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PubMed2005

Human follicular dendritic cells express prostacyclin synthase: a novel mechanism to control T cell numbers in the germinal center.

Stromal cells in the lymphoid organs provide a microenvironment where lymphocytes undergo various biological processes such as development, homing, clonal expansion, and differentiation. Follicular dendritic cells (FDCs) in the primary and secondary follicles of the peripheral lymphoid tissues interact with lymphocytes by contacting directly or producing diffusible molecules. To understand the biological role of human FDC at the molecular level, we developed a mAb, 3C8, that recognizes FDC but not bone marrow-derived cells. Through expression cloning and proteome analysis, we identified the protein that is recognized by 3C8 mAb, which revealed that FDC expresses prostacyclin synthase. The 3C8 protein purified from FDC-like cells indeed displayed the enzymatic activity of prostacyclin synthase and converted PGH2 into prostacyclin. In addition, prostacyclin significantly inhibited proliferation of T cells but delayed their spontaneous apoptosis. These findings may help explain why T cells constitute only a minor population compared with B cells in the germinal center.

Amino Acid Sequence↗
PubMed2007

The duration of exposure to HIV modulates the breadth and the magnitude of HIV-specific memory CD4+ T cells.

The impact of exposure to Ag on the development and maintenance of human CD4(+) memory T cells in general and HIV infection in particular is partially understood. In this study, we measured HIV-specific CD4(+) T cell proliferative responses against HIV proteins and derived peptides one year after highly active antiretroviral therapy initiation in 39 HIV-infected patients who initiated therapy at different times following infection. We show that a brief exposure to HIV of <1 month does not allow the generation of significant detectable frequencies of HIV-specific CD4(+) memory T cells. Patients having prolonged cumulative exposure to high viral load due to therapy failures also demonstrated limited HIV-specific CD4(+) T cell responses. In contrast, patients exposed to significant levels of virus for periods ranging from 3 to 18 mo showed brisk and broad HIV-specific CD4(+) T cell responses 1 year following the onset of therapy intervention. We also demonstrate that the nadir CD4(+) T cell count before therapy initiation correlated positively with the breadth and magnitude of these responses. Our findings indicate that the loss of proliferative HIV-specific CD4(+) T cell responses is associated with the systemic progression of the disease and that a brief exposure to HIV does not allow the establishment of detectable frequencies of HIV-specific memory CD4(+) T cells.

Amino Acid Sequence↗
PubMed1999

Human CD28-CD8+ T cells contain greatly expanded functional virus-specific memory CTL clones.

At birth, almost all human peripheral blood CD8+ T cells express the costimulatory molecule CD28. With increasing age, the proportion of CD8+ T cells that lack CD28 increases. Because the Ag specificity of CD28-CD8+ T cells has not previously been defined, we studied the contribution of CD28-CD8+ T cells to the memory CD8+ CTL response against two human persistent viruses, human CMV (HCMV) and HIV. From PBMC of healthy virus carriers we generated multiple independent CTL clones specific for defined viral peptides and sequenced their TCR beta-chains. We designed clonotypic oligonucleotides complementary to each beta-chain hypervariable sequence and quantified the size of individual immunodominant CTL clones in PBMC. Some individual CTL clones were very large, comprising up to 3.1% of all CD8+ T cells in PBMC, and were generally maintained at a stable level for months. Individual virus-specific CTL clones were consistently more abundant in purified CD28- cells than in the CD8+ population as a whole. Because CD28-CD8+ cells as a population have been reported to proliferate poorly in response to mitogen, we studied the function of these virus-specific CD28- CTL clones by quantifying the frequency of peptide-specific CTL precursors using limiting dilution analysis. CD28-CD8+ T cells contained high frequencies of functional memory CTL precursors specific for peptides of HCMV or HIV, generally higher than in the CD8+ T cell population as a whole. We conclude that in asymptomatic HCMV and HIV infection, human CD28-CD8+ T cells contain high frequencies of functional virus-specific memory CTL clones.

