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Is controversy about 'transfer factor therapy' nearing an end? Gregory B. Wilson and H. Hugh Fudenberg Nearly 30years after Lawrencefirst described 'transfer factor' the therapeutic value of dialysable leukocyte extracts is now being examined in adequately designed trials. The evidence of its efficacy in certain patients in certain circumstances is unmistakable.
Transfer'factor and dialysable leukocyte extracts In the mid 1950s Lawrence first reported that delayedtype cutaneous hypersensitivity ( D T H ) to tuberculin (PPD) or streptococcal M substance could be passively transferred using extracts of leukocytes obtained from skin-test positive, normal human donors to previously skin-test negative, normal human recipients ~'2. In 1963, Lawrence et aL 3 claimed that dialysates of leukocyte extracts (i. e. dialysable leukocyte extracts, DLE) were as effective in transferring D T H as whole leukocyte extracts. The active component(s) was at that time termed 'transfer factor' (TF) as it was thought to transfer D T H . In the succeeding years little was learned about the mechanism of action of T F or its physicochemical properties, except that it was not immunogenic in man or other animals (for a review of the early work see Ref. 4). Because T F was naively thought to be the only biological activity present in DLE, Lawrence and others tended to use the term ' T F ' , rather than ' D L E containing T F activity', when referring to transfers produced by DLE. These semantics led to considerable confusion and controversy, especially when it later became evident that DLE contains several hundred chemical moieties, many of them very active biologically (for review see Refs 5-9). While T F has historically been defined functionally as a biochemical entity in D L E which can transfer antigenspecific reactivity as shown by skintesting, or more recently by more sensitive and more quantitative in-vitro assays which measure the release of lymphokines by antigen-activated T lymphocytes5, it has not yet been completely characterized, and only very recently have any definitive reports been published which appear to demonstrate that T F may indeed act in an antigen-specific fashion (see below). Other components in D L E which have antigen-independent, non-specific effects on cellmediated immunity (CMI) and the inflammatory response include the prostaglandins, nicotinamide, ascorbic acid, histamine, serotonin, T-lymphocyte maturation or differentiation factors (thymosin or thymic humoral factor) 1°, chemoattractants for monocytes, and neutrophil immobilizing factor(s) (reviewed in Refs 5-8). Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, SC 29425, USA.
These components might be expected to produce generalized immunological enhancement or suppression when D L E is evaluated in vitro or in vivo clinically. Beyond the semantic problems were even bigger ones. Until 1970, most immunologists either doubted the existence of T F or questioned its significance. At that time the T F phenomenon could be demonstrated only in man and the relationship of skin-test reactivity to systemic cellular immunity in mammals had only recently become apparent. But in 1970 there was renewed interest when Levin etal. 11demonstrated that D L E containingTF could, in addition to transferring skin-test reactivity, confer (a) the ability to produce lymphokines in response to specific antigens, and (b) resistance to infection in patients with genetically determined immune deficiency. Since 1970, DLE has been used in immunotherapy and/ or immunoprophylaxis in which cell-mediated immune responses are compromised, including inherited or acquired' antigen- specific' o r ' broad spectrum' immunodeficiency diseases, neoplasia, and a variety of viral, fungal and mycobacterial diseases (reviewed in t~.efs 12-15). The success of clinical trials to date has been variable even for a single disease or syndrome, and all of these studies have been uncontrolled, except for four discussed below 16-19. T h e nature and properties of DLE and T F The antigen specificity of T F is a highly controversial matter20 ~swhich may now be approaching resolution with the publication, from three groups z4-27, of compelling evidence that T F does act specifically(at least in vitro). Why the controversy? Table I, compiled from a review of m~/ny clinical studies 22, shows reactivity (as shown by skin-test responses) and the new antigen reactivities that appeared in the recipients of their DLE. Many recipients acquired D T H responses to most but not every one of the antigen specificities to which the donors responded while some failed to respond to any antigen (43 of 200 recipients listed under 'antigen-sensitive donors' in Table I). Even more problematic are the recipients of DLE who became capable of responding to antigens to which the donors were skin-test negative (12 out of 151 times; Table I). Some may feel strongly that such data justify doubts about the specificity of TF. We, however, contend tha~ 1983, Elsevier Science P u b l i s h e r s B V , A m s t e r d a m
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TABLE I. In-vivo evidence for specificityof human transfer facto# Skin-test conversionb Antigenc PPD Coccidioidin SK-SD Candida Mumps KLH Other Total skin tests: Total recipients:
Antigen-sensitive donors
Antigen-insensitive donors
58/74 23/26 9/18 12/17 9/15 25/26 21/23 157/200 (78%) 169
1/26 7/45 1/9 1/11 0/1 1/30 1/29 12/151 (8%) 110
a From Burger et aL 22 b Expressed as the number of responses that became positive after TF/the number of responses that were negative before TF. c PPD: purified protein derivative; SK-SD: streptokinase-streptodornase; KLH: keyhole limpet hemocyanin; 'other' includes diphtheria toxoid, aspergillin, ethylene oxide-treated serum, skin grafts, histoplasmin, trichophytin, lepromin, vaccinia, sarcoma extracts. m u c h of the problem stems from a reliance on skin testing as the sole criterion for antigen responsiveness in both D L E donors a n d recipients 4. Skin testing is neither quantitative nor particularly sensitive and a positive response is highly dependent on the a m o u n t of antigen used. T h e definition of 'responsiveness' is also somewhat arbitrary, different investigators requiring different thickness of induration. I n addition, some D L E donors who were originally skin-test negative to a particular antigen converted to a n antigen-responsive state after donating leucocytes, either because they were primed by skin testing or because they were never non-responsive in the first place. Doubts about the specificity of T F have also been fanned by indications of a generalized non-specific enhancement of i m m u n e reactivity in vitro in some D L E recipients. Clearly, it is these other components of D L E and not T F which are promoting this generalized enh a n c e m e n t of i m m u n e reactivity in some recipients of D L E 5 8.1315. This emphasizes the problems caused by wrongly equating D L E with T F . Fortunately, the sole reliance on skin testing in T F research seems rapidly to be ending as in-vitro assays become available5. Also, since 1970, T F cellular sources ( T lymphocytes) and target cell(s) ( T lymphocytes, natural killer cells, and possibly monocytes-macrophages) have been identified (reviewed in Refs 4, 5, 12, 13). A n i m a l models are available for the study of T F a n d it has been shown to transfer antigen-specific cellular i m m u n e reactivity across m a m m a l i a n species bartiers 12'2s.T h e biochemical n a t u r e of T F is clearer ls'29's°a n d there are hypotheses for its mode(s) of action 5'29'31. For instance, we have shown the presence of two structurally distinct T F moieties in D L E for both the antigen specificities ( P P D a n d coccidioidin) which we have extensively studied 29'31. In addition, a third T F moiety exists for each T F specificity (derived from one T F moiety found in DLE) that is released by i m m u n e lymphocytes pulsed with specific antigen in vitro 3j (see simplest case molecular models shown in Fig. 1). Recently, we have applied some of the above develop-
