Use of live Saccharomyces cerevisiae cells as a biological response modifier in experimental infections

A short-term oral administration of live Saccharomyces cerevisiae cells, strain Sillix Hansen DSM 1883, resulted in enhanced resistance of mice toward infections with K. pneumoniae. S. pneumoniae and S. pyogenes A produced by intranasal inoculation. Yeast pre-treatment also increased the efficacy of antibiotic therapy in bacterial infections and of antiviral drugs in viral infections. Yeast treatment of animals stimulated phagocytosis, activated the complement system and induced interferon which are likely to represent the main mechanisms of action whereby pretreatment of mice with live S. cerevisiae cells increases resistance to infection. It is concluded that preventive administration of live Saccharomyces cerevisiae cells should be used for increasing resistance to bacterial infections, in particular of the respiratory tract, or to viral infections, as well as an adjunct to antibiotic and antiviral drug therapy.     

Use of live Saccharomyces cerevisiae cells as a biological response modifier in experimental infections

Abstract A short-term oral administration of live Saccharomyces cerevisiae cells, strain Sillix Hansen DSM 1883, resulted in enhanced resistance of mice toward infections with K. pneumoniae, S. pneumoniae and S. pyogenes A produced by intranasal inoculation. Yeast pre-treatment also increased the efficacy of antibiotic therapy in bacterial infections and of antiviral drugs in viral infections. Yeast treatment of animals stimulated phagocytosis, activated the complement system and induced interferon which are likely to represent the main mechanisms of action whereby pretreatment of mice with live S. cerevisiae cells increases resistance to infection. It is concluded that preventive administration of live Saccharomyces cerevisiae cells should be used for increasing resistance to bacterial infections, in particular of the respiratory tract, or to viral infections, as well as an adjunct to antibiotic and antiviral drug therapy.     

Immunostimulation and anti-DNA antibody production by backbone modified CpG-DNA

Oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG-DNA) have gained attention as potentially useful therapeutics. However, the phosphorothioate-modified CpG-DNAs (PS-ODN) can induce backbone-related side effects. Here, we compared the immunostimulatory activity of natural phosphodiester CpG-DNA (PO-ODN) from Mycobacterium bovis and PS-ODN in mice. Both PO-ODN and PS-ODN induced production of IL-12. PS-ODN increased spleen weights, spleen cell numbers, and the migration of macrophages into the peritoneal cavity in the mice in a CG sequence-dependent manner. PS-ODN induced anti-PS-ODN antibody production in the mice, and the PS-ODN-specific IgM was cross-reactive with other PS-ODNs in a CG sequence-independent manner. In contrast, PO-ODN did not affect on spleen weights, cell numbers, or IgM production. These results may provide an explanation for the side effects in immunotherapeutic application of PS-ODN. They also suggest that PO-ODN may be more optimal than PS-ODN to enhance innate immune responses without severe side effects.     

Immunostimulation of dendritic cells by cationic liposomes

A nano-aggregate liposome-polycation-DNA (LPD), composed of a cationic lipid, protamine and plasmid DNA was found to effectively deliver a human papillomavirus (HPV)-E7 epitope antigen to the antigen presenting cells of the immune system, eliciting enhanced anti-tumor immune responses in mouse models of cervical carcinoma. Both the cationic liposome and plasmid DNA were essential for the full immunostimulation activity of LPD. Interestingly, cationic liposomes alone could stimulate the antigen presenting dendritic cells (DC) leading to the expression of co-stimulatory molecules, CD80 and CD86. However, cationic lipids could not stimulate DC for the expression of pro-inflammatory cytokines. Moreover, they were unable to enhance the expression of NF-κB, suggesting that dendritic cells stimulation by cationic lipids is signaled through an NF-κB independent mechanism. DC stimulation was specific to cationic lipids, the zwitterionic and anionic lipids showed little or no activity. The ability of different cationic lipids to stimulate the expression of co-stimulatory molecules on DC varied significantly. In general, the cationic lipids bearing ethyl phosphocholine head groups were better stimulants than their trimethylammonium counterparts. In case of the cationic lipids bearing trimethyl ammonium head groups, the ones bearing unsaturated or shorter saturated hydrophobic chains exhibited enhanced immunostimulatory activity. The LPS-induced TNF-α expression by dendritic cells was inhibited by active cationic lipids but not the inactive ones, suggesting the possible involvement of lipopolysaccharide binding protein (LBP) in cationic lipid mediated DC stimulation. Based on the structure-specific activation of dendritic cells by cationic lipids, a model for the immunostimulation of DC by such lipids is proposed.     

