Production of reactive oxygen (H2O2) and nitrogen (NO) intermediates and TNF-α in mice genetically selected for high (H) and low (L) antibody response and experimentally infected with Leptospira serovar pomona

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

MODULATION OF PARASITEMIA AND ANTIBODY-RESPONSE TO TRYPANOSOMA-CRUZI BY CYCLOPHOSPHAMIDE IN CALOMYS-CALLOSUS (RODENTIA, CRICETIDAE)

Calomys callosus a wild rodent, previously described as harboring Trypanosoma cruzi, has a low susceptibility to infection by this protozoan.Experiments were designed to evaluate the contribution of the immune response to the resistance to T. cruzi infection exhibited by C. callosus. Animals were submitted to injections of high (200 mg/kg body weight) and low (20 mg/kg body weight) doses of cyclophosphamide on days -1 or -1 and +5, and inoculated with 4 x 10(3) T. cruzi on day O. Parasitemia, mortality and antibody response as measured by direct agglutination of trypomastigotes were observed. Two hundred mg doses of cyclophosphamide resulted in higher parasitemia and mortality as well as in suppression of the antibody response. A single dose of 20 mg enhanced antibody levels on the 20th day after infection, while an additional dose did not further increase antibody production. Parasitemia levels were not depressed, but rather increased in both these groups as compared to untreated controls. Passive transfer of hyperimmune C. callosus anti-T. cruzi serum to cyclophosphamide immunosuppressed animals resulted in lower parasitemia and mortality rates. These results indicate that the immune response plays an important role in the resistance of C. callosus to T. cruzi.

Antibody response to the 43 kDa glycoprotein of Paracoccidioides brasiliensis as a marker for the evaluation of patients under treatment

Sera of patients with paracoccidioidomycosis contained IgG-, IgA-, and IgM-specific antibodies to a 43 kDa antigen contained in the filtrate of a culture of Paracoccidioides brasiliensis. IgG- and IgA-specific antibodies were present in all observed patients. The IgM response was more frequent in acute cases, and the mean titers of IgG- and IgM-specific antibodies were higher in the acute forms. By the fourth month of chemotherapy, there was a decay of IgG, IgA, and IgM antibody titers to this antigen in acute cases, correlating with clinical improvement. The detection of IgG and IgA antibodies and the sequential determination of antibodies to the 43 kDa glycoprotein may be useful tools for serodiagnosis and evaluation of therapeutic efficacy.

IgG, IgM and IgA antibody response for the diagnosis and follow-up of paracoccidioidomycosis: Comparison of counterimmunoelectrophoresis and complement fixation

IgG, IgM and IgA antibodies to GP43 (glycoprotein fraction of Paracoccidioides brasiliensis) were measured by ELISA in 63 samples from 23 patients with paracoccidioidomycosis before and twice after chemotherapy was started. Antibodies against P. brasiliensis were detected by indirect immunofluorescence (IF) (IgG, IgM and IgA isotypes), counterimmunoelectrophoresis (CIE) and complement fixation. Two control groups composed of 19 healthy individuals and 12 patients with other diseases (six with histoplasmosis, three with tuberculosis and three with other mycoses). The highest efficiency percentages were found with IgG and IgA- ELISA (100%), IgG-IF (96.2%), CIE (94.4%) and the lowest with CF (75.9%). Highest positive and negative predictive values (100%) were observed for IgG and IgA ELISA. IgG and IgM-ELISA antibodies are more often found in patients with acute than chronic disease (P = 0.01). Four to six months after treatment follow-up showed decreased levels of IgG and IgM-ELISA for acute cases and decreased titres of CIE for chronic cases in relation to pretreatment levels. This study suggests that IgG-ELISA anti-GP43 represents a good marker to monitor clinical response to therapy.

A cross-sectional study on canine Leishmania (L.) infantum chagasi infection in Amazonian Brazil ratifies a higher prevalence of specific IgG-antibody response than delayed-type hypersensitivity in symptomatic and asymptomatic dogs

