Effects of anticoagulants and Ficoll on human serum antibody reactivities and functions against Mycobacterium tuberculosis.

The ability to utilize leftover samples containing anticoagulants or Ficoll would provide substantial opportunities for future antibody and biomarker studies. Some anticoagulants might influence antibody reactivity against pathogens, but comprehensive studies investigating effects in the context of TB are lacking. We enrolled 24 individuals with and without history of M. tuberculosis and/or HIV-infection and investigated TB antibody reactivities, function, and other host protein biomarkers in simultaneously obtained serum and plasma from serum separation, EDTA, heparin, acid citrate dextrose (ACD), or mononuclear cell preparation (CPT™) tubes which contain heparin and Ficoll. Antibody isotype reactivities to two mycobacterial antigens, as well as phagocytosis of M. tuberculosis, correlated strongly and significantly between serum and plasma, irrespective of type of anticoagulant or Ficoll present (r ≥ 0.85, p < 0.0001). However, the presence of ACD resulted in slightly lower values than those obtained with serum in both indirect (antibody reactivities to mycobacterial antigens) and Sandwich ELISAs (soluble CD14 measurements). Our data demonstrate that leftover plasma, regardless of containing anticoagulants or Ficoll, can be used in TB antibody or other host protein biomarker studies but suggest the value of a correction factor when using ACD plasma interchangeably with serum in antibody binding studies.

A novel high-throughput assay to quantify the vaccine-induced inhibition of Bordetella pertussis adhesion to airway epithelia.

BACKGROUND: Pertussis or whooping cough is an acute respiratory illness caused by the Gram-negative pathogen Bordetella pertussis. Despite high vaccination coverage whooping cough is currently re-emerging in many developed countries. Although the causes of pertussis resurgence are matter of debate, emerging evidences suggest that acellular vaccines efficiently protect against the hallmark symptoms of pertussis disease but fail to prevent colonization. This presumably impacts on increased risk of bacterial transmission and consequent spread throughout the population. These evidences suggest that improved vaccines may be required for efficient bacterial clearance in the upper respiratory tract. Consequently, there is a need for novel bioassays to evaluate at pre-clinical or clinical level the impact of different vaccines on B. pertussis colonization. RESULTS: We developed a high-throughput bacterial adhesion inhibition (BAI) assay based on human respiratory cell lines and on live bacteria chemically conjugated to a fluorescent dye. Employing A549 cells as model, we evaluated the impact of antibodies elicited by acellular (aP) and whole cell (wP) vaccines on B. pertussis adhesion in vitro. Moreover, we settled the method also on polarized Calu-3 cells grown at air-liquid interface (ALI), showing that this assay can be extended to more complex cell models mimicking the airway epithelium. CONCLUSIONS: We proved that this method is a sensitive, rapid and reproducible system to evaluate the anti-adhesive properties of vaccine-induced antibodies and can be employed to assess improved pertussis vaccines.

Antibodies to Helicobacter pylori and CagA protein are associated with the response to antibacterial therapy in patients with H. pylori-positive API2-MALT1-negative gastric MALT lymphoma.

The aim of this study was to clarify predictive factors for response to eradication therapy in cases of Helicobacter pylori (H. pylori)-positive API2-MALT1-negative gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Sixty-six patients who were examined for H. pylori infection and the presence of the API2-MALT1 chimeric transcript and who underwent H. pylori eradication therapy as first-line therapy, were enrolled in this study. Immunohistochemical markers (p53, Ki-67, and BCL10), microsatellite instability, loss of heterozygosity, serum levels of antibodies (anti-H. pylori and anti-CagA), and markers for gastritis (gastrin and pepsinogens) were examined, and the results were compared between patients whose tumors regressed completely after eradication therapy (responders) and patients whose tumors did not regress (non-responders). Of the 66 patients with localized gastric MALT lymphoma, 47 (71.2%) showed complete remission after eradication therapy. None of the H. pylori-negative (n = 9) and/or API2-MALT1-positive (n = 7) patients responded to antibacterial treatment. Of 44 patients with H. pylori-positive API2-MALT1-negative gastric MALT lymphoma, 38 (86.4%) showed complete remission after eradication therapy. Titers of antibodies against H. pylori and CagA protein were significantly higher in the responders than in the non-responders (P = 0.0235 and 0.0089, respectively). No significant difference between the groups was observed for the other factors. In conclusion, measurement of titers of serum antibodies to H. pylori and CagA protein may be useful for predicting the response to eradication therapy in patients with H. pylori-positive API2-MALT1-negative gastric MALT lymphoma.