ments (particularly the in-vitro assay for T F activity) to the clinical use of D L E containing T F . W e have developed new methods for (a) determining the ' T F potency' of D L E preparations, (b) predicting whether a patient will or will not benefit from i m m u n o t h e r a p y with T F , a n d (c) monitoring the responses of recipients to T F therapy 32'33. Although these methods are only now b e g i n n i n g to be applied to the clinical use of T F , we believe that they will increase the scientific value of immunotherapeutic a n d immunoprophylactic trials, with T F in the future. Lessons from past clinical studies
D L E has been used clinically as a n immunotherapeutic or immunoprophylactic agent in m a n y disorders (Table II). The evaluation of m a n y past reports is difficult because of the small n u m b e r of patients treated, the inclusion of patients with advanced stages of m a l i g n a n t disease (particularly malignancies such as m e l a n o m a a n d Hodgkin's disease), the short duration of follow-ups, a n d the paucity of available laboratory data. Even in the treatment of patients with the same syndrome there are variations in the preparation, type or potency of D L E used, in the methods employed for selecting donors a n d monitoring the progress in recipients, a n d in the clinical complications. Nonetheless, some key lessons have been learned. D L E has remarkably few side-effects either immediate or delayed~2-15-occasional pain or erythema at the site of injection a n d transient fever, without evidence of toxicity. T h e variable results of past clinical studies are, we believe, due to inappropriate selection of recipients a n d
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tuberculin (PPD). Both are polyribonudeopeptides; the TF moiety on the left contains predominantly purine residues while the TF moiety on the right contains mainlypyrimidine residues. The polypeptide segment(except for the phosphate) is thought to be the same in each moietyand probably governs antigen specificity. The two TFs shown are thought to have distinct roles in the process of immune sensitization. The moiety on the right may be strictly an intraceUular product (perhaps a gene deregulator) involved in the synthesis of the other TF moiety, which may be secreted by antigen-activated T lymphocytes and becomes part of a T-cell antigen receptor necessary for antigen responsiveness.Both TFs would be present in DLE, since the cells are disrupted during its preparation2931. Recently, we have found that immune lymphocytes release a third TF moiety when incubated with specificantigen in vitro. This moiety is structuraUysimilar to the TF moiety on the left but lacks the Y-terminal phosphate group31.
159
Immunology Today, vol. 4, No. 6, 1983
donors, failure to evaluate the potency and specificity of D L E preparations used, failure to monitor the i m m u n o logical response to therapy, and failure to provide conventional therapy such as surgery or antibiotics or nutrients to reduce the antigen load. The importance of appropriate selection and evaluation of recipients cannot be overemphasized. Adequate n u m b e r s of target cells (i.e. m a t u r e T cells or their precursors) must be present. The extent of the antigen load (micro-organisms or tumor cell mass) a n d the general health of the patient including nutritional status must be known. The most consistent successes in imm u n o t h e r a p y or immunoprophylaxis with D L E have been in patients with a n antigen-selective defect in T-cellmediated immunity, in w h o m therapy was initiated early in the course of the disease (or the antigen load was reduced prior to D L E therapy), a n d when other forms of therapy were used prior to D L E therapy. Investigators should not expect effective D L E therapy against an infective agent in a patient whose antigen-selective defect in C M I to that agent has allowed the disease to develop (IIA to F in Table II) unless the D L E preparation employed contains T F specific for the etiologic agent in question. However, even patients with broad-spectrum defects have benefited from a generalized e n h a n c e m e n t of i m m u n e reactivity induced by other components in DLE12-14. D L E donor(s) must be selected on the basis of demonstrably strong C M I of specificity desired in the recipient; in patients with chronic mucocutaneous candidiasis a n d osteogenic sarcoma 9'Is's4, improved i m m u n o logical laboratory parameters a n d clinical benefit were seen only when D L E contained T F specific for Candida or osteogenic sarcoma t u m o r antigens, respectively. Because D L E contains, in addition to T F , both imm u n o a d j u v a n t and inhibitory activities which can block (i.e. 'suppress') the potential beneficial effects of T F , some generalized clinical or immunological i m p r o v e m e n t m a y occur in patients receiving D L E of the ' w r o n g ' antigenic specificity or D L E of the 'right' specificity m a y be ineffective. I n patients in whom the infecting organism is u n k n o w n or u n c o m m o n , a n d in malignancies or other disorders of suspected viral etiology (i.e. osteogenic sarcoma, melanoma, Behcet's syndrome), prospective donors should be obtained from household contacts of the patient 3s-37. Household contacts of osteogenic sarcoma patients, for example, show a significantly higher frequency of significant C M I against osteosarcoma cell lines than do r a n d o m l y selected individuals (40% compared to
1%). Genetic factors ( ' i m m u n e response genes') m a y also underlie the finding that different individuals respond differently to the same batch of D L E - so-called 'recipient specificity 'ls'3s'39. In addition, i m m u n e competence m a y be balanced against antigen load, producing either progressive disease or clinical improvement. Thus, the i m m u n e response of each D L E recipient must be monitored regularly by appropriate immunological tests, so that additional injections can be given when the balance begins to shift - analogous to the clinical management of individual insulin-dependent diabetics. The effects of T F m a y be transient in compromised
TABLE II. Disorders in which DLE has been used for immunotherapy or immunoprophylaxis I.
