Prevalence of total hepatitis A antibody among 5 to 7 years old children and their mothers in Cambodia.

This study determined the prevalence of total hepatitis A antibody (anti-HAV) among 5-7 years old children and their mothers in the whole Cambodia, using a nationwide study, and examined the differences between the two cohorts. A total of 4535 dried blood spot-driven (DBS) samples (2021 mothers and their 2514 children of 5-7 years old) and the concomitant 922 whole blood samples (subset of the whole participants) were collected using a multistage random sampling strategy throughout Cambodia in 2017. Total anti-HAV was detected using the chemiluminescence enzyme immunoassay method. Compared to gold standard whole blood samples, the sensitivity and specificity of DBS mediated anti-HAV detection were 94.8% and 98%, respectively. Total anti-HAV prevalence among mothers was 91.2% (95%CI: 90.0-92.5%), and that of their children was 31.5% (95%CI: 29.7-33.3%). In our study, the low prevalence of total anti-HAV among children indicates the country's improvement of safe water and food supply, hygiene and sanitation. If the hygiene and sanitation are consistently improved in Cambodia, the prevalence might be no longer increased when the children become adults.

Eighteen-year follow-up cohort study on hepatitis B and C virus infections related long-term prognosis among hemodialysis patients in Hiroshima.

This study aimed to investigate the prevalence trend of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and their genotype distribution among hemodialysis patients, determining their long-term prognosis and the risk factors to the mortality. This cohort study used both the medical data and the blood samples of hemodialysis patients at nine dialysis centers in Hiroshima from 1999 to 2017. Hepatitis B surface antigen (HBsAg) and anti-HCV were screened and then amplification was done to positive sera by polymerase chain reaction for genotyping. Data were employed for multiple regressions to determine the associated risk factors. A total of 3968 patients were subdivided into three groups: who started hemodialysis before 1990, during 1991 to 2001, and after 2002. The periodic prevalence of HBsAg decreased from 2.8% to 1.3% and that of anti-HCV from 33.3% to 9.5% in the three groups. By multiple regressions, the adjusted hazard ratio of diabetes mellitus (DM) ranges from 1.59 to 2.12 and that of HCV RNA positivity ranges from 1.18 to 1.48 (P < .05). Heart failure is the primary cause of death in all groups. Genotype C2 is predominant for HBV and genotype 1b is predominant for HCV. The decreasing trend of both HBV and HCV was found in the cohort. DM and HCV RNA were the significant risk factors leading to poor prognosis among hemodialysis patients. The similar genotype distribution of both HBV and HCV was found as general population. This alarmed to provide early diagnosis, prompt, and adequate treatment to HCV infection among hemodialysis patients.

Substitution of the CD81 Binding Site and ß-Sandwich Area in E2 of HCV in Cambodia.

The high genetic variability of hepatitis C virus (HCV) is the main obstacle to developing a vaccine. E2 has attracted attention for vaccine development because targeting this protein could potentially overcome issues related to the genetic diversity of HCV. In this study, we analyzed HCV genes in the general population of Cambodia and investigated the E2 locus as a candidate for vaccine development. HCV sero-epidemiological surveys were conducted between the period 2010 and 2014, with an HCV RNA-positive rate of 1.3% (11/868). Follow-up blood samples were collected from four anti-HCV- and HCV RNA- positive patients (genotype 1b: 2 cases, 6e: 1 case, 6r: 1 case) after 4.12 years. Analysis of HCV full-length nucleotide sequences in paired specimens revealed that the mutation rates of HCV genotypes 1b and 6e/6r were 1.61-2.03 × 10-3 and 2.52-2.74 × 10-3 substitutions/site/year, respectively. Non-synonymous substitutions were detected in HVR1, the front layer of the CD81 binding site, and the ß-sandwich, but not in the N-terminal region or adjacent to the CD81 binding site. Therefore, we conclude that the CD81 binding site is a promising locus for HCV vaccine development.

Existence of hepatitis B virus surface protein mutations and other variants: demand for hepatitis B infection control in Cambodia.

