The effects of hypobaric hypoxia on specific B cell responses following immunization in mice and humans.

In order to get some insight on the physiology of the immune system during prolonged exposure to hypobaric hypoxia we evaluated the effects of high altitude on the in vivo immune response to a T-independent antigen. A group of 18 men who participated in a scientific project EV-K2-CNR to Mount Poumori, Nepal for 20 d at 4,930 m (16,174 ft) were immunized with a single subcutaneous dose of antimeningococcal vaccine Menpovax A + C (Sclavo) containing 50 micrograms of polysaccharide A (PsA) and 50 micrograms of polysaccharide C (PsC) of N. meningitidis. A group of 18 men of comparable age were vaccinated at sea level. Antibody titers against both polysaccharides were determined by enzyme-linked immunosorbent assay (ELISA) before and 18 d after vaccination. All subjects examined developed a good antibody response and no statistically significant differences were observed between the two groups. Spectrotypic analysis of antibody response to PsC was also performed by isoelectric focusing. No qualitative differences in the antibody response to PsC were found in the hypoxia-exposed group with respect to the control group. A group of 10 BALB/c inbred mice were kept in a hypobaric chamber at 5,500 m (18,000 ft) for 30 d. After 10 d, the mice were vaccinated with 1 micrograms of Menpovax A + C. Anti-PsA and anti-PsC antibodies were quantified by ELISA in sera collected at day 0 and 30. A control group of 10 mice of the same strain underwent the same study protocol but at sea level. Both groups developed a good antibody response to both polysaccharides and no significant differences were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of specificity in the mechanisms involved in the enhancement of the concanavalin A driven human T lymphocyte stimulation by beta-endorphin: studies on activation marker expression, cell cycle and interleukin release.

We report on the effects of a physiological concentration of Beta-Endorphin (BE) (10(-12)M) on Concanavalin A (ConA) stimulated human peripheral blood T-lymphocytes and monocytes. We evaluated the effect of timing of BE addition to the culture medium on thymidine uptake, the kinetics of expression of activation markers (CD69, CD25 and CD71) on CD4+ and CD8+ lymphocytes, and of class II MHC antigens on CD14+ cells (monocytes), the kinetics of interleukin-1 (IL-1), interleukin-2 (IL-2) and interferon gamma (IFN-gamma) release, and the cell cycle. Data show that BE is able to influence T lymphocyte only when added together with ConA at the beginning of culture, suggesting its major activity is on the early phases of the T cell response. BE did not increase the amount of class II MHC antigens on monocytes and did not preferentially stimulate CD69, CD25 and CD71 antigen expression on either CD4+ or CD8+ lymphocytes. After 24 hours, the relative proportions of CD4+ and CD8+ lymphocyte in S and G2-M phases were not affected by BE, although the opioid did augment the number of cells in the proliferative compartments of the cell cycle, S and G2-M, indicating an actual increase in the number of cells committed to proliferation. BE did not consistently influence the amount of IL-1, IL-2 and IFN-gamma found in the supernatant of ConA stimulated cultures. The mechanism of the enhancing effect on the proliferative response of normal human lymphocytes to ConA by BE, does not seem to be selective for or unique to specific lymphocyte subsets.

Clonotypic analysis of human antibodies specific for Neisseria meningitidis polysaccharides A and C in adults.

Serum antibodies to the capsular polysaccharides A and C (PSA and PSC) of N. meningitidis in healthy adults before and after vaccination with the sole polysaccharides were analysed by isoelectric focusing (IEF). Before vaccination, 49% and 28% had naturally acquired antibodies against PSA and PSC, respectively, whereas 18 days after vaccine administration 84% and 91%, respectively, showed a detectable spectrotypic pattern. Oligoclonality appeared to be the main feature of naturally acquired and vaccine-induced antibodies for both polysaccharides. In all subjects the anti-PSA response, showing dominant bands at the same pH position, was more homogeneous than anti-PSC one. Most subjects with naturally acquired antibodies (25 out of 38 for PSA and 20 out of 22 for PSC) showed a spectrotypic pattern after vaccination, similar to that observed before vaccination (any differences were just related to band intensity), suggesting that PSA and PSC are able to recruit the same B cell clones previously primed with a T-dependent form of the antigen, i.e. the whole bacterium. However, in one-third of subjects with naturally acquired anti-PSA antibodies, the appearance of new alkaline bands after vaccination was observed. Furthermore, in subjects with absence of detectable natural antibodies, the vaccine-induced antibody response started in correspondence of alkaline pH areas, subsequently extending to neutral and acidic areas. Therefore, it may be hypothesized that alkaline antibody-secreting B cell clones are the first to be recruited. The final spectrotype in these subjects was similar to that observed in subjects with naturally acquired antibodies. This observation, together with the above reported data, allow us to conclude that natural (T-dependent pathway) and vaccine (T-independent pathway) immunization induce the expression of the same antibody repertoire, for both meningococcal PSA and PSC.

