The effects of bestatin on humoral response to sheep erythrocytes in non-treated and cyclophosphamide-immunocompromised mice.

The effects of bestatin on humoral immune response to sheep erythrocytes (SRBC) and restoration of the response impaired by a single cyclophosphamide dose (350 mg/kg) were tested on mice. Bestatin (at doses of 10, 1, and 0.1 mg/kg) was administered intraperitoneally (i.p.) 5 or 10 times. The pharmacological immunosuppression was induced by a single i.p. injection of cyclophosphamide (350 mg/kg) administered 24 h before the first bestatin dose. The mice were immunized i.p. with SRBC 24 h after the last dose of bestatin. It was found that multiple administration of bestatin at all three doses potentiated the humoral response to SRBC in non-treated mice, resulting in an increased number of plaque-forming cells (PFC) and 2-mercaptoethanol (2-ME)-resistant anti-SRBC antibodies. However, five times administration of bestatin at the doses under investigation caused further decreases in total anti-SRBC hemagglutinins. A single injection of cyclophosphamide (350 mg/kg) suppressed humoral response of mice to the antigen. Administration of bestatin after pharmacological immunosuppression partially prevented the suppressive action of cyclophosphamide in the in vivo model of the humoral immune response to SRBC. The protective action of bestatin was both dose- and schedule-dependent. Ten times' exposure to a bestatin dose of 0.1 mg/kg after a high cyclophosphamide dose partially reduced the suppressive effect of this drug on humoral response of SRBC-immunized mice, increasing PFC on days 4 and 7 after immunization, which coincided with restored ability of the lymphocytes to produce the 2-ME-resistant hemagglutinins on day 7 and the total anti-SRBC hemagglutinins on day 14 after priming.

Follow-up standard agglutination and 2-mercaptoethanol tests in 175 clinically cured cases of human brucellosis.

BACKGROUND: The standard agglutination (SAT) and 2-mercaptoethanol (2-ME) tests are usually used in the follow-up of treated cases of human brucellosis. The purpose of this study was to monitor the levels of these tests, two years after clinical cure in cases of brucellosis. METHODS: From April 2003 to September 2008, 175 clinically cured cases of brucellosis (103 males, 72 females) were evaluated. Diagnosis of brucellosis was established with a SAT of > or =1:320 and a 2-ME of > or =1:80, with clinical symptoms and signs compatible with brucellosis. SAT and 2-ME were retested at the end of therapy and at 3-monthly intervals for two years. Serologic cure was considered in the event of a SAT titer decrease to < or =1:160 or a 2-ME decrease to<1:80. RESULTS: The mean age of study patients was 31 +/- 13.5 years. At 6, 12, 18, and 24 months after treatment, SAT titers > or =1:320 were seen in 41 (23.4%), 22 (12.6%), 7 (4%), and 6 (3.4%) cases, respectively, whereas 2-ME titers > or =1:80 were seen in 51 (29.1%), 24 (13.7%), 12 (6.9%), and 8 (4.6%) cases, respectively. The probability of serologic cure for patients with SAT titers < or =1:640 was higher than for those >1:640 (95% confidence interval (CI) 2.5-3.47, p=0.023). The probability of serologic cure for patients with 2-ME titers < or =1:320 was higher than for those >1:320 (95% CI 2.48-3.5, p=0.04). CONCLUSIONS: SAT and 2-ME may be found in significant titers in less than 5% of clinically treated cases after two years. Serologic cure for both tests with lower titers were higher than with higher titers.

A proteomic-based approach for the identification of Candida albicans protein components present in a subunit vaccine that protects against disseminated candidiasis.

Candidiasis has become a prevalent infection in different types of immunocompromised patients. The cell wall of Candida albicans plays important functions during the host-fungus interactions. Cell wall (surface) proteins of C. albicans are major elicitors of host immune responses during candidiasis, and represent candidates for vaccine development. Groups of mice were vaccinated subcutaneously with a beta-mercaptoethanol (beta-ME) extract from C. albicans containing cell wall proteins. Vaccinated mice were then infected with a lethal dose of C. albicans. Increased survival and decreased fungal burden were observed in vaccinated mice as compared to a control group, and 75% of vaccinated mice with the beta-ME extract survived this otherwise lethal infection. We used a proteomic approach (2-DE followed by immunoblotting) to demonstrate a complex polypeptidic pattern associated with the beta-ME extract used in the vaccine formulation and to detect immunogenic components recognized by antibodies in immune sera from vaccinated animals. Reactive protein spots were identified by MALDI-TOF-MS and searches in genomic databases. As a conclusion, vaccination strategies using C. albicans cell wall proteins induce protective responses. These antigens can be identified by proteomic approaches and may be used as components of subcellular vaccines against candidiasis.

