Transcriptional activation of TINF2, a gene encoding the telomere-associated protein TIN2, by Sp1 and NF-κB factors.

The expression of the telomere-associated protein TIN2 has been shown to be essential for early embryonic development in mice and for development of a variety of human malignancies. Recently, germ-line mutations in TINF2, which encodes for the TIN2 protein, have been identified in a number of patients with bone-marrow failure syndromes. Yet, the molecular mechanisms that regulate TINF2 expression are largely unknown. To elucidate the mechanisms involved in human TINF2 regulation, we cloned a 2.7 kb genomic DNA fragment containing the putative promoter region and, through deletion analysis, identified a 406 bp region that functions as a minimal promoter. This promoter proximal region is predicted to contain several putative Sp1 and NF-κB binding sites based on bioinformatic analysis. Direct binding of the Sp1 and NF-κB transcription factors to the TIN2 promoter sequence was demonstrated by electrophoretic mobility shift assay (EMSA) and/or chromatin immunoprecipitation (ChIP) assays. Transfection of a plasmid carrying the Sp1 transcription factor into Sp-deficient SL2 cells strongly activated TIN2 promoter-driven luciferase reporter expression. Similarly, the NF-κB molecules p50 and p65 were found to strongly activate luciferase expression in NF-κB knockout MEFs. Mutating the predicted transcription factor binding sites effectively reduced TIN2 promoter activity. Various known chemical inhibitors of Sp1 and NF-κB could also strongly inhibit TIN2 transcriptional activity. Collectively, our results demonstrate the important roles that Sp1 and NF-κB play in regulating the expression of the human telomere-binding protein TIN2, which can shed important light on its possible role in causing various forms of human diseases and cancers.

Lack of peripheral memory B cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study.

Six years have passed since the outbreak of severe acute respiratory syndrome (SARS). Previous studies indicated that specific Abs to SARS-related coronavirus (SARS-CoV) waned over time in recovered SARS patients. It is critical to find out whether a potential anamnestic response, as seen with other viral infections, exists to protect a person from reinfection in case of another SARS outbreak. Recovered SARS patients were followed up to 6 y to estimate the longevity of specific Ab. The specific memory B cell and T cell responses to SARS-CoV Ags were measured by means of ELISPOT assay. Factors in relation to humoral and cellular immunity were investigated. Six years postinfection, specific IgG Ab to SARS-CoV became undetectable in 21 of the 23 former patients. No SARS-CoV Ag-specific memory B cell response was detected in either 23 former SARS patients or 22 close contacts of SARS patients. Memory T cell responses to a pool of SARS-CoV S peptides were identified in 14 of 23 (60.9%) recovered SARS patients, whereas there was no such specific response in either close contacts or healthy controls. Patients with more severe clinical manifestations seemed to present a higher level of Ag-specific memory T cell response. SARS-specific IgG Ab may eventually vanish and peripheral memory B cell responses are undetectable in recovered SARS patients. In contrast, specific T cell anamnestic responses can be maintained for at least 6 y. These findings have applications in preparation for the possible reemergence of SARS.

Clinical and immunological characteristics of patients with 2009 pandemic influenza A (H1N1) virus infection after vaccination.

BACKGROUND: We followed a cohort of 773 individuals who received a monovalent vaccine against 2009 pandemic influenza A (H1N1). Approximately 6 weeks after vaccination, 12 persons developed the disease. METHODS: Three groups of subjects were studied (12 patients who had or had not received previous monovalent vaccine and 1 group of 49 control subjects who had previously been immunized with the same vaccine). For all patients, clinical features were characterized and the causative viruses sequenced for possible mutations. Nasopharyngeal swabs, serum specimens, and peripheral blood monocyte cells (PBMCs) were collected at different time points up to 11 weeks after symptom onset to measure the virus load and humoral and cellular immune responses. Serum samples and PBMCs were also collected from 49 and 16 vaccinated control subjects, respectively. RESULTS: Both patient groups had similar clinical manifestations. No substantial viral mutations were detected. Compared with unvaccinated patients, viral loads in vaccinated patients were initially higher, but the levels decreased faster to undetectable levels. However, the virus became detectable again for 6 of them. Two weeks after infection, vaccinated and unvaccinated patients had similar neutralizing antibody levels as the vaccinated control subjects. Thereafter, the neutralizing antibody levels decreased markedly in vaccinated patients. During the acute phase, memory T cell counts and tumor necrosis factor-α levels were significantly higher in vaccinated than in unvaccinated patients. CONCLUSIONS: Although the clinical consequences of infection are comparable between vaccinated and unvaccinated patients, humoral and cellular immune responses in vaccinated patients are boosted for some weeks, indicating an additional benefit of vaccination against 2009 pandemic influenza A (H1N1) virus.

