged allowing manipulations of genetic material, or, as in the instant case, utilized the genetic material of a unique individual to produce substances of great benefit to humankind. The process involves the joining together of segments of the DNA from the human cells with those of a microorganism such as E. coli bacteria to create “recombinant DNA.” This process of genetic engineering, or gene splicing, produces a cell which divides, and which, in the instant case, produced cells “shown to be capable of continuous culture for an indefinite period of time.” (Patent, Appendix A, p. 516. The patent is not a part of the complaint, but was before the trial court in ruling on the demurrer.)
4. Although defendants refer to the plaintiff's spleen as “diseased,” there is nothing in the complaint or record to support that characterization.
5. See appendix A to this opinion. A genetically engineered microorganism is patentable. (Diamond v. Chakrabarty (1980) 447 U.S. 303, 310, 100 S.Ct. 2204, 2208, 65 L.Ed.2d 144.)
6. The complaint alleges that the products from the Mo cell-line included:“(a) Colony–Stimulating Factor (CSF)․“(b) Erythroid–Potentiating Activity (EPA)․“(c) Immune Interferon (Type II)․“(d) Neutrophil Migration–Inhibitory Factor (NIF–T).“(e) T-cell Growth Factor (TCFG, Interleukin II).“(f) Macrophage–Activating Factor (MAF)․“(g) Factor–Stimulating Fibroblast Growth.“(h) Factor–Stimulating Human Pluripotent Hematopoietic Stem Cell․“(i) Factor–Stimulating Human Leukemic Cells in vitro․”
7. The issues of consent and abandonment will be discussed below.
8. We are not called upon, nor are we attempting, to resolve the complex issues relating to the human fetus.
9. Statutes concerning blood donations are not particularly helpful. Use of blood from paid donors is severely limited (Health & Saf.Code, § 1626) undoubtedly because of public health concerns.
10. This statute was enacted after the splenectomy in the instant case. We make no determination whether the takings of tissue after 1978 came within the definition of “medical experiment” in Health and Safety Code section 24174, subdivision (a): “The severance or penetration or damaging of tissues of a human subject ․ in the practice or research of medicine in a manner not reasonably related to maintaining or improving the health of such subject or otherwise directly benefiting such subject.”
11. The complaint alleges that this technique is not unique or extraordinary science. Biotechnology has been a recognized science for the past two decades.
12. See discussion of damages in Note, Toward the Right of Commerciality, supra, 34 UCLA L.Rev. 207, 257.
13. A simple analogy illustrates the point: “Crude oil may be ruining a farmer's corn crop. The farmer may even be willing to pay an oil refinery company to take it off his land. But the farmer, who would be unable without the refinery's aid to turn the crude oil into a usable commodity, is still entitled to a share of the refinery's profits from his land's product.” (Sherman, The Selling of Body Parts (Dec. 7, 1987) National Law Journal, at p. 1.)
14. The Regents cited an example of research involving the combination of materials from the pituitary glands of hundreds of individuals, claiming this sort of research would be stifled by the onerous record keeping needed if “ownership” had to be tracked. No support from scientific sources was given, and the claim is dubious in light of the meticulous care and planning necessary in serious modern medical research.
15. “The central issue has been stated bluntly by Professor Leon Wofsy, an immunologist at the University of California, Berkeley: ‘The business of business is to make money ․ and the mode is secrecy, a proprietary control of information and the fruits of research. The motive force of the university is the pursuit of knowledge and the mode is open exchange of ideas and unrestricted publication of the results of research.’ ” (Conflict of Interest on the American Campus, The Economist (May 22, 1982) p. 107, col. 1.)
16. Health and Safety Code section 7054.4 states: “Notwithstanding any other provision of law, recognizable anatomical parts, human tissues, anatomical human remains, or infectious waste following conclusion of scientific use shall be disposed of by interment, incineration, or any other method determined by the state department to protect the public health and safety. [¶] As used in this section, ‘infectious waste’ means any material or article which has been, or may have been, exposed to contagious or infectious disease.”
17. For example, see: Michael H. Shapiro and Roy G. Spece, Jr., Cases, Materials and Problems on Bioethics and Law (1981) pages 326–329; Julie Ann Miller, The Clergy Ponder the New Genetics (March 24, 1984) Science News, page 188; Horace Freeland Judson, Thumbprints in Our Clay (Sept. 19–26, 1983) The New Republic, page 12; and Editorial in Christianity Today (Jan. 23, 1981.)
