Quantitative titration of nucleic acids by enzymatic amplification reactions run to saturation

Pannetier, Christophe; Delassus, Sylvie; Darche, Sylvie; Saucier, Cécile; Kourilsky, Philippe

doi:10.1093/nar/21.3.577

Cited by 150 publications

(86 citation statements)

References 32 publications

(22 reference statements)

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“…TCR BV gene segment usage was investigated using a semiquantitative adaptation of the Immunoscope approach (20) after standardization of T cellspecific material between samples by quantification of the number of copies of CD3␦ transcripts by a competitive PCR (21). Briefly, 30 cycles of PCR were performed with each one of the 24 BV-specific primers and a fluorescent (FAM)-labeled constant ␤ segment (BC)-specific primer using 25 or 50 ϫ 10 9 copies of CD3␦-specific cDNA, as determined in preliminary experiments.…”

Section: Tcr Bv Repertoire and Cdr3 Size Spectratyping Analysismentioning

confidence: 99%

Mechanisms of the Natural Reactivity of Lymphocytes from Noninfected Individuals to Membrane-AssociatedLeishmania infantumAntigens

Sassi

Larguèche-Darwaz

Collette

et al. 2005

The Journal of Immunology

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Membrane-associated Leishmania Ags (MLA) or soluble Leishmania Ags were used in vitro to stimulate cord blood or PBMC from healthy donors noninfected by Leishmania parasites. MLA, but not soluble Leishmania Ags, constantly induce strong proliferation of cord blood mononuclear cells and PBMC from noninfected individuals. Responding cells are CD3+, CD4+, TCRαβ+, CD45RO+, and CD45RA+ and secrete IFN-γ and IL-10, but not IL-4. MLA do not activate NK cells nor NKT cells. Membrane Ags also induce purified macrophages from noninfected individuals to secrete IL-10 and TNF-α, but have no effect on IL-1α or IL-12 secretion. The effects of MLA are proteinase K-sensitive and resistant to lipid extraction. The lymphoproliferative responses are inhibited by anti-HLA-DR Abs and require Ag processing by APCs, excluding that the biological effect of MLA could be attributed to a superantigen. Finally, TCR repertoire analysis shows that the T cell expansion induced by MLA uses TCR with various variable β segment rearrangements and CDR3 lengths, features much more characteristic to those observed with a polyclonal activator than with a conventional Ag. These results suggest a particular mechanism developed during the host’s natural response to Leishmania parasites that allows direct activation of naive CD4 lymphocytes by parasite membrane-associated Ags.

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Section: Tcr Bv Repertoire and Cdr3 Size Spectratyping Analysismentioning

confidence: 99%

Mechanisms of the Natural Reactivity of Lymphocytes from Noninfected Individuals to Membrane-AssociatedLeishmania infantumAntigens

Sassi

Larguèche-Darwaz

Collette

et al. 2005

The Journal of Immunology

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“…The expression level of HPRT is constant in most tissues, including the testis, and can therefore serve as an internal RT-PCR standard (Pannetier et al, 1993). …”

Section: Semiquantitative Rt-pcrmentioning

confidence: 99%

VASA Is a Specific Marker for Both Normal and Malignant Human Germ Cells

Zeeman

Stoop

Boter

et al. 2002

Laboratory Investigation

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SUMMARY:VASA is so far the only known gene in mammals whose expression is specific for the germ cell lineage. We investigated the presence of VASA mRNA and protein in a series of germ cell tumors of different histologic subtypes and anatomic location, as well as in nongerm cell tumors such as testicular lymphomas and Leydig cell tumors. We detected VASA mRNA (by quantitative RT-PCR) and protein (by immunohistochemical staining) in normal spermatogenesis, seminoma (both classic and spermatocytic), carcinoma in situ (the precursor of classic seminoma and nonseminoma), dysgerminoma, and gonadoblastoma. VASA immunostaining was relatively weak in seminomas and dysgerminomas compared with spermatocytic seminomas, despite similar mRNA levels, suggesting that VASA is regulated in part by post-transcriptional mechanisms. A higher staining intensity compared with the invasive counterparts was observed in the precursor lesions (ie, carcinoma in situ and gonadoblastoma). No VASA mRNA or protein was detectable in nonseminomatous germ cell tumors (such as embryonal carcinoma, teratoma, and yolk sac tumor) and derived cell lines, or nongerm cell tumors such as lymphoma or Leydig cell tumor. These results provide direct evidence that some germ cell tumors retain germ cell characteristics, whereas other tumors of germ cell origin result from differentiation and loss of germ cell identity. Furthermore, these findings suggest that VASA is likely to serve as a useful and highly specific biomarker for germ cell tumors, particularly classic and spermatocytic seminoma/dysgerminoma, including their precursor stages. (Lab Invest 2002, 82:159 -166).