Antibody transmitting ability of hens from lines of chickens differing in response to SRBC antigen.

1. Hens from White Leghorn lines selected for high (HA) or low (LA) antibody response to sheep red blood cells (SRBC) were inoculated with 0.1 ml of either 0.25% or 2.50% SRBC suspension. Eggs laid over the next 15 d were grouped into 5, 3-d collection periods and incubated. Maternal antibody titres were determined in chicks at hatch and 7 d after hatch. 2. In a subsequent experiment, hens of the 2 lines were inoculated with 0.1 ml of 2.50% suspension of SRBC, and eggs laid on days 10 to 13 after inoculation were incubated. Maternal antibody titres were determined in 15 and 18-d embryos as well as in chicks at 0, 5, 10, 15, 20, and 25 d after hatch. 3. Dosage of SRBC had no effect on the antibody titres in line HA; however, the higher dosage elicited greater antibody titres than the lower dosage in line LA. 4. Maternal antibodies were detected earlier in chicks of line HA (eggs laid on days 7 to 9) than those of line LA (eggs laid on days 10 to 12) regardless of dosage administered to the hens. 5. In both lines, antibodies specific to SRBC were observed on day 15 of incubation, with the frequency of responders greatest at hatch. The high frequency of HA responders was maintained for 15 d after hatch, whereas there was an immediate decline with age in LA responders. 6. It was concluded that lines HA and LA have diverged in the pattern of maternal antibody levels as a result of the divergent selection for antibody response to SRBC.

Expression of increased immunogenicity by thiol-releasing tumor variants.

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cystein or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumorigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.

Protective effect of Porphyromonas gingivalis outer membrane vesicles against bactericidal activity of human serum.

The present study was undertaken to evaluate the effect of Porphyromonas gingivalis outer membrane vesicles on the bactericidal activity of human serum. Human serum was pretreated with extracellular vesicles and then incubated with a cell suspension of Capnocytophaga ochracea. After 2 h at 37 degrees C, the percent viability of C. ochracea was determined by cultivation on blood agar plates. At a final concentration of 0.3 mg/ml, outer membrane vesicles completely inhibited the serum bactericidal activity against C. ochracea. Boiling the vesicles prevented this inhibition. However, partial inhibition of the serum lethal action was obtained when a higher concentration (1.5 mg/ml) of boiled vesicles was used, which indicates the involvement of both heat-labile and heat-stable components associated with vesicles. Combining vesicles at a suboptimal concentration (0.1 mg/ml) with a reducing agent brought back inhibition of the bactericidal activity, whereas combining vesicles at an optimal concentration (0.3 mg/ml) with a thiol-blocking reagent caused a restoration of the bactericidal activity. When a purified preparation of P. gingivalis lipopolysaccharides was used instead of vesicles, inhibition of the bactericidal activity was also observed. These results indicate that the lipopolysaccharides and the proteolytic enzyme(s) associated with P. gingivalis outer membrane vesicles are likely to represent the heat-stable and the heat-labile components, respectively. It is possible that outer membrane vesicles released by P. gingivalis protect other bacterial species from complement action, thus favoring the pathogenic process of periodontal disease.

IgG and IgM responses in high and low antibody-selected lines of chickens.

Kinetics of IgG and IgM as measured by 2-mercaptoethanol-resistant (MER) and susceptible (MES) antibodies to sheep erythrocytes, respectively, were determined as correlated responses in lines of chickens selected for high (HA) and low (LA) antibody response to sheep erythrocytes. Primary response patterns for total, MER, and MES antibody differed according to the genetic line. Total antibody increased rapidly, peaked, and persisted at moderate levels in line HA, whereas both peak and persistency were lower in line LA. Levels of MES peaked and then declined in line HA chickens but persisted at low levels throughout in line LA. Titers of MER antibody were considerably greater in line HA than in line LA both on an absolute basis and as a proportion of total antibody titer. Secondary total titers were greater at five days after injection than at three days and greater for line HA than for line LA chicks. The pattern observed for MER and MES in line LA was similar to that for total antibody, as was MER in line HA. For MES the pattern was reversed in line HA.

Suppression of the in vitro humoral immune response by chrysotile asbestos.