Inhibition of anti-tuberculosis T-lymphocyte function with tumour necrosis factor antagonists.

Reactivation of latent Mycobacterium tuberculosis (Mtb) infection is a major complication of anti-tumour necrosis factor (TNF)-alpha treatment, but its mechanism is not fully understood. We evaluated the effect of the TNF antagonists infliximab (Ifx), adalimumab (Ada) and etanercept (Eta) on anti-mycobacterial immune responses in two conditions: with ex vivo studies from patients treated with TNF antagonists and with the in vitro addition of TNF antagonists to cells stimulated with mycobacterial antigens. In both cases, we analysed the response of CD4+ T lymphocytes to purified protein derivative (PPD) and to culture filtrate protein (CFP)-10, an antigen restricted to Mtb. The tests performed were lymphoproliferation and immediate production of interferon (IFN)-gamma. In the 68 patients with inflammatory diseases (rheumatoid arthritis, spondylarthropathy or Crohn's disease), including 31 patients with a previous or latent tuberculosis (TB), 14 weeks of anti-TNF-alpha treatment had no effect on the proliferation of CD4+ T lymphocytes. In contrast, the number of IFN-gamma-releasing CD4+ T lymphocytes decreased for PPD (p < 0.005) and CFP-10 (p < 0.01) in patients with previous TB and for PPD (p < 0.05) in other patients (all vaccinated with Bacille Calmette-Guérin). Treatments with Ifx and with Eta affected IFN-gamma release to a similar extent. In vitro addition of TNF antagonists to CD4+ T lymphocytes stimulated with mycobacterial antigens inhibited their proliferation and their expression of membrane-bound TNF (mTNF). These effects occurred late in cultures, suggesting a direct effect of TNF antagonists on activated mTNF+ CD4+ T lymphocytes, and Ifx and Ada were more efficient than Eta. Therefore, TNF antagonists have a dual action on anti-mycobacterial CD4+ T lymphocytes. Administered in vivo, they decrease the frequency of the subpopulation of memory CD4+ T lymphocytes rapidly releasing IFN-gamma upon challenge with mycobacterial antigens. Added in vitro, they inhibit the activation of CD4+ T lymphocytes by mycobacterial antigens. Such a dual effect may explain the increased incidence of TB in patients treated with TNF antagonists as well as possible differences between TNF antagonists for the incidence and the clinical presentation of TB reactivation.

Sex and low-level sampling stress modify the impacts of sewage effluent on the rainbow trout (Oncorhynchus mykiss) immune system.

The objective of the present study was to investigate the influence of chronic exposure to municipal sewage treatment effluent at environmentally relevant concentrations on immune parameters in rainbow trout (Oncorhynchus mykiss), including the assessment of potential differences in reactivity between sexually mature male and female fish. Trout were exposed to 1.5 and 15% (v/v) secondary treated municipal sewage effluent for 32 weeks. Fish were injected intra-peritoneally either with inactivated Aeromonas salmonicida to simulate an infection or with PBS as control for this immune challenge 6 weeks prior to sampling. Exposure to effluent resulted in a decrease in A. salmonicida-specific serum antibody level and blood lymphocyte numbers in mature females, but not in male fish. Injection of A. salmonicida resulted in enhanced serum lysozyme activity in mature male trout, which were not exposed to effluent. This stimulating effect of A. salmonicida could not be found in effluent-exposed trout, again potentially revealing a suppressive effect of the effluent. An influence of sampling fish on two consecutive days was observed in many immune parameters, most likely reflecting handling stress. Leucocyte and lymphocyte numbers in peripheral blood were consistently lower in male and female fish on the second sampling day. Phagocytosis in head kidney macrophages from male trout was also influenced by sampling day, whereby a stimulation of this reaction occurred on the second day of sampling. Liver mixed function oxygenase activity was found to be enhanced in mature male trout exposed to 15% effluent. In conclusion, the study showed, that exposure to sewage treatment plant effluent, in surface water relevant concentrations, can lead to potentially adverse effects on selected immune reactions in rainbow trout. However, this study also demonstrated that both handling stress and the sex of mature fish have distinct influences on the immune response detected in male and female fish and are likely to influence measured immune parameters to the extent that subtle effluent induced changes may be difficult to detect.

