52 research outputs found
Highly Sensitive and Quantitative Detection of the H274Y Oseltamivir Resistance Mutation in Seasonal A/H1N1 Influenza Virus
No full textA C-to-T transition mutation in the neuraminidase gene from seasonal A/H1N1 causes a His-to-Tyr mutation at amino acid position 275 (H274Y, universal N2 numbering), conferring resistance against oseltamivir (Tamiflu). This mutation was first detected in clinical samples in Europe during the 2007-2008 influenza season. Viruses with this mutation reached a prevalence of 11 % by the end of the season in North American isolates tested by the CDC. We developed a highly sensitive and specific quantitative real-time reverse transcriptase PCR assay to detect the H274Y mutation. This assay utilizes a 5-methyl-isocytosine (isoC) residue and fluorescent reporters on genotype-specific primers. During PCR, a quencher coupled to isoguanine (isoG) is site-specifically incorporated complementary to the isoC/dye, resulting in loss of fluorescence. Optimization of primers and assay conditions produced a limit of detection of 100 gene copies per reaction for both wild-type and H274Y genotypes. In samples with mixed populations, it can reliably detect as little as a 1% wild-type or 0.1 % H274Y component. This high sensitivity makes the assay usable on samples with viral loads too low for dideoxy or pyrosequencing analysis. Additionally, the assay distinguishes seasonal A/H1N1 from A/H3N2, influenza B, or 2009 pandemic A/H1N1, making it useful for influenza virus subtyping as well as for drug resistance detection. We probed seasonal A/H1N1 samples from the 2005-2006, 2006-2007, and 2007-2008 influenza seasons. Data from the new assay closely matched available drug resistance genotype data previousl
Comparison of DNA polymerase activities between recombinant feline immunodeficiency and leukemia virus reverse transcriptases
No full textAbstractIn this study, we present enzymatic differences found between recombinant RTs derived from feline leukemia virus and feline immunodeficiency virus. Firstly, FIV RT showed low steady state Km values for dNTPs compared to FeLV RT. Consistent with this, FIV RT synthesized DNA more efficiently than FeLV RT at low dNTP concentrations. We observed similar concentration-dependent activity differences between other lentiviral (HIV-1 and SIV) and non-lentiviral (MuLV and AMV) RTs. Second, FeLV RT showed less efficient misincorporation with biased dNTP pools and mismatch primer extension capabilities, compared to FIV RT. In M13mp2 lacZα forward mutation assays, FeLV RT displayed approximately 11-fold higher fidelity than FIV RT. Finally, FeLV RT was less sensitive to 3TCTP and ddATP than FIV RT. This study represents the comprehensive enzymatic characterization of RTs from a lentivirus and a non-lentivirus retrovirus from the same host species. The data presented here support enzymatic divergences seen among retroviral RTs
The acute environment, rather than T cell subset pre-commitment, regulates expression of the human T cell cytokine amphiregulin.
No full textCytokine expression patterns of T cells can be regulated by pre-commitment to stable effector phenotypes, further modification of moderately stable phenotypes, and quantitative changes in cytokine production in response to acute signals. We showed previously that the epidermal growth factor family member Amphiregulin is expressed by T cell receptor-activated mouse CD4 T cells, particularly Th2 cells, and helps eliminate helminth infection. Here we report a detailed analysis of the regulation of Amphiregulin expression by human T cell subsets. Signaling through the T cell receptor induced Amphiregulin expression by most or all T cell subsets in human peripheral blood, including naive and memory CD4 and CD8 T cells, Th1 and Th2 in vitro T cell lines, and subsets of memory CD4 T cells expressing several different chemokine receptors and cytokines. In these different T cell types, Amphiregulin synthesis was