Identification of new candidate drugs for primary Sjögren's syndrome using a drug repurposing transcriptomic approach.

OBJECTIVES: To date, no immunomodulatory drug has demonstrated its efficacy in primary SS (pSS). We sought to analyse potential commonalities between pSS transcriptomic signatures and signatures of various drugs or specific knock-in or knock-down genes. METHODS: Gene expression from peripheral blood samples of patients with pSS was compared with that of healthy controls in two cohorts and three public databases. In each of the five datasets, we analysed the 150 most up- and downregulated genes between pSS patients and controls with regard to the differentially expressed genes resulting from the biological action on nine cell lines of 2837 drugs, 2160 knock-in and 3799 knock-down genes in the Connectivity Map database. RESULTS: We analysed 1008 peripheral blood transcriptomes from five independent studies (868 patients with pSS and 140 healthy controls). Eleven drugs could represent potential candidate drugs, with histone deacetylases and PI3K inhibitors among the most significantly associated. Twelve knock-in genes were associated with a pSS-like profile and 23 knock-down genes were associated with a pSS-revert profile. Most of those genes (28/35, 80%) were interferon-regulated. CONCLUSION: This first drug repositioning transcriptomic approach in SS confirms the interest of targeting interferons and identifies histone deacetylases and PI3K inhibitors as potential therapeutic targets.

Short-hairpin RNAs delivered by recombinant adeno-associated virus inhibited the replication of influenza A viruses in vitro.

Antiviral short-hairpin RNAs (shRNAs) delivered by recombinant adeno-associated virus (rAAV) were investigated for their potential prophylactic and therapeutic applications related to the influenza A virus (IAV). To express shRNAs efficiently, an H1 promoter was inserted into the commercial rAAV2 system. The modified rAAV2 system could express shRNAs, and the purified rAAV was obtained at levels over 1013 viral genomes/ml and 1010 viral infection units/ml. The shNP-1496-n and shM2-925 delivered by rAAV could inhibit the replication of the H1N1 and H5N1 virus by targeting the conserved regions of the IAV nucleoprotein and matrix 2 genes in MDCK cells. The shNP-1496-n and shM2-925 expressed by rAAV could provide potent and long-term anti-H5N1 virus effects in rAAV-shRNA-enriched MDCK cells. Our findings provide a rational basis for developing RNA interference for the prevention and therapy of IAV infection.

Functional dissection of an IFN-alpha/beta receptor 1 promoter variant that confers higher risk to chronic hepatitis B virus infection.

BACKGROUND/AIMS: We previously demonstrated that two linked single nucleotide polymorphisms (SNPs) at -408 and -3 of type I interferon receptor 1 (IFNAR1) promoter are associated with susceptibility to chronic HBV infection. We aimed to elucidate the mechanism by which -3 and/or -408 C/T SNPs had such profound effects. METHODS: A functional SNP in IFNAR1 promoter was defined by reporter gene assay, mutational analysis, flow cytometry analysis and gel shift assay. The nuclear protein binding to the essential polymorphic site was identified and its effect on transcriptional regulation of IFNAR1 was further demonstrated in a series of ex vivo and in vivo experiments. RESULTS: We found C>T change at the -3 locus reduced the transcriptional activity of IFNAR1 promoter. High mobility group B protein 1 (HMGB1) and PARP-1 were co-recruited to the IFNAR1 promoter to regulate its transcription. We demonstrated HMGB1-binding affinity to IFNAR1 promoter was reduced in the -3T variant. Additionally, PARP-1, a cofactor for IFNAR1 transcription activation, was significantly suppressed by HBV. CONCLUSION: Upon HBV infection, decreased binding affinity of HMGB1 to the IFNAR1 promoter -3T variant is aggravated by the suppressed PARP-1 expression caused by HBV, resulting in a further attenuated IFNAR1 expression. This compromises the antiviral and immuno-regulatory effects of IFN-alpha/beta, which may in turn affect the clinical outcome of HBV infection.

A regulatory polymorphism in interferon-gamma receptor 1 promoter is associated with the susceptibility to chronic hepatitis B virus infection.

