Genetically and Antigenically Divergent Influenza A(H9N2) Viruses Exhibit Differential Replication and Transmission Phenotypes in Mammalian Models.

Low-pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Cocirculation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses.IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported, and it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam.

Development of an RNA Strand-Specific Hybridization Assay To Differentiate Replicating versus Nonreplicating Influenza A Viruses.

Replication of influenza A virus (IAV) from negative-sense viral RNA (vRNA) requires the generation of positive-sense RNA (+RNA). Most molecular assays, such as conventional real-time reverse transcriptase PCR (rRT-PCR), detect total RNA in a sample without differentiating vRNA from +RNA. These assays are not designed to distinguish IAV infection versus exposure of an individual to an environment enriched with IAVs but wherein no viral replication occurs. We therefore developed a strand-specific hybridization (SSH) assay that differentiates between vRNA and +RNA and quantifies relative levels of each RNA species. The SSH assay exhibited a linearity of 7 logs with a lower limit of detection of 6.0 × 102 copies of molecules per reaction. No signal was detected in samples with a high load of nontarget template or influenza B virus, demonstrating assay specificity. IAV +RNA was detected 2 to 4 h postinoculation of MDCK cells, whereas synthesis of cold-adapted IAV +RNA was significantly impaired at 37°C. The SSH assay was then used to test IAV rRT-PCR positive nasopharyngeal specimens collected from individuals exposed to IAV at swine exhibitions (n = 7) or while working at live bird markets (n = 2). The SSH assay was able to differentiate vRNA and +RNA in samples collected from infected, symptomatic individuals versus individuals who were exposed to IAV in the environment but had no active viral replication. Data generated with this technique, especially when coupled with clinical data and assessment of seroconversion, will facilitate differentiation of actual IAV infection with replicating virus versus individuals exposed to high levels of environmental contamination but without virus infection.

Shifting Clade Distribution, Reassortment, and Emergence of New Subtypes of Highly Pathogenic Avian Influenza A(H5) Viruses Collected from Vietnamese Poultry from 2012 to 2015.

Whole-genome sequences of representative highly pathogenic avian influenza A(H5) viruses from Vietnam were generated, comprising samples from poultry outbreaks and active market surveillance collected from January 2012 to August 2015. Six hemagglutinin gene clades were characterized. Clade 1.1.2 was predominant in southern Mekong provinces throughout 2012 and 2013 but gradually disappeared and was not detected after April 2014. Clade 2.3.2.1c viruses spread rapidly during 2012 and were detected in the south and center of the country. A number of clade 1.1.2 and 2.3.2.1c interclade reassortant viruses were detected with different combinations of internal genes derived from 2.3.2.1a and 2.3.2.1b viruses, indicating extensive cocirculation. Although reassortment generated genetic diversity at the genotype level, there was relatively little genetic drift within the individual gene segments, suggesting genetic stasis over recent years. Antigenically, clade 1.1.2, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c viruses remained related to earlier viruses and WHO-recommended prepandemic vaccine strains representing these clades. Clade 7.2 viruses, although detected in only low numbers, were the exception, as indicated by introduction of a genetically and antigenically diverse strain in 2013. Clade 2.3.4.4 viruses (H5N1 and H5N6) were likely introduced in April 2014 and appeared to gain dominance across northern and central regions. Antigenic analyses of clade 2.3.4.4 viruses compared to existing clade 2.3.4 candidate vaccine viruses (CVV) indicated the need for an updated vaccine virus. A/Sichuan/26221/2014 (H5N6) virus was developed, and ferret antisera generated against this virus were demonstrated to inhibit some but not all clade 2.3.4.4 viruses, suggesting consideration of alternative clade 2.3.4.4 CVVs.IMPORTANCE Highly pathogenic avian influenza (HPAI) A(H5) viruses have circulated continuously in Vietnam since 2003, resulting in hundreds of poultry outbreaks and sporadic human infections. Despite a significant reduction in the number of human infections in recent years, poultry outbreaks continue to occur and the virus continues to diversify. Vaccination of poultry has been used as a means to control the spread and impact of the virus, but due to the diversity and changing distribution of antigenically distinct viruses, the utility of vaccines in the face of mismatched circulating strains remains questionable. This study assessed the putative amino acid changes in viruses leading to antigenic variability, underscoring the complexity of vaccine selection for both veterinary and public health purposes. Given the overlapping geographic distributions of multiple, antigenically distinct clades of HPAI A(H5) viruses in Vietnam, the vaccine efficacy of bivalent poultry vaccine formulations should be tested in the future.

