De novo initiation of specific cell-mediated immune responsiveness in chickens by transfer factor (specific immunity inducer) obtained from bovine colostrum and milk.

Transfer factors (TF) were prepared from colostrum and milk of bovines previously immunized with antigens obtained from Coccidioides immitis, infectious bovine rhinotracheitis virus, or from the viral agents responsible for avian Newcastle disease, laryngotracheitis disease or infectious bursal disease. The ability of bovine TF to transfer specific cell-mediated immune responsiveness to a markedly xenogenic species was studied using specific pathogen free (SPF) and standard commercial (SC) chickens as model recipients. Cell-mediated immune responsiveness was documented using one or more of the following for each antigen (organism) studied: (a) an in vitro chicken leukocyte (heterophil) migration inhibition assay; (b) delayed-wattle reactivity; or (c) protection from clinical disease. Chicken TFs obtained from spleens of immune donors were evaluated in parallel to bovine TF's in selected comparative studies. Bovine TF also referred to as specific immunity inducer (SII), and chicken TF were found to initiate antigen-specific cell-mediated immunity de novo in previously non-immune SPF chickens as well as in SC chickens despite the presence of maternally acquired humoral antibody which may serve as a "barrier" to immunization of SC chickens when commercially available vaccines are administered by parenteral routes. Bovine TF's specific for laryngotracheitis virus or infectious bursal disease virus afforded protection equal to that found for commercially available vaccines. Bovine TF's action was rapid (less than a day) and of relatively long duration at least 35 days.

Use of mitogen-induced lymphocyte transformation to assess toxicity of aminoglycosides.

The development of suppression of lymphocyte responsiveness during extended clinical use of amikacin promoted studies to evaluate the effects of aminoglycosides and other antibiotics on lymphocyte reactivity. The effects of three commonly used aminoglycosides (gentamicin, kanamycin and amikacin), tetracycline and penicillin on lymphocyte (DNA synthesis) in response to stimulation with phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) were measured. Mitogens were added at suboptimal, optimal and supraoptimal concentrations. The mitogen was added either concomitantly with the drug or 96 hours after preculturing the cells with the drug. Drug concentrations were within therapeutic range. Suppression of lymphocyte DNA synthesis after stimulation with ConA was observed for all the aminoglycosides in cell cultures preincubated with antibiotic. In contrast benzylpenicillin had no effect on lymphocyte transformation. Tetracycline tended to cause suppression of responsiveness to mitogen in all preincubated cultures. Sensitivity of mitogen-induced lymphocyte transformation to aminoglycosides varied between donors. Aminoglycosides appear to selectively suppress or inhibit suppressor immunocytes, particularly when suboptimal concentrations of mitogen are used.

Controlled comparison of plasma and serum for cystic fibrosis protein.

Investigators who have previously reported attempts to utilize the isoelectric focusing (IEF) method to detect the cystic fibrosis protein (CFP) in blood samples from CF homozygotes and heterozygotes, and normal healthy controls have restricted their evaluations to serum samples. The requirement of using serum and the stringent conditions laid down for the collection, processing, and storage of serum samples have been impediments, however, to the successful use of the IEF method for CFP detection. We now report results obtained from controlled ("blind") comparative analyses of matched serum and plasma samples from CF homozygotes and heterozygotes, and normal healthy controls which indicate that the CFP is present in plasma and that heparinized plasma in particular may be a better fluid than serum for use in future studies on the CFP.

Epidermodysplasia verruciformis: response to therapy with dialyzable leukocyte extract (transfer factor) derived from household contacts.

Dialyzable leukocyte extracts (DLE) have been used to treat a variety of antigen selective, and broad spectrum immunodeficiency diseases with sometimes encouraging results. We describe here the clinical and laboratory responses to DLE therapy of 2 patients with epidermodysplasia verruciformis (EV), a chronic cutaneous infection with a variety of human papilloma viruses. One patient with longstanding (30 yr) disease and no improvement to previous therapy showed gradual yet definite resolution of extensive verrucae planae, plaque, tinea-versicolor-like, and tumor lesions scattered over his entire integument. Cessation of DLE therapy for a short time resulted in recurrence of partially regressed lesions and also in the development of new tumors in this patient. The second patient, a grandson of the first patient, with minimal disease showed no progression of the disease during DLE prophylaxis. A third subject (brother of patient number 2) received no DLE and served as a control. All 3 subjects demonstrated severely depressed levels of suppressor T cells, a defect in cell-mediated immunity that has not been hitherto reported in patients with EV. Finally, evidence is presented for a possible X-linked recessive mode of inheritance for susceptibility to EV.