Broad-spectrum defects in cellular mediated immunity A. Congenital defects 1. Wiskott-Aldrichsyndrome 2. Ataxia telangiectasia 3. Severecombined immunodeficiencysyndrome (some types where stem cells are present or where a thymus graft has been given) 4. Partial DiGeorge syndrome (partial absence of thymus)a 5. 'Dysgammaglobulinemia' with defective cellular immunity B. Defects of unknown origin 1. Sarcoidosis 2. Hodgkin's disease II. Antigen-selectivedefects (refractory to standard therapy) A. Fungal 1. Chronic mucocutaneous candidiasis 2. Disseminated histoplasmosis 3. Disseminated coccidioidomycosis B. Viral 1. Cytomegalovirus infection 2. Herpes zoster 3. Measles 4. Others (especiallythose common in immunodeficiency) C. Mycobacteria l. Tuberculosis 2. Leprosy 3. Mycobacteriumfortuitum D. Protozoal 1. Cutaneous leishmaniasis E. Neuro-viro-immunologicaldisorders 1. Multiple sclerosis(?) 2. Guillain-Barr~ syndrome F. Malignancies (especiallyif thought to have viral etiology) 1. Osteosarcoma (human and animal) 2. Breastcancer 3. Hypernephroma 4. Nasopharyngeal carcinoma 5. Others, as evidence for viral etiology in animals or man is obtained III. Antigen-selectiveand/or broad-spectrum defects in cellular immunity A. Autoimmune diseases, non-organ-specific(due to defect in suppressor T cells plus probable antigen-specificdefect for one or another virus) 1. Chronic discoid lupus erythematosus 2. Behcet's syndrome a Of no benefit in 'total DiGeorge syndrome'. hosts but last for several years in normal recipients 4. Thus, immunodeficient patients m a y require weekly, biweekly, or monthly injections 12-~5. In our experience a decrease or complete loss of immunocompetence as shown by in-vitro assays of C M I indicates that-the clinical state of the patient is worsening or will worsen soon 15. In general, after the patient is clinically and i m m u n o logically normal, D L E of k n o w n potency should be given at least every six months for immunoprophylaxis. Three types of in-vitro assays have been widely employed to monitor immunocompetence in patients being treated with DLE: 'active' T-cell rosette formation, lymphocyte activation as measured by the incorporation of labeled precursors into lymphocyte D N A , and assays of mediator production ( M I F and, more recently, LIF) ~4'~5. Active T cells are decreased in acute viral infections for 3-6 weeks following the illness and are low d u r i n g chronic
160 viral infections 4° and in most chronic generalized fungal and mycobacterial infections as well. Thus, enumeration of active T cells is a useful test for monitoring patients who receive DLE for persistent viral, fungal, or atypical mycobacterial infections. It has potential prognostic value: increases in active T cells in the presence of DLE in vitro in patients with metastatic malignancy and with genetically determined immunodeficiencies (e.g. severe combined immunodeficiency, Wiskott-Aldrich syndrome) were seen almost exclusively in those patients who later responded well to DLE therapy 14'15. However, the assay does not indicate the acquisition of antigen-specific reactivity (the only true measure of positive TF effects) and probably measures the effects of other components in DLE such as T-lymphocyte maturation or differentiation factors, for example thymosin; Refs 5,10). Similarly, lymphocyte-activation assays are now known to detect antigen-dependent but non-specific augmentation of lymphocyte responsiveness induced by DLE, rather than the (antigen-specific) effects of T F per se. 5'15 Indeed, in clinical studies in which D L E was used to treat patients with immunodeficiency diseases, clinical improvement or skin-test conversion was usually associated with the capacity of the patients' lymphocytes to show responsiveness to antigen in vitro as measured by MIF and LIF production but not by lymphocyte transformation or DNA-synthesis assaysS'1.'15. Thus, assay(s) of lymphocyte mediator production seem to be both the most suitable and relevant for monitoring T F effects. It is important to note, however, that antigens relevant to the etiologic agent underlying a particular disorder must be used in these tests. Recent clinical trials Four recent double-blind clinical trims of DLE therapy have strongly supported the value of DLE (TF) as an immunotherapeutic or immunoprophylactic agent. Many others are needed. The first involved patients with acute or persistent cutaneous leishmania infection (CLI)16; presumably an antigen-selective immune defect accounts for the persistence of CLI in otherwise healthy individuals. The treatment was placebo (saline), nonspecific DLE (prepared from donors with no history of exposure to leishmania), or leishmania antigen-specific DLE (prepared from individuals who had previously recovered from leishmania infections and presumably had lymphocytes containing TF specific for leishmania). The 8 patients given placebo showed no change; 3 of 11 patients with acute and neither of 2 patients with persistent C L I had significant therapeutic response after receiving non-specific DLE (the 3 patients with acute C L I but none of the others had positive skin-test reactions to leishmania antigens before the initiation of therapy). Twelve patients received antigen-specific DLE: 4 had persistent lesions; early signs of healing were observed in only 2, perhaps because the trial was short. Lesions in all 8 patients with acute CLI began to clear by 6-8 weeks and by 12 weeks had either healed completely or developed dry scabs. This study was perhaps the first adequately designed double-blind clinical trial of DLE. Its design permitted the evaluation of both specific (TF)' and nonspecific effects of DLE in comparison with placebo.
Immunology Today, vol. 4, No. 6, 1983
In a randomized, double-blind, placebo-controlled trial of D L E prophylaxis in a large group with acute childhood leukemia in remission and with susceptibility to varicella zoster ~7, a single injection of pooled D L E from five donors with unusually high in-vitro reactivity to varicella-zoster antigens resulted in long-term prophylaxis against varicella infection. The placebo failed to provide protection. The possibility of non-specific adjuvant effects were not excluded, however, since the placebo was saline, not non-specific DLE. In a controlled, double-blind trial in chronic aggressive hepatitis '8, DLE was prepared from adults who had recovered from acute hepatitis B or from acute non-B viral hepatitis and administered to 5 HBAg-positive subjects; another 4 subjects received placebo (saline). Four of five recipients showed moderate or marked improvement in hepatic histology, clinical status, and serum transaminase levels after 10 weeks but none of the placebo recipients improved (P = 0.048). Here too, the 'adjuvant moiety' or other moieties could have been effective since as noted above the placebo was saline. Basten a al. 19 conducted a 2-year prospective doubleblind trial of DLE therapy in multiple sclerosis (MS) patients. Fifty-eight patients were divided into two groups, one receiving DLE prepared from relatives living in the same house, the other receiving saline placebo. The two patient groups were precisely balanced with respect to numbers, sex ratios, mean disability score, ratio of moderate to severe cases, frequency of HLA-DW2, which has been claimed to be associated with more rapid progression of disease, and for the phenotypic combination, HLA-B7-negative, DW2-positive which may be associated with a more benign prognosis 19. DLE effects in each patient were monitored neurologically and also immunologically using lymphocyte D N A synthesis and macrophage migration inhibition tests with either mumps or herpes simplex viruses (HSV) as antigens in vitro, and skin tests for mumps, Candida, trichophytin and PPD 19. Two DLE pools were used, one from HSV-positive donor leukocytes, the other from HSV-negative donor leukocytes (all donors responded to measles and parainfluenza-3 viruses, and 14 of 20 responded to HSV). In order to maximize the chance of showing an effect to DLE (TF) on an immune variable, only the HSV-positive DLE pool was used for the first 6 months of treatment (21 patients were initially seronegative and did not display significant lymphocyte transformation to HSV). DLE donors with strong immunity to these three viruses were used because of their possible involvement in the etiology of MS 19. DLE significantly retarded but did not reverse progression of the disease but this difference between the groups was not apparent until 18 months after the start of the trial. DLE treatment was effective only in those patients with mild to moderate disease activity. Patients who received HSV-positive D L E for the first 6 months did show increased responsiveness to HSV as judged by macrophage migration inhibition assays conducted at 6 months but not at 12 months or later. This finding agrees with other studies 12 15indicating that the effects of TF (in this case HSV-specific TF) may be transient in compromised hosts. Basten et aL 19 concluded that their study
Immunology Today, vol. 4, No. 6, 1 9 8 3
'has not shown that TF improves the clinical state of MS patients significantly, nor that it prevents the formation of new lesions, but it has shown that over a 2-year period, treatment with TF from relatives living together with the patients slows the rate of progression of the disease in patients with mild to moderate disability'. We agree, although DLE and not 'TF' per se was used; because placebo was saline and not DLE from non-household contacts, the relative contributions of non-TF and TF components in DLE cannot be distinguished. We hope that studies of equal or better quality will be reported in the future. T w o controls should be used: DLE from donors unresponsive to the suspected etiologic agents and saline. Certainly the publication of additional properly performed double-blind evaluations of DLE containing TF in patients with documented antigenselective defects in C M I would permanently put to rest the controversy about the value of TF as an immunotherapeutic or immunoprophylactic modality.
