Differential responsiveness of various substrains of inbred strain 2 guinea pigs to immunotherapy with the methanol extraction residue (MER) of BCG

Summary The immunotherapeutic effects of the methanol extraction residue (MER) of BCG were investigated in strain 2 guinea pigs bearing the transplantable line 10 hepatocarcinoma, a tumor originally induced in guinea pigs at the National Institutes of Health (NIH) by ingestion of the carcinogen diethylnitrosamine. MER was more effective in mediating tumor regression in guinea pigs obtained from the Weizmann Institute of Science (WI), Rehovot, Israel, than in animals obtained from the National Institutes of Health (NIH). These differences indicate the dramatic effects which minor histoincompatibilities between cancer cells and animal substrains may have on experimental results, and highlight the need for immunotherapy experiments to be conducted on laboratory tumors grown in their autochthonous hosts. MER was effective only when injected directly into growing tumor nodules and had no effect on tumor development when administered distally. In contrast, all animals which received both MER and tumor cells developed specific cell-mediated anti-tumor immune responsiveness at higher levels than did non-MER-treated tumor-bearing controls as measured by delayed cutaneous hypersensitivity and in vitro lymphocyte reactivity experiments. Furthermore, the results of the latter but not the former studies suggested that guinea pigs which received MER were able to mount such an immune response more rapidly than their non-treated counterparts. This apparent stimulation of anti-tumor immunity was observed in treated animals regardless of substrain or site of MER injection, and could not be correlated with the outcome of immunotherapy.     

Effects of the methanol extraction residue (MER) tubercle bacillus fraction on the production of antibodies in vitro. III. Consequence of prior sensitization to MER

Mice repeatedly immunized with the methanol extraction residue fraction of tubercle bacilli (MER) in incomplete Freund's adjuvant produced high titers of circulating antibodies against MER, as assessed by the enzyme-linked immunosorbent assay (ELISA) method. Spleen cells derived from these animals failed to respond to the usual nonspecific immunopotentiating influence of MER on the primary production of antibodies (generation of specific plaque-forming cells) in vitro to sheep red blood cells. The defect was expressed by B lymphocytes and splenic macrophages, but not by splenic T lymphocytes or peritoneal exudate macrophagic cells. Impaired responsiveness by spleen cells from MER-immunized animals to nonspecific immunostimulation was also expressed with regard to another, unrelated biological response modifier, lipopolysaccharide. There was no impairment of responsiveness to polyclonal mitogenic stimulation. Possible mechanisms of the effects described are discussed.     

Potentiation and modulation of the immune response of guinea pigs to poorly immunogenic protein-hapten conjugates by pretreatment with the MER fraction of attenuated tubercle bacilli

The MER fraction of attenuated tubercle bacilli of the BCG strain was shown capable of stimulating and modulating the immunological responsiveness of guinea pigs to immunization with DNP conjugates of allogeneic globulin (DNP-GPG) and xenogeneic albumin (DNP-HSA). These antigens are very poorly immunogenic and fail to evoke detectable immune responses following single administration alone.When incorporated in incomplete Freund's adjuvant (IFA) together with the conjugates, MER could substitute for whole tubercle bacilli in the adjuvant mixture, and cause the conjugates to evoke both cellular and humoral reactivity, the former indicated by the development of skin reactions of delayed type (DH) to test injections of the antigens, the latter by the formation of humoral antibodies detected by an indirect hemagglutination (HA) test. When administered in saline together with antigen, MER was ineffective.Pretreatment with MER by any of several different routes 7 or 14 days prior to sensitization enabled a large number of the animals to respond with either DH, circulating antibody formation, or both. Under similar circumstances, pretreatment with Freund's complete adjuvant (FCA) elicited no such preparatory effect. In order to be efficacious in pretreatment, MER had to be given in a saline suspension; activity was lost when it was applied in IFA.MER pretreatment modulated the immune response to subsequent sensitization with the conjugates preferentially towards DH or antibody production, depending on the parameters of treatment and specific immunization. It appeared that when the specific immunogenic stimulus was weak, pretreatment with MER strongly favored DH.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4 dinitro-1-fluorobenzene (DNFB) was induced in BALB/c mice by DNFB skin application. Development of skin CH was suppressed by exposure of the animals after sensitization to the cancer chemotherapeutic drugs cyclophosphamide (CY), sodium methotrexate (MTX), and 5-fluorouracil (5FU). Unresponsiveness to DNFB was also induced in parallel experiments by a single intravenous injection of dinitrobenzenesulfonate (DNBS), either before or concomitant with sensitization. Potentiation of CH skin reactivity was achieved by administration of CY prior to sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of animals exposed to CY, MTX, or 5FU to respond to DNFB sensitization. The agent did not impair the potentiation of CH skin reactivity that could be effected by administration of CY prior to sensitization.MER treatment was not effective in reversing hapten-induced (DNBS) tolerance in mice.These findings favor the assumption that MER, under the conditions tested, stimulates the function of positively reacting T cells and exerts no enhancing or protective action on suppressor T cells.     