Induction of humoral antibody response by soluble polysaccharide of Cryptococcus neoformans

Mice were immunized with purifiedCryptococcus neoformans soluble polysaccharide, or with the polysaccharide coupled to methylated bovine serum albumin or methylated bovine gamma globulin. The polysaccharide-methylated protein complexes were no more immunogenic than the purified polysaccharide when used without Freund's incomplete adjuvant; however, the methylated protein complexes induced higher levels of antibody than purified polysaccharide when emulsified in the adjuvant. Sera from mice were titrated by passive hemagglutination, and maximum antibody titers were observed 14 to 21 days after immunization. Antibody titers declined rapidly after 14 to 21 days in mice immunized with antigen alone; whereas, animals immunized with cryptococcal antigen emulsified in adjuvant remained at peak levels throughout a six week experimental period. All antigens were immunogenic over a wider dosage range when contained in adjuvant. Individual mice immunized with an adjuvant emulsion of purified polysaccharide varied widely in the amount of antibody produced, with some of the animals producing no detectable antibody.     

Kinetics of the antibody response to type III pneumococcal polysaccharide. I. Evidence that suppressor cells function by inhibiting the recruitment and proliferation of antibody-producing cells.

For the first 126 hr after immunization of mice with an optimally immunogenic dose (0.5 mug) of Type III pneumococcal polysaccharide (SSS-III), splenic antibody-forming PFC and serum antibody levels were measured at 2- and 8-hr intervals, respectively. PFC were detected at 28 hr after immunization and then increased through 86 hr after immunization; thereafter, the number of PFC remained nearly constant for the next 20 to 24 hr, and then began to decline. In contrast, serum antibody was first detected 60 hr after immunization. The accumulation of serum antibody continued to lag behind the increase in numbers of PFC by 16 to 20 hr until maximal serum antibody levels were attained; curves fitted to the values obtained for each parameter were nearly parallel.     

Immunostimulation of dendritic cells by cationic liposomes

A nano-aggregate liposome-polycation-DNA (LPD), composed of a cationic lipid, protamine and plasmid DNA was found to effectively deliver a human papillomavirus (HPV)-E7 epitope antigen to the antigen presenting cells of the immune system, eliciting enhanced anti-tumor immune responses in mouse models of cervical carcinoma. Both the cationic liposome and plasmid DNA were essential for the full immunostimulation activity of LPD. Interestingly, cationic liposomes alone could stimulate the antigen presenting dendritic cells (DC) leading to the expression of co-stimulatory molecules, CD80 and CD86. However, cationic lipids could not stimulate DC for the expression of pro-inflammatory cytokines. Moreover, they were unable to enhance the expression of NF-kappaB, suggesting that dendritic cells stimulation by cationic lipids is signaled through an NF-kappaB independent mechanism. DC stimulation was specific to cationic lipids, the zwitterionic and anionic lipids showed little or no activity. The ability of different cationic lipids to stimulate the expression of co-stimulatory molecules on DC varied significantly. In general, the cationic lipids bearing ethyl phosphocholine head groups were better stimulants than their trimethylammonium counterparts. In case of the cationic lipids bearing trimethyl ammonium head groups, the ones bearing unsaturated or shorter saturated hydrophobic chains exhibited enhanced immunostimulatory activity. The LPS-induced TNF-alpha expression by dendritic cells was inhibited by active cationic lipids but not the inactive ones, suggesting the possible involvement of lipopolysaccharide binding protein (LBP) in cationic lipid mediated DC stimulation. Based on the structure-specific activation of dendritic cells by cationic lipids, a model for the immunostimulation of DC by such lipids is proposed.     

Antigen phagocytosis by B cells is required for a potent humoral response

Successful vaccines rely on activating a functional humoral response that results from promoting a proper germinal center (GC) reaction. Key in this process is the activation of follicular B cells that need to acquire antigens and to present them to cognate CD4 T cells. Here, we report that follicular B cells can phagocytose large antigen‐coated particles, a process thought to be exclusive of specialized antigen‐presenting cells such as macrophages and dendritic cells. We show that antigen phagocytosis by B cells is BCR‐driven and mechanistically dependent on the GTPase RhoG. Using Rhog^?/? mice, we show that phagocytosis of antigen by B cells is important for the development of a strong GC response and the generation of high‐affinity class‐switched antibodies. Importantly, we show that the potentiation effect of alum, a common vaccine adjuvant, requires direct phagocytosis of alum–antigen complexes by B cells. These data suggest a new avenue for vaccination approaches by aiming to deliver 1–3 μm size antigen particles to follicular B cells.     

