A Comparative Study of the RAPINA and the Virus-Neutralizing Test (RFFIT) for the Estimation of Antirabies-Neutralizing Antibody Levels in Dog Samples.

The mass vaccination of dogs against rabies is a highly rational strategy for interrupting the natural transmission of urban rabies. According to the World Organization for Animal Health (OIE) and the World Health Organization (WHO), the immunization of at least 70% of the total dog population minimizes the risk of endemic rabies. Knowledge of the virus-neutralizing antibody (VNA) level against the rabies virus (RABV) is required to evaluate protective immunity and vaccine coverage of dogs in the field. The rapid focus fluorescent inhibition test (RFFIT) and the fluorescent antibody virus neutralization (FAVN) test are recommended by OIE and WHO to determine the VNA levels in serum. However, these tests are cell culture based and require the use of live viruses and specialized equipment. The rapid neutralizing antibody test (RAPINA) is a novel, immunochromatographic test that uses inactivated virus to estimate the VNA level qualitatively. It is a simple, rapid and inexpensive, although indirect, assay for the detection of VNA levels. The RAPINA has shown good positive and negative predictive values and a high concordance with the RFFIT results. In this study, we compared the performance of the two tests for evaluating the vaccination status of dogs in the Philippines, Thailand and Japan. A total of 1135 dog sera were analysed by the RAPINA and compared to the VNA levels determined by the RFFIT. The overall positive and negative predictive values of the RAPINA were 96.2-99.3% and 84.5-94.8%, respectively, with a concordance (kappa) of 0.946-0.97 among the three countries. The RAPINA results were highly homologous and reproducible among different laboratories. These results suggest that this test is appropriate to survey vaccination coverage in countries with limited resources.

Bufavirus genotype 3 in Turkish children with severe diarrhoea.

Recently a parvovirus called bufavirus (BuV) has been implicated as a causative agent of diarrhoea. To further reveal the epidemiology and genetic characteristics of BuV, this study was performed in Turkish children with diarrhoea. BuV was detected in 1.4% (8/583) of stool samples. All stool samples from healthy children (n = 148) were negative for BuV. Diarrhoea in BuV-positive patients was severe and occurred mainly during the colder months of the year. Complete genome sequences were generated from four BuVs. Only BuV3 was found, which was genetically and phylogenetically similar to Bhutanese BuV3, indicating that BuV3 is prevalent in Asian countries.

Neurogenic cardiomyopathy in rabbits with experimentally induced rabies.

Cardiomyopathies have been rarely described in rabbits. Here we report myocardial necrosis of the ventricular wall in rabbits with experimentally induced rabies. Myocardial lesions were found only in rabbits with brain lesions, and the severity of the cardiac lesions was proportional to that of the brain lesions. Neither the frequency nor the cumulative dose of anesthesia was related to the incidence or the severity of the myocardial lesions. The myocardial lesions were characterized by degeneration and/or necrosis of myocardial cells and were accompanied by contraction band necrosis, interstitial fibrosis, and infiltration of inflammatory cells. The brain lesions due to rabies virus infection were most prominent in the cerebral cortex, thalamus, hypothalamus, brainstem, and medulla. Rabies virus antigen was not found in the hearts of any rabbits. Based on these findings, the myocardial lesions were classified as neurogenic cardiomyopathy.

Oral administration of an HSP90 inhibitor, 17-DMAG, intervenes tumor-cell infiltration into multiple organs and improves survival period for ATL model mice.

