The role of CD4(+) cells in vivo on the induction of the immune response to Porphyromonas gingivalis in mice

Background: It has previously been suggested that CD4(+) T cells play a pivotal role in regulating the immune response to periodontal pathogens. The aim of the present study therefore was to determine delayed type hypersensitivity (DTH), spleen cell proliferation, serum and splenic anti-Porphyromonas gingivalis antibody levels, and lesion sizes following challenge with viable P. gingiualis in CD4-depleted BALB/c mice immunized with P. gingiualis outer membrane proteins (OMP). Methods: Four groups of BALB/c mice were used. Groups 1 and 2 were injected intraperitoneally (ip) with saline for 3 consecutive days and then weekly throughout the experiment. Groups 3 and 4 were injected ip with rat immunoglobulin and a monoclonal rat anti-mouse CD4 antibody, respectively. Two days later, group 1 mice were injected ip with saline only, while all the other groups were immunized ip with P. gingiualis OMP weekly for 3 weeks. One week later following the last immunization of OMP, 3 separate experiments were conducted to determine: 1) the DTH response to P. gingiualis OMP by measuring footpad swelling; 2) the levels of antibodies to P. gingiualis in serum samples and spleen cell cultures using an enzyme-linked immunosorbent assay, as well as spleen cell proliferation after stimulation with OMP; and 3) the lesion sizes after a subcutaneous challenge with viable P. gingiualis cells. Results: In CD4(+) T-cell-depleted mice (group 4), the DTH response and antigen-stimulated cell proliferation were significantly suppressed when compared to groups 2 and 3. Similarly, the levels of serum and splenic IgM, IgG, and all IgG subclass antibodies to P. gingiualis OMP were depressed. Delayed healing of P. gingivalis-induced lesions was also observed in the CD4(+) T-cell-depleted group. Conclusions: This study has shown that depletion of CD4(+) T cells prior to immunization with P. gingiualis OMP led to the suppression of both the humoral and cell-mediated immune response to this microorganism and that this was associated with delayed healing. These results suggest that the induction of the immune response to P. gingiualis is a CD4(+) T-cell-dependent mechanism and that CD4(+) T cells are important in the healing process.

High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever

Infection with any 1 of 4 dengue viruses produces a spectrum of clinical illness ranging from a mild undifferentiated febrile illness to dengue fever (DF) to dengue hemorrhagic fever (DHF), a potentially life-threatening disease. The morbidity and mortality of DHF can be reduced by early hospitalization and careful supportive care. To determine its usefulness as a predictor of DHF, plasma levels of the secreted dengue virus nonstructural protein NS1 (sNS1) were measured daily in 32 children with dengue-2 virus infections participating in a prospective, hospital-based study. Free sNS1 levels in plasma correlated with viremia levels and were higher in patients with DHF than in those with DF. An elevated free sNS1 level (greater than or equal to600 ng/mL) within 72 h of illness onset identified patients at risk for developing DHF.