Effects of repeated doses of MDMA ("ecstasy") on cell-mediated immune response in humans.

Cell-mediated immune response after the administration of two repeated doses of 100 mg 3,4-methylenedioxymethamphetamine (MDMA) at 4-hour and 24-hour intervals was evaluated in two randomised, double-blind and cross-over clinical trials conducted in healthy male MDMA consumers. MDMA produced a time-dependent decrease in the CD4/CD8 T-cell ratio due to a decrease in the number of CD4 T-helper cells, a decrease in the functional responsiveness of lymphocytes to mitogenic stimulation, and a simultaneous increase in natural killer cells. In case of two 100 mg MDMA doses given 4 hour apart, immune alterations produced by the first dose were strengthened by the second one. At 24 hours after treatment, statistically significant residual effects were observed for all the altered immune parameters after the administration of two MDMA doses if compared to single dose and placebo. In the second clinical trial, the second 100 mg MDMA dose given 24 hours after the first dose produced immunological changes significantly greater than those induced by the initial drug administration and which seemed to show a delayed onset. Significant residual effects were observed for all the immune parameters as late as 48 hours after the second dose. These results show that repeated administration of MDMA with both a short and a long time interval between doses extends the critical period following MDMA administration, already observed after a single dose, in which immunocompetence is severely compromised.     

Chemical modification of macrophages enhances their response to human macrophage activation factor

Human monocyte-derived macrophages pretreated with the esterase inhibitors, antithrombin III and soybean trypsin inhibitor (STI) showed increased responsiveness to limiting concentrations of macrophage activation factor (MAF) as demonstrated by their enhanced cytotoxicity for tumor cells. Other proteins that are not esterase inhibitors did not enhance the effect of MAF on the macrophages. Enhancement of MAF activity was also obtained when macrophages were preincubated with the cell surface reactant diazotized sulfanilic acid (DSA). When MAF was preincubated with untreated, DSA-treated or STI-treated macrophages prior to testing on fresh macrophages, MAF preincubated with the untreated macrophages lost activity whereas the cytotoxicity induced by MAF preincubated with DSA-treated or STI-treated macrophages showed no decrease. These findings suggest that the enhanced responsiveness to MAF is the result of decreased destruction of MAF by macrophage-associated proteinases. Thus, the response of guinea pig and human macrophages to MAF appears to be regulated by similar mechanisms.     

Effects of the methanol extract residue (MER) tubercle bacillus fraction on the production of antibodies in vitro. II. Effects on macrophage and lymphocyte populations

The effect of the methanol extract residue (MER) fraction of BCG tubercle bacilli on the generation of primary antibody responsiveness in vitro to sheep red blood cells (SRBC) was ascertained in cell reconstitution experiments, employing enriched populations of mouse macrophages and of T and B lymphocytes. In each of the antibody generation cultures one or another of the cell fractions had been exposed to MER, either by treatment of the donor animals or by preincubation with the agent for 48 hr in vitro. In some experiments, supernatants of MER-preincubated cells were employed in place of the cells. Macrophages and T cells that had been exposed to MER in vivo or in vitro and their supernatants demonstrated a markedly greater effect than nonexposed cells in the generation of direct specific plaque-forming cells (PFC) upon antigenic stimulation of the cultures with SRBC. In contrast, PFC production was not stimulated in B-lymphocyte populations that had been in contact with the agent.     