Serum antibodies to capsular polysaccharide vaccine of group A and C Neisseria meningitidis in military recruits in Italy.

In this study we evaluated, by a standard bactericidal assay, the antibody response to the meningococcal group A and C polysaccharide vaccine used for the immunization programme of the Italian military recruits, compulsory by law since 1987. The percentage of responders, those who developed a four-fold increase in the bactericidal titre to polysaccharide A and C, was 96% and 98%, respectively. No significant side effects were observed after vaccination.

Antibacterial polysaccharide antibody deficiency after allogeneic bone marrow transplantation.

Allogeneic bone marrow-engrafted adults immunized with meningococcal types A and C and pneumococcal type 14 polysaccharide antigens showed only low antibody titers of the IgM class, no antibody titers of the IgG or IgA classes, and no bactericidal activity in vitro. The analytical isoelectrofocusing showed the appearance of a restricted pattern of clonotypes in a minority of subjects. These observations are consistent with the hypothesis that B cells in bone marrow transplant patients express some characteristics of neonatal B cells and suggest that polysaccharide-protein conjugates, rather than isolated polysaccharide, might be utilized in the setting of bone marrow transplantation.

Flow cytometric evaluation of nitro blue tetrazolium (NBT) reduction in human polymorphonuclear leukocytes.

Oxidative metabolic burst of activated human polymorphonuclear leukocytes (PMN) is most commonly investigated in clinical practice by evaluating nitroblue tetrazolium (NBT) reduction at the single cell level. Reduced NBT precipitates where the redox reaction has taken place and can be visualized as PMN-associated dark blue granules of formazan in light microscopy. Although widely used and not technically demanding, this method remains subjective and labor intensive, especially when large numbers of samples need to be investigated. We developed a new flow cytometry technique in which PMN membrane was rendered fluorescent by a short incubation with fluorescein-conjugated Concanavalin A. PMN were then incubated with NBT and increasing doses of a suitable stimulus, such as phorbol myristate acetate (PMA). Formazan has a distinct peak of absorption at 520 nm that represents the peak of emission of fluorescein. As a consequence, formazan quenches the PMN-associated fluorescence. Data show that a dose-dependent reduction of fluorescence can be obtained using graded amounts of PMA in normal PMN cultures. PMN-associated fluorescence remains unchanged in control patients with chronic granulomatous (CGD) disease, a disorder characterized by a selective impairment of PMN oxidative metabolism. Electronic cell size increases upon PMA incubation in normal PMN, irrespective of the presence of NBT. Conversely, forward light scatter intensity decreases in the presence, but not in the absence, of NBT indicating that the phenomenon is due to the capacity of formazan to absorb/scatter the incident light. The present method for easily detecting NBT reducing activity at single cell level by flow cytometry makes use of commonly available, inexpensive reagents and standard instrumentation. It could become a useful test for clinical purposes.

Prevalence of HBV markers in the Italian Armed Forces and HBV vaccination.

Hepatitis B (HBV) markers were studied in 710 subjects who had been on active duty for over 6 months in the Italian Armed Forces. The prevalence of HBsAg carriers was found to be 4.4%, while 31.6% of the subjects were positive for various HBV antibodies. A total of 137 subjects were vaccinated with an anti-HBV vaccine (HB-VAX, MSD). The percentages of non-responders and low responders were 13.86% and 13.14%, respectively. Boosters administered 3 months post-vaccination schedule, with or without immunostimulatory treatment, resulted in seroconversions and/or substantial increases in HBsAb levels in 50% of these subjects.

A new test for specific IgE to inhalant allergens (Phadiatop) in the screening of immediate respiratory hypersensitivity states.