Assessment of Molecular, Antigenic, and Pathological Features of Canine Influenza A(H3N2) Viruses That Emerged in the United States.

Background: A single subtype of canine influenza virus (CIV), A(H3N8), was circulating in the United States until a new subtype, A(H3N2), was detected in Illinois in spring 2015. Since then, this CIV has caused thousands of infections in dogs in multiple states. Methods: In this study, genetic and antigenic properties of the new CIV were evaluated. In addition, structural and glycan array binding features of the recombinant hemagglutinin were determined. Replication kinetics in human airway cells and pathogenesis and transmissibility in animal models were also assessed. Results: A(H3N2) CIVs maintained molecular and antigenic features related to low pathogenicity avian influenza A(H3N2) viruses and were distinct from A(H3N8) CIVs. The structural and glycan array binding profile confirmed these findings and revealed avian-like receptor-binding specificity. While replication kinetics in human airway epithelial cells was on par with that of seasonal influenza viruses, mild-to-moderate disease was observed in infected mice and ferrets, and the virus was inefficiently transmitted among cohoused ferrets. Conclusions: Further adaptation is needed for A(H3N2) CIVs to present a likely threat to humans. However, the potential for coinfection of dogs and possible reassortment of human and other animal influenza A viruses presents an ongoing risk to public health.

Multiplex assay for subtyping avian influenza A viruses by cDNA hybridization and adapter-mediated amplification.

Multiple subtypes of influenza A viruses circulating in animals must be closely monitored to understand their risk to humans and animal populations. Many molecular-based subtyping methods require constant monitoring of viral genomes for primer and/or probe mismatches and are prone to primer-primer interactions. This report presents a new approach that involves target enrichment through cDNA hybridization followed by adapter-mediated amplification for subtyping influenza virus (AmASIV). As a proof of concept, the AmASIV assay was multiplexed to specifically detect and differentiate influenza A virus subtypes (H5, N5, N7, and N9) in a single reaction without cross-recognition of nontarget subtypes or influenza B virus. The limit of detection (LOD) of AmASIV, as measured by 50 % egg-infective dose per reaction (EID50/reaction), was comparable to that of singleplex TaqMan® qPCR assays with LODs of 10(-0.6) (H5), 10(2) (N5), 10(-0.3) (N7), and 10(-0.5) (N9) EID50/reaction. The AmASIV will strengthen animal influenza virus surveillance and laboratory capacity to improve prevention and control of influenza.

Heme oxygenase-1 gene promoter polymorphism is associated with reduced incidence of acute chest syndrome among children with sickle cell disease.

Sickle cell disease is a common hemolytic disorder with a broad range of complications, including vaso-occlusive episodes, acute chest syndrome (ACS), pain, and stroke. Heme oxygenase-1 (gene HMOX1; protein HO-1) is the inducible, rate-limiting enzyme in the catabolism of heme and might attenuate the severity of outcomes from vaso-occlusive and hemolytic crises. A (GT)(n) dinucleotide repeat located in the promoter region of the HMOX1 gene is highly polymorphic, with long repeat lengths linked to decreased activity and inducibility. We examined this polymorphism to test the hypothesis that short alleles are associated with a decreased risk of adverse outcomes (hospitalization for pain or ACS) among a cohort of 942 children with sickle cell disease. Allele lengths varied from 13 to 45 repeats and showed a trimodal distribution. Compared with children with longer allele lengths, children with 2 shorter alleles (4%; ≤ 25 repeats) had lower rates of hospitalization for ACS (incidence rate ratio 0.28, 95% confidence interval, 0.10-0.81), after adjusting for sex, age, asthma, percentage of fetal hemoglobin, and α-globin gene deletion. No relationship was identified between allele lengths and pain rate. We provide evidence that genetic variation in HMOX1 is associated with decreased rates of hospitalization for ACS, but not pain. This study is registered at www.clinicaltrials.gov as #NCT00072761.