In the peripheral blood leukocytes (PBLs) from the carriers of the human T-lymphotropic virus type-1 (HTLV-1) or the patients with adult T-cell leukemia (ATL), nuclear factor kappaB (NF-κB)-mediated antiapoptotic signals are constitutively activated primarily by the HTLV-1-encoded oncoprotein Tax. Tax interacts with the I κB kinase regulatory subunit NEMO (NF-κB essential modulator) to activate NF-κB, and this interaction is maintained in part by a molecular chaperone, heat-shock protein 90 (HSP90), and its co-chaperone cell division cycle 37 (CDC37). The antibiotic geldanamycin (GA) inhibits HSP90's ATP binding for its proper interaction with client proteins. Administration of a novel water-soluble and less toxic GA derivative, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), to Tax-expressing ATL-transformed cell lines, C8166 and MT4, induced significant degradation of Tax. 17-DMAG also facilitated growth arrest and cellular apoptosis to C8166 and MT4 and other ATL cell lines, although this treatment has no apparent effects on normal PBLs. 17-DMAG also downregulated Tax-mediated intracellular signals including the activation of NF-κB, activator protein 1 or HTLV-1 long terminal repeat in Tax-transfected HEK293 cells. Oral administration of 17-DMAG to ATL model mice xenografted with lymphomatous transgenic Lck-Tax (Lck proximal promoter-driven Tax transgene) cells or HTLV-1-producing tumor cells dramatically attenuated aggressive infiltration into multiple organs, inhibited de novo viral production and improved survival period. These observations identified 17-DMAG as a promising candidate for the prevention of ATL progression.

Human rabies in rural Bangladesh.

Rabies is a major public health problem in Bangladesh, where most of the population live in rural areas. However, there is little epidemiological information on rabies in rural Bangladesh. This study was conducted in 30 upazilas (subdistricts) covering all six divisions of the country, to determine the levels of rabies and animal bites in Bangladesh. The total population of these upazilas was 6 992 302. A pretested questionnaire was used and data were collected by interviewing the adult members of families. We estimated that in Bangladesh, 166 590 [95% confidence interval (CI) 163 350-170 550] people per year are bitten by an animal. The annual incidence of rabies deaths in Bangladesh was estimated to be 1·40 (95% CI 1·05-1·78)/100 000 population. By extrapolating this, we estimated that 2100 (95% CI 1575-2670) people die annually from rabies in Bangladesh. More than three-quarters of rabies patients died at home. This community-based study provides new information on rabies epidemiology in Bangladesh.

Influence of age and duration of infection on bacterial load and immune responses to Helicobacter pylori infection in a murine model.

Using a murine model, we previously showed that Helicobacter pylori infects and colonizes offspring via maternal transmission during the nursing period. The aim of this study was to investigate the influence of age and duration of infection on inflammatory and immune responses to H. pylori in infant and adult mice. During the breast-feeding period, the number of bacteria was significantly suppressed in 1-week-old mice infected with H. pylori at an early stage of nursing, compared with adult mice, suggesting that breast-milk induces such low colonization. In addition, these mice had weaker gastric inflammation, especially Th1 cytokine and humoral responses than in mice infected with H. pylori after weaning in spite of elevated levels of Th1 cytokines. Although infant mice showed low inflammatory responses against H. pylori, they produced H. pylori-specific antibodies following vaccination with oral or parenteral adjuvant. Our results suggest the importance of age at the time of primary infection on bacterial load, gastric inflammation and humoral responses in a murine model of H. pylori infection.

CTLA-4 blockade inhibits induction of Helicobacter pylori-associated gastritis in mice.

The balance between Th1 and Th2 response determines the outcome of Helicobacter pylori infection. Interferon (IFN)-gamma plays an inductive role in gastric inflammation, whereas interleukin (IL)-4 counterbalances Th1 response and suppresses the development of gastritis. Th cell response is regulated by co-stimulatory factors. A co-stimulatory molecule, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), plays an inhibitory role in IL-2-dependent cell growth and mediates an optimal inhibitory signal to Th1 and Th2 cells. We administered anti-CTLA-4 monoclonal antibody (MoAb), which blocks CTLA-4 signalling, to examine the relative role for this signalling during maturation of Th1 and Th2 cells in H. pylori infection in mice. Mice treated by anti-CTLA-4 MoAb within the first week of infection showed an inhibition of gastric inflammation, accompanied by an increasing ratio of H. pylori-specific IgG1/IgG2a in serum following infection. Furthermore, the treatment resulted in the higher ratio of IL-4/IFN-gamma by splenocytes in response to H. pylori antigen at 6 weeks after infection, compared with untreated mice. These results suggest that the predominance of Th2 response by CTLA-4 blockade leads to an inhibition of the development of gastric inflammation. CTLA-4 signalling could contribute to the regulation of Th subsets and the development of gastric inflammation in H. pylori infection.

Childhood Helicobacter pylori infection in a murine model: maternal transmission and eradication by systemic immunization using bacterial antigen-aluminium hydroxide.