The direct addition of UICC chrysotile B asbestos to spleen cell cultures produced a dose-dependent suppression of the antibody-forming cell (AFC) response to the T-dependent antigen, sheep erythrocytes. Concentrations of 0.1, 1, 10, and 100 micrograms/ml, which had no effect on either cell number or viability, suppressed the in vitro response by 12, 43, 71, and 95%, respectively. By separating spleen cells into nonadherent and plastic-adherent populations, the effects of asbestos on each cell type were studied utilizing reconstituted cultures. Treatment of adherent cells for 1 hr with asbestos and reconstitution with untreated nonadherent cells resulted in a dose-dependent suppression of the AFC response similar to that observed when asbestos was added to whole spleen cultures. Reconstitution studies were also conducted with spleen cell populations from animals administered asbestos in vivo. In cultures reconstituted with adherent cells from asbestos-treated mice and nonadherent cells from saline-treated animals, the AFC response was significantly suppressed. The T-dependent AFC response with reconstitution of adherent cells from saline-treated mice and nonadherent cells from asbestos-exposed animals was no different from reconstituted saline controls. Both adherent alveolar and pleural cells were capable of reconstituting the T-dependent AFC response with nonadherent spleen cells and demonstrated reduced immunological capability when exposed to asbestos fibers. The results of this investigation have identified the adherent spleen cell (macrophage) as the target cell type responsible for asbestos-induced immunosuppression of the in vitro T-dependent antibody-forming cell response.

Demonstration of anti-keratin antibodies by ELISA using keratin or thiol-containing compounds in urea as antigens.

During the development of an enzyme-linked immunosorbent assay (ELISA) for the demonstration of anti-keratin antibodies (AKA) it was observed that rabbit anti-keratin antisera also reacted with polystyrene surfaces treated with beta-mercaptoethanol in 8 M urea (ME-urea). Sera from non-immunized rabbits or rabbits immunized with antigens unrelated to keratin failed to react. The specificity of the reaction was further assessed by absorption experiments and by testing affinity-purified AKA. IgM activity against ME-urea could be demonstrated in 62.5% of sera from patients with infectious mononucleosis and in 37.5% of sera from patients with rheumatoid arthritis, and there was a good correlation to the presence of AKA. Coating of the solid phase with compounds containing free SH groups in 4-8 M urea generated the antigen of this ELISA. The exact molecular configuration of this presumptive synthetic antigen is obscure, but the ME-urea ELISA seems to provide a simple way to detect anti-keratin antibodies of a certain specificity.

Human rheumatoid factor-producing cell induction by 2-mercaptoethanol: immunomodulation by a simple thiol compound.

Two-mercaptoethanol (2-ME), a simple 2 carbon thiol compound with a wide variety of in vitro and in vivo immunomodulating effects, was evaluated for its usefulness as a molecular probe of human antibody producing cell activation by adding 2-ME to cultures of peripheral blood mononuclear leukocytes from normal human volunteers. Culturing normal human leukocytes with 2-ME induced a significant number of cells producing rheumatoid factor as measured by a hemolytic plaque forming cell (PFC) assay. Dose response studies revealed 5 X 10(-5)M to be the optimum concentration of 2-ME for the induction of rheumatoid factor plaque forming cells (RF-PFC). This concentration of 2-ME also maximally induced PFC making antibodies to sheep red cells coupled to the trinitrophenyl (TNP) hapten demonstrating that 2-ME is a polyclonal inducer of human PFC. The addition of 5 X 10(-5) M 2-ME to cultures containing maximal concentrations of the polyclonal stimulators, pokeweed mitogen and human heat-aggregrated IgG, increased the number of RF-PFC detected in these cultures by approximately 50%, although both lower and higher concentrations of 2-ME suppressed the RF-PFC response. We conclude that 2-ME induces normal human leukocytes to produce rheumatoid factor as part of a polyclonal activation of antibody producing cells. 2-ME also has immunomodulating effects when added to other polyclonal stimulators of antibody producing cells.

[Importance of cross reactions in evaluating the serological diagnosis of brucellosis in human beings. 1. The results of the examination of chronic brucellosis patients using the agglutination and Coombs' tests with Brucella and Yersinia (serotype 0-9) antigens]. / Znachenie perekrestnykh reaktsii v otsenke serologicheskoi diagnostiki brutselleza u liudei. Soobshchenie 1. Rezul'taty obsledovaniia bol'nykh khronicheskim brutsellezom v reaktsii aggliutinatsii i reaktsii Kumbsa s brutselleznym i iersinioznym (serotop 0-9) antigenami.

The study of blood serum samples obtained from 187 patients with chronic brucellosis revealed that in those cases when blood serum contained specific antibrucella antibodies cross reactions with Y. enterocolitica 0-9 antigen occurred in the agglutination test and Coombs' test. The results thus obtained showed that Y. enterocolitica 0-9 antigen could be used for detecting mainly 2-mercaptoethanol-sensitive complete and incomplete antibodies (IgM). This study indicates that serological cross reactions play an important role in the evaluation of the specific methods for diagnosing brucellosis.

