Dialyzable Leukocyte Extract Research Articles

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.

Adoptive Transfer of Delayed Wattle Reactivity in Chickens with a Dialyzable Leukocyte Extract Containing Transfer Factor

Delayed wattle reactions (DWR) to tuberculin, diptheria toxoid (DT), and keyhole limpet hemocynanin (KLH) were transferred to chickens with dialyzable leukocyte extracts (DLE) prepared from splenic leukocytes of chickens sensitized and reactive by DWR to the tuberculin, DT, or KLH. A DLE prepared from chickens unsensitized and unreactive to tuberculin by DWR failed to transfer DWR to tuberculin in recipients. Only chickens injected with DLE from tuberculin sensitized and reactive chickens exhibited significant (P<.01) DWR to tuberculin. Chickens that received DLE prepared from DT sensitized and reactive chickens exhibited significant (P<.01) DWR to DT but not to KLH. Further, DWR to both DT and KLH was transferred with DLE prepared from chickens sensitized to both antigens. Adoptive transfer of DWR to KLH in comparison to DT was more successful. Finally, the serial transfer of DWR to KLH (P<.05) but not to DT (P>.05) was accomplished using DLE prepared from chickens that previously were recipients of DLE prepared from chickens sensitized to KLH and DT. Results indicate that DLE prepared from chickens contain transfer factor (TF) responsible for adoptive transfer of DWR to tuberculin, DT, and KLH.

Augmentation of delayed-type hypersensitivity in antigen-primed guinea pigs by human dialyzable leukocyte extract. Chromatographic and enzymatic characterization of the active principle.

Column chromatographic purification and sensitivity towards enzymatic treatments of dialyzable transfer factor (TFd), the immunologically specific component of dialyzable leukocyte extract (DLE), have previously been used in its biochemical characterisation. In the present work we studied the effect of enzymes and the Sephadex G-10 chromatographic separation of the components of DLE augmenting delayed-type hypersensitivity. Skin reactivities to streptokinase-streptodornase (SK-SD) and tuberculin PPD were significantly augmented by injecting DLE into antigen-primed guinea pigs. The augmentation caused by DLE treatment correlated to the pre-existing level of immunity in the recipients. Most of the augmentory activity resided in 2 adjacent fractions, eluting early from a Sephadex G-10 column. This augmentation was destroyed by alkaline hydrolysis, by treatment with pronase, proteinase K, ribonuclease, and nuclease P1, but not by alkaline phosphatase or phosphodiesterase II. The observed sensitivities towards these enzymes, except that for ribonuclease, were closely similar to those described for the specific TFd component of DLE. These results are compatible with the idea that either the nonspecific augmenting and the specific TFd molecules are principally similar, or that the TFd molecules, in addition to their capacity to transfer specific immunity, also have an augmenting effect, which needs in its manifestation a sub-threshold dose of immunogen.

Delayed-type dermal hypersensitivity responses in cattle infected or sensitized with Taenia saginata

Steers sensitized with Taenia saginata antigen had greater delayed-type dermal hypersensitivity (DTH) responsiveness than cysticerci-infected steers or those given dialyzable leukocyte extract (DLE, transfer factor, TF) from sensitized or infected steers. DTH responsiveness in sensitized steers persisted longer than in infected animals or those given TF. No correlations existed between DTH responsiveness and the number of cysticerci in the carcasses or the cysticerci live: dead ratio.

Study of the immunopotentiator effects of dialyzable leukocyte extracts obtained out of leukocytes previously used for interferon production.

The existence of immunopotentiator factors able to induce blastogenesis, chemotaxis and able of inhibiting migration of leukocytes was investigated in alcohol precipitates of different batches of dialyzed leukocyte extracts (DLE). These were prepared either out of fresh leukocytes from a large pool of buffy coats obtained from all blood collected from healthy donors with irrelevant antigen sensitiveness (DLE-NS) or from similar leukocytes which were incubated during 19 h for interferon production (DLE-NS-I). Alternatively, the same factors were investigated on a batch of DLE obtained from a donor exquisitely sensitive to Candida antigen (C-DLE-CS). It was observed that all the three batches contained equal amounts of either specific or nonspecific immunological enhancing factors. The similarity of the intensity of specific factors between the so-called nonspecific batches (DLE-NS and DLE-NS-I) and the specific batch (C-DLE-CS) was interpreted to be the result of the random selection of the donors for the preparation of the nonspecific batches, which consequently contain randomized relevant specificities. It is postulated, therefore, that batches of DLE which represent a large waste of leukocytes used for interferon production can be a useful tool for double-blind DLE therapy purposes.