Acknowledgements We thank Charles L. Smith for editorial assistance. This article is publication no. 575 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Research supported in part by U S P H S grant CA-25746.
References 1 Lawrence, H. S. (1955)J. Clin. Invest. 34, 219 2 Lawrence, H. S. and Pappenheimer, A. M., Jr (1956)J. Exp. Med. 104, 321 3 Lawrence, H. S., AI-Askari, S., David, J., Franklin, E. C. and Zweiman, B. (1963) Trans. Assoc. Am. Physicians 76, 84 4 Lawrence, H. S. (1974) Harvey Leer. 68, 239 5 Wilson, G. B. and Fudenberg, H. H. (1981) Lymphok~tes 4, 107 6 Schindler, T. E. and Baram, P. (1980) AllergoL ImmunopathoL 8, 53 7 Schindler, T. E. and Baram, P. (1980) Allergol. ImmunopathoL 8, 125 8 Schindler, T. E. and Baram, P. (1980) Allergol. Immunopathol. 8, 203 9 Fudenberg, H. H. and Wilson, G. B. (1978) Curr. Top. Clin. Chem. 3,228 10 Wilson, G. B., Paddock, G. V., Floyd, E., Newell, R. T. and Dopson, M. H. in Fourth International Transfer Factor Workshop (Kirkpatriek, C. H., Lawrence, H. S. and Burger, D. R., eds), Academic Press, New York (in
press) 11 Levin, A. S., Spitler, L. E., Stites, D. P. and Fudenberg, H. H. (1970) Proc. Natl Acad. Sci. USA 67,821 12 Basten, A. and Croft, S. (1978)in ImmunologicalEngln*,ering(.]irsch, D. W., ed.), p. 83, MTP Press, Lancaster 13 Arala-Chaves, M. P., Horsmanheimo, M., Goust,J. M. and Fudenberg, H. H. (1978)in Immunological Engi~ering (Jirsch, D. W., ed.), p. 35, MTP Press, Lancaster
161 14 Fudenberg, H. H., Wilson, G. B., Goust, J. M., Nekam, K. and Smith, C. L. (1980) in Thymus, Thymic Hormones and TLymphocytes(Aiuti, F. and Wigzell, H., eds), p. 391, Academic Press, London 15 Fudenberg, H. H., Wilson, G. B. and Smith, C. L. (1980) Proc. VirchowHrquet Med. Soc. 34, 3 16 Sharma, M., Firouz, R., Ala, F. and Momtaz, A. (1979) in Immune Regulators in TransferFactor(Khan, A., Kirkpatrick, C. H. and Hill, N. O., eds), p. 563, Academic Press, New York 17 Steele, R. W., Myers, M. G. and Vincent, M. M. (1980) N. Engl. J. Med. 303, 355 18 Shulman, S. T., Hutto, J. H.,Jr, Ayoub, E. M., Howlett, S. A., Scott, B. and McGuigan, J. E. (1979) Cell. Immunol. 43, 352 19 Basten, A., Pollard, J. D., Stewart, G. J., Frith, J. A., McLeod, J. G., Walsh, J. C., Garrick, R. and VanDerBrink, C. M. (1980) Laacet ii, 931 20 Salaman, M. R. (1982) Immunol. Today 3, 4 21 Kirkpatrick, C. H. and Burger, D. R. (1982) in The Lymphokines BiochemisOy and Biological Activity (Hadden, J. W. and Stewart, W. E., II, eds), p. 261, Humana Press Inc., New Jersey 22 Burger, D. R., Vandenbark, A. A., Dunnick, W., Kraybill, W. G. and Vetto, R. M. (1978)J. Reticuloendothel. Soc. 24, 385 23 Bloom, B. R. (1973) A( Engl. J. Med. 288, 908 24 Wilson, G. B., Fudenburg, H. H. and Bahm, V.J. (1978) Trans. Assoc Am. Physicians 91, 294 25 Wilson, G. B., Fudenberg, H. H. and Horsmanheimo, M. (I979)J. Lab. Clin. Med. 93, 800 26 Borkowsky, W. and Lawrence, H. S. (1979)J. Immunol. 123, 1741 27 Sirianni, M. C., Fiorilli, M., Pana, A., Pezzella, M. and Aiuti, F. (1979) Clin. ImmunoL Irnmunopathol. 14, 300 28 Klesius, P. H., Fudenberg, H. H. and Smith, C. L. (1980) Comp. ImmunoL Microbiol. Infect. D/s. 3, 247 29 Wilson, G. B., Paddock, G. V. and Fudenberg, H. H. (1981) Thymus 2, 257 30 Burger, D. R., Vandenbark, A. A. and Vetto, R. M. (1980) in Thymus, Thymic Hormons and TLymphocytes (Aiuti, F. and Wigzell, H., eds), p. 391, Academic Press, London 31 Wilson, G. B., Fudenberg, H. H., Paddock, G. V., Tsang, K Y., Williams, A. M. and Floyd, E. in Fourth International Transfer Factor Workshop (Kirkpatrick, C. H., Lawrence, H. S. and Burger, D. R., eds), Academic Press, New York (in press) 32 Wilson, G. B., Metcalf, J. F. and Fudenberg, H. H. (1982) C[in. Immunol. Immunopathol. 23, 478 33 Fudenberg, H. H., Wilson, G. B., Keller, R. H., Metcalf, J. F., Paulling, E. E., Stuart, E.J. and Floyd, E. in: Fourth International Trarufer Factor Workshop(Kirkpatriek, C. H., Lawrence, H. S. and Burger, D. R., eds), Academic Press, New York (in press) 34 Littman, B. H., Rocklin, R. E., Parkman, R. and David, J. R. (1978) Clin. Irnmunol. Immunopathol. 9, 97 35 Fudenberg, H. H. (1976)Ann. NYAcad. Sci. 277, 545 36 Thor, D. E., Cottman, C. A., Bearden, J. D., Williams, T. E. and Flippen, J. H. (1976) in Transfer Factor: Basic Properties and Clinical Applications (Aseher, M. S., Gottlieb, A. A. and Kirkpatrick, C. H., eds), p. 563, Academic Press, New York 37 Byers, V. S., Levin, A. S., Hackett, A.J. and Fudenberg, H. H. (1975) J. Clin. Invest. 55, 500 38 Arala-Chaves, M. P. and Fudenberg, H. H. (1976)Nature (London)362, 155 39 Arala-Chaves, M. P., Silva, A., Porto, M. T., Picoto, A., Ramos, M. T. F. and Fudenberg, H. H. (1977) Clin. Immunol. Immunopathol. 8, 430 40 Wybran, J. and Fudenberg, H. H. (1973)J. Clin. Invest. 51, 2537
157
Is controversy about 'transfer factor therapy' nearing an end? Gregory B. Wilson and H. Hugh Fudenberg Nearly 30years after Lawrencefirst described 'transfer factor' the therapeutic value of dialysable leukocyte extracts is now being examined in adequately designed trials. The evidence of its efficacy in certain patients in certain circumstances is unmistakable.