In humans, transmission of Helicobacter pylori is thought to occur largely during childhood. Infected mothers are generally considered to be the main source of the pathogen. However, little is known about when and how often maternal transmission of H. pylori occurs during childhood. In the present study, we examined these issues in an experimental murine model. Pregnant C57BL/6 mice, infected experimentally with H. pylori, delivered and nursed their litters. The stomachs of the infants were isolated and assessed for transmission of H. pylori. We also investigated the effect of systemic immunization using H. pylori antigen-aluminium hydroxide (AlOH) with regard to providing anti-H. pylori immunity and eradicating the maternally transmitted bacteria in infants. Polymerase chain reaction (PCR) was used to examine the presence of transmitted bacteria and their eradication. Maternal transmission of H. pylori varied widely during the nursing period, but almost all litters showed bacterial transmission at 2 weeks postpartum. Systemic immunization with bacterial antigen-AlOH eradicated the bacteria in most litters; this immunization induced a local decrease of Th2 cytokines and a local increase of Th1 cytokines in the gastric tissue, as determined by ELISA. Our results indicate that our H. pylori vaccine provides not only protection, but also eradication of the already transmitted H. pylori.

Comparison of genomic structures and antigenic reactivities of orthologous 29-kilodalton outer membrane proteins of Helicobacter pylori.

We purified a 29-kDa Helicobacter pylori outer membrane protein (Omp29 protein) and cloned the gene encoding the protein from H. pylori strain ATCC 43504. The Omp29 gene corresponded to the reported JHP73 and the HP78-79 genes of H. pylori strains. A corresponding nucleotide fragment was detected in all 150 tested H. pylori clinical isolates by PCR or Southern blotting. The amplified Omp29-corresponding fragments were categorized into a ca. 770-bp-long group and a larger-fragment group. Sequence analysis indicated that the larger fragments were likely synthesized from the 770-bp fragments by insertion of an irrelevant fragment via 17-bp-long repeat sequences. Immunoblot analysis implies that the ca. 770-bp fragment is responsible for the protein homologous to Omp29, whereas the larger fragments are not responsible for those proteins or encoding antigenically distinct proteins. We postulate that the H. pylori outer membrane protein Omp29 can alter its antigenicity through gene modifications mediated by nucleotide transfer.

Hepatitis B virus harboring nucleotide deletions in the core promoter region and genotype B correlate with low viral replication activity in anti-HBe positive carriers.

BACKGROUND: Emergence of anti-HBe following seroconversion of HBe antigen indicates reduced hepatitis B virus (HBV) replication in the liver and low infectivity in the natural course of infection. However, some patients show continued replication or reactivation even in the presence of anti-HBe. OBJECTIVE: To clarify the cause of HBV replication, we investigated genotype differences and mutations in the core promoter and precore region in relation to virus titer. STUDY DESIGN: Using quantification of HBV DNA, nucleotide sequencing of the core promoter and precore region, and genotyping with the S gene by restriction fragment length polymorphism (RFLP), we analyzed sera of 26 anti-HBe positive carriers (28 serum samples). RESULTS: Various mutations were detected including C to T point mutation at nt 1653, A to T and G to A contiguous point mutations at nt 1762 and 1764 in the core promoter region, and G to A point mutation at nt 1896 in the precore region, but no common mutations were detected that were directly related to the virus titer from earlier reported mutations. In contrast, the mean titer of genotype B virus was 1.5 x 10(5) copies per ml and that of mutant HBV of genotype C having 8 base pairs (8-bp) deletion (nt 1768-1775) in the core promoter region was 7.9 x 10(4) copies per ml (mean titer). These titers showed commonly lower than that of genotype C virus without 8-bp deletion (median titer 5.0 x 10(6) copies per ml). Transition of genotype from C to B after viral reactivation and reduction of proportion of 8-bp deletion mutant at reactivation period was observed in a patient who demonstrated exacerbation of liver dysfunction due to immunosuppressive therapy and increased viral replication. CONCLUSIONS: These results confirm those of our earlier study describing low replication ability of 8-bp deletion mutant HBV in vitro, and also indicate that the presence of genotype B correlates with reduced titer of HBV.

Effects of zinc acetate on splenocytes of endotoxemic mice: enhanced immune response, reduced apoptosis, and increased expression of heat shock protein 70.