Guidelines for immunotherapy of antigen-specific defects with transfer factor.

Dialyzable leukocyte extracts (DLE) containing transfer factor (TF) with documented specificity for one or more microbial antigens have shown previously variable clinical effectiveness in treating many infectious diseases caused by viruses, fungi, protozoa and mycobacteria. The efficacy has sometimes been strong, and at other times dubious, in treating patients with inherited or presumably "acquired" immunodeficiency diseases refractory to standard therapy. The recent development of assays for screening leukocyte donors of DLE, for monitoring recipients, and especially for determining the potency of various DLE preparations containing antigen-specific TF and for predicting the clinical course of disease have, in our hands, greatly improved the likelihood of successful immunotherapy with TF. Two representative cases are reported, one involving a patient with an antigen selective defect to Candida, and another involving a patient with an antigen selective defect to Mycobacterium fortuitum. Both patients responded as judged by laboratory tests and clinical improvement when treated with certain DLE preparations but not with others. Finally, certain DLE preparations appeared to suppress cell-mediated immunity in vivo and this suppression could be predicted by in vitro tests. Based on these results, guidelines for optimal therapy with DLE are proffered .

Dialyzable leukocyte extracts contain thymosin.

Trypsinization of human T-lymphocytes removes surface receptors which bind to sheep erythrocytes (E). Human dialyzable leukocytes extracts (DLE) and thymosin (Fraction V) have been shown to significantly increase the rate of regeneration of T-lymphocyte E-receptors. Both physical-chemical and immunochemical results reported herein indicate that the enhancing effect of human DLE preparations on the rate of regeneration of T-lymphocyte E-receptors is due at least in part to the presence of thymosin alpha 1-peptide in these preparations. Thymosin alpha 1-peptide purified from thymosin Fraction V and putative thymosin alpha 1 preparations purified from human DLEs were each active not only in increasing the rate of regeneration of T-lymphocyte E-receptors removed by trypsinization but also were active in vitro in markedly increasing the number of E-rosetting cells in two patients with immunodeficiency disease manifested in part as a reduction in the normal percentage of mature T-lymphocytes capable of forming E-rosettes.

Is controversy about 'transfer factor therapy' nearing an end?

Nearly 30-years 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.

Does a primary host defense abnormality involving monocytes-macrophages underlie the pathogenesis of lung disease in cystic fibrosis?

The primary cause of morbidity and mortality in cystic fibrosis (CF) patients is chronic pulmonary disease. Pulmonary disease in CF is characterized in part by: (a) obstruction of the bronchi and bronchioles by inspissated secretions (mucus is hypersecreted and may also be abnormal), (b) recurrent or persistent bacterial infections, and (c) a chronic inflammatory state. We propose herein that much of the pathophysiology of lung disease in CF stems from a genetically inherited metabolic defect in monocyte-macrophages (M-Mphi), and we review evidence which indicates that CF M-Mphi are innately metabolically abnormal. Once activated by various stimuli, CF M-Mphi become metabolically hyperactive and hypersecretory as evidenced by the production of excessive levels of a variety of mediators which could have definite roles in both the initiation of pulmonary obstruction and the accelerated development of a chronic inflammatory response in CF. Evidence is also reviewed which indicates that other CF M-Mphi functions crucial to the afferent and efferent phases of the immune response to bacterial infections in the lung may be adversely affected. Mechanisms proposed to explain the abnormal production of mediators by CF M-Mphi are discussed, and it is concluded that hyperproduction of mediators by CF M-Mphi and their metabolic hyperactivity probably result from a defect in autoregulation. The nature of the metabolic defect in CF M-Mphi indicates that CF should be classified as a "new" primary host defense abnormality or alternatively as a "new" primary immune deficiency disease.