Transfer'factor and dialysable leukocyte extracts In the mid 1950s Lawrence first reported that delayedtype cutaneous hypersensitivity ( D T H ) to tuberculin (PPD) or streptococcal M substance could be passively transferred using extracts of leukocytes obtained from skin-test positive, normal human donors to previously skin-test negative, normal human recipients ~'2. In 1963, Lawrence et aL 3 claimed that dialysates of leukocyte extracts (i. e. dialysable leukocyte extracts, DLE) were as effective in transferring D T H as whole leukocyte extracts. The active component(s) was at that time termed 'transfer factor' (TF) as it was thought to transfer D T H . In the succeeding years little was learned about the mechanism of action of T F or its physicochemical properties, except that it was not immunogenic in man or other animals (for a review of the early work see Ref. 4). Because T F was naively thought to be the only biological activity present in DLE, Lawrence and others tended to use the term ' T F ' , rather than ' D L E containing T F activity', when referring to transfers produced by DLE. These semantics led to considerable confusion and controversy, especially when it later became evident that DLE contains several hundred chemical moieties, many of them very active biologically (for review see Refs 5-9). While T F has historically been defined functionally as a biochemical entity in D L E which can transfer antigenspecific reactivity as shown by skintesting, or more recently by more sensitive and more quantitative in-vitro assays which measure the release of lymphokines by antigen-activated T lymphocytes5, it has not yet been completely characterized, and only very recently have any definitive reports been published which appear to demonstrate that T F may indeed act in an antigen-specific fashion (see below). Other components in D L E which have antigen-independent, non-specific effects on cellmediated immunity (CMI) and the inflammatory response include the prostaglandins, nicotinamide, ascorbic acid, histamine, serotonin, T-lymphocyte maturation or differentiation factors (thymosin or thymic humoral factor) 1°, chemoattractants for monocytes, and neutrophil immobilizing factor(s) (reviewed in Refs 5-8). Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, SC 29425, USA.
These components might be expected to produce generalized immunological enhancement or suppression when D L E is evaluated in vitro or in vivo clinically. Beyond the semantic problems were even bigger ones. Until 1970, most immunologists either doubted the existence of T F or questioned its significance. At that time the T F phenomenon could be demonstrated only in man and the relationship of skin-test reactivity to systemic cellular immunity in mammals had only recently become apparent. But in 1970 there was renewed interest when Levin etal. 11demonstrated that D L E containingTF could, in addition to transferring skin-test reactivity, confer (a) the ability to produce lymphokines in response to specific antigens, and (b) resistance to infection in patients with genetically determined immune deficiency. Since 1970, DLE has been used in immunotherapy and/ or immunoprophylaxis in which cell-mediated immune responses are compromised, including inherited or acquired' antigen- specific' o r ' broad spectrum' immunodeficiency diseases, neoplasia, and a variety of viral, fungal and mycobacterial diseases (reviewed in t~.efs 12-15). The success of clinical trials to date has been variable even for a single disease or syndrome, and all of these studies have been uncontrolled, except for four discussed below 16-19. T h e nature and properties of DLE and T F The antigen specificity of T F is a highly controversial matter20 ~swhich may now be approaching resolution with the publication, from three groups z4-27, of compelling evidence that T F does act specifically(at least in vitro). Why the controversy? Table I, compiled from a review of m~/ny clinical studies 22, shows reactivity (as shown by skin-test responses) and the new antigen reactivities that appeared in the recipients of their DLE. Many recipients acquired D T H responses to most but not every one of the antigen specificities to which the donors responded while some failed to respond to any antigen (43 of 200 recipients listed under 'antigen-sensitive donors' in Table I). Even more problematic are the recipients of DLE who became capable of responding to antigens to which the donors were skin-test negative (12 out of 151 times; Table I). Some may feel strongly that such data justify doubts about the specificity of TF. We, however, contend tha~ 1983, Elsevier Science P u b l i s h e r s B V , A m s t e r d a m
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TABLE I. In-vivo evidence for specificityof human transfer facto# Skin-test conversionb Antigenc PPD Coccidioidin SK-SD Candida Mumps KLH Other Total skin tests: Total recipients:
Antigen-sensitive donors
Antigen-insensitive donors
58/74 23/26 9/18 12/17 9/15 25/26 21/23 157/200 (78%) 169
1/26 7/45 1/9 1/11 0/1 1/30 1/29 12/151 (8%) 110
a From Burger et aL 22 b Expressed as the number of responses that became positive after TF/the number of responses that were negative before TF. c PPD: purified protein derivative; SK-SD: streptokinase-streptodornase; KLH: keyhole limpet hemocyanin; 'other' includes diphtheria toxoid, aspergillin, ethylene oxide-treated serum, skin grafts, histoplasmin, trichophytin, lepromin, vaccinia, sarcoma extracts. m u c h of the problem stems from a reliance on skin testing as the sole criterion for antigen responsiveness in both D L E donors a n d recipients 4. Skin testing is neither quantitative nor particularly sensitive and a positive response is highly dependent on the a m o u n t of antigen used. T h e definition of 'responsiveness' is also somewhat arbitrary, different investigators requiring different thickness of induration. I n addition, some D L E donors who were originally skin-test negative to a particular antigen converted to a n antigen-responsive state after donating leucocytes, either because they were primed by skin testing or because they were never non-responsive in the first place. Doubts about the specificity of T F have also been fanned by indications of a generalized non-specific enhancement of i m m u n e reactivity in vitro in some D L E recipients. Clearly, it is these other components of D L E and not T F which are promoting this generalized enh a n c e m e n t of i m m u n e reactivity in some recipients of D L E 5 8.1315. This emphasizes the problems caused by wrongly equating D L E with T F . Fortunately, the sole reliance on skin testing in T F research seems rapidly to be ending as in-vitro assays become available5. Also, since 1970, T F cellular sources ( T lymphocytes) and target cell(s) ( T lymphocytes, natural killer cells, and possibly monocytes-macrophages) have been identified (reviewed in Refs 4, 5, 12, 13). A n i m a l models are available for the study of T F a n d it has been shown to transfer antigen-specific cellular i m m u n e reactivity across m a m m a l i a n species bartiers 12'2s.T h e biochemical n a t u r e of T F is clearer ls'29's°a n d there are hypotheses for its mode(s) of action 5'29'31. For instance, we have shown the presence of two structurally distinct T F moieties in D L E for both the antigen specificities ( P P D a n d coccidioidin) which we have extensively studied 29'31. In addition, a third T F moiety exists for each T F specificity (derived from one T F moiety found in DLE) that is released by i m m u n e lymphocytes pulsed with specific antigen in vitro 3j (see simplest case molecular models shown in Fig. 1). Recently, we have applied some of the above develop-