Characterization of Avian Influenza Viruses Detected in Kenyan Live Bird Markets and Wild Bird Habitats Reveal Genetically Diverse Subtypes and High Proportion of A(H9N2), 2018-2020.

Following the detection of highly pathogenic avian influenza (HPAI) virus in countries bordering Kenya to the west, we conducted surveillance among domestic and wild birds along the shores of Lake Victoria. In addition, between 2018 and 2020, we conducted surveillance among poultry and poultry workers in live bird markets and among wild migratory birds in various lakes that are resting sites during migration to assess introduction and circulation of avian influenza viruses in these populations. We tested 7464 specimens (oropharyngeal (OP) and cloacal specimens) from poultry and 6531 fresh fecal specimens from wild birds for influenza A viruses by real-time RT-PCR. Influenza was detected in 3.9% (n = 292) of specimens collected from poultry and 0.2% (n = 10) of fecal specimens from wild birds. On hemagglutinin subtyping, most of the influenza A positives from poultry (274/292, 93.8%) were H9. Of 34 H9 specimens randomly selected for further subtyping, all were H9N2. On phylogenetic analysis, these viruses were genetically similar to other H9 viruses detected in East Africa. Only two of the ten influenza A-positive specimens from the wild bird fecal specimens were successfully subtyped; sequencing analysis of one specimen collected in 2018 was identified as a low-pathogenicity avian influenza H5N2 virus of the Eurasian lineage, and the second specimen, collected in 2020, was subtyped as H11. A total of 18 OP and nasal specimens from poultry workers with acute respiratory illness (12%) were collected; none were positive for influenza A virus. We observed significant circulation of H9N2 influenza viruses in poultry in live bird markets in Kenya. During the same period, low-pathogenic H5N2 virus was detected in a fecal specimen collected in a site hosting a variety of migratory and resident birds. Although HPAI H5N8 was not detected in this survey, these results highlight the potential for the introduction and establishment of highly pathogenic avian influenza viruses in poultry populations and the associated risk of spillover to human populations.

Antigenic Characterization of Circulating and Emerging SARS-CoV-2 Variants in the U.S. throughout the Delta to Omicron Waves.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into numerous lineages with unique spike mutations and caused multiple epidemics domestically and globally. Although COVID-19 vaccines are available, new variants with the capacity for immune evasion continue to emerge. To understand and characterize the evolution of circulating SARS-CoV-2 variants in the U.S., the Centers for Disease Control and Prevention (CDC) initiated the National SARS-CoV-2 Strain Surveillance (NS3) program and has received thousands of SARS-CoV-2 clinical specimens from across the nation as part of a genotype to phenotype characterization process. Focus reduction neutralization with various antisera was used to antigenically characterize 143 SARS-CoV-2 Delta, Mu and Omicron subvariants from selected clinical specimens received between May 2021 and February 2023, representing a total of 59 unique spike protein sequences. BA.4/5 subvariants BU.1, BQ.1.1, CR.1.1, CQ.2 and BA.4/5 + D420N + K444T; BA.2.75 subvariants BM.4.1.1, BA.2.75.2, CV.1; and recombinant Omicron variants XBF, XBB.1, XBB.1.5 showed the greatest escape from neutralizing antibodies when analyzed against post third-dose original monovalent vaccinee sera. Post fourth-dose bivalent vaccinee sera provided better protection against those subvariants, but substantial reductions in neutralization titers were still observed, especially among BA.4/5 subvariants with both an N-terminal domain (NTD) deletion and receptor binding domain (RBD) substitutions K444M + N460K and recombinant Omicron variants. This analysis demonstrated a framework for long-term systematic genotype to antigenic characterization of circulating and emerging SARS-CoV-2 variants in the U.S., which is critical to assessing their potential impact on the effectiveness of current vaccines and antigen recommendations for future updates.

Acute chest syndrome is associated with single nucleotide polymorphism-defined beta globin cluster haplotype in children with sickle cell anaemia.

