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.

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.

[Circulating immunocomplexes in patients with various morbid conditions]. / Immunocomplessi circolanti in pazienti con varie condizioni morbose.

The polyethylenglycol (PEG) precipitation technique has been employed for the measurement of immune complexes in the circulation of 100 normal subjects and in 14 patients suffering from a variety of diseases (systemic lupus erythematodes, nephrosic syndrome, cryoglobulinaemia, Buckley's syndrome). Values higher than 0.80 UA on the absorption scale were considered pathological, namely 2 standard deviations above the mean (0.32 UA) in the subjects examined; in the patients, values between a minimum of 0.98 UA and a maximum of 2.38 UA were observed. Longitudinal study of these cases also pointed to the disappearance of immune complexes during therapy. The results suggest that the PEG precipitation technique can play an important part as a screening test in situations in which circulating IC pathology is suspected; it is also a sensitive means of monitoring treatment.

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)

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.

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.

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.

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.

In vitro effect of ketoconazole on human neutrophil chemotaxis and Candida albicans killing.

Ketoconazole, an antifungal agent, was investigated to discover its possible enhancing role on polymorphonuclear chemotactic and Candida killing functions in vitro. Polymorphonuclear leukocytes from 10 healthy subjects and two different strains of Candida albicans were used. At concentrations of 1 microgram/ml Ketoconazole showed no distinct synergistic effect on either of the above-mentioned polymorphonuclear cellular functions, but, on the other hand it operated directly on Candida cells. In addition, this drug did not demonstrate any chemotactic activity on granulocytes. These data could suggest that host cellular defence and Ketoconazole exert parallel rather than synergistic activity.

In vitro and in vivo effect of sisomicin and gentamycin on polymorphonuclear chemotaxis and phagocytosis.

Ten patients with acute bacterial infections were studied to demonstrate the influence of two aminoglycosides, sisomicin and gentamycin on polymorphonuclear (PMN) chemotactic and phagocytic functions. Five of these subjects were treated with gentamycin and five with sisomicin both at a dosage of 2.5 mg kg-1 day-1. The assays were performed before and after 5 days of therapy. Before beginning treatment, the in vitro action of the aminoglycoside antibiotics (20 micrograms ml-1) was also tested. Granulocytes from 8 healthy people were used as controls. After therapy, no significant difference in basic values was found in either the gentamycin treated or in the sisomicin treated group. Similar results were obtained in incubation tests on both patients and healthy subjects. In conclusion, we conclude that the use of these mentioned aminoglycoside antibiotics, at the therapeutical doses employed, does not interfere with the PMN functions.

Study of chemotaxis by a modified method of migration under agarose gel.

In this paper the standardization of a method for studying chemotaxis of polymorphonuclear cells under agarose gel in vitro on 20 adult normal subjects is reported. An original method of reading is emphasized in which the maximum migration distance and the number of migrated cells are held in consideration.

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.

Serum and salivary IgA levels in normal subjects: comparison between tonsillectomized and non-tonsillectomized subjects.

Serum IgA and secretory IgA (SIgA) were determined in 1,000 apparently healthy subjects; 274 were tonsillectomized. Four groups were identified in this study: (1) 0.3% of subjects had no serum IgA and SIgA; (2) 1.6% of subjects showed partial serum and SIgA deficiency; (3) 27.4% of individuals were tonsillectomized and had partial serum IgA deficiency but normal SIgA, and (4) 71.4% were normal, both with respect to serum IgA and SIgA. There results stress the relatively high incidence of IgA deficiency in the normal population and confirm the role of the tonsils in maintaining serum IgA levels.

In vitro studies on cellular and humoral chemotaxis in Crohn's disease using the under agarose gel technique.

The locomotor function of polymorphonuclear cells (cellular chemotaxis) and serum chemotactic activity (humoral chemotaxis) were studied in 51 patients with Crohn's disease using a method of migration under agarose gel. To study cellular chemotaxis patient's polymorphonuclear cells were challenged against normal Zymosan activated serum and humoral chemotaxis was evaluated testing the patient's Zymosan activated serum against normal polymorphonuclear cells. Cellular chemotaxis in the Crohn's disease group was normal (although 30% of the 51 patients had migration values out of the normal range), while humoral chemotaxis was significantly lower in Crohn's disease patients than in the control group. However, the value of humoral chemotaxis in the group of Crohn's disease patients treated with steroids was lower than that of patients not treated, thus accounting for the low mean value observed inthe Crohn's disease-group as a whole. The present results suggest that a defective chemotactic response may occur in some Crohn's disease patients, particularly during steroid treatment. These findings might be related either to a defective generation of complement derived chemotactic factors or to the presence of circulating inhibitors.

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.

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.

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.

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.