Effects of the MER tubercle bacillus fraction on the production of antibodies in vitro: I. Effect on the primary response

The effect of the methanol extraction residue (MER) fraction of BCG tubercle bacilli on the primary antibody response in vitro to sheep red blood cells (SRBC), TNP conjugates, and the monovalent hapten DNP-glycine was studied. Addition of MER to whole splenocyte cultures simultaneously with antigen presentation potentiated the antibody response to SRBC and TNP-SRBC, and facilitated reactivity to DNP-glycine; there was no effect on the response to the T-independent entity TNP-LPS (lipopolysaccharide from E. coli 055-B5). Immunopotentiating activity of MER for SRBC and DNP-glycine was also evident in macrophage-depleted cultures. Peritoneal exudate cells (PEC) taken from MER-treated donors were more efficient than PEC from untreated donors in reconstituting antibody formation to SRBC by macrophage-depleted spleen cell populations. The results obtained indicate that activation of both macrophages and of certain lymphocyte population(s) by MER may play a role in the potentiation of antibody responsiveness in vitro by this agent.     

Different photoperiods affect proliferation of lymphocytes but not expression of cellular,humoral, or innate immunity in hamsters

In seasonal mammals, photoperiod change is associated with a suite of alterations in physiology. It has recently been proposed that the immune response is one of the systems regulated by changes in photoperiod, although this hypothesis has not been rigorously challenged by assays of functional immune responses. The aim of this study was to test the hypothesis that photoperiod modulates immune responsiveness in Syrian (Mesocricetus auratus) and Siberian (Phodopus sungorus) hamsters. Consistent with previously reported data, short-day-housed (SD) animals exhibited a significant increase in lymph node cell (LNC) numbers and increased cellular proliferation in response to the polyclonal mitogen concanavalin A compared to long-day-housed (LD) animals. In contrast, LNC numbers from intact or gonadectomized SD animals that had been sensitized with the antigen dinitrofluorobenzene (DNFB) exhibited a reduced ex vivo proliferative response and reduced production of interleukin-6 (IL-6) compared to LD animals. In vivo studies of the contact hypersensitivity response of animals that had previously been sensitized, and subsequently challenged, with DNFB were similar in SD and LD animals, as was the proliferative activity of LNC recovered from these animals. There were also no photoperiodic differences in the antidinitrophenyl antibody response of animals sensitized with DNFB, or the anti-sheep red blood cell (srbc) response of animals immunized with srbc. Furthermore, no differences could be detected in the activity of natural killer cells from spleens of LD and SD Siberian hamsters, or in lipopolysaccharide-induced IL-6 production by LD and SD Syrian hamsters in vivo. Thus, although photoperiod is able to influence factors regulating the gross number and non-antigen-specific proliferation of lymphocytes in seasonally breeding mammals, day length does not directly influence activation of an effective immune response. The authors conclude, therefore, that expression of the immune response is not directly modified or compromised by photoperiod in these seasonally breeding hamster species.     

Increase of regulatory T cells in the peripheral blood of dogs with metastatic tumors

It is well known that lymphocytes from patients with advanced-stage cancer have impaired immune responsiveness and that type1 T lymphocyte subsets in tumor bearing hosts are suppressed. Treg have been reported to comprise a subgroup which inhibits T cell mediated immune responses. In the present study, the percentage of Treg, Th1 and Tc1 in the peripheral blood of tumor bearing dogs with or without metastases was evaluated. The percentages of Th1 and Tc1 in dogs with metastatic tumor were significantly less, and that of Treg was significantly greater, than those of dogs without metastatic tumor. The percentage of Treg showed an inverse correlation with that of Th1 and Tc1 in tumor bearing dogs. It was concluded that an increase in Treg in the peripheral blood of dogs with metastatic tumor may induce suppression of tumor surveillance by the Type1 immune response and lead to metastasis of tumor[0][0].[0]     

Effects of intravenous silica on immune and non-immune functions of the murine host.