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

Equine respiratory viruses in foals in New Zealand

AIMS: To identify the respiratory viruses that are present among foals in New Zealand and to establish the age at which foals first become infected with these viruses. METHODS: Foals were recruited to the study in October/ November 1995 at the age of 1 month (Group A) or in March/ April 1996 at the age of 4-6 months (Groups B and C). Nasal swabs and blood samples were collected at monthly intervals. Nasal swabs and peripheral blood leucocytes (PBL) harvested from heparinised blood samples were used for virus isolation; serum harvested from whole-blood samples was used for serological testing for the presence of antibodies against equine herpesvirus (EHV)-1 or -4, equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). Twelve foals were sampled until December 1996; the remaining 19 foals were lost from the study at various times prior to this date. RESULTS: The only viruses isolated were EHV 2 and EHV 5. EHV 2 was isolated from 155/157 PBL samples collected during the period of study and from 40/172 nasal swabs collected from 18 foals. All isolations from nasal swabs, except one, were made over a period of 2-4 months from January to April (Group A), March to April (Group B) or May, to July (Group C). EHV 5 was isolated from either PBL, nasal swabs, or both, from 15 foals on 32 occasions. All foals were positive for antibodies to EHV 1 or EHV 4, as tested by serum neutralisation (SN), on at least one sampling occasion and all but one were positive for EHV 1 antibodies measured by enzyme-linked immunosorbent assay (ELISA) on at least one sampling occasion. Recent EHV 1 infection was evident at least once during the period of study in 18/23 (78%) foals for which at least two samples were collected. SN antibodies to ERBV were evident in 19/23 (83%) foals on at least one sampling occasion and 15/23 foals showed evidence of seroconversion to ERBV Antibodies to ERAV were only detected in serum samples collected from foals in Group A and probably represented maternally-derived antibodies. Haemagglutination inhibition (HI) antibody titres greater than or equal to 1:10 to EAdV-1 were evident in 21/23 (91%) foals on at least one sampling occasion and 16/23 foals showed serological evidence of recent EAdV-1 infection. None of the 67 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. There was no clear association between infection with any of the viruses isolated or tested for and the presence of overt clinical signs of respiratory disease. CONCLUSIONS: There was serological and/or virological evidence that EHV-1, EHV-2, EHV-5, EAdV-1 and ERBV infections were present among foals in New Zealand. EHV-2 infection was first detected in foals as young as 3 months of age. The isolation of EHV-2 from nasal swabs preceded serological evidence of infection with other respiratory viruses, suggesting that EHV-2 may predispose foals to other viral infections.

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand. METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV 2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR) = 2.67, 95% CI = 1.59-4.47, p = 0.0171]. CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV 2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.

Comparison of an ELISA and an LC/MS/MS method for measuring tacrolimus concentrations and making dosage decisions in transplant recipients

This study compared an enzyme-linked immunosorbent assay (ELISA) to a liquid chromatography-tandem mass spectrometry (LC/MS/MS) technique for measurement of tacrolimus concentrations in adult kidney and liver transplant recipients, and investigated how assay choice influenced pharmacokinetic parameter estimates and drug dosage decisions. Tacrolimus concentrations measured by both ELISA and LC/MS/MS from 29 kidney (n = 98 samples) and 27 liver (n = 97 samples) transplant recipients were used to evaluate the performance of these methods in the clinical setting. Tacrolimus concentrations measured by the two techniques were compared via regression analysis. Population pharmacokinetic models were developed independently using ELISA and LC/MS/MS data from 76 kidney recipients. Derived kinetic parameters were used to formulate typical dosing regimens for concentration targeting. Dosage recommendations for the two assays were compared. The relation between LC/MS/MS and ELISA measurements was best described by the regression equation ELISA = 1.02 . (LC/MS/MS) + 0.14 in kidney recipients, and ELISA = 1.12 . (LC/MS/MS) - 0.87 in liver recipients. ELISA displayed less accuracy than LC/MS/MS at lower tacrolimus concentrations. Population pharmacokinetic models based on ELISA and LC/MS/MS data were similar with residual random errors of 4.1 ng/mL and 3.7 ng/mL, respectively. Assay choice gave rise to dosage prediction differences ranging from 0% to 30%. ELISA measurements of tacrolimus are not automatically interchangeable with LC/MS/MS values. Assay differences were greatest in adult liver recipients, probably reflecting periods of liver dysfunction and impaired biliary secretion of metabolites. While the majority of data collected in this study suggested assay differences in adult kidney recipients were minimal, findings of ELISA dosage underpredictions of up to 25% in the long term must be investigated further.