Destructive arthritis in vaccinated interferon gamma-deficient mice challenged with Borrelia burgdorferi: modulation by tumor necrosis factor alpha.

We found that Borrelia burgdorferi-vaccinated gamma interferon-deficient (IFN-gamma(0)) mice challenged with B. burgdorferi developed prominent chronic destructive osteoarthropathy. When these mice were treated with anti-tumor necrosis factor alpha (TNF-alpha) antibody, the severity of the destructive osteoarthritis was enhanced and affected the mobility of the animals. In addition, extensive swelling of the hind paws occurred. In contrast, treatment of B. burgdorferi-vaccinated, challenged IFN-gamma(0) mice with recombinant TNF-alpha (rTNF-alpha) inhibited the development of arthritis, including swelling of the hind paws. Moreover, treatment of vaccinated, challenged IFN-gamma(0) mice with anti-TNF-alpha inhibited fourfold the production of an antibody that kills B. burgdorferi, while treatment of vaccinated, challenged IFN-gamma(0) mice with rTNF-alpha slightly elevated the level of the borreliacidal antibody. These results suggest that the level of TNF-alpha directly or indirectly regulates the production of borreliacidal antibody and the development of vaccine-induced destructive Lyme osteoarthritis. Studies are in progress to determine the mechanism by which TNF-alpha-dependent cytokines generate the destructive arthritis.

Noninvasive stool antigen assay can effectively screen Helicobacter pylori Infection and assess success of eradication therapy in hemodialysis patients.

Helicobacter pylori (H pylori) stool antigen (HpSA), serological antibody against H pylori (immunoglobulin G [IgG]), and urea breath test (UBT) are noninvasive methods used to detect H pylori infection that can allow a patient to avoid the discomfort and risk of invasive endoscopy. However, because the UBT has proven not highly reliable in patients with end-stage renal disease (ESRD), this study attempts to investigate the diagnostic efficacy of HpSA and IgG for H pylori detection in 80 patients with ESRD and 80 dyspeptic patients without renal function impairment as a control group. All patients in both study groups underwent panendoscopy to obtain gastric biopsy specimens for histological examination and H pylori culture. With H pylori infection defined as a positive result on either histological examination or culture, we evaluated the reliability of HpSA and serum IgG in detecting H pylori infection. Forty of the patients with ESRD (50%) and 48 patients in the control group (60%) were proven to be infected with H pylori. To eradicate H pylori infection, these patients were administered a 1-week course of triple therapy. To evaluate the success of H pylori eradication, 38 patients in the ESRD group and 44 patients in the control group underwent a follow-up endoscopy and provided stool samples for HpSA 6 to 8 weeks later. Success of H pylori eradication was found in 86.8% of the patients with ESRD (33 of 38 patents) and 84.1% of the control patients (37 of 44 patients). Before therapy, HpSA for H pylori detection was 97.5% sensitive and 97.5% specific in patients with ESRD, as effective as that in the control group. After therapy, HpSA was 100% sensitive and more than 96% specific to detect the failure of H pylori eradication therapy in both the ESRD and control groups. Conversely, the use of IgG as a screening method for H pylori infection proved to be less effective because it showed a sensitivity of 87.5% and specificity of 80% in this study. Monitoring the success of triple therapy, IgG had a specificity of only 21.9% in the ESRD group and 24.3% in the control group. In summary, HpSA is a noninvasive and reliable tool to screen H pylori infection before therapy and assess the success of eradication therapy in patients with ESRD.

Effects of ethyl carbamate and its metabolites on the antibody response in splenocyte cultures from female Balb/C mice.

To investigate a possible role by cytochrome P450 (P450) in ethyl carbamate-induced immunosuppression, an attempt to assess the ability of ethyl carbamate, its metabolites produced by P450 (i.e., ethyl N-hydroxycarbamate and vinyl carbamate), and methyl carbamate to suppress the polyclonal antibody response induced by bacterial lipopolysaccharide was made in splenocyte cultures isolated from female Balb/C mice. The results showed that vinyl carbamate and ethyl N-hydroxycarbamate were more immunosuppressive compared to ethyl carbamate. A structurally related analogue, methyl carbamate, did not suppress the antibody response. These results indicate that metabolism of ethyl carbamate by P450 may produce more immunosuppressive metabolites as in ethyl carbamate-induced carcinogenicity. A pre-incubation study with phenobarbital-induced liver microsomes in the presence of NADPH-generating system showed that the antibody response was suppressed by ethyl carbamate when splenocytes were pre-incubated with ethyl carbamate and microsomes simultaneously. Moreover, the suppression was completely recovered by the addition of a P450 inhibitor, aminoacetonitrile, in the pre-incubation. Taken together, the present results indicate that metabolism of ethyl carbamate by P450 enzyme(s) may be an important pathway to cause immunosuppression.