Silica, an agent toxic for macrophages, administered i.v. to DBA/2 mice rapidly depresses the clearance of colloidal carbon by the reticuloendothelial system and reduces the in vitro phagocytic activity of peritoneal macrophages harvested 3 days after silica injection. Silica blocks the humoral immune response to sheep erythrocytes and the cell-mediated immune response to allogeneic fibroblasts when given before antigen. Silica also induces complex alterations in spleen cell responsiveness to concanavalin A involving both local and serum factors. Silica had no significant effect on the induction of interferon by statolon or Newcastle disease virus. No unequivocal evidence was obtained that silica has a direct depressive effect on cells other that macrophages, but indirect effects on lymphocytes were produced most likely by factors released from silica-lysed macrophages. Intravenous silica may prove useful for the separation of interferon induction and immune response stimulation in studies of host resistance to infection and oncogenesis. Considerable variation exists in the immunodepressive effects of different preparations of silica.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4-dinitro-1-fluorobenzene (DNFB) was induced in guinea pigs and mice by DNFB skin application. Development of CH was suppressed in both species either by cyclophosphamide (CY) treatment after sensitization or by single intravenous injection of dinitrobenzene-sulfonate (DNBS) before sensitization (hapten-induced tolerance). Additional treatment schedules were employed in guinea pigs, with the following results: Suppression of CH by injection of DNBS concomitant with sensitization; abrogation of hapten-induced tolerance by administration of CY before sensitization; and potentiation of CH skin reactivity by administration of CY before sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of CY treated animals to respond to DNFB sensitization. In contrast, administration of MER either by one injection before sensitization, concomitant with DNFB, or after sensitization did not prevent immunosuppression by CY.MER treatment was not effective in reversing hapten-induced tolerance in mice, and had only an occasional effect on this process in guinea pigs. Abrogation of hapten-induced tolerance and potentiation of DNFB sensitization by CY in guinea pigs were also not influenced by MER treatment.Supported by Contract NO1-CM-12127 from the NCI and by research grants from Concern Foundation, Inc., the Lautenberg Endowment, the National Council for Research and Development, Israel, and the GSF Munich, Germany, and the Leukemia Research Foundation, Inc.     

Immune reactivity in cattle with ocular squamous cell carcinoma after intralesional BCG immunotherapy

Summary Lymphocyte stimulation with Con A and specific immune reactivity to BCG (antibody formation to BCG and DTH reaction to PPD) were determined in BCG-treated, surgically treated and untreated cows with ocular squamous cell carcinoma. In tumor-bearing cows the Con A-induced proliferation of lymphocytes was reduced when compared to healthy controls. This suppression consisted of a reduced blastogenic response to Con A of lymphocytes from tumor-bearing cows, and the presence of a factor in the sera of these animals, as these sera suppressed the blastogenic response of lymphocytes from healthy cows. BCG had only a minor influence on the suppressive activity. Antibodies to BCG were demonstrated in 50% of the BCG-treated animals. The formation of antibodies was not influenced by intradermal injection of PPD of Mycobacterium bovis. Absorption of a BCG antibody containing serum with BOSCC tumor extracts did not reveal the existence of cross reacting antigens between BCG and BOSCC. Pretherapeutic and posttherapeutic Con A reactivity could not be correlated with clinical response. Of the 30 BCG treated cows 29 developed a positive DTH reaction to PPD. Correlation between clinical response and immune reactivity was seen only with regard to the DTH reaction to PPD: this reaction remained positive for a longer period after treatment in animals with a favorable clinical outcome than in nonresponding animals.Animals were maintained under the guidelines laid down by the Faculty of Veterinary Medicine, State University, Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation) Abbreviations used: BCG, Bacillus Calmette-Guerin; BOSCC, bovine ocular squamous cell carcinoma PBL peripheral blood leukocytes; PPD, purified protein derivative of Mycobacteria; DTH, delayed type hypersensitivity Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen     

Suppression of lymphokine production: II. Macrophage-dependent inhibition of production of macrophage activating factor