The antiviral cascade triggered by interferon-gamma (IFN-gamma) represents a vital event for eradicating hepatitis B virus (HBV) in experimental animals. IFN-gamma signaling is mediated through the ligand binding to IFN-gamma receptor 1 (IFNGR1). Control of IFNGR1 expression level is one of the mechanisms by which cells modulate the potency of IFN-gamma signaling. In this study, we comprehensively investigated the single nucleotide polymorphisms (SNPs) in IFNGR1 gene and correlated their occurrence to susceptibility to HBV infection in a Chinese population. A total of 983 participants, including 361 chronic hepatitis B patients, 256 individuals who had spontaneously recovered from HBV infection, and 366 healthy control subjects, were enrolled in the study. Polymerase chain reaction-restriction fragment length polymorphism was used to identify seven SNPs (-611A/G, -56C/T, 40G/A, 95C/T, 130A/G, 20685A/G, 21227T/C) in IFNGR1 gene. We found that -56C and -56T allele were associated with viral clearance and viral persistence, respectively (P = 0.014). In a reporter-driven assay, we validated that the promoter variant with -56C exhibited a higher transcription level than that with -56T in HepG2 cells in a cell-type-specific pattern. We conclude that a functional -56C/T SNP in IFNGR1 promoter is associated with the clinical outcome of HBV infection in this Chinese population.

Environmental loadings of Active Pharmaceutical Ingredients from manufacturing facilities in Canada.

Recent evidence has revealed that cities with pharmaceutical manufacturers have elevated concentrations of Active Pharmaceutical Ingredients (APIs) in their receiving water bodies. The purpose of this study was to gather information on direct sewer discharges of APIs during their manufacturing and processing from five pharmaceutical manufacturing facilities in Ontario, Canada. Drug classes and maximum reported concentrations (ng/L) for which APIs were directly discharged included: antidepressants (paroxetine - 3380 and sertraline - 5100); mood stabilizer (carbamazepine - 575,000); antibiotics (penicillin - 14,300); analgesics (acetaminophen - 461,000; codeine - 49,200; ibuprofen - 344,000; naproxen - 253,000 and oxycodone 21,000); cardiovascular drugs (atorvastatin - 893 and metoprolol - 7,333,600) and those drugs used for blood pressure control (amlodipine - 22,900; diltiazem - 1,160,000; furosemide - 1,200,000 and verapamil - 7340). Based on flow and water usage data from the individual facilities, the maximum concentrations for acetaminophen, ibuprofen, carbamazepine, diltiazem and metoprolol correlate to approximately 200, 220, 390, 420 and 14,200 g respectively, of lost product being directly discharged to the sewers daily during active manufacturing. This survey demonstrates that direct point source discharges from pharmaceutical manufacturers represent a key source of pharmaceutical pollution to receiving sewersheds. Onsite recovery of product or treatment at pharmaceutical manufacturing or processing facilities to reduce the sewage loadings to receiving treatment plants, product loss and potential environmental loadings is strongly recommended.

An H5N1-based matrix protein 2 ectodomain tetrameric peptide vaccine provides cross-protection against lethal infection with H7N9 influenza virus.

In March 2013, a patient infected with a novel avian influenza A H7N9 virus was reported in China. Since then, there have been 458 confirmed infection cases and 177 deaths. The virus contains several human-adapted markers, indicating that H7N9 has pandemic potential. The outbreak of this new influenza virus highlighted the need for the development of universal influenza vaccines. Previously, we demonstrated that a tetrameric peptide vaccine based on the matrix protein 2 ectodomain (M2e) of the H5N1 virus (H5N1-M2e) could protect mice from lethal infection with different clades of H5N1 and 2009 pandemic H1N1 influenza viruses. In this study, we investigated the cross-protection of H5N1-M2e against lethal infection with the new H7N9 virus. Although five amino acid differences existed at positions 13, 14, 18, 20, and 21 between M2e of H5N1 and H7N9, H5N1-M2e vaccination with either Freund's adjuvant or the Sigma adjuvant system (SAS) induced a high level of anti-M2e antibody, which cross-reacted with H7N9-M2e peptide. A mouse-adapted H7N9 strain, A/Anhui/01/2013m, was used for lethal challenge in animal experiments. H5N1-M2e vaccination provided potent cross-protection against lethal challenge of the H7N9 virus. Reduced viral replication and histopathological damage of mouse lungs were also observed in the vaccinated mice. Our results suggest that the tetrameric H5N1-M2e peptide vaccine could protect against different subtypes of influenza virus infections. Therefore, this vaccine may be an ideal candidate for developing a universal vaccine to prevent the reemergence of avian influenza A H7N9 virus and the emergence of potential novel reassortants of influenza virus.