Immune function is markedly attenuated in endotoxemia. Zinc is involved in the regulation of cellular functions and maintenance of immune function, and its level in the serum is low in endotoxemia. We mainly investigated the effects of zinc acetate (ZA) on splenocytes in mice with endotoxemia. After we confirmed increased plasma zinc level by ZA treatment, C57BL/6 mice were randomly divided into four groups: 10 control mice received 500 microL saline solution as vehicle; 10 control mice received ZA at 3 mg/kg body weight; 20 endotoxemic mice received a 40 mg/kg lethal dose of lipopolysaccharide (LPS); 20 mice received ZA followed by LPS as the above dose. In vivo, we confirmed that ZA pretreatment did not significantly affect the plasma cytokine level in endotoxemic mice. In vitro, splenocytes from ZA-plus-LPS mice showed drastic effects, in that ZA abrogated LPS-induced suppression of cellular proliferation and production of interleukin-2 and interferon-gamma. The percentage of apoptotic splenocytes was significantly reduced in ZA-plus-LPS mice (23.4%) as compared with LPS mice (41.6%). Furthermore, the expression of HSP-70 mRNA in splenocytes was strongly enhanced in both ZA and ZA-plus-LPS mice, especially in the latter group. Finally, studies monitoring survival rates for 6 days showed that LPS caused 100% mortality while ZA-plus-LPS mice showed 75% survival. Our results suggest that zinc normalized the immune response and reduced apoptosis of splenocytes. These changes were probably caused by increased synthesis of HSP-70 by splenocytes, which might enhance survival of mice with LPS-induced endotoxemia.

Immunoglobulin G and M hepatitis C virus core antibody (JCC.2) response in chimpanzees infected experimentally with hepatitis C virus.

OBJECTIVE: To evaluate changes in serum immunoglobulin M (IgM) and G (IgG) hepatitis C virus (HCV) core antibodies after HCV infection in acute hepatitis C. METHODS: Serum HCV RNA and IgM and IgG HCV core antibodies were investigated using sera sequentially sampled from three chimpanzees experimentally infected with HCV. Serum IgG HCV core antibody titer was measured using a JCC.2 enzyme-linked immunosorbent assay (ELISA) kit (Chemo-Sera-Therapeutic Research Center, Kumamoto, Japan). IgM core antibody titer was measured using horseradish peroxidase-labeled monoclonal anti-human IgM as the secondary antibody for the JCC.2 ELISA kit. Serum HCV RNA was detected using the 5' noncoding region as the primer according to the reverse transcriptase (RT) nested polymerase chain reaction (PCR) and competitive RT-PCR method. RESULTS: IgM JCC.2 antibody was detected when alanine aminotransferase (ALT) peaked, showing the closest correlation with the changes in ALT. A period during which IgM JCC.2 antibody was positive but HCV RNA as determined by RT-nested PCR was negative was observed after the elevation of ALT level. CONCLUSION: These results indicate the usefulness of detection of serum IgM JCC.2 antibody in making a definitive diagnosis of acute hepatitis C and the follow-up observation of hepatitis C.

Reduced transcription and progeny virus production of hepatitis B virus containing an 8-bp deletion in basic core promoter.

We have demonstrated previously the presence of an 8-bp deletion mutant, spanning from nt. 1768 to nt. 1775 in the basic core promoter region of hepatitis B virus (HBV) in patients with anti-HBe positive asymptomatic phase before developing acute exacerbation after immunosuppressive treatment. The transcription and progeny virus production activities of the mutant were examined by transfection of the recombinant plasmid [pUC Del(2)] containing the head-to-tail dimer DNA of the mutant into HepG2 cells. The amounts of hepatitis B surface antigen (HBsAg) and HBe antigens secreted into the culture medium were markedly reduced. Southern blotting of DNAs extracted from the culture medium also showed reduced mutant activity to produce progeny virus. Northern blotting and RNase protection assay of RNAs extracted from transfected cells demonstrated that the transcription of both precore mRNA and pregenome RNA was reduced significantly compared to that of wild-type HBV. The promoter activity examined by transfection of the CAT plasmid containing deletion mutant DNA was much lower than that of wild type. Co-transfection experiments, however, of the CAT plasmid containing wild-type DNA with pUC Del(2) reduced CAT activity induced by wild-type, suggesting that truncated X protein produced by the mutant does not possess a sufficient transactivating activity. Gel shift assay using HepG2 nuclear extract and a probe containing four TA-rich regions in CP and various competitors suggested that the lack of the third TA-rich region was responsible for the transcription reduction of precore mRNA and pregenome RNA. The possible mechanisms are discussed.