The ability of different populations of macrophages to affect the production of macrophage activating factor (MAF) by stimulated T lymphocytes was investigated. We found that activated macrophages, infiltrating MSV-induced regressing tumors or macrophages recovered from the peritoneum of mice injected with Corynebacterium parvum, were able to actively suppress the production of MAF. MAF production by antigen-stimulated MSV-immune or -alloimmune spleen cells and by normal spleen cells stimulated by Con A was susceptible to macrophage-dependent suppression to a similar extent. In contrast, resident macrophages or those elicited by light mineral oil or proteose-peptone did not affect MAF production. While suppressor macrophages added at the time of the lymphocyte stimulation inhibited MAF production, the same cells added 4–6 hr after stimulation were ineffective. Therefore, it seems that the macrophages suppressed the early events of lymphocyte activation leading to MAF production. Suppressor macrophages, by inhibiting MAF production, may limit the expansion of the cytotoxic activity. This regulation of macrophage functions, mediated by the effects of suppressor macrophages on T lymphocytes, could be responsible for an insufficient antitumor cytotoxic response by macrophages.     

Study of the immunotropic activity of aminoglycoside antibiotics

The action of some aminoglycoside antibiotics on the immune system was studied on both intact mice and the animals with immune deficiency caused by administration of cyclophosphamide. The following tests were used: local hemolysis (the Herne test), lymphocyte transformation (LT), delayed hypersensitivity to sheep red blood cells and the local graft versus host reaction (GVHR). Amikacin was shown to have no significant action on the activity of lymphocytes in the intact mice and stimulated both cellular (LT and GVHR) and humoral (the Herne test) immunity in the animals with lowered immunological reactivity. Sisomicin had no significant action on the immune system of the animals. Gentamicin suppressed the immune response only in the intact mice. Kanamycin and streptomycin induced inhibition of humoral and cellular immunity in both the intact mice and animals with immune deficiency. On the basis of the results it was concluded that gentamicin, amikacin and sisomicin may be used in the treatment of diseases developing in the presence of immune deficiency whereas streptomycin and kanamycin should be recommended when inhibition of the immunity is needed.     

Ascorbic acid blockade of muscle contractions by neurotransmitters and 2-aminoethanol.

Rats given intracisternal administration of 5–6 dihydroxytryptamine (5,6 DHT) 5 days after birth were tested at two subsequent periods for alterations in locomotor activity and responsiveness to dl-amphetamine and scopolamine. Later in life they were given amphetamine while performing on an FR-6 operant schedule for food reward. Biochemical analyses revealed that the 5,6 DHT treatment produced reductions in forebrain serotonin content. There was no alteration in the treated animals in locomotor activity or in responsiveness to amphetamine in the open field. However, animals given the highest dose (75 μg) of 5,6 DHT neonatally showed open field activity increases at high doses of scopolamine significantly more frequently than the other groups of animals. Although treatment with 5,6 DHT did not affect response rates on the FR task under no-drug conditions, the rate-suppressive effects of amphetamine on the FR task were greatly enhanced in the treated animals.     

The role of circulating interferon in the modifications of immune responsiveness by mouse hepatitis virus (MHV-3).

MHV-3 modifies the humoral immune response to SRBC. During acute infections timing was critical: infecting mice before antigen administration led to immunodepression. Simultaneous injection with virus and SRBC resulted in immunostimulation. Persistent MHV-3 infections were associated with a chronic immunodepression. The presence of circulating interferon (IF) was well correlated with these modifications. IF peaking before antigen was associated with immunodepression whereas IF secretion after antigen was associated with immunostimulation. Low, permanent levels of IF were associated with chronic immunodepression. Since IF is, up to now, the only product of activated lymphocytes that has been shown to modulate immune responses, our results suggest that induction of IF by MHV-3 may be the main mechanism by which this virus modifies immune responsiveness. Moreover, we have shown that MHV-3 infection in susceptible mice diminishes the secretion of lymphocyte IF in response to Sendai virus. In these animals, the thymus cortex was profoundly depleted although the thymus medulla remained unchanged. The MHV-3 infection may, therefore, interfere with a subpopulation of IF-secreting lymphocytes. The possible physiologic role of such lymphocyte subpopulation in terms of host-virus relationships is discussed.     