Three hundred recruits were classified as either allergic or nonallergic to inhalant allergens on the basis of history, physical examination, skin testing, and RAST. Forty-nine (16.3%) were classified as allergic as they were affected by oculorhinitis and/or asthma. This diagnosis was compared with results obtained with PRIST and with a new commercially available multi-RAST test for inhalant allergy, Phadiatop (Pharmacia). Sixty subjects (20%), including all 49 allergic subjects, scored positive in the Phadiatop assay (100% sensitivity) while PRIST (cut-off 220 IU/mL) identified only 20/49 allergics (40.8% sensitivity). The correlation between RAST and Phadiatop was excellent. Phadiatop is much more sensitive than PRIST; furthermore, Phadiatop is easier, less expensive, and as reliable as RAST for use in mass screening programs.

A comparative in vitro study of low pH and enzyme treated immunoglobulin preparation for intravenous use.

Three not yet commercially available immunoglobulin preparations, intended for intravenous administration, were tested for their purity, physical integrity and in vitro functional activity. Preparations had been factory treated either at pH 4 and with insolubilized pepsin or at pH 4.25 only. Nephelometry revealed a high degree of isotypic purity (IgG greater than 99%). Gel filtration and SDS-PAGE analysis showed only trace amounts of aggregates and/or contaminants, particularly in one of the pepsin treated preparations. Protein A binding inhibition test and phagocytosis of Candida albicans by normal human polymorphonuclear leukocytes, after opsonization of the yeast with different concentrations of immunoglobulin preparations, showed the functional integrity of both the Fc and F(ab)2 regions in the three IgG preparations. Compared to previously reported methods of preparation which produce structural alterations of the Ig molecule, present data indicate the suitability of preparative methods aimed at preserving the integrity of the Ig molecule. Preparation and storage at pH 4.25 without any other treatment appear to be sufficient to obtain pure, aggregate free and in vitro functionally active Ig preparations.

Intravenous immunoglobulin preparations: a comparative in vitro study of Fc mediated functions.

An ideal immunoglobulin (Ig) preparation intended for clinical use should be safe and efficacious. Efficacy depends on suitable levels of protective antibodies against pathogens and the functional integrity of the Ig molecule. In the present paper, the functional integrity of the Ig molecule was investigated in eight intravenous Ig preparations commercially available in our country and compared to an intramuscular preparation. The experimental approach included protein A and rheumatoid factor binding, complement activation and opsonic activity. Ultracentrifugation profiles were obtained for all Ig preparations in order to ascertain the presence of components other than the expected 7S monomeric IgG. Immune complexes were investigated with C1q solid phase and conglutinin assays. Results show that chemical treatments such as sulfonation or reduction-alkylation, and enzymatic treatment such as plasmin digestion, variably but consistently impair Fc-mediated functions. The present data emphasize the use of in vitro tests for assessing the suitability of Ig preparations for intravenous administration.

Cellular and humoral modifications during response to HBsAg vaccine in healthy subjects: comparison between French and American vaccines.

Two groups of 18 healthy adults, chosen from a risk population, were vaccinated against hepatitis B virus with French (Hevac B, Pasteur) or American (HB-VAX, MSD) vaccine; immunological and biochemical parameters (HBsAb titers, Ig, IgM-rheumatoid factor (IgM-RF) levels, autologous mixed lymphocyte reaction (AMLR), T4/T8 ratio, lymphocyte migration inhibition test (LMIT) with specific antigen and transaminase levels) were compared at preselected time intervals. The vaccines were found to be clinically safe, as documented by the absence of demonstrable side effects and changes in serum transaminase levels. The positivity of the LMIT test demonstrated an early immunization on day 45 in all subjects tested, along with a marked reduction in AMLR, independent of the vaccine used. A slightly higher, though not statistically significant, percentage of seroconversion was observed for the Pasteur vaccine. This same preparation induced a significant increase in the main Ig classes and in Ig;-RF levels on day 45. This data indicates that aspecific modification of the immune system occur to a greater extent with Hevac B, presumably for its lower degree of purification. Thus, despite the slightly lower efficacy, the HB-VAX might be the preparation of choice in subjects with immunological disturbances.

Comparative analysis of immunological responses to oral (Ty21a) and parenteral (TAB) typhoid vaccines.