Malaria vectors on Buka and Bougainville islands, Papua New Guinea

Anophelines were sampled from 82 locations oil Buka and Bougainville islands in Papua New Guinea by larval collections, carbon dioxide-baited Mosquito traps, and human biting catches. Anopheles farauti s.s. was collected in larval Surveys but infrequently in mosquito traps on both islands; on Buka Island this species was readily collected in human biting catches. Anopheles faraunti 2 was commonly collected in larval surveys on both islands however. it was not collected in either mosquito traps or human biting catches. Anopheles punctulatus was found only on Buka Island, where it was commonly collected as larvae, but rarely in human biting catches and mosquito traps. Anopheles lungae was collected Lis larvae from only I site on Bougainville. Anopheles farauti s.s. led consistently throughout the night (1900-0600 h): small peaks at midnight and dawn were not statistically significant. Of 1,156 An. farauti s.s. specimens examined by enzyme-linked immunosorbent assay for malaria sporozoites. 20 were found to be positive: 12 were positive for Plasmodium falciparum and 8 were positive for P. vivax (247 variant = 5: 210 variant = 3). Anopheles farauti s.s. seems to be the major malaria vector on these islands, whereas An. punctulatus may play a minor role on Buka Island. Anophele farauti 2 is unlikely to be involved in malaria transmission on Buka or Bougainville islands.

Clarithromycin and prednisolone inhibit cytokine production in chronic rhinosinusitis

Objectives. Long-term, low-dose macrolide therapy is effective in the treatment of chronic rhinosinusitis. The mechanism of its anti-inflammatory effect and how this differs from corticosteroids remains unclear. The effect of clarithromycin and prednisolone on interleukin-5, interleukin-8, and granulocyte-macrophage colony-stimulating factor production by cultured chronic sinusitis nasal mucosa was examined in the study. Study Design and Methods. Nasal mucosa was obtained from 11 patients with chronic sinusitis. This tissue was cultured for 24 hours in the presence of clarithromycin or prednisolone at a variety of concentrations. Cytokine levels were determined by enzyme-linked immunoassay. Results. Clarithromycin and prednisolone each produced significant reductions in interleukin-5, interleukin-8, and granulocyte-macrophage colony-stimulating factor production. There was no significant difference between the effects of clarithromycin and prednisolone. Conclusion: Macrolide antibiotics are capable of inhibiting pro-inflammatory cytokine production in vitro and are as potent as prednisolone. This mechanism is likely to be at least partly responsible for the clinical efficacy of macrolide antibiotics in chronic rhinosinusitis. Key Words. Macrolide, prednisolone, sinusitis, enzyme-linked immunosorbent assay, cytokine.

Enhancing the immunogenicity and modulating the fine epitope recognition of antisera to a helical group A streptococcal peptide vaccine candidate from the M protein using lipid-core peptide technology

A conserved helical peptide vaccine candidate from the M protein of group A streptococci, p145, has been described. Minimal epitopes within p145 have been defined and an epitope recognized by protective antibodies, but not by autoreactive T cells, has been identified. When administered to mice, p145 has low immunogenicity. Many boosts of peptide are required to achieve a high antibody titre (> 12 800). To attempt to overcome this low immunogenicity, lipid-core peptide technology was employed. Lipid-core peptides (LCP) consist of an oligomeric polylysine core, with multiple copies of the peptide of choice, conjugated to a series of lipoamino acids, which acts as an anchor for the antigen. Seven different LCP constructs based on the p145 peptide sequence were synthesized (LCP1-->LCP7) and the immunogenicity of the compounds examined. The most immunogenic constructs contained the longest alkyl side-chains. The number of lipoamino acids in the constructs affected the immunogenicity and spacing between the alkyl side-chains increased immunogenicity. An increase in immunogenicity (enzyme-linked immunosorbent assay (ELISA) titres) of up to 100-fold was demonstrated using this technology and some constructs without adjuvant were more immunogenic than p145 administered with complete Freund's adjuvant (CFA). The fine specificity of the induced antibody response differed for the different constructs but one construct, LCP4, induced antibodies of identical fine specificity to those found in endemic human serum. Opsonic activity of LCP4 antisera was more than double that of p145 antisera. These data show the potential for LCP technology to both enhance immunogenicity of complex peptides and to focus the immune response towards or away from critical epitopes.