This was a cross-sectional study which analyzed the prevalence and the clinical and immunological spectrum of canine Leishmania (L.) infantum chagasi infection in a cohort of 320 mongrel dogs living in an endemic area of American visceral leishmaniasis in the Amazonian Brazil by using, mainly, the indirect fluorescence antibody test (IFAT-IgG) and the delayed-type hypersensitivity (DTH), and the parasite research by the popliteal lymph node aspiration. The IFAT and DTH reactivity recognized three different immune response profiles: (1) IFAT((+))/DTH(-) (107 dogs), (2) IFAT((-))/DTH(+) (18 dogs), and (3) IFAT((+))/DTH(+) (13 dogs), providing an overall prevalence of infection of 43 % (138/320). Thus, the specific prevalence of IFAT ((+)) /DTH ((-)) 33.4 % (107/320) was higher than those of IFAT ((-)) /DTH ((+)) 5.6 % (18/320) and IFAT ((+)) /DTH ((+)) 4.0 % (13/320). Moreover, the frequency of these profiles among 138 infected dogs showed that the IFAT ((+)) /DTH ((-)) rate of 77.5 % (107/138) was also higher than those of 13.0 % (18/138) of IFAT ((-)) /DTH ((+)) and 9.5 % (13/138) of IFAT ((+)) /DTH ((+)) rates. The frequency of asymptomatic dogs (76 %-105) was higher than those of symptomatic (16.6 %-23) and oligosymptomatic ones (7.4 %-10). A total of 16 (11.6 %) L. (L.) i. chagasi isolates were obtained from infected dogs, all from the IFAT ((+)) /DTH ((-)) profile: 41 % (9/22) from symptomatic, 33.3 % (3/9) from oligosymptomatic, and 5.2 % (4/76) from asymptomatic dogs. These findings strongly suggested that despite the higher frequency of asymptomatic dogs (76 %-105), the majority (72.4 %-76) was characterized by the IFAT ((+)) /DTH ((-)) profile with a doubtful immunogenetic resistance against infection.

Natural antibody response to Plasmodium falciparum merozoite antigens MSP5, MSP9 and EBA175 is associated to clinical protection in the Brazilian Amazon

BACKGROUND: Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. METHODS: Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients' plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. RESULTS: 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X2(DF=1) = 9.26/p = 0.0047) and MSP5 (X2(DF=1) = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X2(DF=1) = 6.41/p = 0.0206, Fisher's exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p < 0.03), MSP5 (U = 212, p < 0.004), MSP9 (U = 189.5, p < 0.002) and EBA175 (U = 197, p < 0.014, Mann-Whitney's U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR = 0.112, IC95% = 0.021-0.585) and MSP9 (OR = 0.125, IC95% = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC95% = 1.29-69.25) and 5.7 (IC95% = 1.12-29.62, logistic regression), respectively, with an asymptomatic status. CONCLUSIONS: Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms.

Analysis of the antibody response to an immunodominant epitope of the envelope glycoprotein of a lentivirus and its diagnostic potential

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.

The antibody response in experimental ocular toxoplasmosis

BACKGROUND: The dynamics of the humoral immune response in ocular toxoplasmosis (OT) are poorly understood. We therefore investigated this process in a rabbit model of the disease. MATERIALS AND METHODS: Of 24 infection-naïve adult rabbits, 12 were left untreated and 12 were systematically infected with 5,000 tachyzoites of the non-cyst-forming BK strain of Toxoplasma gondii. Three months later, all rabbits were inoculated transvitreally with 5,000 tachyzoites of Toxoplasma gondii. Paired samples of aqueous humor and serum were analyzed temporally for their total and specific IgG contents. RESULTS: In infection-naïve rabbits with primary OT, specific IgG reached detectable levels in the inoculated eyes between 5 and 15 days after inoculation. In infection-immunized rabbits with secondary OT, a significant increase in specific IgG was regularly detected after 5 days. The antibody ratio C was diagnostic (>/=3) from day 15 onward in primary OT and from day 21 onward in secondary OT. In the uninfected partner eyes, the antibody ratio C was found sporadically diagnostic from day 15 onward in primary OT, but at no time in secondary OT. Specific IgG persisted both locally and in the serum until the end of the monitoring period (100 days). CONCLUSION: Our findings relating to the rabbit model of OT reveal three features of clinical relevance: a diagnostic window precedes the establishment of a humoral immune response; specific antibodies persist long after the cessation of disease activity; and in primary OT, the antibody ratio C may also increase in the uninfected partner eye.

Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response

Inquilinus limosus is a novel Gram-negative bacterium of the subdivision alpha-Proteobacteria recently found in the airways of patients with cystic fibrosis (CF). Here, the authors report on the clinical courses of six CF patients colonized with I. limosus. Five patients suffered from either an acute respiratory exacerbation or a progressive loss of pulmonary function, whereas one patient was in a stable clinical situation. This study focused on two aims: (i) the clonal analysis of I. limosus isolates by random amplified polymorphic DNA (RAPD)-PCR, and (ii) the clarification of whether the presence of I. limosus in the respiratory tract is associated with a specific serum antibody response. Serum IgG was detected by immunoblotting using I. limosus whole-cell-lysate proteins as antigens. Sera from healthy blood donors (n=10) and from CF patients colonized with Pseudomonas aeruginosa (n=10) were found to be immunoblot negative. All six Inquilinus-positive patients raised serum IgG antibodies against various I. limosus antigens. Surprisingly, in one patient, a specific I. limosus serum antibody response was already detected 1 year prior to Inquilinus-positive sputum cultures. Two prominent antigens were characterized by MALDI-MS: a 23 kDa protein revealed homology to the outer membrane lipoprotein OmlA of Actinobacillus pleuropneumoniae, and an 18 kDa protein to a protein-tyrosine phosphatase of Burkholderia cepacia. In conclusion, detection of I. limosus is accompanied by a specific serum antibody response and may reflect the infectious/pathogenic potential of I. limosus. Moreover, IgG immunoblotting may be useful to detect early infection with I. limosus and may support the selective cultivation of this novel emerging pathogen.

Antibody Response to Inactivated Viral Vaccines Administered to Calves at Weaning

Respiratory disease in beef calves has been associated with the stress of weaning. Management practices commonly delay vaccination of calves to this time, and weaning stress could potentially suppress the immune response. To reduce this stress we have been experimenting with a procedure termed “pasture weaning” in which the dams are removed and the calves remain on pasture. Observation suggests that calves weaned with this approach adapt to the weaned state much better than those held in drylot. Consequently, one would expect less stress-mediated effects including those on the immune system. Calves were weaned and assigned to groups that were pasture or drylot weaned, and calves within the groups were vaccinated with one of two inactivated virus vaccines by either the intramuscular or subcutaneous route. Weaning placement did not affect antibody responses to the viruses included in the vaccines. The route of administration did not influence responses with subcutaneous injection inducing responses equivalent to the intramuscular site. Utilization of this route for vaccination could be advantageous because it precludes the tissue damage and hidden abscessation that sometimes results from intramuscular injections. A distinct difference was noted in the immunogenicity of the vaccines with the Vira Shield product yielding significantly better responses to all viral entities.

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs.

BACKGROUND Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.

Catalytic antibody response to the Staphylococcus aureus virulence factor Efb

Staphylococcus aureus is a globally prevalent pathogen that can cause a wide variety of acute and chronic diseases in both adults and children, in both immune susceptible populations and healthy individuals. Its ability to cause persistent infections has been linked to multiple immune evasion strategies, including Efb-mediated complement inhibition. As new multi-drug-resistant strains emerge, therapeutic alternatives to traditional antibiotics must be developed. These experiments assessed the ability of healthy patient immunoglobulin to cleave Efb and disable the complement-inhibitory properties of Efb in vitro. Levels of immunoglobulin-mediated Efb catalysis varied both between immunoglobulin isoform/isotype and between individuals. Serum IgG showed the strongest catalytic activity of the immunoglobulin isotypes tested. Additionally, IgG hydrolyzed the virulence factor in a way that enabled only minimal binding to the complement component C3b, effectively blocking Efb-mediated inhibition of complement lysis. Salivary IgA and serum IgM did not block Efb-mediated inhibition of complement. Catalytic IgG selectively cleaved Efb and showed no cleavage of a variety of other proteins tested. Catalytic activity of IgG was inhibited by serine protease inhibitors, but not by other protease inhibitors, suggesting a serine-protease mechanism of catalysis. It is proposed that varying concentrations and activity levels of catalytic IgG between healthy individuals and those with current or recurrent S. aureus infections in both adult and pediatric populations be studied in order to assess the potential effectiveness of passive immunization therapy with catalytic monoclonal IgG. ^

Antibody response to Panton-Valentine Leukocidin, a Staphylococcus aureus virulence factor, in pediatric patients from Texas Children's Hospital in Houston, Texas

Staphylococcus aureus is an important human pathogen of global health significance, whose frequency is increasing and whose persistence and versatility allow it to remain established in communities worldwide. An observed significant increase in infections, particularly in children with no predisposing risk factors or medical conditions, led to an investigation into pediatric humoral immune response to Panton-Valentine Leukocidin (PVL) and to other antigens expressed by S. aureus that represent the important classes of virulence activities. Patients who were diagnosed with staphylococcal infections were enrolled (n=60), and serum samples collected at the time of admission were analyzed using ELISA and Western blot to screen for immune response to the panel of recombinant proteins. The dominant circulating immunoglobulin titers in this pediatric population were primarily IgG, were specific, and were directed against LukF and LukS, while suppression of other important virulence factors in the presence of PVL was suggested. Patients with invasive infections (osteomyelitis, pneumonia or myositis) had higher titers against LukF and LukS compared to patients with non-invasive infections (abscesses, cellulitis or lymphadenitis). In patients with osteomyelitis, antibody responses to LukF and LukS were higher than antibody responses to any other virulence factor examined. This description of immune response to selected virulence factors of S. aureus caused by isolates of the USA300 lineage in children is novel. Antibody titers also correlated with markers of inflammation. The significance of these correlations remains to be understood.^