ments (particularly the in-vitro assay for T F activity) to the clinical use of D L E containing T F . W e have developed new methods for (a) determining the ' T F potency' of D L E preparations, (b) predicting whether a patient will or will not benefit from i m m u n o t h e r a p y with T F , a n d (c) monitoring the responses of recipients to T F therapy 32'33. Although these methods are only now b e g i n n i n g to be applied to the clinical use of T F , we believe that they will increase the scientific value of immunotherapeutic a n d immunoprophylactic trials, with T F in the future. Lessons from past clinical studies
D L E has been used clinically as a n immunotherapeutic or immunoprophylactic agent in m a n y disorders (Table II). The evaluation of m a n y past reports is difficult because of the small n u m b e r of patients treated, the inclusion of patients with advanced stages of m a l i g n a n t disease (particularly malignancies such as m e l a n o m a a n d Hodgkin's disease), the short duration of follow-ups, a n d the paucity of available laboratory data. Even in the treatment of patients with the same syndrome there are variations in the preparation, type or potency of D L E used, in the methods employed for selecting donors a n d monitoring the progress in recipients, a n d in the clinical complications. Nonetheless, some key lessons have been learned. D L E has remarkably few side-effects either immediate or delayed~2-15-occasional pain or erythema at the site of injection a n d transient fever, without evidence of toxicity. T h e variable results of past clinical studies are, we believe, due to inappropriate selection of recipients a n d
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tuberculin (PPD). Both are polyribonudeopeptides; the TF moiety on the left contains predominantly purine residues while the TF moiety on the right contains mainlypyrimidine residues. The polypeptide segment(except for the phosphate) is thought to be the same in each moietyand probably governs antigen specificity. The two TFs shown are thought to have distinct roles in the process of immune sensitization. The moiety on the right may be strictly an intraceUular product (perhaps a gene deregulator) involved in the synthesis of the other TF moiety, which may be secreted by antigen-activated T lymphocytes and becomes part of a T-cell antigen receptor necessary for antigen responsiveness.Both TFs would be present in DLE, since the cells are disrupted during its preparation2931. Recently, we have found that immune lymphocytes release a third TF moiety when incubated with specificantigen in vitro. This moiety is structuraUysimilar to the TF moiety on the left but lacks the Y-terminal phosphate group31.
159
Immunology Today, vol. 4, No. 6, 1983
donors, failure to evaluate the potency and specificity of D L E preparations used, failure to monitor the i m m u n o logical response to therapy, and failure to provide conventional therapy such as surgery or antibiotics or nutrients to reduce the antigen load. The importance of appropriate selection and evaluation of recipients cannot be overemphasized. Adequate n u m b e r s of target cells (i.e. m a t u r e T cells or their precursors) must be present. The extent of the antigen load (micro-organisms or tumor cell mass) a n d the general health of the patient including nutritional status must be known. The most consistent successes in imm u n o t h e r a p y or immunoprophylaxis with D L E have been in patients with a n antigen-selective defect in T-cellmediated immunity, in w h o m therapy was initiated early in the course of the disease (or the antigen load was reduced prior to D L E therapy), a n d when other forms of therapy were used prior to D L E therapy. Investigators should not expect effective D L E therapy against an infective agent in a patient whose antigen-selective defect in C M I to that agent has allowed the disease to develop (IIA to F in Table II) unless the D L E preparation employed contains T F specific for the etiologic agent in question. However, even patients with broad-spectrum defects have benefited from a generalized e n h a n c e m e n t of i m m u n e reactivity induced by other components in DLE12-14. D L E donor(s) must be selected on the basis of demonstrably strong C M I of specificity desired in the recipient; in patients with chronic mucocutaneous candidiasis a n d osteogenic sarcoma 9'Is's4, improved i m m u n o logical laboratory parameters a n d clinical benefit were seen only when D L E contained T F specific for Candida or osteogenic sarcoma t u m o r antigens, respectively. Because D L E contains, in addition to T F , both imm u n o a d j u v a n t and inhibitory activities which can block (i.e. 'suppress') the potential beneficial effects of T F , some generalized clinical or immunological i m p r o v e m e n t m a y occur in patients receiving D L E of the ' w r o n g ' antigenic specificity or D L E of the 'right' specificity m a y be ineffective. I n patients in whom the infecting organism is u n k n o w n or u n c o m m o n , a n d in malignancies or other disorders of suspected viral etiology (i.e. osteogenic sarcoma, melanoma, Behcet's syndrome), prospective donors should be obtained from household contacts of the patient 3s-37. Household contacts of osteogenic sarcoma patients, for example, show a significantly higher frequency of significant C M I against osteosarcoma cell lines than do r a n d o m l y selected individuals (40% compared to
1%). Genetic factors ( ' i m m u n e response genes') m a y also underlie the finding that different individuals respond differently to the same batch of D L E - so-called 'recipient specificity 'ls'3s'39. In addition, i m m u n e competence m a y be balanced against antigen load, producing either progressive disease or clinical improvement. Thus, the i m m u n e response of each D L E recipient must be monitored regularly by appropriate immunological tests, so that additional injections can be given when the balance begins to shift - analogous to the clinical management of individual insulin-dependent diabetics. The effects of T F m a y be transient in compromised
TABLE II. Disorders in which DLE has been used for immunotherapy or immunoprophylaxis I.