Prophylactic intervention in radiation-leukemia-virus-induced murine lymphoma by the biological response modifier polysaccharide K

Polysaccharide K (PSK) is a biological response modifier used for adjuvant immunotherapy of malignant diseases. We studied the potential applicability of PSK for preventing tumor progression using an experimental model of murine lymphoma. Mice inoculated with the radiation leukemia virus (RadLV) develop thymic lymphomas after a latency of 3–6 months. However, 2 weeks after virus inoculation, prelymphoma cells can already be detected in the thymus. We found that PSK treatment induced hyperresponsiveness to concanavalin A and heightened production of interleukin-2 (IL-2) and IL-4 in spleen cells of both control and prelymphoma mice. The response was transient and was accompanied with a dominant usage of T cells expressing V8, but other T cell subsets were also stimulated by PSK. T lymphoma cells expressing V8.2 underwent apoptosis when incubated with PSK. Treatment of RadLV-inoculated mice with PSK delayed the onset of overt lymphoma (and mortality) but could not protect the mice from the disease. Combined treatment with PSK and a RadLV-specific immunotoxin prevented synergistically the progression of the prelymphoma cells to frank lymphoma. The results suggest that PSK contains a superantigen-like component that selectively activates V8⁺ T cells. Its administration prelymphoma mice interfered with the process of lymphoma progression.     

Prophylactic intervention in radiation-leukemia-virus-induced murine lymphoma by the biological response modifier polysaccharide K

Polysaccharide K (PSK) is a biological response modifier used for adjuvant immunotherapy of malignant diseases. We studied the potential applicability of PSK for preventing tumor progression using an experimental model of murine lymphoma. Mice inoculated with the radiation leukemia virus (RadLV) develop thymic lymphomas after a latency of 3–6 months. However, 2 weeks after virus inoculation, prelymphoma cells can already be detected in the thymus. We found that PSK treatment induced hyperresponsiveness to concanavalin A and heightened production of interleukin-2 (IL-2) and IL-4 in spleen cells of both control and prelymphoma mice. The response was transient and was accompanied with a dominant usage of T cells expressing Vβ8, but other T cell subsets were also stimulated by PSK. T lymphoma cells expressing Vβ8.2 underwent apoptosis when incubated with PSK. Treatment of RadLV-inoculated mice with PSK delayed the onset of overt lymphoma (and mortality) but could not protect the mice from the disease. Combined treatment with PSK and a RadLV-specific immunotoxin prevented synergistically the progression of the prelymphoma cells to frank lymphoma. The results suggest that PSK contains a superantigen-like component that selectively activates Vβ8⁺ T cells. Its administration prelymphoma mice interfered with the process of lymphoma progression.     

Augmentation of natural killer cell activity by ImuVert: a biological response modifier derived from Serratia marcescens

The natural killer (NK) cell activity of peripheral blood mononuclear cells (PBMC) from healthy human volunteers was studied following in vitro incubation with ImuVert, a biological response modifier derived from the bacterium Serratia marcescens. Exposure of these cells to ImuVert for as little as 10 minutes followed by an additional incubation in vitro of at least 12 hours and optimally 18 hours resulted in a substantial, consistent, and dose-dependent augmentation of NK cell activity against K562 tumor cells. Additional studies indicate that the augmented cell expressed the leu 11 cell surface marker and that peripheral blood monocytes were essential in the induction of augmented NK cell activity but were not the effector cell of NK activity.     

Deficient humoral antibody response of the spontaneously diabetic Chinese hamster

The antibody responses of spontaneously diabetic Chinese hamsters to sheep erythrocytes was investigated. After primary challenge, total antibody production of the diabetics was only 50% or less than that of nondiabetic controls, and little or no switchover to IgG production occurred. After second challenge, total antibody of diabetics was still reduced compared with controls. Some switchover to IgG did occur after second challenge but was only 40% that of controls. Poor antibody response was not related to abnormal glucose metabolism since low titers and lack of IgG was found in euglycemic prediabetics. Additionally, genetic nondiabetics rendered hyperglycemic by streptozotocin responded normally, similar to euglycemic controls. The impaired humoral antibody response of the diabetic hamster is similar to that reported for some human diabetics and makes this animal model attractive for studying the consequences and possible causes of diabetes-dependent impairment of immune responses.     