Effect of in vivo and in vitro treatment with dialyzable leukocyte extracts on human natural killer cell activity

The effects of in vivo and in vitro treatment with human dialyzable leukocyte extracts (DLE) on natural killer (NK) cell activity were studied in 12 patients with malignancies in an allogenic test system using K-562 target cells. A marked increase in NK cell activity was observed in 7 patients with originally low cytotoxic capacity, while originally normal NK cell activity of other tumor patients and of healthy subjects was not significantly influenced by DLE treatment. The observed stimulation of NK cell activity by DLE may be a possible mechanism of its therapeutic effects.

Secretory/excretory antigens produced by tetraphyridia of mesocestoides corti defined by SDS—PAGE and HPLC

Antigens derived from culture medium in which metacestodes of Mesocestoides corti Hoeppli, 1925, had been maintained were studied by sodium-dodecyl-sulfate—polyacrylamide gel electrophoresis (SDS—PAGE) and high performance liquid chromatography (HPLC). By SDS—PAGE a minimum of 18 Coomassie-staining bands were discerned, of which 4 major bands and 4 major peaks by HPLC of similar molecular weights were observed. The HPLC eluate peaks were analyzed for antigenic activity in vitro by double diffusion in two dimensions, and in vivo in a rabbit. The rabbit had been artificially sensitized with a dialyzable leukocyte extract, showing transfer-factor-like activity, from peritoneal exudate cells removed from mice infected with tetrathyridia. All of the HPLC fractions reacted with rabbit antibody prepared against secretory/excretory antigens, but the sensitized rabbit responded only to two fractions. It is now possible by HPLC to fractionate complex antigens without denaturing them and to elucidate further the role they play during infection.

Treatment of Mycobacterium fortuitum pulmonary infection with “transfer factor” (TF): New methodology for evaluating TF potency and predicting clinical response

The successful clinical use of dialyzable leukocyte extracts (DLE) containing transfer factor (TF) has been hampered in the past by the lack of an in vitro assay for TF activity. We have shown previously that the leukocyte migration inhibition (LMI) assay can be used to detect and quantitate TF in DLE in vitro. The development of an in vitro assay for TF has in turn permitted the development of a new method for determining the TF potency in different preparations of DLE. We report herein the first application of the LMI assay to the clinical use of DLE. As an example we present a case study in which DLE was successfully employed to treat a patient with pulmonary disease associated with an “antigen-selective” defect in cell-mediated immunity to Mycobacterium fortuitum. The clinical applications of the LMI assay illustrated in this report include: (1) documentation of the antigen-selective defect in cell-mediated immunity; (2) selection of donors for the preparation of DLE for immunotherapy; (3) determination of the TF potency of different DLE preparations; (4) prediction of the patient's in vivo response to DLE therapy; (5) determining dosages of DLE for immunotherapy and immunoprophylaxis; and (6) monitoring the patient's response to treatment with DLE.

Dialyzable leukocyte extract induces mast cell secretion

Dialyzable leukocyte extract (DLE) transfers the ability to elicit delayed-type hypersensitivity, an action which may be due to components that are antigen specific and those that are nonspecific. In the present study we have found that human DLE is capable of inducing secretion of serotonin and histamine from rat mast cells. Release of mediators was noncytotoxic, dose-dependent, and time-dependent and required energy and calcium. In addition, DLE facilitated mast cell secretion caused by IgE and anti-IgE antibody when mast cells were preincubated in doses of DLE below those capable of causing secretion directly. Similar secretory effects were also observed with purified mast cells, and DLE caused mast cell degranulation in vivo. Fractionation of DLE showed that the mast cell-activating components were mostly in the 500–3500 molecular weight fraction in which other nonspecific activities of DLE have been found, and not in the 3500-10,000 molecular weight fraction in which the ability to transfer antigen-specific delayed reactions has been reported to be found. We suggest that some nonspecific effects of DLE may be due to induction or augmentation of secretion from mast cells, and that in some instances the participation of mast cells in delayed-type hypersensitivity could be mediated by such leukocyte-derived factors.