ANXA3/JNK Signaling Promotes Self-Renewal and Tumor Growth, and Its Blockade Provides a Therapeutic Target for Hepatocellular Carcinoma.

Frequent tumor relapse in hepatocellular carcinoma (HCC) has been commonly attributed to the presence of residual cancer stem cells (CSCs) after conventional treatments. We have previously identified and characterized CD133 to mark a specific CSC subset in HCC. In the present study, we found endogenous and secretory annexin A3 (ANXA3) to play pivotal roles in promoting cancer and stem cell-like features in CD133+ liver CSCs through a dysregulated JNK pathway. Blockade of ANXA3 with an anti-ANXA3 monoclonal antibody in vitro as well as in human HCC xenograft models resulted in a significant reduction in tumor growth and self-renewal. Clinically, ANXA3 expression in HCC patient sera closely associated with aggressive clinical features. Our results suggest that ANXA3 can serve as a novel diagnostic biomarker and that the inhibition of ANXA3 may be a viable therapeutic option for the treatment of CD133+ liver-CSC-driven HCC.

A novel CCR5 mutation selectively affects immunoreactivity and fusogenic property of the HIV co-receptor.

A Nepalese heterozygous carrier of a CCR5 mutant, designated 118delF, was characterized. There was a 3 basepair deletion at 352-354 in the CCR5 open reading frame, resulting in the deletion of the phe-118 residue located in the third transmembrane domain. The mutant protein has retained antigen specificity near the third extra-cellular loop (ECL3), but that of ECL2 is markedly reduced. The mutation has also abrogated HIV co-receptor activity. Clinically, the HIV disease had progressed slowly.

Wild type and mutant 2009 pandemic influenza A (H1N1) viruses cause more severe disease and higher mortality in pregnant BALB/c mice.

BACKGROUND: Pregnant women infected by the pandemic influenza A (H1N1) 2009 virus had more severe disease and higher mortality but its pathogenesis is still unclear. PRINCIPAL FINDINGS: We showed that higher mortality, more severe pneumonitis, higher pulmonary viral load, lower peripheral blood T lymphocytes and antibody responses, higher levels of proinflammatory cytokines and chemokines, and worse fetal development occurred in pregnant mice than non-pregnant controls infected by either wild type (clinical isolate) or mouse-adapted mutant virus with D222G substitution in hemagglutinin. These disease-associated changes and the lower respiratory tract involvement were worse in pregnant mice challenged by mutant virus. Though human placental origin JEG-3 cell line could be infected and proinflammatory cytokines or chemokines were elevated in amniotic fluid of some mice, no placental or fetal involvement by virus were detected by culture, real-time reverse transcription polymerase chain reaction or histopathological changes. Dual immunofluorescent staining of viral nucleoprotein and type II alveolar cell marker SP-C protein suggested that the majority of infected alveolar epithelial cells were type II pneumocytes. CONCLUSION: The adverse effect of this pandemic virus on maternal and fetal outcome is largely related to the severe pulmonary disease and the indirect effect of inflammatory cytokine spillover into the systemic circulation.

Polymorphisms of type I interferon receptor 1 promoter and their effects on chronic hepatitis B virus infection.

BACKGROUND/AIMS: Exposure to HBV leads to a distinct clinical course which is partially pertained to host genetic variability. We aimed to study polymorphisms of type I interferon receptor 1 (IFNAR1) promoter and their potential effects on chronic HBV infection. METHODS: Polymorphisms of IFNAR1 promoter were identified in 320 chronic hepatitis B patients, 148 spontaneously recovered individuals, 148 healthy Chinese donors and 114 Caucasians. Their functional capability in driving reporter gene expression was analyzed. RESULTS: Four polymorphic alleles were identified at loci -568, -408, -77 and -3. Association analysis revealed that carriers of alleles -568G, -408C and their related haplotype I were less susceptible to chronic HBV infection whereas those of alleles -568C, -408T and related haplotype III were significantly associated with higher risk to chronic hepatitis B (P<0.01). In a reporter-driven system, the promoter variants with alleles -408C and -3C could drive higher expression of the reporter gene than those with alleles -408T and -3T (P<0.01). Interestingly, an allele with 9 GT repeats at -77 that was rarely found in Chinese but prevalent in Caucasian exhibited the highest transcriptional ability. CONCLUSIONS: Our results showed that polymorphisms of IFNAR1 promoter may affect, at least in part, the outcomes of HBV infection.