BACKGROUND: This study aimed to detect Hepatitis B virus (HBV) genome sequences and their variants as of nationwide scale using dried blood spot (DBS) samples and to provide up-to-date reference data for infection control and surveillance in Cambodia. METHOD: Among 2518 children age 5-7 years and their 2023 mothers participated in 2017 Cambodia nationwide sero-survey on hepatitis B surface antigen (HBsAg) prevalence using multistage random sampling strategy, 95 mothers and 13 children positive to HBsAg were included in this study. HBV DNA was extracted from DBS, then performed polymerase chain reaction. HBV genotypes and potential variants were examined by partial and full length genomic analysis. RESULTS: HBsAg positive rate was 4.7% (95/2023) in mothers and 0.52% (13/2518) in their children. Genotype C (80.49%) was abundantly found throughout the whole Cambodia whilst genotype B (19.51%) was exclusively found in regions bordering Vietnam. S gene mutants of HBV were found in 24.29% of mothers and 16.67% of children with HBV DNA positive sera. Full-length genome analysis revealed the homology of 99.62-100% in each mother-child pair. Genotype B was clarified to recombinant genotype B4/C2 and B2/C2. Double (48.39%) and combination mutation (32.26%) were observed in core promoter region of HBV C1 strains. CONCLUSIONS: This study showed the capable of DBS for large-scale molecular epidemiological study of HBV in resource limited countries. Full-genome sequences yield the better understanding of sub-genotypes, their variants and the degree of homology between strains isolated from mother-child pairs calls for effective strategies on prevention, control and surveillance of mother-to-child HBV transmission in Cambodia.

Evaluation of the efficiency of dried blood spot-based measurement of hepatitis B and hepatitis C virus seromarkers.

Although hepatitis B (HBV) and C (HCV) virus infections are still global health issues, measuring sero-markers by standard venipuncture is challenging in areas limited with the adequate human resources and basic infrastructure. This study aimed to inform the usefulness of dried blood spot (DBS) sampling technique for epidemiological study of HBV and HCV in the resources limited areas. We compared specimen recovery rate expressed as analytical sensitivity ratio of HBsAg, HBcAb and anti-HCV between serum specimens and DBS samples (HemaSpot vs Whatman903). Sensitivity ratio was calculated as the ratio of the measured value from DBS to the measured value from serum. Then both the qualitative and quantitative comparisons of HBsAg detection by DBS were done using Cambodian samples. HBsAg, HBcAb and anti-HCV sensitivity ratios for the highest sample dilution (8-fold) were 31.2:1, 38.9:1 and 32.0:1 for Whatman903 card and 17.6:1, 23.5:1 and 26.3:1 for HemaSpot respectively. Detection efficacy of HemaSpot (80%) was not inferior to Whatman903 (60%) after 1 month storage, and no significant difference in any hepatitis virus sero-markers was observed in HemaSpot-spotted patient samples stored for 2 weeks at -25 °C and 29 °C. All reference HemaSpot -spotted 400 HBsAg sero-negative samples showed negative. Sensitivity and specificity of HBsAg in HemaSpot were 92.3% and 100%. The recovery expressed as analytical sensitivity ratio of HBsAg, HBcAb and anti-HCV of HemaSpot specimen were not inferior to Whatman903. Therefore, DBS with its usefulness proved as an acceptable tool for large epidemiological study of HBV and HCV in resources limited remote area.

The changing epidemiology of hepatitis B and C infections in Nanoro, rural Burkina Faso: a random sampling survey.

BACKGROUND: This study sought to provide up-to-date hepatitis B (HBV) and C (HCV) seroprevalence in rural Burkina Faso decade after hepatitis B vaccine was introduced in the national immunization scheduled for children. METHODS: In 2018, a community-based, random sampling strategy with probability proportional to population size was conducted in Nanoro to investigate the prevalence of viral hepatitis in children and their mothers. Sociodemographic, vaccination history and risk factors were assessed by interview and health books. HBsAg rapid tests were done by finger prick and Dried Blood Spots (DBS) were collected for hepatitis seromarkers by chemiluminescence enzyme immunoassay. Positive samples underwent confirmatory PCR and phylogenetic analysis. RESULTS: Data were presented on 240 mother-child pairs. HBsAg Prevalence was 0.8% in children and 6.3% in mothers. Hepatitis B core antibody positivity was 89.2% in mothers, 59.2% in children and was associated with age, sex and scarification. Hepatitis B surface antibodies prevalence was 37.5% in children and 5.8% in mothers. Good vaccination coverage was limited by home delivery. Phylogenetic analysis of HBV strains based on full genome sequences (n = 7) and s-fragment sequences (n = 6) revealed genotype A, E, and recombinant A3/E. Viral genome homology was reported in one mother-child pair. Anti-HCV prevalence was 5.4% in mothers, 2.1% in children and strains belonged to genotype 2. CONCLUSIONS: In Nanoro, HBsAg prevalence was low in children, intermediate in mothers and mother-to-child transmission persists. Home delivery was a limiting factor of Hepatitis B vaccination coverage. HBV genotype E was predominant and genotype A3/E is reported for the first time in Burkina Faso.