Cellular responses to Schistosoma japonicum cathepsin D aspartic protease

Lymphocyte proliferation and cytokine production were measured in groups of mice vaccinated (but not subsequently challenge infected) with recombinant forms of Schistosoma japonicum cathepsin D aspartic protease, rSjASP1 (expressed in bacteria; enzymatically inactive) and rSjASP2 (expressed in insect cells; enzymatically active). Both forms of the schistosome enzyme induced significant proliferation of splenocytes recovered from vaccinated mice, and expression of interferon (IFN)-gamma, interleukin (IL)-4 and IL-10 mRNA in these cells was detected using reverse transcriptase-polymerase chain reaction. Secretion of IFN-gamma, IL-4 and IL-10 by splenocytes from vaccinated mice was confirmed and quantified using enzyme-linked immunosorbent assay. IFN-gamma was the most abundant cytokine produced, followed by IL-4 and IL-10 in rank order. These findings indicated that vaccination of mice with the schistosome protease induces a mixed Th1/Th2 cytokine response, which may explain the modest level of protection after challenge infection in cathepsin d-vaccinated mice, reported previously.

Acquisition and loss of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Prevotella intermedia over a 5-year period: effect of a triclosan/copolymer dentifrice

Objectives: The present study describes the natural history of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Prevotella intermedia over a 5-year period and the effect of a triclosan/copolymer dentifrice on these organisms in a normal adult population. Material and Methods: Subgingival plaque samples were collected from 504 adult volunteers. Probing pocket depths (PPD) and relative attachment levels were measured using an automated probe. Participants were matched for disease status (CPI), plaque index, age and gender, and allocated to receive either a triclosan/copolymer or placebo dentifrice. Re-examination and subgingival plaque sampling was repeated after 1, 2, 3, 4 and 5 years. P. gingivalis, A. actinomycetemcomitans and P. intermedia were detected and quantitated using an enzyme linked immunosorbent assay. Logistic regression and generalised linear modelling were used to analyse the data. Results: This 5-year longitudinal study showed considerable volatility in acquisition and loss (below the level of detection) of all three organisms in this population. Relatively few subjects had these organisms on multiple occasions. While P. gingivalis was related to loss of attachment and to PPD greater than or equal to3.5 mm, there was no relationship between A. actinomycetemcomitans or P. intermedia and disease progression over the 5 years of the study. Smokers with P. gingivalis had more PPD greater than or equal to3.5 mm than smokers without this organism. There was no significant effect of the triclosan dentifrice on P. gingivalis or A. actinomycetemcomitans . Subjects using triclosan were more likely to have P. intermedia than those not using the dentifrice; however this did not translate into these subjects having higher levels of P. intermedia and its presence was uniform showing no signs of increasing over the course of the study. Conclusion: The present 5-year longitudinal study has shown the transient nature of colonisation with P. gingivalis , A. actinomycetemcomitans and P. intermedia in a normal adult population. The use of a triclosan-containing dentifrice did not lead to an overgrowth of these organisms. The clinical effect of the dentifrice would appear to be independent of its antimicrobial properties.

Avaliação de dois métodos laboratoriais para diagnóstico de hidatidose

A hidatidose, vulgarmente conhecida por quisto hidático, é causada pelos estados larvares do parasita Echinococcus granulosus. O seu diagnóstico baseia-se na clínica, na epidemiologia e nas técnicas imagiológicas, sendo suportado por testes serológicos. O tratamento mais comum é o cirúrgico, sendo importante o diagnóstico definitivo da doença antes da cirurgia, para se prevenir a disseminação dos quistos que pode ocorrer durante a mesma. Neste trabalho comparam-se dois métodos imunoserológicos para o diagnóstico da hidatidose: Enzyme-linked Immunosorbent Assay (ELISA) para pesquisa de Imunoglobulina G; e Fluoro-enzyme Immunoassay (FEIA) para pesquisa de Imunoglobulina E. Dos 55 indivíduos incluídos no estudo, todos em fase pré-operatória, 31% possuíam quisto hidático calcificado, 54,5% quisto hidático não calcificado e 14,5% não possuíam hidatidose, mas quistos simples. Os testes apresentaram a mesma especificidade (87,5%), sendo a sensibilidade do ELISA IgG mais baixa (63,8%) do que a do FEIA IgE (76,6%). Foi demonstrada uma boa correlação entre os dois métodos (r = 0,726, p < 0,05).