Modification of an experimental mouseCandidainfection by human dialyzable leukocyte extract

Human dialyzable leukocyte extract (10(7) - 10(8) leukocyte equivalents, containing transfer factor) was administered intraperitoneally to CFW mice the day of and 2 days after intravenous infection with Candida albicans. Tissue Candida populations were determined immediately after and 2, 4, 7 and 14 days after infection. Kidney populations were significantly reduced on 27% of the days studied. Similar reduction in C. albicans census was obtained after injection of leukocyte extracts from donors skin test-positive to Candida antigens or donors negative to Candida antigens by skin test and migration inhibition analyses. There was no evidence of a dose-response relationship for leukocyte extract in the range 10(5) - 10(9) leukocyte equivalents. When mice were primed with C. albicans antigen 4 weeks prior to challenge the efficacy of leukocyte extracts was not augmented. There was no evidence that the infection-reducing effects were related to augmented polymorphonuclear leukocyte mobilization, increased mononuclear clearance of C. albicans, or to a direct toxic effect on C. albicans blastospores. These studies suggest that the reduction in Candida populations was non-specific and give further impetus to the use of the dialyzable leukocyte extracts as non-specific supplements to antibiotics in overwhelming or recalcitrant infections in man.

Study of the Immunopotentiator Effects of Dialyzable Leukocyte Extracts Obtained Out of Leukocytes Previously Used for Interferon Production

The existence of immunopotentiator factors able to induce blastogenesis, chemotaxis and able of inhibiting migration of leukocytes was investigated in alcohol precipitates of different batches of dialyzed leukocyte extracts (DLE). These were prepared either out of fresh leukocytes from a large pool of buffy coats obtained from all blood collected from healthy donors with irrelevant antigen sensitiveness (DLE-NS) or from similar leukocytes which were incubated during 19 h for interferon production (DLE-NS-I). Alternatively, the same factors were investigated on a batch of DLE obtained from a donor exquisitely sensitive to Candida antigen (C-DLE-CS). It was observed that all the three batches contained equal amounts of either specific or nonspecific immunological enhancing factors. The similarity of the intensity of specific factors between the so-called nonspecific batches (DLE-NS and DLE-NS-I) and the specific batch (C-DLE-CS) was interpreted to be the result of the random selection of the donors for the preparation of the nonspecific batches, which consequently contain randomized relevant specificities. It is postulated, therefore, that batches of DLE which represent a large waste of leukocytes used for interferon production can be a useful tool for double-blind DLE therapy purposes.

Atypical mycobacterium infection treated with dialyzable leukocyte extracts: Evidence for antigenic specificity

A patient had infection with Mycobacterium intracellulare and anergy. Antituberculous chemotherapy proved ineffective. Repeated administration of dialyzable leukocyte extract (DLE) produced a sequential appearance of delayed type hypersensitivity in response to several antigens, as well as definitive clinical improvement. Unfortunately, the patient died from an Aspergillus fumigatus brain abscess. At autopsy, no evidence of active mycobacterial infection was found. DLE therapy may be indicated for the treatment of M. intracellulare infections which are unresponsive to chemotherapy. The disparity between the onset and course of skin test reactivities to different antigens suggests that DLE enhanced cellular immunity on an antigen-specific basis.

Dialyzable leukocyte extract stimulates cAMP in T gamma lymphocytes.

Dialyzable leukocyte extract (DLE) from human blood leukocytes gave a 3- to 8-fold increase in the cAMP concentration after addition to purified lymphocytes. No significant change in cGMP was observed. When purified lymphocytes were separated into T cells and non-T cells by an E-AET rosette technique the cAMP response was most pronounced in T cells. When further separating T lymphocytes into T cells with Fc receptors for IgG and T cells with Fc receptors for IgM (T gamma and T mu lymphocytes), the increase in cAMP following stimulation was specifically conferred to T gamma lymphocytes. Our findings may support the concept that DLE activates T gamma lymphocytes, leading to an increase in suppressor cell function.

Dialyzable leukocyte extract therapy in immunodepressed patients with cutaneous leishmaniasis

Immunological studies were performed in 80 patients with different types of American cutaneous leishmaniasis (ACL). Cellular immunodeficiency was found in seven of the patients studied, all seven resistant to standard drug therapy for this disease. The seven patients were treated with dialyzable leukicyte extract (DLE) obtained from healthy donors with previous ACL, all of whom were positive for skin and in lymphocyte DNA synthesis tests to leishmanial extracts. The seven immunodeficient patients were treated with DLE prior to the administration of the chosen drug for each patient and for up to 18 weeks after the drug was stopped. DLE treatment resulted in a clinical, parasitological, and immunological improvement in four of the seven patients. In the remaining three patients, who had had the typical syndrome of diffuse cutaneous leishmaniasis (DCL) for more than 10 years, no clinical or immunological improvement could be observed.