High possibility of hepatocarcinogenesis in HBV genotype C1 infected Cambodians is indicated by 340 HBV C1 full-genomes analysis from GenBank.

Approximately 75% of hepatocellular carcinomas (HCC) occur in Asia; core promoter mutations are associated with HCC in HBV genotype C, the dominant genotype in Cambodia. We analyzed these mutations in Cambodian residents and compared them with HBV full genomes registered in GenBank. We investigated the characteristics of 26 full-length HBV genomes among 35 residents positive for hepatitis B surface antigen in Siem Reap province, Cambodia. Genotype C1 was dominant (92.3%, 24/26), with one case of B2 and B4 each. Multiple mutations were confirmed in 24 Cambodian C1 isolates, especially double mutation at A1762T/G1764A in 18 isolates (75.0%), and combination mutation at C1653T and/or T1753V and A1762T/G1764A in 14 isolates (58.3%). In phylogenetic analysis, 16 of 24 isolates were located in the cluster with Laos, Thailand, and Malaysia. In 340 GenBank-registered C1 strains, 113 (33.2%) had combination mutation amongst which 16.5%, 34.2%, and 95.2% were found in ASC, chronic hepatitis, and liver cirrhosis (LC)/HCC respectively (P < 0. 001). Mutations were abundantly found in 24 Cambodian C1 isolates, and 340 C1 strains from GenBank showed mutation in genotype C1 brings high possibility of LC/HCC occurrence. Therefore, we suggest that Cambodian people infected with HBV genotype C1 have high possibility of hepatocarcinogenesis.

Hepatitis B surface antigen seroprevalence among pre- and post-vaccine cohorts in Cambodia, 2017.

BACKGROUND: Hepatitis B virus (HBV) infection is highly endemic in most low income countries including Cambodia. This nationwide serosurvey was conducted to assess the impact of hepatitis B vaccination and to determine whether Cambodia met the WHO regional 2017 target of hepatitis B surface antigen (HBsAg) seroprevalence less than 1% in five-year-old children. METHODS: A cross-sectional multi-stage cluster survey was conducted among children born during 2010-2012 and their mothers in Cambodia. HBsAg prevalence was estimated by rapid point-of-care testing, and demographic data, including vaccination history, was collected. Vaccine coverage in children and the prevalence of HBsAg among children and mothers was calculated taking into account the complex survey design. Factors associated with children's failure to receive timely (within 24 h) vaccination were analysed by multivariate logistic analysis. FINDINGS: A total of 2,520 children 5-7 years old and 2,028 mothers were recruited. In total, 78.4% of children received hepatitis B vaccination birth-dose (HepB-BD); of these, 58.7% were administered ≤ 24 h. Birth at home or "other" location were independent risk factors for children's failure to receive timely HepB-BD. Overall HBsAg seroprevalence was 4.39% (95%CI: 3.53%-5.45%) among mothers and 0.56% (95%CI: 0.32%-0.98%) among children. The prevalence among children without hepatitis B vaccination was 4.62% (95%CI: 1.31%-14.97%). Among children with a HBsAg-positive mother, prevalence was 10.11% (95%CI: 5.41%-18.11%). INTERPRETATION: Having achieved the 2017 target of less than 1% HBsAg prevalence among 5 years old children, Cambodia can now focus on eliminating mother-to-child transmission of HBV. Moreover, the high HBsAg prevalence among mothers suggests that routine screening with proper linkage to care and treatment is needed. Strengthening measures to improve vaccination coverage further and eliminate mother-to-child transmission by coordinated programming with other services offering additional HBV interventions will help move towards the global goal of hepatitis B elimination by 2030. FUNDING: As per sources of funding.