inhibited by an antagonist of protein kinase A, a downstream component of the cAMP signaling pathway, and enhanced by ligands that increased cAMP or directly activated protein kinase A. Prostaglandin E2 and adenosine, natural ligands that stimulate adenylyl cyclase activity, also enhanced Amphiregulin synthesis while reducing synthesis of most other cytokines. Thus, in contrast to mouse T cells, Amphiregulin synthesis by human T cells is regulated more by acute signals than pre-commitment of T cells to a particular cytokine pattern. This may be appropriate for a cytokine more involved in repair than attack functions during most inflammatory responses
J Infect Dis
No full textBackgroundThe etiology of acute watery diarrhea remains poorly characterized, particularly after rotavirus vaccine introduction.MethodsWe performed quantitative polymerase chain reaction for multiple enteropathogens on 878 acute watery diarrheal stools sampled from 14643 episodes captured by surveillance of children <5 years of age during 2013\u20132014 from 16 countries. We used previously developed models of the association between pathogen quantity and diarrhea to calculate pathogen-specific weighted attributable fractions (AFs).ResultsRotavirus remained the leading etiology (overall weighted AF, 40.3% [95% confidence interval {CI}, 37.6%\u201344.3%]), though the AF was substantially lower in the Americas (AF, 12.2 [95% CI, 8.9\u201315.6]), based on samples from a country with universal rotavirus vaccination. Norovirus GII (AF, 6.2 [95% CI, 2.8\u20139.2]), Cryptosporidium (AF, 5.8 [95% CI, 4.0\u20137.6]), Shigella (AF, 4.7 [95% CI, 2.8\u20136.9]), heat-stable enterotoxin-producing Escherichia coli (ST-ETEC) (AF, 4.2 [95% CI, 2.0\u20136.1]), and adenovirus 40/41 (AF, 4.2 [95% CI, 2.9\u20135.5]) were also important. In the Africa Region, the rotavirus AF declined from 54.8% (95% CI, 48.3%\u201361.5%) in rotavirus vaccine age-ineligible children to 20.0% (95% CI, 12.4%\u201330.4%) in age-eligible children.ConclusionsRotavirus remained the leading etiology of acute watery diarrhea despite a clear impact of rotavirus vaccine introduction. Norovirus GII, Cryptosporidium, Shigella, ST-ETEC, and adenovirus 40/41 were also important. Prospective surveillance can help identify priorities for further reducing the burden of diarrhea.20172017-06-21T00:00:00Z001/World Health Organization/InternationalK23 AI114888/AI/NIAID NIH HHS/United States28838152PMC5853801820
TCR activation induced AR expression in human PBMC T cells.
No full text<p>(A) PBMC were treated as indicated and analyzed by ICS. The upper panels show the gating strategy to identify activated (CD69+) CD4 or CD8 T cells expressing AR. The lower panels show the induction of AR by different stimuli in CD4 or CD8 T cells. (B) AR and IL-2 mRNA were measured by RT-PCR in purified CD4 and CD8 T cells after activation by anti-CD3+anti-CD28 beads. Results in (A) and (B) are representative of at least three experiments.</p
Use of quantitative molecular diagnostic methods to assess the aetiology, burden, and clinical characteristics of diarrhoea in children in low-resource settings: a reanalysis of the MAL-ED cohort study
No full textAUTHORS - James A Platts-Mills*, Jie Liu*, Elizabeth T Rogawski, Furqan Kabir, Paphavee Lertsethtakarn, Mery Siguas, Shaila S Khan, Ira Praharaj,
Arinao Murei, Rosemary Nshama, Buliga Mujaga, Alexandre Havt, Irene A Maciel, Timothy L McMurry, Darwin J Operario, Mami Taniuchi,
Jean Gratz, Suzanne E Stroup, James H Roberts, Adil Kalam, Fatima Aziz, Shahida Qureshi, M Ohedul Islam, Pimmada Sakpaisal, Sasikorn Silapong,
Pablo P Yori, Revathi Rajendiran, Blossom Benny, Monica McGrath, Benjamin J J McCormick, Jessica C Seidman, Dennis Lang, Michael Gottlieb,
Richard L Guerrant, Aldo A M Lima, Jose Paulo Leite, Amidou Samie, Pascal O Bessong, Nicola Page, Ladaporn Bodhidatta, Carl Mason,
Sanjaya Shrestha, Ireen Kiwelu, Estomih R Mduma, Najeeha T Iqbal, Zulfiqar A Bhutta, Tahmeed Ahmed, Rashidul Haque, Gagandeep Kang,