Recombinant adeno-associated virus expressing the receptor-binding domain of severe acute respiratory syndrome coronavirus S protein elicits neutralizing antibodies: Implication for developing SARS vaccines.

Development of an effective vaccine for severe acute respiratory syndrome (SARS) remains to be a priority to prevent possible re-emergence of SARS coronavirus (SARS-CoV). We previously demonstrated that the receptor-binding domain (RBD) of SARS-CoV S protein is a major target of neutralizing antibodies. This suggests that the RBD may serve as an ideal vaccine candidate. Recombinant adeno-associated virus (rAAV) has been proven to be an effective system for gene delivery and vaccine development. In this study, a novel vaccine against SARS-CoV was developed based on the rAAV delivery system. The gene encoding RBD was cloned into a pAAV-IRES-hrGFP plasmid. The immunogenicity induced by the resulting recombinant RBD-rAAV was evaluated in BALB/c mice. The results demonstrated that (1) a single dose of RBD-rAAV vaccination could induce sufficient neutralizing antibody against SARS-CoV infection; (2) two more repeated doses of the vaccination boosted the neutralizing antibody to about 5 times of the level achieved by a single dose of the immunization and (3) the level of the antibody continued to increase for the entire duration of the experiment of 5.5 months. These results suggested that RBD-rAAV is a promising SARS candidate vaccine.

Functional analysis of naturally occurring mutations in the open reading frame of CCR5 in HIV-infected Chinese patients and healthy controls.

We studied polymorphism of the HIV coreceptor CC chemokine receptor (CCR) 5 in 1099 Chinese adults residing in Hong Kong, including 785 HIV-negative healthy donors and 314 HIV-positive patients. Ten mutants in the CCR5 open reading frame were identified, 7 of which were nonsynonymous. The frequencies of these alleles did not show a significant difference between HIV patients and healthy controls. G106R, Delta32, R223Q, 299(FS), and S336I were cloned from prevalent mutant genes, and their effects on HIV infection were analyzed by a series of in vitro experiments to determine their transcription levels, expression levels, conformational changes, and HIV coreceptor function. R223Q is the most prevalent CCR5 mutant in ethnic Chinese, with a frequency of 0.046, which does not affect HIV infection in vitro, however. The S336I mutant also does not affect its transcription, expression, or HIV coreceptor function. Similar to 299(FS), the mutant G106R located in the third transmembrane domain results in diminished HIV coreceptor function in vitro through conformation changes in ECL2.

Interobserver variation of prostatic volume estimation with digital rectal examination by urological staffs with different experiences.

PURPOSE: To compare the accuracy of estimating prostatic volume with digital rectal examination (DRE) by urological staffs with different experiences. Measurement of prostatic volume with transrectal ultrasonography (TRUS) serves as the reference standard. MATERIALS AND METHODS: Thirty-nine consecutive male patients admitted with acute urinary retention had their prostatic volume estimated with DRE by a urology junior trainee, a urology higher trainee and a trained urologist. All patients had TRUS to measure their prostatic volumes. Pearson correlation coefficients (r) were used to assess the relationships between the prostatic volume measured with TRUS and that estimated with DRE by the 3 urological staffs. Wilcoxon signed ranks tests were used to compare the discrepancies between the prostatic volume measured with TRUS and that estimated with DRE for the 3 Urological staffs, and to assess the inter-observer differences of these discrepancies. RESULTS: The correlation coefficients for the 3 urological staffs were r = 0.573 for the urology junior trainee, r = 0.541 for the urology higher trainee, and r = 0.640 for the trained urologist. The median discrepancies between the prostatic volume measured with TRUS and that estimated with DRE were -9.1 mL for the urology junior trainee, -1.3 mL for the urology higher trainee and 0.9 mL for the trained urologist. These discrepancies were statistically significant only in the case of urology junior trainee (p = 0.015, Wilcoxon signed ranks test). The difference in these discrepancies was statistically significant only between the urology junior trainee and the trained urologist (p = 0.003, Wilcoxon signed ranks test). CONCLUSIONS: The trained urologist was more accurate in estimating prostatic volume with DRE than the urology junior trainee.