Anti-glomerular basement membrane glomerulonephritis: Characterization of an epitope, antibody response, and the potential role of IL-4Ralpha in disease progression

Anti-Glomerular Basement Membrane Glomerulonephritis (anti-GBM GM) is one of the earliest described autoimmune disorders. Patients present with proteinuria, anti-GBM antibodies, and renal failure. Studies have implicated a T Helper 1 (TH1) response in disease induction and a T Helper 2 (TH2) response for disease progression. A 13 amino acid long peptide sequence spanning residues 28 through 40 [pCol(28–40)] of the Collagen IV α3 non-collagen domain (Col IV α3 NCD) is immunogenic and induces anti-GBM GN. In order to fully understand disease initiation, this peptide was further characterized. Peptides were created containing one amino acid substitution for the entire length of pCol(28–40) and induction of anti-GBM GN was monitored. When residues 31, 33, or 34 contained the substitution, anti-GBM GN was unable to be induced. Thus, residues 31, 33, and 34 of pCol(28–40) are required for induction of anti-GBM. Glomerular injury is observed as early as 14 days post anti-GBM GN induction. However, the presence of anti-GBM antibodies is not observed until 20 days post immunization. An enlarged lymph node adjacent to the diseased kidney exhibits B cell activation after renal injury and produces antibodies toward GBM. Thus, anti-GBM antibodies are a consequence of the initial renal injury. Differences between disease susceptible and disease resistant rat strains exist in the expression of IL-4Rα, a major player in the TH2 response. IL-4Rα signaling is regulated by soluble IL-4Rα (sIL-4Rα). Low expression levels of sIL-4Rα result in the stabilization of IL-4 binding, while elevated expression sequesters IL-4. Quantitative PCR experiments noted low siL-4Rα expression levels in disease susceptible rats. Induction of an immune response toward sIL-4Rα in this strain was responsible for delayed disease progression in 15 out of the 17 experimental animals. Antibody transfer and in vivo biological activity experiments confirmed that delayed disease development was due to anti-sIL-4Rα antibodies. Together these experiments indicate that a T-cell epitope is required for activation of a TH1 autoimmune response and anti-GBM antibodies are a consequence of renal injury. More importantly, a role for IL-4Rα signaling is implicated in the progression of anti-GBM GN. ^

Disease risk factors and humoral antibody response to periodontal-associated pathogens: A model to evaluate periodontal disease in older adults

Periodontal diseases (PD) are infectious, inflammatory, and tissue destructive events which affect the periodontal ligament that surround and support the teeth. Periodontal diseases are the major cause of tooth loss after age 35, with gingivitis and periodontitis affecting 75% of the adult population. A select group of bacterial organisms are associated with periodontal pathogenesis. There is a direct association between oral hygiene and prevention of PD. The importance of genetic differences and host immune response capabilities in determining host, susceptibility or resistance to PD has not been established. This study examined the risk factors and serum (humoral) immune response to periodontal diseased-associated pathogens in a 55 to 80+ year old South Texas study sample with PD. This study sample was described by: age, sex, ethnicity, the socioeconomic factors marital status, income and occupation, IgG, IgA, IgM immunoglobulin status, and the autoimmune response markers rheumatoid factor (RF) and antinuclear antibody (ANA). These variables were used to determine the risk factors associated with development of PD. Serum IgG, IgA, IgM antibodies to bacterial antigens provided evidence for disease exposure.^ A causal model for PD was constructed from associations for risk factors (ethnicity, marital status, income, and occupation) with dental exam and periodontitis. The multiple correlation between PD and ethnicity, income and dental exam was significant. Hispanics of low income were least likely to have had a dental exam in the last year and most likely to have PD. The etiologic agents for PD, as evidenced by elevated humoral antibody responses, were the Gram negative microorganisms Bacteroides gingivalis, serotypes FDC381 and SUNYaBA7A1-28, and Wolinella recta. Recommendation for a PD prevention and control program are provided. ^