[Monthly dynamics of the antitoxic immunity against diphtheria and tetanus and of normal antibodies to fraction I of the plague microbe in adults]. / Pomesiachnaia dinamika antitoksicheskogo immuniteta protiv difterii i stolbniaka i normal'nykh antitel k fraktsii I chumnogo mikroba u vzrosylkh.

Monthly characteristics of antitoxic immunity to diphtheria and tetanus toxoids in 3,334 adults in Ryazan were determined. As a result, the following differences were established: the characteristics of antidiphtheria immunity were somewhat higher in the autumn-winter period and dropped to the minimal level in the winter-spring period; the maximal characteristics of antitetanus immunity clearly coincided with the hot season, its minimal characteristics with the cold season. For the first time the frequency and titers of normal antibodies to fraction I of Pasteurella pestis in humans were determined. Their monthly dynamics proved to be parallel to the curve indicating the characteristics of antitetanus immunity, but on a lower level: 1-10%. The presence of normal antibodies was accompanied by higher titers to tetanus toxoid, but not to diphtheria toxoid. According to the results of earlier studies, normal antibodies were shown to be the sign of homologous reactivity in inbred animals. Our data indicate that these antibodies can serve as the indirect sign of heterologous reactivity in humans.

Immunization of mouse spleen cell cultures in the absence of serum and its proteins using SiO2 and 2-mercaptoethanol.

High recoveries of plaque-forming cells were obtained in a serum-free medium using 2-mercaptoethanol and SiO2. Under such conditions an increase in the number of plaque-forming cells occurred between the fourth and fifth day of incubation, as compared with cultures with foetal calf serum. The addition of glutathione to the medium had a stimulating effect, it was however not absolutely necessary for a positive response. The recovery of plaque-forming cells was strongly influenced by an interdependence of cell density and amount of SiO2 gel particles.

An absolute requirement for 2-mercaptoethanol in the in vitro primary immune response in the absence of serum.

Using a serum-free medium, high recoveries of plaque-forming cells (PFCs) were obtained up to 5 days of incubation in the in vitro primary immune response. Under these conditions, the presence in the medium of 2-mercaptoethanol was an absolute requirement. In order to obtain maximal recoveries of PFCs, the antigen and 2-mercaptoethanol had to be present from the start of the culture, while the addition of fetuin could be delayed for 24 h without any loss of PFCs. The 2-mercaptoethanol could be replaced by (dialysed) human serum.

Function of 2-mercaptoethanol as a macrophage substitute in the primary immune response in vitro.

The mechanism by which 2-ME acts as a macrophage-substitute for the induction of a primary PFC response to SRC in vitro was studied in macrophage-depleted mouse spleen cell cultures. 2-ME could replace macrophages only in FCS-supplemented cultures. Evidence is presented that the function of 2-ME is independent of residual macrophages. Neither normal nor macrophage-depleted spleen cell cultures from congenitally athymic nude mice supplemented with 2-ME, with or without FCS, could give rise to a primary in vitro anti-SRC immune response. 2-ME, at an optimal concentration of 10(-5) M, induced DNA synthesis in normal and macrophage-depleted spleen cells in both FCS-containing and serum-free cultures. The peak response occurred on day 3. The stimulation was accompanied by a polyclonal B cell activation to antibody secretion which was much more pronounced in FCS-containing than in serum-free cultures. Spleen cells from nude mice showed a weaker DNA stimulation than did cells from normal mice in FCS-containing cultures, and nearly no response under serum-free conditions. T cells obtained by a nylon column adherence method from normal mouse spleen cells showed good DNA synthetic responses in FCS-containing, but no response in serum-free cultures. These results show that 2-ME has weak mitogenic activity for B cells, and in combination with FCS, strong mitogenic activity for T cells. Since the macrophage provides stimulation to the T cell in the primary anti-SRC PFC response in vitro, these results suggest that the direct mitogenic activity of 2-ME with FCS on T cells provides the functional substitution for macrophages.

[Intrauterine sensitization to benzylpenicillin in an experiment on rabbits]. / Vnutriutrobnaia sensibilizatsiia k benzilpenitsillinu v éksperimente na krolikakh

Antibodies of benzylpenicillin in the posterity of various ages of female rabbits were found with reaction of passive hemagglutination (RPHA) and degranulation of the basophiles after their immunization with the antibiotic. This provided an assumption of intrauterine sensitization. Treatment of the sera of the sensitized animals with 2-mercaptoethanol partially decreased their activity in the RPHA which indicated to the presence of the antibodies of classes IgG and IgM.