Genetic diversity at the human ß-globin locus has been implicated as a modifier of sickle cell anaemia (SCA) severity. However, haplotypes defined by restriction fragment length polymorphism sites across the ß-globin locus have not been consistently associated with clinical phenotypes. To define the genetic structure at the ß-globin locus more thoroughly, we performed high-density single nucleotide polymorphism (SNP) mapping in 820 children who were homozygous for the sickle cell mutation (HbSS). Genotyping results revealed very high linkage disequilibrium across a large region spanning the locus control region and the HBB (ß-globin gene) cluster. We identified three predominant haplotypes accounting for 96% of the ß(S) -carrying chromosomes in this population that could be distinguished using a minimal set of common SNPs. Consistent with previous studies, fetal haemoglobin level was significantly associated with ß(S) -haplotypes. After controlling for covariates, an association was detected between haplotype and rate of hospitalization for acute chest syndrome (ACS) (incidence rate ratio 0·51, 95% confidence interval 0·29-0·89) but not incidence rate of vaso-occlusive pain or presence of silent cerebral infarct (SCI). Our results suggest that these SNP-defined ß(S) -haplotypes may be associated with ACS, but not pain or SCI in a study population of children with SCA.

Avian influenza A(H5) virus circulation in live bird markets in Vietnam, 2017-2022.

BACKGROUND: Highly pathogenic avian influenza A(H5) human infections are a global concern, with many A(H5) human cases detected in Vietnam, including a case in October 2022. Using avian influenza virus surveillance from March 2017-September 2022, we described the percent of pooled samples that were positive for avian influenza A, A(H5), A(H5N1), A(H5N6), and A(H5N8) viruses in live bird markets (LBMs) in Vietnam. METHODS: Monthly at each LBM, 30 poultry oropharyngeal swab specimens and five environmental samples were collected. Samples were pooled in groups of five and tested for influenza A, A(H5), A(H5N1), A(H5N6), and A(H5N8) viruses by real-time reverse-transcription polymerase chain reaction. Trends in the percent of pooled samples that were positive for avian influenza were summarized by LBM characteristics and time and compared with the number of passively detected avian influenza outbreaks using Spearman's rank correlation. RESULTS: A total of 25,774 pooled samples were collected through active surveillance at 167 LBMs in 24 provinces; 36.9% of pooled samples were positive for influenza A, 3.6% A(H5), 1.9% A(H5N1), 1.1% A(H5N6), and 0.2% A(H5N8). Influenza A(H5) viruses were identified January-December and at least once in 91.7% of sampled provinces. In 246 A(H5) outbreaks in poultry; 20.3% were influenza A(H5N1), 60.2% A(H5N6), and 19.5% A(H5N8); outbreaks did not correlate with active surveillance. CONCLUSIONS: In Vietnam, influenza A(H5) viruses were detected by active surveillance in LBMs year-round and in most provinces sampled. In addition to outbreak reporting, active surveillance for A(H5) viruses in settings with high potential for animal-to-human spillover can provide situational awareness.

Magnetic resonance angiography-defined intracranial vasculopathy is associated with silent cerebral infarcts and glucose-6-phosphate dehydrogenase mutation in children with sickle cell anaemia.

Silent cerebral infarct (SCI) is the most commonly recognized cause of neurological injury in sickle cell anaemia (SCA). We tested the hypothesis that magnetic resonance angiography (MRA)-defined vasculopathy is associated with SCI. Furthermore, we examined genetic variations in glucose-6-phosphate dehydrogenase (G6PD) and HBA (α-globin) genes to determine their association with intracranial vasculopathy in children with SCA. Magnetic resonance imaging (MRI) of the brain and MRA of the cerebral vasculature were available in 516 paediatric patients with SCA, enrolled in the Silent Infarct Transfusion (SIT) Trial. All patients were screened for G6PD mutations and HBA deletions. SCI were present in 41·5% (214 of 516) of SIT Trial children. The frequency of intracranial vasculopathy with and without SCI was 15·9% and 6·3%, respectively (P < 0·001). Using a multivariable logistic regression model, only the presence of a SCI was associated with increased odds of vasculopathy (P = 0·0007, odds ratio (OR) 2·84; 95% Confidence Interval (CI) = 1·55-5·21). Among male children with SCA, G6PD status was associated with vasculopathy (P = 0·04, OR 2·78; 95% CI = 1·04-7·42), while no significant association was noted for HBA deletions. Intracranial vasculopathy was observed in a minority of children with SCA, and when present, was associated with G6PD status in males and SCI.

Legionella nagasakiensis sp. nov., isolated from water samples and from a patient with pneumonia.