Bovine 'transfer factor': an oligoribonucleopeptide which initiates antigen-specific lymphocytes responsiveness.

Bovine transfer factor (TF)--active in initiating specific responsiveness in human thymus-derived (T) lymphocytes to purified protein derivative from Mycobacterium tuberculosis (PPD) in vitro--was partially purified from the dialyzable portion of medium from immune lymph node cells (DLNE). Its physiochemical properties and structure were determined by methods previously employed to characterize human PPD-specific TF isolated from dialyzable leukocyte extracts (DLE). Bovine TF had a molecular weight (MW) of 1100-3000, was destroyed by heating at 56 or 80 degrees C for 30 min, was soluble in water but not in phenol or ether, and could be precipitated with ethanol. Bovine TF activity eluted as a single peak after high-pressure reverse-phase liquid chromatography (HPLC); the active moiety contained at least one free co-planar cis-diol group, as shown by boronate affinity chromatography. Additional structural features were deduced by evaluating TF activity after incubation with various endonucleases, exonucleases, and peptidases, a phosphatase, and a protease. The combined results indicate that bovine TF specific for PPD is an oligoribonucleopeptide. A simplest case molecular model was constructed on the basis of the data obtained. A comparative evaluation of the physicochemical properties and structural features of bovine TF and human TF specific for PPD indicated striking similarities and some differences.

Mycobacterium fortuitum pulmonary infection associated with an antigen-selective defect in cellular immunity.

In this study we describe the first example of a well documented case of pulmonary infection caused by Mycobacterium fortuitum shown to be associated with an antigen-selective defect in cell-mediated immunity to this organism. Immunologic parameters were evaluated before, during and after antibiotic treatment with amikacin. A defect in cellular immunity to purified protein derivative from Myco. fortuitum, shown to be antigen-selective as indicated by normal responsiveness to purified protein derivative from Mycobacterium tuberculosis and several other common recall antigens, accompanied the prolonged infection by this organism. During the first three months of treatment with amikacin, the patient's clinical status improved coincident with the eradication of the organism from the sputum. During the next three months of therapy with amikacin, however, a generalized defect in cellular immunity developed, and the lung disease again progressed. The deteriorating clinical condition was presumably related to a generalized cellular immune anergy or hyporesponsiveness induced by the amikacin therapy. After three more months of treatment, the organism became resistant to the drug and reappeared in sputum cultures. Since amikacin therapy was discontinued, the patient's general immune responsiveness returned to normal. He did, however, remain unresponsive to purified protein derivative from Mycobacterium fortuitum.

Cystic fibrosis ciliary dyskinesia substances and pulmonary disease. Effects of ciliary dyskinesia substances on neutrophil movement in vitro.

Cultured mononuclear cells (MNC) from individuals homozygous or heterozygous for the defective gene causing the inherited disease cystic fibrosis (CF) synthesize three unusual "mediators" termed ciliary dyskinesia substances (CDS), which markedly affect tracheal mucociliary systems in vitro. MNC cultures from normal healthy controls do not accumulate any CDS, whereas MNC cultures from non-CF patients controls with pulmonary disease synthesized at least one CDS. The possible involvement of the CDS in pulmonary disease is being investigated. In this study, we sought to determine whether the CDS could be chemoattractants for polymorphonuclear neutrophils (PMN), since they have characteristics in common with known chemoattractants generated by alveolar macrophages. Our analyses of crude MNC culture supernates indicated that cultures from both CF genotypes accumulate significantly higher levels of PMN chemoattractants than do analogous cultures from normal healthy controls. CF homozygote MNC also generated more activity than MNC from patient controls with chronic pulmonary disease. Fractionation of MNC culture supernates by gel permeation chromatography and characterization of active fractions demonstrated six distinct PMN chemoattractants in cultures from CF genotypes; five were also present in patient control and four in normal healthy control cultures. The excessive chemoattractant activity in MNC cultures from CF genotypes and patient controls was due to several different substances produced by monocytes: (a) two components of 1,000-3,500 mol wt. (b) two fragments of C5, and (c) a fragment of C3. One C5 fragment had ciliary dyskinesia activity, the other did not. The C3 fragment chemoattractant also had ciliary dyskinesia activity and was not found in MNC cultures from patient controls. A third CDS, Which is CF-specific (5,000 mol wt), was neither chemotactic not chemokinetic and did not inhibit random PMN migration; however, fractions containing this CF-specific CDS completely inhibited PMN chemotaxis in response to three different chemoattractants. We conclude that all of the CDS can potentially play a role in the pathophysiology of lung disease, as judged by their effects on PMN movement in vitro.