Induction by an immunogenic immunomodulating agent of nonspecific T cell suppression of lymphocyte responsiveness in MLR but not of antibody production

Summary Spleen cells derived from BALB/c mice that had been repeatedly immunized with the methanol extraction residue (MER) fraction of tubercle bacilli exhibited a depressed capacity to act as responder cells in allogeneic and syngeneic mixed lymphocyte reactions (MLR). Previously reported studies revealed that such spleen cells are also defective in the in vitro generation of antibodies. In order to determine the nature of the cells responsible for the depressed MLR reactivity, purified populations of splenic macrophages, B lymphocytes, T lymphocytes originating from normal and from MER-immunized mice, and cell culture supernatants were added to MLR mixtures consisting of normal mouse splenocytes. Macrophages originating from MER-immunized mice and their culture supernatants exerted a significantly higher suppressive effect on MLR than that of corresponding preparation from normal mice. Splenic T cells originating from MER-immunized mice and their supernatants also significantly suppressed the MLR response. However, the same T cell populations that were inhibitory in MLR failed to suppress the in vitro generation of antibodies against sheep red blood cells in the presence of either MER or 2-mercaptoethanol. These and previously reported findings indicate that a nonspecific immunomodulating agent, MER, can, under certain conditions of treatment, elicit the induction of nonspecific suppressor T cells for MLR but not for antibody production, and, accordingly, can inhibit cellular and humoral immunological responsiveness by different mechanisms.     

Immunoregulation in experimental filariasis. I. In vitro suppression of mitogen-induced blastogenesis by adherent cells from Jirds chronically infected with Brugia pahangi

Nonspecific immunoregulatory events were examined in inbred jirds chronically infected with Brugia pahangi. The responsiveness of spleen cells from infected animals to the T cell mitogens PHA and Con A and to the B cell mitogens, LPS and PWM, was found to be suppressed by as much as 90% when compared with the reactivity of lymphocytes from normal animals. Furthermore, spleen cells from infected jirds were capable of suppressing the mitogen reactivity of normal spleen cells. Depletion of cells adherent to nylon wool, glass wool, or plastic alleviated the regulatory activity exerted by spleen cells from infected jirds. Addition of indomethacin, an inhibitor of prostaglandin synthetase, to cultures of spleen cells from infected animals did not alter the suppression observed. In contrast, lymphocytes from the peripheral lymph nodes of infected jirds did not exhibit depressed T cell mitogen reactivity and were incapable of suppressing the PHA or Con A responsiveness of normal lymph node cells. However, the reactivity of lymph node cells from infected jirds to B cell mitogens, LPS and PWM, was suppressed. These results imply the existence of multiple regulatory mechanisms, at least one of which is restricted to the spleen. The relevance of nonspecific regulation to development of parasite-specific immunologic reactivity and to the infection is discussed.     

Effects of chronic leptin administration on nitric oxide production and immune responsiveness of greenfinches

Leptin and nitric oxide (NO) are both important messengers in intra- and intercellular communication systems in vertebrates. Several studies have demonstrated an involvement of both substances in the immune response. Here we tested the effects of chronic leptin and anti-leptin treatments on the NO production and phytohaemagglutinin- (PHA) induced cutaneous inflammatory response in a wild passerine, the greenfinch (Carduelis chloris). Plasma leptin levels of individual birds were consistent in time but could be still temporarily increased by administration of recombinant chicken leptin. Increase of plasma leptin was also induced by administration of anti-leptin, which can be most likely explained by increased endogenous leptin production due to disruption of signalling pathways. Contrary to previous findings in mammals, leptin administration reduced systemic NO production. Leptin increased cutaneous swelling response to PHA. This immune-enhancing effect was observable despite the similar plasma leptin levels of leptin-treated and control birds at the time of measurement of immune responses, i.e., 9 days after start of the treatments. This provides evidence for a delayed or long-term potentiation of the cells and cytokines involved. The effects of leptin administration on NO production and immune responsiveness were age-dependent, which indicates the complexity of underlying regulatory mechanisms.