Amino Acid Sequence↗
PubMed1992

[HIV-1 proviral DNA sequences in the saliva of patients with HIV infection].

In order to understand the significance of presence of HIV-1 in saliva, we searched for by PCR HIV-1 proviral sequences in the saliva cells of 49 HIV-1 infected patients. Seven out 49 specimens resulted positive, 4 of which were from patients with PGL, 1 with ARC and 2 with AIDS. Four patients had a CD4+ lymphocyte counts < 200/cmm and in 3 patients the CD4+ lymphocyte count ranged from 200 to 400/cmm. Two patients were treated with AZT, 1 with DDI and 4 had no antiretroviral treatment. In conclusion, although HIV-1 proviral sequences have been found in saliva of HIV-1 infected patients, a larger group of patients should be investigated to define more precisely the role of HIV-1 in saliva.

Base Sequence↗
PubMedDate not supplied

Effects of two different alpha-interferon regimens on clinical and virological findings in mixed cryoglobulinemia.

OBJECTIVE: As previous studies have shown a good response of mixed cryoglobulinemia (MC) to alpha-interferon (IFN) therapy, we investigated the efficacy and tolerability of two IFN regimens in a group of 36 patients affected by MC. METHODS: The patients, diagnosed on the basis of standard clinical and laboratory criteria, were randomly divided into 2 groups: group A (18 cases) received alpha 2b-IFN 3 M.U. thrice a week for six months, while group B (18 cases) received alpha 2b-IFN thrice a week for 1 year. The patients were followed for six months after the end of therapy. Liver function tests, cryoglobulin determinations and a clinical examination were performed each month. HCV serology and HCV-RNA detection by PCR were performed before therapy and at the end of the follow-up period. RESULTS: The two study groups were comparable in age, male/female ratio, purpura score, cryoglobulin level, mean ALT serum activity and liver histology. 32 patients (89%) were positive for anti-HCV antibodies and 29 (81%) for HCV-RNA. During therapy all patients showed a significant (p < 0.001) decrease in their cryoglobulin level as well as improvement (p < 0.05) in their purpura score. In group A, five patients (28%) showed normalized ALT, but three later relapsed. In group B seven patients (39%) responded to treatment but three relapsed after suspension of the drug. Two patients from group B developed severe side effects (hypothyroidism and depression) and therapy was discontinued after 9 and 11 months, respectively. In all the non-responders and relapsed patients, purpura, ALT, and cryoglobulins rose to pre-treatment levels within a few months. At the end of follow-up, two patients from group A (11%) and four in group B (22%) had achieved complete remission. CONCLUSION: This study indicates that IFN is useful in MC and that viral replication can be considered the target of the therapy. Despite the absence of a statistical difference in the response rate between the two regimens (due to the low number of subjects), the one-year therapy course seemed to show a better efficacy, although associated with higher toxicity.

Adult↗
PubMed1999

Dose-dependent induction of distinct anergic phenotypes: multiple levels of T cell anergy.

T cell anergy has been proposed as one of the mechanisms underlying peripheral T cell tolerance. In recent years, the functional relevance of T cell anergy has been studied extensively in vitro and in vivo, using different species, cell systems, and ways to induce anergy. Although these studies concurred about the induction of unresponsiveness, conflicting findings were obtained with respect to the function of anergic T cells and to the persistence of T cell anergy. In the present study, T cell anergy was induced through T-T presentation of the specific Ag by rat MHC class II+ T cells in the absence of professional APC. We show that, depending on the Ag dose with which T cells were incubated, distinct anergic phenotypes were induced. Incubation of T cell clones with a low (suboptimal) Ag dose induced hyporesponsiveness. Incubation with a higher (optimal) Ag dose induced an anergic state capable of exerting immunoregulatory effects. Incubation with a high (supraoptimal) Ag dose led to an anergic suppressive phenotype that was persistent and was not reversed by APC, Ag, and rIL-2. These findings demonstrate that T cell anergy is not confined to a single state of functional inactivation. Instead, multiple levels of T cell anergy exist. Thus, anergic T cells can contribute to the regulation of the immune response either in a persistent and active manner or in a passive manner, depending on their level of T cell anergy.