Margaret N Kosek, Eric R Houpt, and The MAL-ED Network Investigators† - AFFILIATIONS - Division of Infectious Diseases
and International Health
(J A Platts-Mills MD, J Liu PhD,
E T Rogawski PhD,
D J Operario PhD,
M Taniuchi PhD, J Gratz MS,
S E Stroup MS, R L Guerrant MD,
Prof E R Houpt MD) and
Department of Public Health
Sciences (E T Rogawski,
T L McMurry PhD, J H Roberts),
University of Virginia,
Charlottesville, VA, USA;
Aga Khan University, Karachi,
Pakistan (F Kabir MSc,
A Kalam MSc, F Aziz MSc,
S Qureshi MSc, N T Iqbal PhD,
Prof Z A Bhutta PhD); Armed
Forces Research Institute of
Medical Sciences (AFRIMS),
Bangkok, Thailand
(P Lertsethtakarn PhD,
P Sakpaisal MSc,
S Silapong B BSc,
L Bodhidatta MD, C Mason MD);
Asociación Benéfica PRISMA,
Iquitos, Peru (M Siguas BSc,
P P Yori MPH, M N Kosek MD);
International Centre for
Diarrhoeal Disease Research,
Dhaka, Bangladesh
(S S Khan MSc, M O Islam MSc,
Prof T Ahmed MBBS,
R Haque PhD); Christian Medical
College, Vellore, India
(I Praharaj MD, R Rajendiran MSc,
B Benny MSc, Prof G Kang MD);
University of Venda,
Thohoyandou, South Africa (A Murei BSc, A Samie PhD,
Prof P O Bessong PhD); Haydom
Global Health Institute,
Haydom, Tanzania
(R Nshama BSc,
E R Mduma MPH); Kilimanjaro
Clinical Research Institute,
Moshi, Tanzania (B Mujaga BSc,
I Kiwelu PhD); Federal
University of Ceara, Fortaleza,
Brazil (A Havt PhD,
Prof A A M Lima PhD); Fundação
Oswaldo Cruz (Fiocruz), Rio de
Janeiro, Brazil (I A Maciel PhD,
J P Leite PhD); Bloomberg
School of Public Health, Johns
Hopkins University, Baltimore,
MD, USA (P P Yori MPH,
M McGrath ScD, M N Kosek);
Fogarty International Center,
National Institutes of Health,
Bethesda, MD, USA
(M McGrath,
B J J McCormick DPhil,
J C Seidman PhD); Foundation
for the National Institutes of
Health, Bethesda, MD, USA
(D Lang PhD, M Gottlieb PhD);
National Institute for
Communicable Diseases,
Johannesburg, South Africa
(N Page PhD); Walter
Reed/AFRIMS Research Unit,
Nepal, Kathmandu, Nepal
(S Shrestha MD); and University
of Bergen, Bergen, Norway
(S Shrestha).Optimum management of childhood diarrhoea in low-resource settings has been hampered by
insufficient data on aetiology, burden, and associated clinical characteristics. We used quantitative diagnostic methods
to reassess and refine estimates of diarrhoea aetiology from the Etiology, Risk Factors, and Interactions of Enteric
Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study
AR is produced by T cell subsets expressing different chemokine receptors and surface markers.
No full text<p>PBMC were treated with medium alone, anti-CD3+ anti-CD28 antibodies, or SEB in the presence of TAPI-1 for 8 hours. Cells were stained for AR and cell-surface markers and analyzed by flow cytometry. Representative of two experiments.</p
TCR and cAMP synergize to induce AR production in human CD4 T cells.
No full text<p>Purified CD4 T cells were incubated with or without TCR stimulation (anti-CD3/CD28 beads) and the cAMP agonist. (A) AR and HB-EGF mRNA expression was measured by RT-PCR. (B) The concentrations of AR in the supernatant and cell lysates were measured by ELISA. (C) Enriched CD45RA+CD45RO- (naïve) and CD45RA-CD45RO+ (memory) CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of cAMP agonist (1 ∼ 1000 µM). The concentration of AR in the supernatant at 24 hours was measured by ELISA. (D) Purified CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of the cAMP-modifying agents shown. RNA was extracted at 4 hours, and AR mRNA was measured by RT-PCR. The concentration of AR in the 24-hour supernatant was measured by ELISA. (E) PBMC were treated with anti-CD3+ anti-CD28 antibodies in the presence or absence of cAMP agonist or antagonist for 8 hours. CD4 T cells were purified by cell sorting and RNA was extracted. The mRNA levels of AR and other cytokines were measured by RT-PCR. All results are representative of at least three experiments.</p
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