Lyme Disease: antibodies against Borrelia burgdorferi in farm workers in Argentina

Lyme Disease is a tick-borne (specially by Ixodes ticks) immune-mediated inflammatory disorder caused by a newly recognize spirochete, Borrelia burgdorferi. Indirect fluorescent antibody (IF) staining methods and enzyme-linked immunosorbent assay are frequently relied upon to confirm Lyme borreliosis infections. Although serologic testing for antibodies has limitations, it is still the only practical means of confirming B. burgdorferi infections. Because we have no previous report of Lyme disease in human inhabitants in Argentina, a study was designed as a seroepidemiologic investigation of the immune response to B. burgdorferi in farm workers of Argentina with arthritis symptoms. Three out of 28 sera were positive (#1,5 and 9). Serum # 1 was positive for Immunoglobulin G at dilution 1:320, serum # 5 and # 9 both to dilution 1:160; while for Immunoglobulin M all (#1, 5 and 9) were positive at low dilution (1:40) using IF. The results showed that antibodies against B. burgdorferi are present in an Argentinian population. Thus caution should be exercised in the clinical interpretation of arthritis until the presence of B. burgdorferi be confirmed by culture in specific media.

Infectivity of cysts of the ME-49 Toxoplasma gondii strain in bovine milk and homemade cheese

OBJECTIVE: Analyze the infectivity and storage resistance of cysts of the ME-49 strain of Toxoplasma gondii in artificially infected bovine milk and homemade fresh cheese. METHODS: Pasteurized bovine milk was infected with 10 cysts/ml of the ME-49 strain of T.gondii and inoculated in different groups of mice, immediately or after storage at 4ºC for 5, 10 and 20 days. Homemade fresh cheese was prepared with artificially infected milk, and also tested in groups of mice, using the same storage process. Infection was identified by the presence of cysts in the brain or serological testing in challenged mice after 5 weeks, confirmed by Western Blot and histology. RESULTS: The infectivity of cysts of the ME-49 strain of T.gondii was maintained in the milk even after storage for 20 days at refrigerator temperatures. Cysts were also able to survive the production process of homemade fresh cheese and storage for a period of 10 days in the same conditions. CONCLUSIONS: These data demonstrated that milk and dairy products could be an important source of T.gondii in human contamination, reinforcing the importance of milk pasteurization before any processing or ingestion.

Cytomegalovirus seroepidemiology in an urban community of São Paulo, Brazil

INTRODUCTION: After the era of rubella vaccine, cytomegalovirus (CMV) infection is one of the most frequently causes of mental retardation and congenital deafness. Seroepidemiological studies are necessary to understand the transmission dynamics of the disease. The purpose of the study was to quantify the transmission rate of CMV disease in a community in the state of São Paulo, Brazil. METHODS: Using ELISA test (IgG), a retrospective serological survey looking for CMV antibodies was performed in an non-immunized community. Frozen sera from 443 individuals, randomly selected by cluster sampling technique in the town of Caieiras, São Paulo, were collected from November 1990 to January 1991. Seroprevalence was stratified by age (0-40 years). Mathematical techniques were applied to determine the age-dependent decay function of maternal antibodies during the first year of life, the age-dependent seroprevalence function and the force of infection for CMV in this community. RESULTS: It was observed a descending phase of seropositivity in the first 9 months, but changes in antibody titration were observed between 8 months old and one year of age. The average age of the first infection was 5.02 months of age and 19.84 years, when the age-dependent seroprevalence and the force of infection were analyzed between 10 months of age and 10 years of age and from 10 to 40 years old, respectively. CONCLUSION: CMV infection is highly prevalent among the population studied and infection occurs in the first year of life. This study shows that most women at reproductive age are vulnerable to the first infection, increasing the risk for congenital infection.