Amino Acid Sequence↗
PubMed2003

Identification of a novel Cryptosporidium genotype in pigs.

Over a 3-year period, a total of 646 fecal samples from pigs in 22 indoor and outdoor herds from Western Australia were screened for Cryptosporidium spp. by microscopy. Results revealed that 39 of 646 samples (6.03%) were positive for Cryptosporidium. Cryptosporidium was much more common in outdoor herds (17.2%) than in indoor herds (0.5%) and was more common in animals between the ages of 5 and 8 weeks (69.2%) than in younger animals (P < 0.0001). Molecular characterization of the positive samples at the 18S ribosomal DNA locus identified two distinct genotypes of Cryptosporidium: the previously identified pig genotype I and a novel pig genotype (pig genotype II), both of which warrant species status.

Animals↗
PubMed2006

Expression of vasa (vas)-related genes in germ cells and specific interference with gene functions by double-stranded RNA in the monogenean, Neobenedenia girellae.

Neobenedenia girellae, a monogenean, is an important pathogen in marine cultured fish such as yellowtail and amberjack. An effective control method is required but none has yet been established. Aiming to establish a new control method by interfering with the gametogenesis of N. girellae, we focused on vasa (vas)-related genes that are expressed exclusively in the germline granules in Drosophila, Caenorhabditis elegans and other animals. Three vas-related genes (N. girellae vasa-like gene, Ngvlg1, Ngvlg2 and Ngvlg3) were isolated by PCR and Ngvlg1 and Ngvlg2 were shown to be expressed only in germ cells. We demonstrated that introduction of double-stranded Ngvlg1 or Ngvlg2 RNA by soaking resulted in partial or complete loss of germ cells. Moreover, the hatching rate of eggs from animals showing partial loss of germ cells decreased significantly. These results suggest that Ngvlg1 and Ngvlg2 are essential genes for germ cell quantity and quality. The possibility that a new control method can be developed by controlling gametogenesis of N. girellae was proven, because sterilised N. girellae could be produced.

Amino Acid Sequence↗
PubMed2002

A multiplex allele specific polymerase chain reaction (MAS-PCR) on the dihydrofolate reductase gene for the detection of Cryptosporidium parvum genotypes 1 and 2.

A multiplex allele specific polymerase chain reaction (MAS-PCR) based on the Cryptosporidium parvum dihydrofolate reductase (dhfr) gene sequence differentiates genotype 1 ('Human') from 2 ('Cattle') in a 1-step reaction. The MAS-PCR was validated on a panel of 34 microscopically positive C. parvum faecal samples of human and animal origin in comparison with 2 published PCR-restriction fragment length polymorphism (RFLP) methods targeting dhfr and the oocyst wall protein (cowp) genes. A validation panel of 37 negative faecal samples of human and animal origin was also tested in comparison with the cowp PCR-RFLP. MAS-PCR was found to be as sensitive for species detection as the most sensitive of the other tests, and detected more mixed genotype infections than the two other tests combined. In addition the MAS-PCR showed equivalent detection sensitivity in comparison with a published nested RFLP targeting the SSU rRNA gene, on a panel of prepared mixed genotype samples. The 1-step reaction is simpler and less expensive to perform than the RFLP methods, while the C. parvum specific amplicons and those for genotypes 1 and 2 (575, 357 and 190 bp respectively) can be easily distinguished on agarose gel.

Alleles↗
PubMed2026

Uniform processing and analysis of IGVF massively parallel reporter assay data with MPRAsnakeflow.

