Biomedical subjects

R Cambronero

Publications and source records attributed to R Cambronero.

6 recordsLinked to original sources

PubMed2003

Three novel mutations reflect the variety of defects causing phenotypically diverse X-linked hyper-IgM syndrome.

X-linked hyper-IgM syndrome (HIGM1) (MIM musical sharp 308230), is a severe primary immunodeficiency caused by mutations in the gene coding for CD40 ligand (CD40L or CD154), a member of the tumour necrosis factor (TNF) superfamily. The interaction of this protein with its ligand, CD40, mediates crucial processes in the immune response. The variety of defects that have been described in HIGM1 patients range from a complete lack of CD40L protein expression to missense mutations that interfere with its interaction with CD40L. In this study we describe three families - a total of seven HIGM1 patients and carriers, presenting a spectrum of severity in clinical evolution. In two of these families, patient DNA samples were available for genetic studies. In the third, carrier detection was performed on female family members. The results of immunological studies - the different patterns of CD40L expression and binding capacity as measured by flow cytometry - and molecular diagnosis are presented. Three novel mutations were identified: an intron mutation that partially interferes with the splicing process (intron 3, position + 5 G/T); a missense mutation (Ser222 Phe) located in the molecular region which interacts with the receptor and which abrogates binding capacity; and a 14 base pair deletion leading to a frameshift and a premature truncated mutation (del I 171 X 195). An attempt to correlate protein expression and function of the CD40L mutants with clinical disease evolution is described.

Base Sequence↗

PubMed2002

Characterization of Herpesvirus saimiri-transformed T lymphocytes from common variable immunodeficiency patients.

Common variable immunodeficiency (CVID) is a very frequent but heterogeneous syndrome of antibody formation. The primary defect remains unknown, but many reports describe peripheral blood T lymphocyte dysfunctions in a substantial proportion of CVID patients, which may impair T--B cell collaboration. In order to investigate whether such putative defects were intrinsic to T cells or, rather, secondary to quantitative differences in T cell subset distribution, or to other described disorders, we have used Herpesvirus saimiri (HVS) for the targeted transformation of CVID CD4+ and CD8+ T cells and subsequent functional evaluation by flow cytometry of their capacity to generate cell surface (CD154, CD69) or soluble (IL-2, TNF-alpha, IFN-gamma) help after CD3 engagement. Unexpectedly, the results showed that 40 different CVID blood samples exposed to HVS gave rise with a significantly increased frequency to transformed CD4+ T cell lines, compared to 40 age-matched controls (27% versus 3%, P < or = 0.00002) suggesting the existence of a CVID-specific signalling difference which affects CD4+ cell transformation efficiency. The functional analysis of 10 CD4+ and 15 CD8+ pure transformed T cell lines from CVID patients did not reveal any statistically significant difference as compared to controls. However, half of the CD4+ transformed cell lines showed CD154 (but not CD69) induction (mean value of 46.8%) under the lower limit of the normal controls (mean value of 82.4%, P < or = 0.0001). Exactly the same five cell lines showed, in addition, a significantly low induction of IL-2 (P < or = 0.04), but not of TNF-alpha or IFN-gamma. None of these differences were observed in the remaining CD4+ cell lines or in any of the transformed CD8+ cell lines. We conclude that certain CVID patients show selective and intrinsic impairments for the generation of cell surface and soluble help by CD4+ T cells, which may be relevant for B lymphocyte function. The transformed T cell lines will be useful to establish the biochemical mechanisms responsible for the described impairments.

Adolescent↗

PubMed2000

Up-regulation of IL-12 in monocytes: a fundamental defect in common variable immunodeficiency.

We show that LPS-stimulated circulating CD14-positive monocytes from patients with common variable immunodeficiency (CVID) express a higher proportion of intracellular IL-12-positive cells than monocytes from patients with X-linked agammaglobulinemia or normal subjects. We used four-color flow cytometry and measured IL-12 with an Ab to the p40 subunit following stimulation with LPS. The raised IL-12 is associated with an increased frequency of IFN-gamma-positive T cells, but not of IFN-gamma-positive CD56+ NK cells. These increases in frequency of cytokine-positive cells are due to a decrease in the absolute numbers of circulating monocytes and T cells that are negative for IL-12 and IFN-gamma, respectively. The increased frequency of IL-12-positive monocytes appears to be selective because TNF-alpha was not increased, and is thus unlikely to reflect a general activation. Chronic infection is also unlikely to explain our data since cells from X-linked agammaglobulinemia patients with a similar Ig deficiency do not show these changes. Our data suggest a fundamental abnormality in the IL-12/IFN-gamma circuit in CVID, with up-regulation of IL-12 being the "primary" factor. This imbalance is likely to skew the immune response away from Ab production and also explains the failure of CVID T cells to make Ag-specific memory cells and the chronic inflammatory and granulomatous complications that are a feature of CVID. This disease appears to be a rare example of a polarized Th1-type response and may in part be due to a genetic defect in the control of IL-12 production.