Very low prevalence of anti-HAV in Japan: high potential for future outbreak.

Since the early 21st century, almost all developed countries have had a very low hepatitis A virus antibody (anti-HAV) sero-prevalence profile, as sanitation conditions and health care facilities have been optimized to a universal standard. There has not been a report on anti-HAV prevalence among a large scale population in Japan since 2003. Therefore, this study aimed to investigate the current HAV status among the general population in Hiroshima. From each age and sex specific group, a total of 1,200 samples were randomly selected from 7,682 stocked serum samples from residents' and employees' annual health check-ups during 2013-2015. Total anti-HAV was detected using Chemiluminescent Enzyme Immunoassay. The overall anti-HAV sero-prevalence was 16.8%. In both males and females, anti-HAV prevalence among individuals between 20-59 years of age was as low as 0.0-2.0%, whilst that among 70 s was as high as 70.0-71.0%. A large number of residents aged under 60 are now susceptible to HAV infection. The cohort reduction trend of anti-HAV in Japan exposes the high possibility of mass outbreak in the future. HAV vaccine especially to younger generation and high risk population may prevent outbreak in Japan.

Acquisition rate of antibody to hepatitis B surface antigen among medical and dental students in Japan after three-dose hepatitis B vaccination.

BACKGROUND: Health care workers (HCWs) are at high risk of contracting blood-borne infections including hepatitis B virus (HBV) infection. In Japan, all HCWs are required to receive HB vaccination before beginning work. This study aimed to investigate the dynamics of the HB surface antibody (anti-HBs) titer after a three-dose HB vaccination in HCWs and to determine effective scheduling of HB vaccination for non-responders. METHODS: Subjects included 832 medical and dental students who had received a three-dose HB vaccination (Bimmugen® 0.5 ml/vial). Anti-HBs was measured three times (before the third dose and 1 and 5 months after the third dose) using the CLIA method. The cut-off value of anti-HBs acquisition was 10 mIU/mL. After booster doses (three maximum) were administered to non-responders, the anti-HBs titers were measured again. RESULTS: Out of 832 students, 491 were analyzed, of which 58.9% (289) were male. Anti-HBs-positive rates before the third dose and 1 and 5 months later were 47.9%, 95.9%, and 89.0%, respectively. The relationship between the antibody titer at one month (x) and 5 months (y) was estimated by log10y = log10x - 0.134 (P < 0.0001). Twelve non-responders were followed-up, all of which acquired a protective anti-HBs titer after revaccination with a three-dose booster. CONCLUSION: Anti-HBs titer decreases by an average of 20% within 4 months between the 1st and 5th month after the third dose. Therefore, anti-HBs titer should be measured periodically after completing the three-dose vaccination. Additionally, results suggested that booster doses are effective if administered with the same schedule as primary vaccination.

Cleavage of Toll-Like Receptor 9 Ectodomain Is Required for In Vivo Responses to Single Strand DNA.

Mouse toll-like receptor 9 (TLR9) is an endosomal sensor for single-stranded DNA. TLR9 is transported from the endoplasmic reticulum to endolysosomes by a multiple transmembrane protein Unc93 homolog B1, and proteolytically cleaved at its ectodomain. The structure of TLR9 and its biochemical analyses have shown that the proteolytic cleavage of TLR9 ectodomain enables TLR9-dimerization and TLR9 activation. However, the requirement of TLR9 cleavage in vivo has not been studied. We here show that the 13 amino acids deletion at the cleavage site made TLR9 resistant to proteolytic cleavage. The deletion mutation in the Tlr9 gene impaired TLR9-dependent cytokine production in conventional dendritic cells from the mutant mice. Not only in vitro, in vivo production of inflammatory cytokines (TNF-α and IL-12p40), chemokine (CCR5/RANTES), and type I interferon (IFN-α) induced by administration of TLR9 ligand was also impaired. These results demonstrate that the TLR9 cleavage is required for TLR9 responses in vivo.

Pilot study for hepatitis virus screening among employees as an effective approach to encourage employees who screened positive to receive medical care in Japan.