As researchers and clinicians seek to identify human genomic alterations relevant to traits and disorders, identifying and aggregating evidence providing mechanistic support for associations between alterations and phenotypes remains challenging. In particular, the study of noncoding genomic variation remains a major challenge because of the lack of accurate functional annotation for activity in a given context and across alleles. Experimental evidence is critical for prioritizing and interpreting functional effects of genetic alterations. Massively parallel reporter assays (MPRAs) have emerged as a powerful high-throughput approach, enabling quantification of regulatory element activity and allelic effects, as well as systematic dissection of gene regulatory logic and variant effects across different contexts. However, the diversity of MPRA designs, lack of standardized formats, and many potential processing parameters hamper data integration, reproducibility, and meta-analyses across studies. To address these challenges, the Impact of Genomic Variation on Function (IGVF) Consortium established an MPRA focus group to develop community standards, including harmonized file formats, and robust analysis pipelines for a wide range of library types and experimental designs. Here, we present these formats and comprehensive computational tools, MPRAlib and MPRAsnakeflow, for uniform processing from raw sequencing reads to counts, processing, and visualization. Using diverse MPRA data sets, we investigated technical variability sources including barcode sequence bias, outlier barcodes, and delivery method (episomal vs. lentiviral). Our results establish best practices for MPRA data generation and analysis, facilitating robust, reproducible research and large-scale integration. The presented tools and standards are publicly available, providing a foundation for future collaborative efforts in regulatory genomics.

Humans↗
PubMed2005

Parasite burden and constitution of major histocompatibility complex in the Malagasy mouse lemur, Microcebus murinus.

We investigated the importance of the major histocompatibility complex (MHC) constitution on the parasite burden of free-ranging mouse lemurs (Microcebus murinus) in four littoral forest fragments in southeastern Madagascar. Fourteen different MHC class II DRB-exon 2 alleles were found in 228 individuals with high levels of sequence divergence between alleles. More nonsynonymous than synonymous substitutions in the functional important antigen recognition and binding sites indicated selection processes maintaining MHC polymorphism. Animals from the four forest fragments differed in their infection status (being infected or not), in the number of different nematode morphotypes per individual (NNI) as well as in the fecal egg counts (FEC) values. Heterozygosity in general was uncorrelated with any of these measures of infection. However, a positive relationship was found between specific alleles and parasite load. Whereas the common allele Mimu-DRB*1 was more frequently found in infected individuals and in individuals with high NNI and FEC values (high parasite load), the rare alleles Mimu-DRB*6 and 10 were more prevalent in uninfected individuals and in individuals with low NNI and FEC values (low parasite load). These three alleles associated with parasite load had unique amino acid motifs in the antigen binding sites. This distinguished them from the remaining 11 Mimu-DRB alleles. Our results support the hypothesis that MHC polymorphism in M. murinus is maintained through pathogen-driven selection acting by frequency-dependent selection. This is the first study of the association of MHC variation and parasite burden in a free-ranging primate.

Analysis of Variance↗
PubMed1998

Antigenic homology of HIV-1 GP41 and human platelet glycoprotein GPIIIa (integrin beta3).

Fifty-eight of 89 serum samples (65.17%) from HIV-1-infected individuals at various disease stages contain antibodies that react with a platelet peptide located in the cytoplasmic domain of integrin beta3, glycoprotein GPIIIa (aa749-761; sequence DRKEFAKFEEERA). Rabbit polyclonal antibodies raised against the synthetic platelet peptide also react with the structurally homologous HIV-1 gp41-derived peptide (EKNEQELLELDKW(A)) and bind to a Western blot band with molecular weight corresponding to HIV-1 gp41. These findings point to molecular mimicry between HIV-1 and a human membrane protein found in platelets and other cells that could be of pathologic consequence.

Amino Acid Sequence↗
PubMed2001

Long-term intake of Korean red ginseng in HIV-1-infected patients: development of resistance mutation to zidovudine is delayed.