The clinical and immunological responses to typhoid vaccination with parenteral (TAB) and oral (Ty21a) vaccines in two groups of 30 adult male subjects were studied. Parameters monitored included specific anti-Salmonella typhi cell-mediated immunity and total and specific antilipopolysaccharide fecal immunoglobulin A (IgA) titers in Ty21a-vaccinated subjects. Peripheral blood lymphocytes antibacterial activity was significantly increased only in Ty21a-vaccinated subjects. Serum arming activity and results of human F(ab')2 anti-IgG and -IgA inhibition tests suggest antibody-dependent cellular cytotoxicity mediated by IgA in those vaccinated with Ty21a. Interestingly enough, the cells of TAB-vaccinated subjects were able to mediate IgG-dependent cellular cytotoxicity, as was observable from the results of blocking experiments. Moreover, total and specific antilipopolysaccharide fecal IgA levels were observed to be significantly increased with Ty21a, up to 8 months post-vaccination schedule. An early-onset, transitory increase in serum IgM rheumatoid factor was also found, exclusively in subjects treated with TAB, and was no longer detectable on day 240. Ty21a was well tolerated and free of side effects, whereas 65% of subjects administered TAB reported fever, headache, malaise, and local tenderness at the injection site. Our data show that the two typhoid vaccines induce different cell-mediated specific immune responses. The role of these responses in protection against Salmonella infection, however, requires further investigation.

Complement activation is variably affected by fibronectin preparations obtained through different procedures.

Human plasma fibronectin (FN) containing preparations (FNCP) were prepared by means of gelatin-affinity chromatography of normal plasma cryoprecipitate (FNCP-a), and by affinity chromatography of cryoprecipitate on a rabbit anti-human FN antiserum Sepharose column (FNCP-b) or of normal citrated plasma (FNCP-c). These preparations were then tested for their ability to influence certain complement (C')-dependent polymorphonuclear phagocyte activities. FNCP-a consistently inhibited humoral chemotaxis, whereas FNCP-b and -c did not. Measurement of C' activation by the C3 conversion assay revealed that only FNCP-a displayed inhibitory activity on classical and alternative pathways. Ouchterlony and immunoelectrophoretic analyses indicated substantial identity of all FNCPs. FNCP-a contained a consistent amount of lower MW contaminants (between 40 and 60 KD) which were shown not to be immunologically related to FN by Western blot analysis. Immunoblotting also revealed a single band in FNCP-a (MW approx. 210 KD) instead of the anticipated two bands which were evident in the other FNCPs. Thus, a slight modification of the FN molecule and/or the presence of low MW contaminants in the gelatin-purified preparation seem able to induce inhibitory effects on C'-dependent polymorphonuclear phagocytic activities.

Rapidly progressive periodontitis. Neutrophil chemotaxis inhibitory factors associated with the presence of Bacteroides gingivalis in crevicular fluid.

In the last few years several bacteriological and immunological studies have investigated the role of bacteria and immune defects in order to establish the etiopathogenesis of periodontal disease. With regard to the immune system, a defect in polymorphonuclear neutrophil (PMN) chemotaxis has been frequently reported in patients with rapidly progressive or juvenile periodontitis. The purpose of this study was to investigate in five patients with rapidly progressive periodontitis and normal chemotaxis of peripheral blood PMNs the presence of chemotaxis inhibitory activity in gingival fluid and to relate such activity to three types of bacteria, often involved in rapidly evolving periodontal lesions, that are able to inhibit in vitro PMN chemotaxis: Bacteroides gingivalis, Capnocytophaga sp., and Actinobacillus actinomycetemcomitans. We found strong inhibitory activity in three of these patients. This activity was consistently associated with the finding of B. gingivalis in gingival pockets. We cannot rule out, however, that other substances not of bacterial origin could be responsible for such inhibitory activity. The strict association with B. gingivalis, known to secrete blocking factors, is highly suggestive, although this data must be considered preliminary.

Raised serum levels of IgM-rheumatoid factor and anti-F(ab')2 autoantibodies in patients with active inflammatory bowel disease.