Agammaglobulinemia↗

PubMed1999

Internalization of factor J and cellular signalization after factor J-cell interaction.

Factor J (FJ) is a cationic glycoprotein with inhibitory activity in vitro against both classical and alternative pathways of complement activation. Recently FJ has been implicated in adhesion to several cell lines, through a membrane receptor identified as nucleolin. In the present work we study the events that follow the binding of FJ to cells. After incubation of K562 with FJ, this protein was internalized actively and localized in the cytoplasm and nucleus. Adhesion to immobilized FJ induced tyrosine phosphorylation of several intracellular proteins in Jurkat cell line with a similar pattern to that induced by fibronectin (FN), an extracellular matrix protein. This effect was maximal at 5 min and decreased after 10 min, and inhibited by anti-FJ monoclonal antibody (mAb). These results suggest that the binding of FJ to cells may play an important role in transduction of biochemical signals across the plasma membrane to the cell interior.

Antibodies, Monoclonal↗

PubMed1999

In vivo modulation of cytokine synthesis by intravenous immunoglobulin.

We examined the effects of intravenous immunoglobulin (IVIG) on cytokine regulation in vivo using samples taken before and after replacement-dose (200-400 mg/kg) IVIG in a group of patients with common variable immunodeficiency (CVID) and X-linked agammaglobulinaemia (XLA). The intracellular cytokine content of CD4+ and CD8+ lymphocytes, and their CD28+/- subsets, were measured following in vitro activation with phorbol myristate acetate (PMA) and ionomycin. The cytokines IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), and the early activation marker CD69, were assessed by four-colour flow cytometry of whole blood cultures taken before and after IVIG infusion. There was a significant increase in IL-2 expression in CD4+ (and CD4+28-) cells and an increase in TNF-alpha expression in CD8+28- cells following IVIG in CVID, but not in XLA patients. IFN-gamma and CD69 expression were not affected by IVIG infusion. This increase in TNF-alpha and IL-2, combined with unchanged IFN-gamma expression, is evidence against the putative 'anti-inflammatory' role of IVIG, and may explain the failure of resolution of granulomata in CVID patients treated with IVIG alone.

Adult↗

PubMed1998

Cellular adhesion mediated by factor J, a complement inhibitor. Evidence for nucleolin involvement.

Factor J (FJ) is a complement inhibitor that acts on the classical and the alternative pathways. We demonstrated FJ-cell interactions in fluid phase by flow cytometry experiments using the cell lines Jurkat, K562, JY, and peripheral blood lymphocytes. FJ bound to plastic plates was able to induce in vitro adhesion of these cells with potency equivalent to fibronectin. As evidence for the specificity of this reaction, the adhesion was blocked by MAJ2, an anti-FJ monoclonal antibody, and by soluble FJ. Attachment of the cells required active metabolism and cytoskeletal integrity. The glycosaminoglycans heparin, heparan sulfate, or chondroitin sulfates A, B, and C inhibited to varying degrees the binding of FJ to cells, as did treatment with chondroitinase ABC. In the search for a putative receptor, a protein of 110 kDa was isolated by affinity chromatography, and microsequence analysis identified this protein as nucleolin. Confocal microscopy evidenced the presence of nucleolin in cell membrane by immunofluorescence with monoclonal (D3) and polyclonal anti-nucleolin antibodies in Jurkat cells. The interaction FJ-nucleolin was evidenced by Western blot and enzyme-linked immunosorbent assay. Furthermore, purified nucleolin and D3 inhibited adhesion of Jurkat cells to immobilized FJ, suggesting that the interaction was specific and that nucleolin mediated the binding.

Antibodies, Monoclonal↗