Mixed infections of pandemic H1N1 and seasonal H3N2 viruses in 1 outbreak.

BACKGROUND: In early September 2009, an outbreak of influenza occurred at a college campus in Beijing, China, in which both pandemic H1N1 and seasonal H3N2 viruses were detected. METHODS: Outbreak investigation was performed in the campus. Epidemiologic, clinical data were collected by interviewing patients and retrieving medical records. Individual contact tracing was performed for detailed contact information. Viruses were identified by reverse-transcription polymerase chain reaction assays followed by sequence analysis. The hemagglutination inhibition test was used to detect antibodies to both viruses for paired serum samples. RESULTS: Forty of 45 people with influenza-like illness had laboratory-confirmed influenza A infection; 22 of these 40 people were infected with pandemic H1N1 virus, 12 were infected with seasonal H3N2 virus, and 6 were coinfected with both viruses. In the subsequent generation of cases with mixed infection, we detected pandemic H1N1 virus infection more often than seasonal H3N2 virus infection. The clinical patterns were essentially similar for patients with different virus infections. No substantial differences in sequences were observed in either pandemic H1N1 or seasonal H3N2 virus between patients with mixed and single infection. Sequence analyses revealed that all of the detected viruses were susceptible to oseltamivir but resistant to adamantane. Hemagglutination inhibition tests of paired serum samples confirmed mixed infection in the outbreak. CONCLUSIONS: Cocirculation of pandemic H1N1 virus and seasonal H3N2 virus led to a mixed infection in patients. Pandemic H1N1 virus, however, took prevalence over seasonal influenza virus in the course of transmission. Therefore, competitive circulation of seasonal influenza A virus with the pandemic H1N1 virus seems less likely.

Detection of Francisella tularensis in ticks and identification of their genotypes using multiple-locus variable-number tandem repeat analysis.

BACKGROUND: Tularemia was reported in China over 50 years ago, however, many epidemical characteristics remain unclear. In the present study, the prevalence of Francisella tularensis in ticks was investigated during an epidemiological surveillance in China and then we measured their genetic diversity by conducting multiple-locus variable- number tandem repeat analysis (MLVA). RESULTS: 1670 ticks from 2 endemic areas (Inner Mongolia Autonomous Region and Heilongjiang Province) and 2 non-endemic areas (Jilin and Fujian Provinces) were collected and tested for evidence of tularemia by nested PCR. The prevalence of Francisella tularensis in ticks averaged 1.98%. The positive rates were significantly different among tick species, with Dermacentor silvarum and Ixodes persulatus responsible for all positive numbers. All F. tularensis that were detected in ticks belonged to F. tularensis subsp. holarctica and MLVA disclosed genetic diversity. One subtype was identified in 17 of 33 positive tick samples in three different study areas. Another subtype belonging to F. tularensis subsp. holarctica genotype was described for the first time in the current study. CONCLUSION: The study showed two tick species, D. silvarum and I. persulatus harboring the pathogen of tularemia in natural environment, indicating these two tick species might have a role in tularemia existence in China. MLVA results disclosed the genetic diversity F. tularensis and identified one genotype as the most prevalent among the investigated ticks in China.

NF-kappaB signaling differentially regulates influenza virus RNA synthesis.