Broad-spectrum defects in cellular mediated immunity A. Congenital defects 1. Wiskott-Aldrichsyndrome 2. Ataxia telangiectasia 3. Severecombined immunodeficiencysyndrome (some types where stem cells are present or where a thymus graft has been given) 4. Partial DiGeorge syndrome (partial absence of thymus)a 5. 'Dysgammaglobulinemia' with defective cellular immunity B. Defects of unknown origin 1. Sarcoidosis 2. Hodgkin's disease II. Antigen-selectivedefects (refractory to standard therapy) A. Fungal 1. Chronic mucocutaneous candidiasis 2. Disseminated histoplasmosis 3. Disseminated coccidioidomycosis B. Viral 1. Cytomegalovirus infection 2. Herpes zoster 3. Measles 4. Others (especiallythose common in immunodeficiency) C. Mycobacteria l. Tuberculosis 2. Leprosy 3. Mycobacteriumfortuitum D. Protozoal 1. Cutaneous leishmaniasis E. Neuro-viro-immunologicaldisorders 1. Multiple sclerosis(?) 2. Guillain-Barr~ syndrome F. Malignancies (especiallyif thought to have viral etiology) 1. Osteosarcoma (human and animal) 2. Breastcancer 3. Hypernephroma 4. Nasopharyngeal carcinoma 5. Others, as evidence for viral etiology in animals or man is obtained III. Antigen-selectiveand/or broad-spectrum defects in cellular immunity A. Autoimmune diseases, non-organ-specific(due to defect in suppressor T cells plus probable antigen-specificdefect for one or another virus) 1. Chronic discoid lupus erythematosus 2. Behcet's syndrome a Of no benefit in 'total DiGeorge syndrome'. hosts but last for several years in normal recipients 4. Thus, immunodeficient patients m a y require weekly, biweekly, or monthly injections 12-~5. In our experience a decrease or complete loss of immunocompetence as shown by in-vitro assays of C M I indicates that-the clinical state of the patient is worsening or will worsen soon 15. In general, after the patient is clinically and i m m u n o logically normal, D L E of k n o w n potency should be given at least every six months for immunoprophylaxis. Three types of in-vitro assays have been widely employed to monitor immunocompetence in patients being treated with DLE: 'active' T-cell rosette formation, lymphocyte activation as measured by the incorporation of labeled precursors into lymphocyte D N A , and assays of mediator production ( M I F and, more recently, LIF) ~4'~5. Active T cells are decreased in acute viral infections for 3-6 weeks following the illness and are low d u r i n g chronic
160 viral infections 4° and in most chronic generalized fungal and mycobacterial infections as well. Thus, enumeration of active T cells is a useful test for monitoring patients who receive DLE for persistent viral, fungal, or atypical mycobacterial infections. It has potential prognostic value: increases in active T cells in the presence of DLE in vitro in patients with metastatic malignancy and with genetically determined immunodeficiencies (e.g. severe combined immunodeficiency, Wiskott-Aldrich syndrome) were seen almost exclusively in those patients who later responded well to DLE therapy 14'15. However, the assay does not indicate the acquisition of antigen-specific reactivity (the only true measure of positive TF effects) and probably measures the effects of other components in DLE such as T-lymphocyte maturation or differentiation factors, for example thymosin; Refs 5,10). Similarly, lymphocyte-activation assays are now known to detect antigen-dependent but non-specific augmentation of lymphocyte responsiveness induced by DLE, rather than the (antigen-specific) effects of T F per se. 5'15 Indeed, in clinical studies in which D L E was used to treat patients with immunodeficiency diseases, clinical improvement or skin-test conversion was usually associated with the capacity of the patients' lymphocytes to show responsiveness to antigen in vitro as measured by MIF and LIF production but not by lymphocyte transformation or DNA-synthesis assaysS'1.'15. Thus, assay(s) of lymphocyte mediator production seem to be both the most suitable and relevant for monitoring T F effects. It is important to note, however, that antigens relevant to the etiologic agent underlying a particular disorder must be used in these tests. Recent clinical trials Four recent double-blind clinical trims of DLE therapy have strongly supported the value of DLE (TF) as an immunotherapeutic or immunoprophylactic agent. Many others are needed. The first involved patients with acute or persistent cutaneous leishmania infection (CLI)16; presumably an antigen-selective immune defect accounts for the persistence of CLI in otherwise healthy individuals. The treatment was placebo (saline), nonspecific DLE (prepared from donors with no history of exposure to leishmania), or leishmania antigen-specific DLE (prepared from individuals who had previously recovered from leishmania infections and presumably had lymphocytes containing TF specific for leishmania). The 8 patients given placebo showed no change; 3 of 11 patients with acute and neither of 2 patients with persistent C L I had significant therapeutic response after receiving non-specific DLE (the 3 patients with acute C L I but none of the others had positive skin-test reactions to leishmania antigens before the initiation of therapy). Twelve patients received antigen-specific DLE: 4 had persistent lesions; early signs of healing were observed in only 2, perhaps because the trial was short. Lesions in all 8 patients with acute CLI began to clear by 6-8 weeks and by 12 weeks had either healed completely or developed dry scabs. This study was perhaps the first adequately designed double-blind clinical trial of DLE. Its design permitted the evaluation of both specific (TF)' and nonspecific effects of DLE in comparison with placebo.
Immunology Today, vol. 4, No. 6, 1983
In a randomized, double-blind, placebo-controlled trial of D L E prophylaxis in a large group with acute childhood leukemia in remission and with susceptibility to varicella zoster ~7, a single injection of pooled D L E from five donors with unusually high in-vitro reactivity to varicella-zoster antigens resulted in long-term prophylaxis against varicella infection. The placebo failed to provide protection. The possibility of non-specific adjuvant effects were not excluded, however, since the placebo was saline, not non-specific DLE. In a controlled, double-blind trial in chronic aggressive hepatitis '8, DLE was prepared from adults who had recovered from acute hepatitis B or from acute non-B viral hepatitis and administered to 5 HBAg-positive subjects; another 4 subjects received placebo (saline). Four of five recipients showed moderate or marked improvement in hepatic histology, clinical status, and serum transaminase levels after 10 weeks but none of the placebo recipients improved (P = 0.048). Here too, the 'adjuvant moiety' or other moieties could have been effective since as noted above the placebo was saline. Basten a al. 19 conducted a 2-year prospective doubleblind trial of DLE therapy in multiple sclerosis (MS) patients. Fifty-eight patients were divided into two groups, one receiving DLE prepared from relatives living in the same house, the other receiving saline placebo. The two patient groups were precisely balanced with respect to numbers, sex ratios, mean disability score, ratio of moderate to severe cases, frequency of HLA-DW2, which has been claimed to be associated with more rapid progression of disease, and for the phenotypic combination, HLA-B7-negative, DW2-positive which may be associated with a more benign prognosis 19. DLE effects in each patient were monitored neurologically and also immunologically using lymphocyte D N A synthesis and macrophage migration inhibition tests with either mumps or herpes simplex viruses (HSV) as antigens in vitro, and skin tests for mumps, Candida, trichophytin and PPD 19. Two DLE pools were used, one from HSV-positive donor leukocytes, the other from HSV-negative donor leukocytes (all donors responded to measles and parainfluenza-3 viruses, and 14 of 20 responded to HSV). In order to maximize the chance of showing an effect to DLE (TF) on an immune variable, only the HSV-positive DLE pool was used for the first 6 months of treatment (21 patients were initially seronegative and did not display significant lymphocyte transformation to HSV). DLE donors with strong immunity to these three viruses were used because of their possible involvement in the etiology of MS 19. DLE significantly retarded but did not reverse progression of the disease but this difference between the groups was not apparent until 18 months after the start of the trial. DLE treatment was effective only in those patients with mild to moderate disease activity. Patients who received HSV-positive D L E for the first 6 months did show increased responsiveness to HSV as judged by macrophage migration inhibition assays conducted at 6 months but not at 12 months or later. This finding agrees with other studies 12 15indicating that the effects of TF (in this case HSV-specific TF) may be transient in compromised hosts. Basten et aL 19 concluded that their study
Immunology Today, vol. 4, No. 6, 1 9 8 3
'has not shown that TF improves the clinical state of MS patients significantly, nor that it prevents the formation of new lesions, but it has shown that over a 2-year period, treatment with TF from relatives living together with the patients slows the rate of progression of the disease in patients with mild to moderate disability'. We agree, although DLE and not 'TF' per se was used; because placebo was saline and not DLE from non-household contacts, the relative contributions of non-TF and TF components in DLE cannot be distinguished. We hope that studies of equal or better quality will be reported in the future. T w o controls should be used: DLE from donors unresponsive to the suspected etiologic agents and saline. Certainly the publication of additional properly performed double-blind evaluations of DLE containing TF in patients with documented antigenselective defects in C M I would permanently put to rest the controversy about the value of TF as an immunotherapeutic or immunoprophylactic modality.