A novel Legionella species was identified based on analysis of 16S rRNA and mip (macrophage infectivity potentiator) gene sequences, cellular fatty acids, isoprenoid quinones, biochemical reactions, antigens and quantitative DNA-DNA hybridization. Strain CDC-1796-JAP-E(T) was isolated from well water at the Nagasaki Municipal Medical Center, Japan. Two strains, CDC-3041-AUS-E and CDC-3558-AUS-E, were isolated from water samples during an outbreak of legionellosis in South Australia. Strain CDC-5427-OH-H was isolated from a 66-year-old female patient diagnosed with Legionnaires' disease in the US. Cells from these four strains were gram-negative, non-fluorescent, rod-shaped, and positive for alkaline phosphatase, esterase, leucine arylamidase, catalase, gelatinase, ß-lactamase and tyrosine browning assay. Phylogenetic analysis of 16S rRNA and mip genes revealed that the four strains formed a distinct cluster within the genus Legionella. The bacteria contained branched-chain fatty acids and quinones that are typical of members of the genus Legionella. Slide agglutination tests demonstrated no cross-reaction with 52 previously described members of the Legionellaceae. DNA-DNA hybridization studies indicated that DNAs from the four strains were highly related (78-84 %) but they showed 29 % relatedness to Legionella oakridgensis ATCC 33761(T) and less than 10 % to strains of other Legionella species tested. These characterizations suggest that the isolates represent a novel species, for which the name Legionella nagasakiensis sp. nov. is proposed; the type strain is CDC-1796-JAP-E(T) ( = ATCC BAA-1557(T) = JCM 15315(T)).

Application of TaqMan low-density arrays for simultaneous detection of multiple respiratory pathogens.

The large and growing number of viral and bacterial pathogens responsible for respiratory infections poses a challenge for laboratories seeking to provide rapid and comprehensive pathogen identification. We evaluated a novel application of the TaqMan low-density array (TLDA) cards for real-time PCR detection of 21 respiratory-pathogen targets. The performance of the TLDA was compared to that of individual real-time PCR (IRTP) assays with the same primers and probes using (i) nucleic acids extracted from the 21 pathogen strains and 66 closely related viruses and bacteria and (ii) 292 clinical respiratory specimens. With spiked samples, TLDA cards were about 10-fold less sensitive than IRTP assays. By using 292 clinical specimens to generate 2,238 paired individual assays, the TLDA card exhibited 89% sensitivity (95% confidence interval [CI], 86 to 92%; range per target, 47 to 100%) and 98% specificity (95% CI, 97 to 99%; range per target, 85 to 100%) overall compared to IRTP assays as the gold standard with a threshold cycle (C(T)) cutoff of 43. The TLDA card approach offers promise for rapid and simultaneous identification of multiple respiratory pathogens for outbreak investigations and disease surveillance.

Laboratory evaluation of two point-of-care detection systems for early and accurate detection of influenza viruses in the Lao People's Democratic Republic.

BACKGROUND: We evaluated molecular-based point-of-care influenza virus detection systems in a laboratory prior to a field evaluation of on-site specimen testing. METHODS: The performance characteristics of 1) insulated isothermal polymerase chain reaction (PCR) on a POCKIT™ device and 2) real-time reverse transcription-PCR (rRT-PCR) on a MyGo Mini™ device were evaluated using human clinical specimens, beta-propiolactone-inactivated influenza viruses, and RNA controls. The rRT-PCR carried out on a CXF-96™ real-time detection system was used as a gold standard for comparison. RESULTS: Both systems demonstrated 100% sensitivity and specificity and test results were in 100% agreement with the gold standard. POCKIT™ only correctly identified influenza A (M gene) in clinical specimens due to the unavailability of typing and subtyping reagents for human influenza viruses, while MyGo Mini™ had either a one log higher or the same sensitivity in detecting influenza viruses in clinical specimens compared to the gold standard. For inactivated viruses and/or viral RNA, the analytic sensitivity of POCKIT™ was shown to be comparable to, or more sensitive, than the gold standard. The analytic sensitivity of MyGo Mini™ had mixed results depending on the types and subtypes of influenza viruses. CONCLUSIONS: The performance of the two systems in a laboratory is promising and supports further evaluation in field settings.

Detection of highly pathogenic avian influenza A(H5N6) viruses in waterfowl in Bangladesh.