Therapeutic oral vaccination induces mucosal immune response sufficient to eliminate long-term Helicobacter pylori infection.

We examined the efficacy of therapeutic oral vaccination using Helicobacter pylori-whole cell sonicate and cholera toxin (CT) in mice persistently infected with H. pylori. Efficacy was determined by bacterial culture and microscopic examination of gastric tissues for the persistence of bacteria at 6 weeks after the last vaccination. Vaccination of H. pylori-whole cell sonicate combined with CT eradicated bacteria in 10/16 mice (62.5%). Interestingly, oral vaccination with CT alone also eliminated the bacteria in 8/17 mice (47.1%). However, a therapeutic intraperitoneally administered vaccine failed to eradicate H. pylori from the stomach (1/17 mice, 5.9%). Identification of the type of immunity involved in the eradication process showed that oral vaccination enhanced the antigen-specific IgA in the feces and saliva. The efficacy of eradication of H. pylori correlated well with increases in IgA secretion in mucosal tissue and a higher labeling index of IgA-positive lumina of pyloric glands. Moreover, the expression of IL-4 mRNA in the stomach of mice with eradicated bacteria was higher than in the uneradicated group. Our results suggest that the efficacy of vaccination depends on the mucosal IgA response in the gastrointestinal tract against H. pylori via Th2 cell activation and that therapeutic oral vaccination induces a mucosal immune response sufficient to eradicate long-term infection with H. pylori.

Analysis of p53 gene mutations in Helicobacter pylori-associated gastritis mucosa in endoscopic biopsy specimens.

BACKGROUND: No previous report has shown the relationship between Helicobacter pylori infection and a direct sequence analysis of p53 gene mutation in a biopsy sample of human gastric mucosa. METHODS: A total of 60 endoscopic biopsies samples (21 patients with H. pylori-positive gastritis and 9 patients with H. pylori-negative gastritis), including antral mucosa and corpus mucosa, were used in this study. Direct DNA sequencing of exons 5, 6, 7, and 8 of the p53 gene was performed by the dyedeoxy terminator method. RESULTS: Mutations in the p53 gene were identified in non-hot spot codons in exon 7 and 8 in 11 of 21 samples (52.4%) from H. pylori-positive gastritis patients. There was no mutation in H. pylori-negative gastritis patients. CONCLUSIONS: This finding shows that H. pylori infection can induce p53 point mutations and appears to be involved in the pathway leading to dysplasia or carcinoma.

Local secretory immunoglobulin A and postimmunization gastritis correlate with protection against Helicobacter pylori infection after oral vaccination of mice.

C57BL/6 mice were orally immunized with five weekly doses of 2 mg, 200 microgram, or 2 microgram of Helicobacter pylori (Sydney strain) whole-cell sonicate combined with cholera toxin. One week after the last vaccination, mice were challenged with 5 x 10(7) CFU of live H. pylori three times at 2-day intervals. At 6 or 18 weeks after the challenge, mice were sacrificed and bacterial cultures and histological studies of the stomach were performed. Vaccination with 2 mg/session or 200 microgram/session inhibited H. pylori colonization by 90 and 100%, respectively. These mice were considered protected. Lower levels of H. pylori-specific immunoglobulin A (IgA) were detected in fecal and saliva samples before challenge. However, a significant increase in IgA secretion in mucosal tissue and a higher labeling index for IgA-positive lumina of pyloric glands were noted in these mice in response to challenge and in a vaccine dose-dependent manner. In protected mice, however, severe gastritis characterized by marked infiltration of inflammation mononuclear cells was noted at 6 weeks after challenge, compared with the gastritis seen in unprotected mice or nonvaccinated, ordinarily infected mice. Marked expression of gamma interferon mRNA was detected in the stomach of all protected mice, and 50% of these mice expressed interleukin 4 (IL-4) or IL-5 mRNA. Our findings suggest that local secretory IgA antibody and severe postimmunization gastritis correlate well with protection of mice against H. pylori infection.