Cutting edge: CTLA-4 (CD152) differentially regulates mitogen-activated protein kinases (extracellular signal-regulated kinase and c-Jun N-terminal kinase) in CD4+ T cells from receptor/ligand-deficient mice.

Although CTLA-4 (CD152) has potent inhibitory effects on T cell function, the signaling events affected by this coreceptor remain to be fully defined. Mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) act as crucial regulators of multiple aspects of cell function. Ab ligation studies have reported an inhibitory effect of CTLA-4 on TCR-induced ERK and JNK activation. In this study, we have re-examined the specificity of CTLA-4 inhibition of MAPKs by using natural ligand with ex vivo-purified CD4(+) T cells deficient in CD80 and CD86 (double knockout), or CTLA-4, CD80, and CD86 (triple knockout). Under these conditions, CTLA-4 ligation was found to up-regulate and sustain JNK activation, while inhibiting ERK activity. At the same time, JNK activation could not account for CTLA-4 induction of TGF-beta production. Our findings demonstrate that CTLA-4 cosignaling is more complex than previously appreciated, with an ability to differentially regulate members of the MAPK family in T cells.

EBV specific antibody-based and DNA-based assays in serologic diagnosis of nasopharyngeal carcinoma.

We assessed 5 EBV specific assays for their capacity to effect serologic diagnosis of suspected NPC. The assays were the immunofluorescent assays, VCA IgA and EA IgA, the enzyme-linked immunosorbent assays specific for EBNA 1 IgA or zta IgG and an EBV DNA assay. Serum samples were taken from 218 symptomatic NPC patients presenting consecutively at a public hospital in Hong Kong, 51 of whom were subsequently diagnosed as having NPC; 4 had EBV-associated lung cancer with similar serology as NPC. The remaining patients included 23 who had other cancers and 140 who had other diseases. Objectives of serodiagnosis under such clinical settings, therefore, are to both exclude and predict a diagnosis of NPC. None of the assays individually can meet both requirements adequately, however. The difficulty was best overcome by combining EBNA 1 IgA and zta IgG. It was shown that 68.3% of the patients gave a confirmed test results, negative or positive, by both tests. A confirmed negative result was associated with a negative predictive value of 99.1%, providing a clear indication to exclude a diagnosis of NPC; a confirmed positive result was associated with a positive predictive value of 86.8%, providing a clear indication to proceed with diagnostic work-up of NPC. The remaining patients gave equivocal test results, being positive for one or the other test, which were associated with a positive predictive value of 43.3% and 24.2%, respectively.

Interobserver variation of prostatic volume estimation with digital rectal examination by urological staffs with different experiences

PURPOSE: To compare the accuracy of estimating prostatic volume with digital rectal examination (DRE) by urological staffs with different experiences. Measurement of prostatic volume with transrectal ultrasonography (TRUS) serves as the reference standard. MATERIALS AND METHODS: Thirty-nine consecutive male patients admitted with acute urinary retention had their prostatic volume estimated with DRE by a urology junior trainee, a urology higher trainee and a trained urologist. All patients had TRUS to measure their prostatic volumes. Pearson correlation coefficients (r) were used to assess the relationships between the prostatic volume measured with TRUS and that estimated with DRE by the 3 urological staffs. Wilcoxon signed ranks tests were used to compare the discrepancies between the prostatic volume measured with TRUS and that estimated with DRE for the 3 Urological staffs, and to assess the inter-observer differences of these discrepancies. RESULTS: The correlation coefficients for the 3 urological staffs were r = 0.573 for the urology junior trainee, r = 0.541 for the urology higher trainee, and r = 0.640 for the trained urologist. The median discrepancies between the prostatic volume measured with TRUS and that estimated with DRE were -9.1 mL for the urology junior trainee, -1.3 mL for the urology higher trainee and 0.9 mL for the trained urologist. These discrepancies were statistically significant only in the case of urology junior trainee (p = 0.015, Wilcoxon signed ranks test). The difference in these discrepancies was statistically significant only between the urology junior trainee and the trained urologist (p = 0.003, Wilcoxon signed ranks test). CONCLUSIONS: The trained urologist was more accurate in estimating prostatic volume with DRE than the urology junior trainee.