Effects of dialyzable leukocyte extracts with transfer factor activity on leukocyte migration in vitro. V. Antigen-specific lymphocyte responsiveness can be initiated by two structurally distinct polyribonucleopeptides.

Human transfer factors (TF) active in specifically inducing responsiveness in human thymus-derived (T) lymphocytes previously nonresponsive to purified protein derivative from Myobacterium tuberculosis (PPD) or to Coccidioides immites (Cocci) in vitro were isolated from the dialyzable portion of extracts of immune leukocytes (DLE). Each TF segregated into two active fractions after high-pressure reverse-phase liquid chromatography (HPLC), suggesting the presence of two TF components in DLE for each antigen specificity. Determination of the structures of both TF components specific for PPD was accomplished by evaluating their activity after incubation with various endonucleases, exonucleases, phosphatases, peptidases and a protease. The results indicated that both PPD-specific TF components are oligoribonucleopeptides but that they are structurally distinct. Simplest-case molecular models were constructed on the basis of the data obtained.

Synthesis and secretion of cystic fibrosis ciliary dyskinesia substances by purified subpopulations of leukocytes.

Cultured peripheral blood leukocytes (PBL) from individuals homozygous or heterozygous for the defective gene causing the inherited disease cystic fibrosis (CF) secrete three different ciliary dyskinesia substances (CDS), which can be detected by their activity in vitro in a rabbit mucociliary bioassay. Their PBL also release substances that promote mucus expulsion and destruction of the ciliated epithelium. In the present study the relative numbers of lymphocytes (T, B, and null), monocytes-macrophages (Mphi), and polymorphonuclear neutrophils were found to be normal in subjects with the CF gene, as were the responses of their PBL to phytohemagglutinin and pokeweed mitogen. Using purified subpopulations of leukocytes, we obtained evidence that both monocytes and T lymphocytes can secrete CDS in vitro with no requirement for cooperation with other lymphocyte subsets, whereas B and "null" lymphocytes probably require either differentiation or cellular cooperation for optimal secretion of CDS. Mucus expulsion and tissue destruction were produced by substances released primarily from polymorphonuclear neutrophils and secondarily from Mphi. Using cycloheximide and actinomycin D, we obtained evidence that CDS accumulation requires active protein synthesis and is not dependent on newly synthesized RNA, at least in short-term cultures. Gel filtration chromatography of active culture supernates showed that T lymphocytes synthesized only a CF-specific CDS, whereas Mphi synthesized all three CDS found in PBL cultures. Evidence is presented that one CDS is related structurally to C3a, since it can be removed with rabbit antisera specific for human C3a.

Chorioretinitis with a combined defect in T and B lymphocytes and granulocytes. A new syndrome successfully treated with dialyzable leukocyte extracts (transfer factor).

A patient with immune deficiency, recurrent pyogenic infections and active chorioretinitis is described; in addition to agammaglobulinemia, both quantitative and qualitative T-cell deficiencies were documented. Furthermore, the patient's granulocytes (polymorphonuclear leukocytes), although normal in their bactericidal capacity for Staphylococcus, responded poorly to both leukocyte migration inhibition factor and neutrophil immobilizing factor obtained from normal cells. The immunologic features of this patient appear to comprise a new syndrome. Remarkable diminution of the ocular lesions and increased visual acuity occurred within two months after the initiation of therapy with dialyzable leukocyte extracts (transfer factor). Concurrent testing of the patient's cell-mediated immunity showed increased numbers of circulating T lymphocytes and improved T-cell function following dialyzable leukocyte extract [DLE] therapy. The dramatic clinical results indicate that similar therapy may prove to be beneficial in other patients with chorioretinitis and T-cell deficiency.