We have observed that CD4+ T cell counts in human immunodeficiency virus (HIV)-1-infected patients treated with only Korean red ginseng (KRG) are maintained or even increased for a prolonged period. In the present study, we investigated whether the development of resistance mutations in reverse transcriptase (RT) to zidovudine (ZDV) is delayed by combined therapy with KRG and ZDV. Nested polymerase chain reaction (PCR) and direct sequencing methods were used to define RT codons 41, 67, 70, 210, 215 and 219 of the HIV-1 pol gene in DNA from peripheral blood mononuclear cells (PBMC) samples from 18 patients. Nine of these eighteen patients were in the KRG group and had been treated with KRG for 60 +/- 15 months (range: 38-82) and ZDV, and nine were in the control group and had been treated with ZDV only. The patients in the KRG group had been treated with ZDV for 75 +/- 24 months, and CD4+ T cell counts were maintained from 239 +/- 85 to 234 +/- 187 microliters-1 (P > 0.05) during the study period, whereas the patients in the control group had been treated with ZDV for 51 +/- 31 months, and their CD4+ T cell counts decreased from 272 +/- 97 to 146 +/- 154 microliters-1 (P < 0.01). In samples within 24 months of ZDV therapy, the overall incidence of 6 resistance mutations to ZDV was 4.2% and 47% in the KRG and control group (P < 0.01), respectively. In samples after 24 months of therapy, the incidence was 21.7% and 56.3% in the KRG and control group (P < 0.01), respectively. These data suggest that the maintenance of CD4+ T cell counts by ZDV and KRG-intake for a prolonged period might be indirectly associated with delayed development of resistance to ZDV by KRG-intake.

Adult↗
PubMed2004

An empirical Bayesian significance test of cDNA library data.

Automated high-throughput sequencing of cDNA clones from numerous libraries has generated a wealth of information about both genome sequence and relative transcript abundances. A common statistical challenge in the analysis of library sequences is to infer whether there is differential expression for the same transcript under two different conditions, such as normal and diseased tissue. In contrast to the continuously variable intensity measurements from microarray experiments, data from cDNA library sequencing presents itself as a discrete count of the incidence of some clone or transcript in a finite sample. In this paper, we first propose a statistical model for data generated from cDNA library sequencing efforts. The model is based on the Poisson mixed with generalized inverse Gaussian (PGIG), introduced by Sichel (1971, 1975). PGIG has been used in modeling population abundance, ecological studies, word frequencies in publications, etc. Using data from the literature, we show that the proposed model provides a good fit to the observed data. Using this new model for cDNA library data, we developed an empirical Bayesian significance test (EBST) for inferring the statistical significance of differential gene expression from discrete data.

Bayes Theorem↗
PubMed2005

Molecular analysis of astacin-like metalloproteases of Ostertagia ostertagi.

In this study, we describe the molecular analysis of zinc-metalloproteases from the abomasal nematode Ostertagia ostertagi which were exclusively recognized by local antibodies of immune cattle. Full-length or partial coding sequences of 4 different zinc-metalloprotease cDNAs of Ostertagia (met-1, -2, -3 and -4) were amplified using gene-specific primers using the 3'- and 5'-Rapid Amplification of cDNA Ends (RACE) technique. Sequence analysis identified the cDNAs as encoding zinc-metalloproteases, which showed between 62% and 70% homology to a metalloprotease 1 precursor of Ancylostoma caninum. The full-length cDNA of met-1 consists of an open reading frame (ORF) of 586 amino acids which contains 5 potential N-glycosylation sites and a predicted zinc-binding domain (HEBXHXBGFXHEXXRXDRD). The complete coding sequence of met-3 contains an ORF of 508 aa and the same conserved zinc-binding domain. These domains are signature sequences of the astacin family of the superfamily of metzincin metalloproteases. The presence of a threonine amino acid after the third histidine in MET-1 and MET-3, however, may place them in a new family or subfamily. Real-time PCR analysis of L3, exsheathed L3, L4 and adult cDNA identified transcription of the 4 metalloproteases in different life-stages. The protein MET-1 was expressed in insect cells using the baculovirus expression system but the immunization of calves with this molecule did not lead to protection against challenge infection.

Amino Acid Sequence↗