18. We note the comments reported in Science, February 7, 1986, from participants in a conference on January 17, 1986, of leading medical ethicists, lawyers and researchers. Many of the participants “supported the idea that a physician should not be involved with both the research and the therapy of the patient at the same time. The duties should be divided among different individuals to eliminate the possibility that a patient might feel coerced into participating in the research․” One participant was quoted as saying, “I don't want researchers to be called vultures.” (Id., at pp. 543–544.)
19. In passing, however, we agree that by sustaining the demurrers without leave to amend, the trial court conserved judicial resources by allowing for the consolidation of the appeals.
20. Plaintiff alleged that: “․ each of the defendants was the agent, joint venturer and employee of each of the other remaining defendants, and is jointly liable for the acts of every other defendant and in doing the things hereinafter alleged, each was acting within the course and scope of said agency, employment, partnership and joint venture with the advance knowledge, acquiescence or subsequent ratification of each and every remaining defendant, and that each defendant joined together with every other defendant and conspired to do and carry out all of the acts herein alleged and that each defendant had a fiduciary duty to the plaintiff, and each acted in concert with every other defendant in violating their duties to plaintiff.”Further, plaintiff alleged that Dr. Golde acted in all his conduct respecting plaintiff with the knowledge and support of all the other defendants. All the defendants “․ knew or should have known of the physician-patient relationship with plaintiff, the means by which Defendant Golde had obtained access to, and continued to have access to, plaintiff's Blood and Bodily Substances, and through their actions and compensation of Defendant Golde, ratified or approved Defendant Golde's activities in this regard.”
1. All further statutory references are to the Civil Code unless otherwise noted.
2. Corey v. Struve (1915) 170 Cal. 170, 173, 149 P. 48 (disapproved on another point in Maben v. Rankin (1961) 55 Cal.2d 139, 144, 10 Cal.Rptr. 353, 358 P.2d 681).
3. In Lugosi v. Universal Pictures (1979) 25 Cal.3d 813, 818–819, 160 Cal.Rptr. 323, 603 P.2d 425, the court found it unnecessary to reach the issue whether the deceased had a property interest in his own name and likeness, concluding that the right to exploit them commercially was personal to him and could not pass by inheritance to his surviving family members. (Cf. § 990, enacted in 1984.)
4. The majority asserts, “Although defendants refer to the plaintiff's spleen as ‘diseased,’ there is nothing in the complaint or record to support that characterization.” (Majority opn., ante, p. 500, fn. 4.)The complaint refers at length to the abstract of invention appended to defendants' patent and comprising appendix A of the majority's opinion. This document indicates that prior to the removal of his spleen, “On presentation to a physician the patient had massive splenomegaly [enlargement of the spleen].” The same document describes the cells removed from plaintiff's spleen after the splenectomy as possessing a quality “characteristic of hairy-cell leukemia.”
5. Plaintiff's third amended complaint concedes he signed medical experimentation consent forms on the occasion of his last two medical visits, on April 11, 1983, and September 20, 1983. The form signed on the latter occasion was referred to in the third amended complaint and comprises appendix B of the majority opinion, ante. That form, approved by the UCLA Human Subject Protection Committee and entitled Informed Consent for Use of Blood and Bone Marrow Tissue for Medical Research Concerned with “Growth of Human Hematopoietic Cells in Vitro,” informed plaintiff among other advisements that the “research” involving plaintiff's bodily substances “will be ongoing for four years” and “may not benefit” plaintiff. By signing the consent form, plaintiff acknowledged his understanding that defendant Dr. David Golde “will answer any questions I may have at any time concerning details of the procedures performed as part of this study,” and further “acknowledge[d] receiving a copy of the form, as well as a copy of the ‘Subject's Bill of Rights.’ ” In signing the form on September 20, 1983, plaintiff circled the words “do not,” preceding a provision relinquishing his “rights ․ in any cell line or any other potential product which might be developed from the blood and/or bone marrow obtained from [plaintiff].”
6. In 1984, in the aftermath of Lugosi v. Universal Pictures, supra, 25 Cal.3d 813, 160 Cal.Rptr. 323, 603 P.2d 425, the Legislature enacted detailed provisions establishing “property rights, freely transferable,” in a “personality's name, voice, signature, photograph, or likeness.” (§ 990.)
ROTHMAN *, Associate Justice. FN* Assigned by the Chairperson of the Judicial Council.
WOODS, P.J., concurs.