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“…DNA standards were essentially constructed as previously described. 19 For construction of CD95L, CD95tm, CD95sol, CD3␦ chain, and HPRT DNA standards, the respective 5Ј and 3Ј PCR primers (see below) were used to amplify a specific fragment in a human peripheral blood lymphocyte-derived cDNA. A 1000-fold dilution of this product was reamplified using the respective 3Ј PCR primer (see below) and an additional construct primer containing a four-nucleotide deletion compared with the wild-type sequence.…”

Section: Quantitative Competitive Reverse Transcriptionpolymerase Chamentioning

confidence: 99%

Involvement of Soluble CD95 in Churg-Strauss Syndrome

Müschen

Warskulat

Perniok

et al. 1999

The American Journal of Pathology

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Deficiency of CD95 (Apo-1/Fas)-mediated apoptosis has recently been found in some autoimmune lymphoproliferative disorders due to inherited mutations of the CD95 gene. In this study , impairment of CD95 ligand-mediated killing of lymphocytes and eosinophils in Churg-Strauss Syndrome (CSS) , which was a result of variation of CD95 receptor isoform expression , is demonstrated. Compared to those from healthy individuals , peripheral blood lymphocytes from eight CSS patients exhibit a switch from the membrane-bound CD95 receptor expression to its soluble splice variant , which protects from CD95L-mediated apoptosis. In five out of seven CSS patients recurrent oligoclonal T cell expansions were found, all using a V␤-gene from the V␤21 family associated with similar CDR3 motifs , indicating the predominance of T cell clones of a similar specificity in the CSS patients. In two of them , the effect of immunosuppressive therapy was studied. In both cases aberrant overexpression of the soluble CD95 receptor isoform and deviations from normal TCR V␤-gene usage normalized in parallel with the clinical improvement. Furthermore , soluble CD95 was identified as a survival factor for eosinophils rescuing eosinophils from apoptosis in the absence of growth factors in vitro. Churg-Strauss Syndrome (CSS) was for the first time described by Lotte Strauss and Jacob Churg in 1951 in The American Journal of Pathology, 1 defining a new entity with systemic vasculitis, blood and tissue eosinophilia, and a long-term history of asthma. Systemic vasculitis in this rare syndrome mainly affects the lower respiratory tract, kidneys, skin, and nervous system. 2 The gastrointestinal tract is involved in about 35% of the CSS cases. 2 Infiltrating eosinophils are frequently found in granulomatous lesions 3 and the fraction of eosinophils in the peripheral blood correlates with the course of the disease, 3,4 suggestive of a role of eosinophils in the pathogenesis of CSS. 5 On the other hand, T cells were frequently implicated in autoreactivity and inflammatory lesions. 6 However, a potential role of T lymphocytes in CSS and the molecular mechanisms underlying this disease have not yet been studied.One tool to analyze T cell populations in clinical samples at the molecular level is the Immunoscope technique. 5 This approach is based on the hypothesis that the expression of unique, rearranged T cell receptor (TCR) genes reflects the specificity of a given T cell. 7 Each TCR-␤ chain consists of a variable (V), diversity (D), joining (J), and a constant region, the first three determining the antigen specificity in their VDJ-junction, including complementarity-determining region III (CDR3). During T cell differentiation, unique variable region genes are created by recombination of V, D and J segments for the TCR-␤ locus. More than 70 V␤ gene segments have been characterized and are classified into 24 gene families 8 ( Figure 1A).In T cells the CD95/CD95 ligand system is a major pathway of apoptotic cell death and thus essential for prevention of lymp...

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Quantitative titration of nucleic acids by enzymatic amplification reactions run to saturation

Cited by 150 publications

References 32 publications

Mechanisms of the Natural Reactivity of Lymphocytes from Noninfected Individuals to Membrane-AssociatedLeishmania infantumAntigens

Mechanisms of the Natural Reactivity of Lymphocytes from Noninfected Individuals to Membrane-AssociatedLeishmania infantumAntigens

VASA Is a Specific Marker for Both Normal and Malignant Human Germ Cells

Involvement of Soluble CD95 in Churg-Strauss Syndrome

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