Divergence of virulence factors of Helicobacter pylori among clinical isolates does not correlate with disease specificity.

The aim of this study was to examine the relation between disease specificity and the virulence factors of Helicobacter pylori isolated from patients with gastric cancer (GC), duodenal ulcer (DU), and gastritis (GS). Altogether 18 isolates obtained from patients with GC, 28 isolates from DU patients, and 13 isolates from GS patients were analyzed. All isolates were tested for the presence of the cagA gene, and genotyping of the vacA gene was done by the polymerase chain reaction. Production of VacA protein and expression of vacuolating cytotoxic activity in the H. pylori culture supernatant were examined. The serum antibody titers against purified VacA and CagA proteins were determined by enzyme-linked immunosorbent assay (ELISA). Interleukin-8 (IL-8) production by AGS cells in response to H. pylori isolates was measured by an hIL-8 ELISA kit. Genetic analysis of vacA revealed that most of the clinical isolates were classified into the S1a type by signal sequence typing. There were no differences in cagA detection rates, vacuolating cytotoxin activity, or mean antibody titers against VacA and CagA protein among the three groups. The mean IL-8 concentrations in the supernatants of AGS cells were similar in the three groups. In this study, there was no difference in virulence factors of H. pylori among isolates from GC, DU, and GS.

In vitro cytotoxic effects of vacuolating cytotoxin produced by clinical isolates of Helicobacter pylori.

The vacuolating cytotoxin produced by Helicobacter pylori is considered to be one virulence factor causing peptic ulceration. In this study, we examined the activity of vacuolating cytotoxin in induction of intracellular vacuolation of rabbit gastric epithelial cells (RGECs). We used culture supernatants of H. pylori as a source of vacuolating cytotoxin and quantitated cytotoxic activity by the MTT method. Intracellular vacuolation of RGECs was observed in the presence of 36 of 57 (63%) clinically isolated H. pylori strains. However, there were no differences in the incidence of H. pylori strains with positive vacuolating cytotoxin (Tox+) among patients with gastritis, gastric ulcers or duodenal ulcers. The MTT assay showed that the cytotoxic activity of H. pylori supernatants obtained from patients with gastric ulcers was significantly higher than in patients with gastritis (p < 0.01), but was not different to duodenal ulcer patient supernatants. Similar results were also observed in Tox+ isolates, however, there were no significant differences between patients with regard to the incidence of vacuolating cytotoxin-negative isolates. Although our data may not indicate a clear correlation between prevalence of vacuolating cytotoxin and clinical manifestations, they suggest that H. pylori harboring vacuolating cytotoxin may particularly induce damage to the gastric epithelium in patients with gastric ulcers.

Serological assessment of the early response to eradication therapy using an immunodominant outer membrane protein of Helicobacter pylori.

Eradication of Helicobacter pylori infection cures gastritis and prevents recurrence of peptic ulcers. Endoscopy is usually used to evaluate the effectiveness of eradication therapy. We designed a new noninvasive assay system for the early evaluation of eradication of H. pylori infection in which a crude H. pylori outer membrane protein preparation (HPOmp) is used as an antigen, and we determined the sensitivity and specificity of the serological assay system. Immunoblot analysis showed that anti-HPOmp antibodies reacted to a protein with a molecular mass of approximately 29 kDa. In those patients who responded to therapy, the anti-HPOmp immunoglobulin G (IgG) titers measured by enzyme-linked immunosorbent assay (ELISA) at 1 month after the end of therapy were significantly lower than those before treatment (34.8% reduction; P < 0.001), and the posttreatment reduction in the antibody titer was significantly greater than that of the titer measured with a commercially available anti-H. pylori IgG ELISA (34.8% versus 16.1%; P < 0.001). When a 25% reduction of anti-HPOmp IgG titer at 1 month after the end of treatment was taken as the cutoff value for H. pylori eradication, the sensitivity and specificity of our new assay were 75% (51 of 68 treatment responders) and 96% (22 of 23 nonresponders), respectively. Our results indicate that the novel serological test with HPOmp might be a clinically useful tool for assessment of eradication of H. pylori.