Resynthesis of trypsinized sheep red blood cell receptors on human lymphocytes: Comparison of the effects of immunopotentiators of biological and synthetic origin in vitro

Five immunostimulants—thymosin, dialyzable leukocyte extracts containing transfer factor (DLE), isoprinosine, BM 12 531 (azimexon), and levamisole—were compared separately and in various combinations for their ability to increase the binding of sheep erythrocytes by trypsinized human peripheral blood lymphocytes in vitro. Levamisole produced the greatest enhancement when used alone, DLE produced a smaller increase, and isoprinosine, thymosin, and BM 12 531 were less effective. When combining two of these agents, the greatest increase was seen when one of the components was thymosin and the other either DLE, isoprinosine, or BM 12 531. These results indicate possible synergistic mechanisms and the possible advantage of combined therapeutic use of selected combinations of immunostimulants. In the majority of the combinations, however, inhibition rather than stimulation of rosette formation was observed.

Effect of treatment with glutaurine on human antibody-dependent cell-mediated cytotoxicity (ADCC).

The effects of in vitro and in vivo treatment with glutaurine, a newly discovered parathyroid hormone possessing immunostimulative activity, on human antibody-dependent cell-mediated cytotoxicity (ADCC) were studied in 15 tumor patients with healthy subjects in a xenogeneic test system using chicken erythrocytes as target cells. A marked increase in "K" cell activity was observed in 8 tumor patients with originally low cytotoxic capacity, while originally normal ADCC activity of other tumor patients and healthy subjects was not significantly influenced by glutaurine treatment. The changes in cytotoxicity were not accompanied by changes in lymphocyte populations. Incubation of effector cells with glutaurine in vitro caused no change in ADCC activity in lymphocyte populations. Some similarities between the effects of glutaurine treatment of ADCC and that of dialyzable leukocyte extracts are discussed.

Isolation and characterization of immunopeptide from dialyzable leukocyte extract

The dialyzable leukocyte extract was fractionated using high-pressure liquid chromatography through an anion exchange column (preparative). Twenty-one fractions were obtained. Fraction 1, which gave maximum enhancement of E rosettes, was further purified by thin-layer chromatography on silica gel plates. Five ninhydrin-positive bands were seen. The area under each band was scraped from the plates and eluted. The fastest moving component was labeled S1 and the slowest moving S5. Fraction S3 had given maximum enhancement of E rosettes and was designated as immunopeptide. The immunopeptide was further characterized. It contained protein, ribose, and hexose. The molecular weight was determined to be 1870 by sedimentation equilibrium method. It contained 15 amino acid residues. The immunopeptide enhanced E-rosette formation in vivo when given in doses of 5 units/m 2, to four individuals with low E rosettes.

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.

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

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

Effects of dialyzable leukocyte extracts (DLE) with transfer factor activity on leukocyte migration in vitro: IV. Two distinct effects of DLE on leukocyte migration can be produced by prostaglandins

Dialyzable leukocyte extracts (DLE) prepared from peripheral blood leukocytes obtained from normal human donors have several effects on the migration of polymorphonuclear neutrophils (PMN). Previously, we proposed that one or more of these effects might be mediated at least in part by prostaglandins (PG). In the present study we present results which indicate that an antigen-independent enhancement of PMN migration (ELM) induced by a still unidentified component in DLE may be caused by de novo synthesis of PG. This ELM was abolished by preincubation of PMN with noncytotoxic concentrations of indomethacin, mefenamic acid, ibuprofen, or eicosatetraynoic acid, and in agreement with other studies, we found that PGEs or PGFs could enhance random PMN migration. We also found that an antigen-dependent ELM can be produced by PGA. The levels of PGA and of PGE and F in crude DLE preparations were determined by radioimmunoassay and found to be within the range of concentrations required for the in vitro effects noted. The two effects produced by PGA and PGE or F might tend to suppress or otherwise modify antigen-dependent LMI activity in vivo, especially at local inflammatory foci produced by injection of DLE when used for immunotherapy. Future studies seem warranted to determine whether PG levels should be altered in DLE for more effective immunotherapy, and to further investigate the effects of PG or related metabolites on LMI and associated aspects of cell-mediated immunity.