The NF-kappaB signaling pathway has previously been shown to be required for efficient influenza A virus replication, although the molecular mechanism is not well understood. In this study, we identified a specific step of the influenza virus life cycle that is influenced by NF-kappaB signaling by using two known NF-kappaB inhibitors and a variety of influenza virus-specific assays. The results of time course experiments suggest that the NF-kappaB inhibitors Bay11-7082 and ammonium pyrrolidinedithiocarbamate inhibited an early postentry step of viral infection, but they did not appear to affect the nucleocytoplasmic trafficking of the viral ribonucleoprotein complex. Instead, we found that the levels of influenza virus genomic RNA (vRNA), but not the corresponding cRNA or mRNA, were specifically reduced by the inhibitors in virus-infected cells, indicating that NF-kappaB signaling is intimately involved in the vRNA synthesis. Furthermore, we showed that the NF-kappaB inhibitors specifically diminished influenza virus RNA transcription from the cRNA promoter but not from the vRNA promoter in a reporter assay, a result which is consistent with data obtained from virus-infected cells. The overexpression of the p65 NF-kappaB molecule could not only eliminate the inhibition but also activate influenza virus RNA transcription from the cRNA promoter. Finally, using p65-specific small interfering RNA, we have shown that p65 knockdown reduced the levels of influenza virus replication and vRNA synthesis. In summary, we have provided evidence showing, for the first time, that the NF-kappaB host signaling pathway can differentially regulate influenza virus RNA synthesis, which may also offer some new perspectives into understanding the host regulation of RNA synthesis by other RNA viruses.

IL-12 RB1 genetic variants contribute to human susceptibility to severe acute respiratory syndrome infection among Chinese.

BACKGROUND: Cytokines play important roles in antiviral action. We examined whether polymorphisms of interleukin (IL)-12 receptor B1 (IL-12RB1) affect the susceptibility to and outcome of severe acute respiratory syndrome (SARS). METHODS: A case-control study was carried out in Chinese SARS patients and healthy controls. The genotypes of 4SNPs on IL-12 RB1 gene, +705A/G,+1158T/C, +1196G/C and +1664 C/T, were determined by PCR-RFLP. Haplotypes were estimated from the genotype data using the expectation-maximisation algorithm. RESULTS: Comparison between patients and close contacts showed that individuals with the +1664 C/T (CT and TT) genotype had a 2.09-fold (95% confidence interval [CI], 1.90-7.16) and 2.34-fold (95% CI, 1.79-13.37) increased risk of developing SARS, respectively. For any of the other three polymorphisms, however, no significant difference can be detected in allele or genotype frequencies between patients and controls. Additionally, estimation of the frequencies of multiple-locus haplotypes revealed potential risk haplotypes (GCCT) for SARS infection. CONCLUSIONS: Our data indicate that genetic variants of IL12RB1confer genetic susceptibility to SARS infection, but not necessary associated with the progression of the disease in Chinese population.

Functional characterization of yeast telomerase RNA dimerization.

Telomerase is the cellular RNA-dependent DNA polymerase (i.e. reverse transcriptase) that uses an integral RNA template to synthesize telomeric DNA repeats at the ends of linear chromosomes. Human telomerase RNA (hTERC) is thought to function as a dimeric complex consisting of two RNAs that interact with each other physically as well as genetically. We show here for the first time that the yeast Saccharomyces cerevisiae telomerase RNA TLC1 likewise forms dimers in vitro. TLC1 dimerization depends on a unique 6-base self-complementary sequence, which closely mimics palindromic sequences that mediate functional dimerization of HIV-1 and other retroviral genomes. We found that dissimilar but comparably located TLC1 palindromes from other sensu stricto yeasts can functionally substitute for that of S. cerevisiae. Yeast cells expressing dimerization-defective TLC1 alleles have shorter telomeres than those with wild-type TLC1. This study, therefore, highlights dimerization as a functionally conserved feature of the RNA templates utilized by reverse transcriptases of both viral and cellular origins.

Functional characterization of natural telomerase mutations found in patients with hematologic disorders.

Human telomerase hTERC RNA serves as a template for the catalytic hTERT protein to synthesize telomere repeats at chromosome ends. We have recently shown that some patients with bone marrow failure syndromes are heterozygous carriers for hTERC or hTERT mutations. These sequence variations usually lead to a compromised telomerase function by haploinsufficiency. Here, we provide functional characterization of an additional 8 distinct hTERT sequence variants and 5 hTERC variants that have recently been identified in patients with dyskeratosis congenita (DC) or aplastic anemia (AA). Among the mutations, 2 are novel telomerase variants that were identified in our cohort of patients. Whereas most of the sequence variants modulate telomerase function by haploinsufficiency, 2 hTERC variants with sequence changes located within the template region appear to act in a dominant-negative fashion. Inherited telomerase gene mutations, therefore, operate by various mechanisms to shorten telomere lengths, leading to limited marrow stem cell reserve and renewal capacity in patients with hematologic disorders.