Acknowledgements We thank Charles L. Smith for editorial assistance. This article is publication no. 575 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Research supported in part by U S P H S grant CA-25746.
References 1 Lawrence, H. S. (1955)J. Clin. Invest. 34, 219 2 Lawrence, H. S. and Pappenheimer, A. M., Jr (1956)J. Exp. Med. 104, 321 3 Lawrence, H. S., AI-Askari, S., David, J., Franklin, E. C. and Zweiman, B. (1963) Trans. Assoc. Am. Physicians 76, 84 4 Lawrence, H. S. (1974) Harvey Leer. 68, 239 5 Wilson, G. B. and Fudenberg, H. H. (1981) Lymphok~tes 4, 107 6 Schindler, T. E. and Baram, P. (1980) AllergoL ImmunopathoL 8, 53 7 Schindler, T. E. and Baram, P. (1980) Allergol. ImmunopathoL 8, 125 8 Schindler, T. E. and Baram, P. (1980) Allergol. Immunopathol. 8, 203 9 Fudenberg, H. H. and Wilson, G. B. (1978) Curr. Top. Clin. Chem. 3,228 10 Wilson, G. B., Paddock, G. V., Floyd, E., Newell, R. T. and Dopson, M. H. in Fourth International Transfer Factor Workshop (Kirkpatriek, C. H., Lawrence, H. S. and Burger, D. R., eds), Academic Press, New York (in
press) 11 Levin, A. S., Spitler, L. E., Stites, D. P. and Fudenberg, H. H. (1970) Proc. Natl Acad. Sci. USA 67,821 12 Basten, A. and Croft, S. (1978)in ImmunologicalEngln*,ering(.]irsch, D. W., ed.), p. 83, MTP Press, Lancaster 13 Arala-Chaves, M. P., Horsmanheimo, M., Goust,J. M. and Fudenberg, H. H. (1978)in Immunological Engi~ering (Jirsch, D. W., ed.), p. 35, MTP Press, Lancaster
161 14 Fudenberg, H. H., Wilson, G. B., Goust, J. M., Nekam, K. and Smith, C. L. (1980) in Thymus, Thymic Hormones and TLymphocytes(Aiuti, F. and Wigzell, H., eds), p. 391, Academic Press, London 15 Fudenberg, H. H., Wilson, G. B. and Smith, C. L. (1980) Proc. VirchowHrquet Med. Soc. 34, 3 16 Sharma, M., Firouz, R., Ala, F. and Momtaz, A. (1979) in Immune Regulators in TransferFactor(Khan, A., Kirkpatrick, C. H. and Hill, N. O., eds), p. 563, Academic Press, New York 17 Steele, R. W., Myers, M. G. and Vincent, M. M. (1980) N. Engl. J. Med. 303, 355 18 Shulman, S. T., Hutto, J. H.,Jr, Ayoub, E. M., Howlett, S. A., Scott, B. and McGuigan, J. E. (1979) Cell. Immunol. 43, 352 19 Basten, A., Pollard, J. D., Stewart, G. J., Frith, J. A., McLeod, J. G., Walsh, J. C., Garrick, R. and VanDerBrink, C. M. (1980) Laacet ii, 931 20 Salaman, M. R. (1982) Immunol. Today 3, 4 21 Kirkpatrick, C. H. and Burger, D. R. (1982) in The Lymphokines BiochemisOy and Biological Activity (Hadden, J. W. and Stewart, W. E., II, eds), p. 261, Humana Press Inc., New Jersey 22 Burger, D. R., Vandenbark, A. A., Dunnick, W., Kraybill, W. G. and Vetto, R. M. (1978)J. Reticuloendothel. Soc. 24, 385 23 Bloom, B. R. (1973) A( Engl. J. Med. 288, 908 24 Wilson, G. B., Fudenburg, H. H. and Bahm, V.J. (1978) Trans. Assoc Am. Physicians 91, 294 25 Wilson, G. B., Fudenberg, H. H. and Horsmanheimo, M. (I979)J. Lab. Clin. Med. 93, 800 26 Borkowsky, W. and Lawrence, H. S. (1979)J. Immunol. 123, 1741 27 Sirianni, M. C., Fiorilli, M., Pana, A., Pezzella, M. and Aiuti, F. (1979) Clin. ImmunoL Irnmunopathol. 14, 300 28 Klesius, P. H., Fudenberg, H. H. and Smith, C. L. (1980) Comp. ImmunoL Microbiol. Infect. D/s. 3, 247 29 Wilson, G. B., Paddock, G. V. and Fudenberg, H. H. (1981) Thymus 2, 257 30 Burger, D. R., Vandenbark, A. A. and Vetto, R. M. (1980) in Thymus, Thymic Hormons and TLymphocytes (Aiuti, F. and Wigzell, H., eds), p. 391, Academic Press, London 31 Wilson, G. B., Fudenberg, H. H., Paddock, G. V., Tsang, K Y., Williams, A. M. and Floyd, E. in Fourth International Transfer Factor Workshop (Kirkpatrick, C. H., Lawrence, H. S. and Burger, D. R., eds), Academic Press, New York (in press) 32 Wilson, G. B., Metcalf, J. F. and Fudenberg, H. H. (1982) C[in. Immunol. Immunopathol. 23, 478 33 Fudenberg, H. H., Wilson, G. B., Keller, R. H., Metcalf, J. F., Paulling, E. E., Stuart, E.J. and Floyd, E. in: Fourth International Trarufer Factor Workshop(Kirkpatriek, C. H., Lawrence, H. S. and Burger, D. R., eds), Academic Press, New York (in press) 34 Littman, B. H., Rocklin, R. E., Parkman, R. and David, J. R. (1978) Clin. Irnmunol. Immunopathol. 9, 97 35 Fudenberg, H. H. (1976)Ann. NYAcad. Sci. 277, 545 36 Thor, D. E., Cottman, C. A., Bearden, J. D., Williams, T. E. and Flippen, J. H. (1976) in Transfer Factor: Basic Properties and Clinical Applications (Aseher, M. S., Gottlieb, A. A. and Kirkpatrick, C. H., eds), p. 563, Academic Press, New York 37 Byers, V. S., Levin, A. S., Hackett, A.J. and Fudenberg, H. H. (1975) J. Clin. Invest. 55, 500 38 Arala-Chaves, M. P. and Fudenberg, H. H. (1976)Nature (London)362, 155 39 Arala-Chaves, M. P., Silva, A., Porto, M. T., Picoto, A., Ramos, M. T. F. and Fudenberg, H. H. (1977) Clin. Immunol. Immunopathol. 8, 430 40 Wybran, J. and Fudenberg, H. H. (1973)J. Clin. Invest. 51, 2537