Bangladesh has reported repeated outbreaks of highly pathogenic avian influenza (HPAI) A(H5) viruses in poultry since 2007. Because of the large number of live poultry markets (LPM) relative to the population density of poultry throughout the country, these markets can serve as sentinel sites for HPAI A(H5) detection. Through active LPM surveillance during June 2016-June 2017, HPAI A(H5N6) viruses along with 14 other subtypes of influenza A viruses were detected. The HPAI A(H5N6) viruses belonged to clade 2.3.4.4 and were likely introduced into Bangladesh around March 2016. Human infections with influenza clade 2.3.4.4 viruses in Bangladesh have not been identified, but the viruses had several molecular markers associated with potential human infection. Vigilant surveillance at the animal-human interface is essential to identify emerging avian influenza viruses with the potential to threaten public and animal health.

Transcriptional repression of the Neurofibromatosis-1 tumor suppressor by the t(8;21) fusion protein.

Von Recklinghausen's disease is a relatively common familial genetic disorder characterized by inactivating mutations of the Neurofibromatosis-1 (NF1) gene that predisposes these patients to malignancies, including an increased risk for juvenile myelomonocytic leukemia. However, NF1 mutations are not common in acute myeloid leukemia (AML). Given that the RUNX1 transcription factor is the most common target for chromosomal translocations in acute leukemia, we asked if NF1 might be regulated by RUNX1. In reporter assays, RUNX1 activated the NF1 promoter and cooperated with C/EBPalpha and ETS2 to activate the NF1 promoter over 80-fold. Moreover, the t(8;21) fusion protein RUNX1-MTG8 (R/M), which represses RUNX1-regulated genes, actively repressed the NF1 promoter. R/M associated with the NF1 promoter in vivo and repressed endogenous NF1 gene expression. In addition, similar to loss of NF1, R/M expression enhanced the sensitivity of primary myeloid progenitor cells to granulocyte-macrophage colony-stimulating factor. Our results indicate that the NF1 tumor suppressor gene is a direct transcriptional target of RUNX1 and the t(8;21) fusion protein, suggesting that suppression of NF1 expression contributes to the molecular pathogenesis of AML.

Loss of neurofibromatosis-1 and p19(ARF) cooperate to induce a multiple tumor phenotype.

Inactivation of the neurofibromatosis-1 (NF1) gene de-regulates RAS and cooperates with mutation or loss of the p53 tumor suppressor to induce tumorigenesis. p19(ARF) acts upstream of p53 in an oncogene checkpoint to induce apoptosis in response to activated RAS and other factors that stimulate proliferation. Therefore, we bred p19(ARF-/-) to NF1(+/-) mice to determine if loss of these genes collaborates in tumorigenesis. As expected from the embryonic lethality of NF1 null mice, no mice lacking both p19(ARF) and NF1 were born. Unexpectedly, the loss of one allele of NF1 did not greatly shorten the time to tumor formation in a p19(ARF) null background. The tumor types observed were characteristic of p19(ARF) null animals, not those associated with neurofibromatosis or those observed with NF1(+/-)/p53(+/-) mice. However, seven out of 12 animals developed multiple tumors, some with metastases. This multiple tumor phenotype was not previously observed with p19(ARF)-null mice and suggests a distinct form of cooperation between the loss of these tumor suppressors.

Role of Epithelial-Mesenchyme Transition in Chlamydia Pathogenesis.