Selection of novel nickel-binding peptides from flagella displayed secondary peptide library.

Nickel (Ni) performs its biological or toxic functions in nickel-protein coordination form. Novel Ni-binding peptides were isolated from a random dodecapeptide library displayed on the flagella of Escherichia coli against immobilized ions. On the basis of isolated sequences rich in histidine residues, two secondary libraries were constructed respectively. By consequent selection, more Ni-chelating peptides were identified and the consensus motif RHXHR (where X was always H) was deduced. The result suggested that not only histidine, but also arginine, play an important role in Ni-binding. Furthermore, two selected clones (1035 and 2022) were chosen for further identification. They exhibited similar relative binding affinity, which was about nine times that of the original library derived clones and statistically much more significant than the positive control with polyhistidine insert. Free nickel ions could almost completely inhibit the binding of the clones 1035 and 2022 to immobilized nickel, implicating that the peptides were able to chelate nickel ions. These studies reveal that bacterial surface displayed peptide libraries may have promising future potential for the development of metal bioadsorbents. Furthermore, novel Ni-binding peptides may provide lead molecules for Ni-chelation and applications thereof.

Genetic variation in telomeric repeat binding factors 1 and 2 in aplastic anemia.

OBJECTIVE: Abnormal telomere shortening has been observed in a subset of individuals with aplastic anemia (AA). We hypothesized that genetic variation in two genes critical in telomere biology, TERF1 and TERF2, could be a risk factor for AA. METHODS: The proximal promoter and all coding regions of TERF1 and TERF2 were sequenced in 47 individuals with acquired AA. Regions with genetic variation were sequenced in an additional 95 AA patients and 289 healthy controls. Single nucleotide polymorphism (SNP) frequencies were analyzed using co-dominant and dominant models and haplotypes determined. Functional studies evaluated telomerase activity, telomere and telomeric overhang lengths, and TRF2 protein expression in select patients. RESULTS: Two nonsynonymous amino acid changes were detected, one in exon 9 of TERF1 and another in exon 6 of TERF2. These sequence variants resulted in conservative amino acid changes and were not predicted to alter TRF1 or TRF2 protein expression or function. SNP and haplotype analyses in acquired AA patients suggested that one variant allele, in intron 9 of TERF1, and haplotype could be associated with increased risk for aplastic anemia (OR 1.59, 95% confidence interval 1.06-2.39, p = 0.033). TERF2 SNPs and haplotypes were not significantly associated with aplastic anemia. CONCLUSIONS: It is possible that a common genetic variant in TERF1 is associated with risk for AA but additional studies are required. Highly penetrant, non-synonymous, or insertion-deletion mutations in TERF1 and TERF2 were not identified and therefore are not likely to be major genetic risk factors for the development of AA.

Francisella tularensis in rodents, China.

A total of 420 rodents in China were examined for Francisella tularensis by polymerase chain reaction. The infection rates were 4.76% in total, and 11.65%, 10.00%, 6.56%, 1.77%, and 0% in Jilin, Xinjiang, Heilongjiang, Inner Mongolia, and Zhejiang, respectively. Sequence analysis showed that all the detected agents belonged to F. tularensis subsp. holarctica.

Two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome.

In a cohort study of 56 convalescent patients with severe acute respiratory syndrome (SARS), titers of immunoglobulin G (IgG) antibodies and neutralizing antibodies (NAbs) against SARS-associated coronavirus were assessed at regular intervals (at 1, 4, 7, 10, 16, and 24 months after the onset of disease) by use of enzyme-linked immunosorbent assay and neutralization assay. IgG antibody and NAb titers were highly correlated, peaking at month 4 after the onset of disease and decreasing thereafter. IgG antibodies remained detectable in all patients until month 16, and they became undetectable in 11.8% of patients at month 24. The finding that NAbs remained detectable throughout follow-up is reassuring in terms of protection provided against reinfection; however, NAb titers decreased markedly after month 16.

Molecular epidemiology of SARS-associated coronavirus, Beijing.