AIM: Countermeasures against hepatitis B and C virus (HBV, HCV) infection at work sites in Japan have not yet been implemented. This study aimed to determine the status of viral hepatitis infection among employees in Japan. METHODS: We undertook a workplace-based cross-sectional study from 2011 to 2016 in Hiroshima, Japan. Hepatitis B virus and HCV markers were identified during a routine checkup of employees in 15 enterprises. The screening results were sent to employees directly and not to employers. A thorough examination of the participants who screened positive was encouraged by forwarding to them a referral letter by our research group to specialized medical institutions. RESULTS: Of the 3015 employees, 2420 (80.3%) underwent hepatitis virus screening. Of these, 13.8% had been screened for hepatitis virus before this survey. The prevalence of hepatitis B surface antigen was 0.95% (n = 23; 95% confidence interval, 0.6-1.3%). The prevalence of hepatitis B core antibody was as high as 31.5% at age 60-69 years, and 41.5% at age 70 years and over. The HCV carrier rate was 0.45% (n = 11; 0.2-0.7%) and 54.5% of them had genotype 2. Thirty-four carriers were detected, and 44.1% of them were detected for the first time; 53.3% of the newly detected carriers visited medical institutions with the referral, and underwent a periodic follow-up or treatment. CONCLUSION: Promoting hepatitis virus screening for employees may help detect carriers who are unaware of their infection and require treatment. Submitting the results to employees with a referral letter to medical institutions at the time of positive diagnosis may be effective.

A seroepidemiological survey of the effect of hepatitis B vaccine and hepatitis B and C virus infections among elementary school students in Siem Reap province, Cambodia.

AIM: This study aimed to survey the prevalence and incidence of hepatitis B (HBV) and hepatitis C virus (HCV) infection among elementary school students in Siem Reap province, Cambodia and to evaluate the effects of a national infant HBV vaccination program introduced in 2001. METHODS: Students in 3rd grade during the 2011, 2012, and 2013 academic years were enrolled in this study; at the time of the second examination, in the 2014-2015 academic year, the students were in 5th or 6th grade. The incidence and prevalence rates of HBV and HCV infection were estimated and full HBV sequences were analyzed. RESULTS: Among 248 students (107 male and 141 female) born between 1999 and 2005, five students were HBV surface antigen (HBs-Ag) positive (2.02%), and all of them were infected with genotype C. Among them, subgenotype C1 was found in four students and, unexpectedly, complete genetic sequence identity of subgenotype C1 was found in two students from different families. The anti-HBV core (HBc) and anti-HBs prevalence rates were 10.89% and 16.13%, respectively. Twenty-five students were positive for anti-HBs and negative for both HBsAg and anti-HBc (10.08%; estimated serological vaccination rate); this rate increased significantly with the birth year (P = 0.0229). Prevalence of anti-HCV was 2.82%, and HCV RNA was not detected. The estimated incidence of HBV and HCV infection were both 0/1000 person-years (PY) (95% confidence interval, 0-20.61/1000 PY and 0-14.50/1000 PY, respectively). CONCLUSION: Hepatitis B virus full-genome sequencing and serological analysis revealed the possibility of horizontal transmission of HBV among Cambodian schoolchildren. However, the anti-HBc positivity rate decreased along with increasing age and estimated serological vaccination rates.

Reduced prevalence of hepatitis B surface antigen positivity among pregnant women born after the national implementation of immunoprophylaxis for babies born to hepatitis B virus-carrier mothers in Japan.

AIM: We aimed to estimate hepatitis B surface antigen (HBsAg) positivity among birth year-stratified pregnant women in Hiroshima, Japan, and compare prevalence rates between women born before and after implementation of a national immunoprophylactic vaccination program for babies born to hepatitis B virus (HBV) carrier mothers in Japan. METHODS: Pregnant women who gave birth at all delivery hospitals/clinics in Hiroshima prefecture between 1 April 2010 and 31 March 2011 were eligible. Lists collected from each institution included survey items such as age (pregnant woman's birth year) and HBsAg and hepatitis C virus (HCV) antibody (anti-HCV) test results, which were posted anonymously and non-consolidated from medical records. We calculated the HBsAg and anti-HCV prevalence in our cohort according to the mothers' birth year. RESULTS: In 41 of 58 hospitals/clinics, 15 233 and 15 035 pregnant women underwent HBsAg and anti-HCV testing, corresponding to 59.6% and 58.9% of 25 546 births in the 2010 fiscal year, respectively. The overall HBsAg positive rate was 0.51% (95% confidence interval [CI], 0.40-0.63%), and an extremely low prevalence (0.10%; 95% CI, 0.00-0.25%) was observed among pregnant women born after 1986. However, the prevalence in this study was slightly higher than the nationwide value (0.31%) and the Chugoku region-specific value (0.46%) among first-time blood donors at Japanese Red Cross blood centers between 2001 and 2006. No significant difference in anti-HCV positivity was observed. CONCLUSION: Only two pregnant women born after the preventive program implementation were HBsAg-positive. Perinatal HBV transmission is estimated to be almost completely inhibited in the next generation.