[The effect of recombinant tumor necrosis factor on antibody formation in mice immunized with a preparation of the outer membranes from Francisella tularensis]. / Vliianie rekombinantnogo faktora nekroza opukholi na antitelogenez u myshei, immunizirovannykh preparatom vneshnikh membran Francisella tularensis.

The influence of exogenic human recombinant tumor necrosis factor (TNF-alpha) on antibody production in mice immunized with the preparation of F.tularensis outer membranes (OM) was studied. TNF-alpha was injected into mice in doses of 0.001-10 I.U. before and simultaneously with the injection of the preparation of F.tularensis OM. The levels of tularemia antibodies, determined by ELISA techniques, and the number of antibody-producing cells (APC) were studied. The study revealed that recombinant TNF-alpha in the range of doses used in this investigation stimulated the formation of humoral immunity. The injection of TNF-alpha in a dose of 0.001 I.U. was found to produce the most pronounced effect on the level of tularemia antibodies and the number of antibody-producing cells. The use TNF-alpha as immunomodulator made it possible to decrease the dose of the preparation of F.tularensis OM introduced as immunogenic agent without essential changes in the number of splenic APC and in the level of tularemia antibodies.

[Immunotherapy in the combined treatment of chronic destructive pulmonary tuberculosis]. / Immunokorrektsiia v kompleksnoi terapii khronicheskogo destruktivnogo tuberkulëza legkikh.

The paper provides the results of treatment of chronic pulmonary tuberculosis patients by using immunoactive drugs (thymalin and T-activin) and prednisolone, which has been performed by monitoring immunological parameters. The patients with fibrous and cavernous tuberculosis display a combination of antituberculous antibody hyperproduction and specific cell-mediated response inhibition. Immunological hormone therapy allows normalization of the immunological status (an increase in the count and functional activity of T-lymphocytes) and relative stabilization of the process with cessation of bacterial isolation in shorter periods than in the controls, which is favourable to the reduction in hospital treatment periods.

[The use of plasma from donor convalescent patients in the combined treatment of peritonitis]. / Primenenie plazmy donorov-rekonvalestsentov v kompleksnom lechenii peritonita.

The article analyses the treatment of 12 patients with diffuse purulent peritonitis in whom the titers of specific antibodies against microflora causing peritonitis were corrected in the period of secondary immunodeficiency. The authors suggest a new method for the treatment of peritonitis which is based on inclusion of convalescents' plasma after peritonitis in the therapeutic complex. This plasma contains specific antibodies against the cultured microflora in high titers and is a polyvalent hyperimmune medium which makes it possible to cause an effect on the level of humoral immunity. This improved the results of treatment: the titres of specific antibodies against the cultured microflora grew, such complications of diffuse peritonitis as the formation of circumscribed abscesses in the abdominal cavity and suppuration of the postoperative wound were encountered less frequently, mortality reduced by 16.6% as compared to the control group. The efficacy of the suggested methods was also proved in experiments on 164 animals.

[The enhancement of the immunogenicity of streptococcal group-A M protein by conjugation with a synthetic polyelectrolyte]. / Povyshenie immunogennosti M-belka streptokokkov gruppy A pri kon''iugatsii s sinteticheskim poliélektrolitom.

Immunization with the polypeptide fragment of group A streptococcal protein M conjugated with the copolymer of acrylic acid and N-vinylpyrrolidone in complete Freund's adjuvant has been found to lead to a sharp increase in the level of antibodies to the type-specific determinants of protein M, detected in the enzyme immunoassay (EIA). The possibility of the application of such sera to preliminary typing of streptococci in EIA with the use of whole microbial cells as antigens has been shown. The data on high activity of the sera thus obtained in the bactericidal test with streptococci of the homologous type are presented. Recommendations on the use of sera obtained by the above method for highly precise typing of the virulent cultures of group A streptococci in the bactericidal test are given.