Altered humoral immune response to sheep red blood cells by sheep erythrocyte-soluble hemolysate.

Adult C3H/He mice were rendered unresponsive to a primary injection of sheep red blood cells (SRBC) by pretreatment with sheep hemolysate supernatant (SHS) or subfractions of SHS isolated by column chromatography. The following effects of SHS on the immune response were observed: SHS did not kill antigen-reactive cells, it did not prevent the release of antibody by cells actively synthesizing and secreting antibody, and SHS-induced tolerance was not inhibited or abrogated by methods which terminate or abolish tolerance. In addition, cell-mediated responses were not affected in animals whose humoral responses were suppressed; however, the secondary plaque-forming cell (PFC) response was enhanced by SHS treatment. SDS gel electrophoresis revealed SHS to contain several proteins ranging from 12,000 to approximately 500,000 daltons.     

The neutrophil as an information transmitter in tumor inhibition by a streptococcal biological response modifier,OK-432

 Effective treatment of a rat transplanted ascites tumor by i. p. injection of a streptococcal biological response modifier, OK-432, was abrogated by selective in vivo depletion of neutrophils by a monoclonal antibody, RP-3. The mechanisms by which neutrophils participate in the therapeutic action of OK-432 were studied with Winn’s assay using peritoneal exudate cells periodically obtained from rats i. p. injected with this biological response modifier. Intraperitoneal resident macrophages were first activated with OK-432, and within 3 h, tumor-inhibitory activity had moved to the early exuded neutrophils. However, 6 h after injection, exuded macrophages were the only cells involved in tumor inhibition. Considered together with other findings, it is likely that, in this system, neutrophils may transmit information from resident macrophages to exuded inflammatory macrophages in a series of responses induced by i. p. injection of OK-432. Received: 29 April 1996 / Accepted: 27 July 1996     

Characterisation of gilthead seabream acidophilic granulocytes by a monoclonal antibody unequivocally points to their involvement in fish phagocytic response

The various cell types involved in fish phagocytic defence have not been properly established because of the morphological heterogeneity of leucocytes and the lack of appropriate cell-surface markers. In this study, we report the production and characterisation of a monoclonal antibody, G7, which specifically recognises gilthead seabream acidophilic granulocytes, as assayed by immunofluorescence and immunoelectron microscopy. The antibody reacted with 40%-50% of head-kidney and peritoneal exudate leucocytes and 10%-20% of spleen and peripheral blood leucocytes. More importantly, G7(+) acidophils constituted 85% of the head-kidney leucocytes showing phagocytic activity towards the fish pathogenic bacterium Vibrio anguillarum. The results are discussed in relation to the role played by this cell type in fish immune responses.     

Sensitivity of ovarian tumor cells to effector cells generated by various biological response modifiers

The sensitivity of freshly derived human ovarian tumors (FOT) to various allogeneic cytotoxic effector cells stimulated by recombinant interleukin 2 (rIL-2), recombinant interferon alpha 2 (rIFN-alpha 2), OK-432, and concanavalin A was examined using the 51Cr release assay. Peripheral blood lymphocytes (PBL) of normal female donors were used as source of effector cells. Incubation of PBL with these biological response modifiers for 24 h generated effector cells with high natural killer activity, and only 20% (1/5) of the FOT examined were susceptible to lysis. By contrast, 83% (5/6) of the FOT were sensitive to lymphokine-activated killer (LAK) cells generated by rIL-2. OK-432 and concanavalin A activation of PBL also generated cytotoxic cells, though the cytotoxic activity against FOT was much less than that obtained by LAK cells. The addition of OK-432 to LAK culture medium containing rIL-2 generated effector cells with higher cytotoxicity against FOT than cultures with IL-2 alone. However, the addition of rIFN-alpha 2 in LAK culture medium resulted in the generation of effector cells with lower cytotoxicity. The addition of rIL-2, rIFN-alpha 2, or OK-432 to LAK cells during the in vitro cytotoxicity assay had no significant effect. When FOT target cells were pretreated with OK-432 they became more sensitive to LAK than nontreated tumor cells. However, pretreatment with rIL-2 or rIFN-alpha 2 did not influence cytolysis. These results suggest that the generation of LAK cells in vitro using rIL-2 plus OK-432 may be a more effective way to prepare these cells for adoptive immunotherapy in the treatment of ovarian cancer.