Chlamydia trachomatis genital infection in women causes serious adverse reproductive complications, and is a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma. We tested the hypothesis that Chlamydia induces epithelial-mesenchyme transition (EMT) involving T cell-derived TNF-alpha signaling, caspase activation, cleavage inactivation of dicer and dysregulation of micro-RNA (miRNA) in the reproductive epithelium; the pathologic process of EMT causes fibrosis and fertility-related epithelial dysfunction, and also provides the co-factor function for HPV-related cervical epithelial carcinoma. Using a combination of microarrays, immunohistochemistry and proteomics, we showed that chlamydia altered the expression of crucial miRNAs that control EMT, fibrosis and tumorigenesis; specifically, miR-15a, miR-29b, miR-382 and MiR-429 that maintain epithelial integrity were down-regulated, while miR-9, mi-R-19a, miR-22 and miR-205 that promote EMT, fibrosis and tumorigenesis were up-regulated. Chlamydia induced EMT in vitro and in vivo, marked by the suppression of normal epithelial cell markers especially E-cadherin but up-regulation of mesenchymal markers of pathological EMT, including T-cadherin, MMP9, and fibronectin. Also, Chlamydia upregulated pro-EMT regulators, including the zinc finger E-box binding homeobox protein, ZEB1, Snail1/2, and thrombospondin1 (Thbs1), but down-regulated anti-EMT and fertility promoting proteins (i.e., the major gap junction protein connexin 43 (Cx43), Mets1, Add1Scarb1 and MARCKSL1). T cell-derived TNF-alpha signaling was required for chlamydial-induced infertility and caspase inhibitors prevented both infertility and EMT. Thus, chlamydial-induced T cell-derived TNF-alpha activated caspases that inactivated dicer, causing alteration in the expression of reproductive epithelial miRNAs and induction of EMT. EMT causes epithelial malfunction, fibrosis, infertility, and the enhancement of tumorigenesis of HPV oncogene-transformed epithelial cells. These findings provide a novel understanding of the molecular pathogenesis of chlamydia-associated diseases, which may guide a rational prevention strategy.

Nuclear Factors Enhance the Binding of Estrogen Receptor to Estrogen Response Element.

We obtained the full length of human estrogen receptor (hER) through the in vitro translation. It was shown that the translated product could bind to the estrogen response element (ERE). The nuclear extract prepared from the rat uterus after ovariectomy could enhance the binding of hER-ERE in an estrogen dependent manner. However, the enhancing effect was sharply decreased when the nuclear extract was pre-incubated at 50 degrees for 15 minutes before being used for the binding reaction. These results indicated the presence in the rat uterus extracts after ovariectomy of a heat labile factor that can enhance the binding of hER-ERE in an estrogen-dependent manner. The DNA binding domain of estrogen receptor (ER DBD) fused to the Scistosoma japonicam glutathione S-transferase (GST) was expressed in the E. coli. The expression product also could bind the ERE. However, the binding was not affected by the uterine extract, indicating that the heat-sensitive nuclear factors may interact with hER outside the DBD to enhance the binding of ER to ERE.

Physiological functions and clinical implications of fibrinogen-like 2: A review.

Fibrinogen-like 2 (FGL2) encompasses a transmembrane (mFGL2) and a soluble (sFGL2) form with differential tertiary structure and biological activities. Typically, mFGL2 functions as prothrombinase that is capable of initiating coagulation in tissue without activation of the blood clotting cascade, whereas sFGL2 largely acts as an immunosuppressor that can repress proliferation of alloreactive T lymphocytes and maturation of bone marrow dendritic cells. Protein sequences of FGL2 exhibit evolutionary conservation across wide variety of species, especially at the carboxyl terminus that contains fibrinogen related domain (FRED). The FRED of FGL2 confers specificity and complexity in the action of FGL2, including receptor recognition, calcium affiliation, and substrate binding. Constitutive expression of FGL2 during embryogenesis and in mature tissues suggests FGL2 might be physiologically important. However, excessive induction of FGL2 under certain medical conditions (e.g., pathogen invasion) could trigger complement activation, inflammatory response, cellular apoptosis, and immune dysfunctions. On the other hand, complete absence of FGL2 is also detrimental as lack of FGL2 can cause autoimmune glomerulonephritis and acute cellular rejection of xenografts. All these roles involve mFGL2, sFGL2, or their combination. Although it is not clear how mFGL2 is cleaved off its host cells and secreted into the blood, circulating sFGL2 has been found correlated with disease severity and viral loading among patients with human hepatitis B virus or hepatitis C virus infection. Further studies are warranted to understand how FGL2 expression is regulated under physiological and pathological conditions. Even more interesting is to determine whether mFGL2 can fulfill an immunoregulatory role through its FRED at carboxyl end of the molecule and, and vice versa, whether sFGL2 is procoagulant upon binding to a target cell. Knowledge in this area should shed light on development of sFGL2 as an alternative immunosuppressive agent for organ transplantation or as a biomarker for predicting disease progression, monitoring therapeutic effects, and targeting FGL2 for repression in ameliorating fulminant viral hepatitis.