Clustering infection of hepatitis B virus genotype B4 among residents in Vietnam, and its genomic characters both intra- and extra-family.

Vietnam has a high rate of hepatitis B virus (HBV) infection and a high mortality rate from hepatocellular carcinoma. We performed a detailed genetic analysis of 48 residents and four families from Binh Thuan Province, a southern coastal area of Vietnam. The route of infection and genomic characteristics related to hepatocellular carcinoma (HCC) were studied in HBV spread among carriers that we detected in our previous hepatitis survey. The HBV genotype was B4 in 91.7% and C1 in 8.3% of the cases. The intra-family's HBV sequence homology was high at 96.8-99.4%. However, it was also high at 99.4-99.8% among residents of the same age and sex as family members. In addition, full genome analysis was performed in 21 cases. The core region of all 20 isolates with genotype B4 was a recombinant of genotype C, and pre-S deletion was found in 20% of cases. The promoter mutation G1613A was found in 13.6% of cases, and a 24 bp insertion from nt1673 in the X region was found in 6.3% of cases. The phylogenetic tree and homology analysis of the HBV full genome suggested the probability and its possibility of horizontal transmission not only within families nor vertical transmission but within cohorts of the same generation in the population. Moreover, the HBV genotype B4 isolates were found not only to be recombinants of genotype C, which results in a high cancer risk, but also to have other risk of HCC, pre-S deletions, the G1613A mutation, and X region insertions corresponding to the promoter. These genomic characters were suggested to be one of the factors to explain the high HCC mortality rate in Vietnam.

Selectivity of Human TLR9 for Double CpG Motifs and Implications for the Recognition of Genomic DNA.

TLR9 acts as a first-line host defense against pathogens recognizing DNA comprising unmethylated CpG motifs present in bacteria and viruses. Species- and sequence-specific recognition differences were demonstrated for TLR9 receptors. Activation of human (h)TLR9 requires a pair of closely positioned CpG motifs within oligodeoxyribonucleotides (ODNs), whereas mouse TLR9 is effectively activated by an ODN with a single CpG motif. Molecular model-directed mutagenesis identified two regions, site A and site B, as important for receptor activation. Amino acid residues Gln346 and Arg348 within site A contribute to the sequence-specific recognition by hTLR9 in determining the bias for two appropriately spaced CpG motifs within immunostimulatory ODNs. Mutation of Gln562 at site B, in combination with Gln346 and Arg348 mutations of mouse counterparts, increased activation of hTLR9 by mouse-specific ODN, mammalian genomic DNA, and bacterial DNA. We propose that the double CpG motif sequence-specificity of hTLR9 results in decreased activation by ODNs with a lower frequency of CpG motifs, such as from mammalian genomic DNA, which increases hTLR9 selectivity for pathogen versus host DNA.

Species-Specific Minimal Sequence Motif for Oligodeoxyribonucleotides Activating Mouse TLR9.

Synthetic oligodeoxyribonucleotides (ODNs) containing unmethylated CpG recapitulate the activation of TLR9 by microbial DNA. ODNs are potent stimulators of the immune response in cells expressing TLR9. Despite extensive use of mice as experimental animals in basic and applied immunological research, the key sequence determinants that govern the activation of mouse TLR9 by ODNs have not been well defined. We performed a systematic investigation of the sequence motif of B class phosphodiester ODNs to identify the sequence properties that govern mouse TLR9 activation. In contrast to ODNs activating human TLR9, where the minimal sequence motif for the receptor activation comprises a pair of closely positioned CpGs we found that the mouse TLR9 requires a single CpG positioned 4-6 nt from the 5'-end. Activation is augmented by a 5'TCC sequence one to three nucleotides from the CG. The distance of the CG dinucleotide of four to six nucleotides from the 5'-end and the ODN's length fine-tunes activation of mouse macrophages. Length of the ODN <23 and >29 nt decreases activation of dendritic cells. The ODNs with minimal sequence induce Th1-type cytokine synthesis in dendritic cells and confirm the expression of cell surface markers in B cells. Identification of the minimal sequence provides an insight into the sequence selectivity of mouse TLR9 and points to the differences in the receptor selectivity between species probably as a result of differences in the receptor binding sites.