The serum levels of IgM-Rheumatoid Factor and of anti-F(ab')2 autoantibodies were investigated in patients with inflammatory Bowel Disease (IBD) by sensitive radioimmunoassays. Serum levels of the 2 autoantibodies were significantly increased in active IBD. In patients with Crohn's Disease raised titers of the 2 antibodies appeared to be also related to colonic involvement. There was, in Crohn's Disease, a significant association between concordantly positive results with the 2 assays and the occurrence of systemic complications. Immunocomplexes detected by the C1q-SP method were higher in sera of Crohn's Disease patients with raised IgM-RF than in the others. Data from the present investigation indicate that in active IBD and particularly in Crohn's Disease autoantibodies directed against different parts of the immunoglobulin molecule may be produced. These findings add support to the concept that an in vivo polyclonal B-cell activation may occur in these patients.

Salivary IgA levels in normal children.

Salivary IgA (SlgA) levels were determined in 1,539 normal children, aged between 8 months and 6 years. In four children (0.26%), an absence and in 25 children (1.62%) a relative defect, (less than 1 mg/dl) were found. These values are similar to those encountered in an adult male population. Conversely, the mean SlgA in all age groups we examined was much lower in comparison to the adult mean. This difference is particularly great for SlgA and much smaller for total salivary proteins. There was no difference between males and females, and no relation with personal or familial history of allergic and/or infectious episodes. The conclusions of this study are as follows: Absolute and relative SlgA defects, when found, are present from birth, The complete maturation of SlgA occurs after 6 years of age, The role of SlgA in the protection against allergic or infectious episodes perhaps ought to be revised.

[In vitro effects of lysozyme on some specific and non-specific functions of the immune system]. / Gli effetti in vitro del lisozima su alcune funzioni specifiche e non specifiche del sistema immunitario.

In this paper the in vitro effects of lysozyme on several immunological function was investigated. Henn-egg-white lysozyme (at concentrations ranging between 1 and 10 micrograms/ml) has shown an inhibitory action on mitogen-induced lymphoblastigenesis and autologus mixed lymphocyte reaction. On the other hand, NK activity against K 562 cell line resulted to be increased in the presence of lysozyme. In addition lysozyme has shown to behave as chemokinetic factor increasing PMN random locomotion and inhibiting PMN response only towards complement derived chemotaxins, whereas there was no effect against other chemotactic factors. Then, in addition to its enzymatic activity, a role of lysozyme as modulator of the inflammatory response may be proposed.

Adriamycin treatment for hepatocellular carcinoma. Experience with 109 patients.

One hundred nine patients with hepatocellular carcinoma were treated with intravenous (IV) Adriamycin (doxorubicin). Cumulative survival rate was 34% at 6 months and 13% at 1 year. Survival was positively related to a good performance status and to alpha-fetoprotein less than 50 ng/ml, not influenced by hepatitis B surface antigen (HBsAg) and by presence of clear cells in the tumor. Partial response (alpha-fetoprotein decrease by greater than or equal to 50% of the initial value) was observed in 10 patients and complete response in 1 patient, always within the fourth dose, with a 10% response rate. Twenty of 75 symptomatic patients (27%) achieved improvement in performance and/or pain reduction. Withdrawal of treatment became necessary for side effects in six patients. In conclusion, IV Adriamycin in hepatocellular carcinoma has only limited efficacy. Because of its early activity, treatment can be stopped after three doses if there is no evidence of response.

Humoral inhibition of neutrophil chemotaxis in Crohn's disease.

In patients with Crohn's disease (CD) we investigated the C3 conversion of zymosan-activated serum (ZAS) and looked for the occurrence of chemotactic factor inactivation (CFI). We also studied the cell-directed inhibitory effect (CDI) of the CD patients' plasma and, in the same group, complement activation and complement-mediated deactivation. The mean value of ZAS C3 conversion in CD was no different from that of healthy controls, but in steroid-treated patients it was lower than in untreated CD. CFI occurred in 1 of the 23 CD sera tested, and CDI was observed in 6 out of the 22 patients tested. EDTA C3 conversion was present in 12 patients, and complement-mediated deactivation was associated with high values of EDTA C3 conversion. Our findings indicate that complement dysfunction and inhibitory factors of neutrophil chemotaxis are present in CD. These findings could explain the defective neutrophil migration into skin windows. Whether they are relevant to the pathogenesis of tissue injury or of infectious complications and are specific for CD, however, remains to be established.