Single nucleotide variations (SNVs) at 5 loci (17564, 21721, 22222, 23823, and 27827) were used to define the molecular epidemiologic characteristics of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) from Beijing patients. Five fragments targeted at the SNV loci were amplified directly from clinical samples by using reverse transcription-polymerase chain reaction (RT-PCR), before sequencing the amplified products. Analyses of 45 sequences obtained from 29 patients showed that the GGCTC motif dominated among samples collected from March to early April 2003; the TGTTT motif predominanted afterwards. The switch from GGCTC to TGTTT was observed among patients belonging to the same cluster, which ruled out the possibility of the coincidental superposition of 2 epidemics running in parallel in Beijing. The Beijing isolates underwent the same change pattern reported from Guangdong Province. The same series of mutations occurring in separate geographic locations and at different times suggests a dominant process of viral adaptation to the host.

A subtractive fluorescence-activated cell-sorting strategy to identify mimotopes of HBV-preS protein from bacterially displayed peptide library.

A novel subtractive fluorescence-activated cell-sorting (FACS) strategy using a model system is described here to identify disease-specific (DS) epitopes from a bacterially displayed random peptide library. In this process, preimmune serum was used as "Driver " to block any common binding sites on the bacterial surface and the labeled anti-preS IgG polyclonal antibodies from immunized serum were used as "Tester" to enrich preS-specific mimotopes. Bacterial clones were identified out of this pool through an "antigen-independent" procedure only using both different sera samples. After four rounds of sub-FACS screening, 41 out of 50 bacterial clones were identified as reacting with the immunized serum but not reacting with the pre-immune one. Two motif sequences HQLD and DPAF were obtained from 13 clones. Immunization of mice with two representative bacterial clones elicited a strong specific response against native preS antigen in comparison with the control. This technique may provide a useful technology platform for high-throughput screening of disease-related epitope which is of importance to develop vaccine against some infectious diseases whose pathogen or immunodominant antigen is still unknown.

Long-term SARS coronavirus excretion from patient cohort, China.

This study investigated the long-term excretion of severe acute respiratory syndrome-associated coronavirus in sputum and stool specimens from 56 infected patients. The median (range) duration of virus excretion in sputa and stools was 21 (14-52) and 27 (16-126) days, respectively. Coexisting illness or conditions were associated with longer viral excretion in stools.

Mechanisms of inactivation of hepatitis A virus in water by chlorine dioxide.

In this study, to elucidate the mechanisms of inactivation of hepatitis A virus (HAV) by chlorine dioxide, cell culture, enzyme-linked immunosorbent assay (ELISA), and long-overlapping RT-PCR were used to detect the infectivity, antigenicity, and entire genome of HAV before and after disinfection. The results revealed the complete inactivation of infectivity after a 10-min exposure to 7.5mg of chlorine dioxide per liter; and the highest level of sensitivity in the 5'non-translated regions (5'NTR) (the sequence from bp 1 to 671), inactivation of which took as much time as the inactivation of infectivity of HAV by chlorine dioxide; the complete destruction of antigenicity after a 10-min exposure to 7.5mg of chlorine dioxide per liter. It is suggested that the inactivation mechanism of HAV by chlorine dioxide was due to the loss of the 5'NTR and/or destruction of the antigenicity, which is not similar to that of chlorine (Appl Environ Microbiol 68: 4951).

Identification of anti-TNFalpha peptides with consensus sequence.

Phage displayed peptide library was used to select tumor necrosis factor alpha (TNFalpha) binding peptides. After three sequential rounds of biopanning, some linear TNFalpha-binding peptides were identified from a 12-mer peptide library. A consensus sequence (L/M)HEL(Y/F)(L/M)X(W/Y/F), where X might be variable residue, was deduced from sequences of these peptides. The phages bearing these peptides showed specific binding to immobilized TNFalpha, with over 80% of phages bound being competitively eluted by free TNFalpha. To confirm the binding activity and to explore further functional properties, three peptides with typical structure were selected and expressed as GST-fused protein. These recombinant peptides effectively competed for [125I]TNFalpha binding to TNFR1 in a dose-dependent manner, with IC(50) from 10 to 160 microM. Furthermore, the GST-fused derivatives showed inhibitory effects on TNFalpha-induced cytotoxicity. Taken together, these data demonstrate that the TNFalpha-binding peptides are effective antagonists of TNFalpha and the deduced motif might be useful in development of novel low molecular weight anti-TNFalpha drugs.