Essential role for Toll-like receptor 7 (TLR7)-unique cysteines in an intramolecular disulfide bond, proteolytic cleavage and RNA sensing.

Toll-like receptor 7 (TLR7) an innate immune sensor for microbial RNA, erroneously responds to self-derived RNA. To avoid autoimmune responses, TLR7 is suggested to be silenced until the N-terminal half of the TLR7 ectodomain (TLR7N) is cleaved off. Resultant truncated TLR7 (TLR7C) is thought to signal microbial RNA. We here show that TLR7N remains associated with TLR7C through a disulfide bond. By N-terminal amino acid sequencing, TLR7C was found to start at 461E or 462A. The newly established monoclonal anti-TLR7N showed that endogenous TLR7 in bone marrow-derived dendritic cells was almost all cleaved and cleaved TLR7N remained in endolysosomes. TLR7N in endolysosomes was linked with TLR7C by a disulfide bond. In contrast, TLR9 did not have a disulfide bond between TLR9N and TLR9C fragments. Among the cysteines unique to the ectodomain of TLR7 but not TLR9 (Cys98, Cys445, Cys475 and Cys722), Cys98 in TLR7N and Cys475 in TLR7C were required for an intramolecular disulfide bond. These cysteines were also needed for proteolytic cleavage of and RNA sensing by TLR7, but not for TLR7 trafficking from endoplasmic reticulum to endosomes. No response was seen in TLR7 mutants lacking the proteolytic cleavage site or TLR7C alone. These results demonstrate requirement for proteolytic cleavage and TLR7N in TLR7 responses and indicate RNA sensing by TLR7N + TLR7C.

Signal-transducing adaptor protein-2 modulates Fas-mediated T cell apoptosis by interacting with caspase-8.

We found that an adaptor protein, signal-transducing adaptor protein (STAP)-2, is a new member of the Fas-death-inducing signaling complex and participates in activation-induced cell death in T cells. STAP-2 enhanced Fas-mediated apoptosis and caspase-8 aggregation and activation in Jurkat T cells. Importantly, STAP-2 directly interacted with caspase-8 and Fas, resulting in enhanced interactions between caspase-8 and FADD in the Fas-death-inducing signaling complex. Moreover, STAP-2 protein has a consensus caspase-8 cleavage sequence, VEAD, in its C-terminal domain, and processing of STAP-2 by caspase-8 was crucial for Fas-induced apoptosis. Physiologic roles of STAP-2 were confirmed by observations that STAP-2-deficient mice displayed impaired activation-induced cell death and superantigen-induced T cell depletion. Therefore, STAP-2 is a novel participant in the regulation of T cell apoptosis after stimulation.

Involvement of STAP-2 in Brk-mediated phosphorylation and activation of STAT5 in breast cancer cells.

Signal-transducing adaptor protein (STAP)-2 is a recently identified adaptor protein that contains Pleckstrin homology and Src homology 2-like domains, and is also known to be a substrate of breast tumor kinase (Brk). In a previous study, we found that STAP-2 upregulated Brk-mediated activation of signal transducer and activator of transcription (STAT) 3 in breast cancer cells. Here, we examined the involvement of STAP-2 in Brk-mediated STAT5 activation in breast cancer cells. Ectopic expression of STAP-2 induced Brk-mediated transcriptional activity of STAT5. Furthermore, STAP-2-knockdown in T47D breast cancer cells induced a marked decrease in proliferation that was as strong as that after Brk- or STAT5b-knockdown. Regarding the mechanism, the Pleckstrin homology domain of STAP-2 is likely to participate in the process by which Brk phosphorylates and activates STAT5. Taken together, our findings provide insights toward the development of novel therapeutic strategies as well as novel prognostic values in breast carcinomas.