[Immunohistochemical expression of microvascular density and carbonic anhidrase IX in renal carcinoma. Relation to histological type and tumoral progression]. / Expresión inmunohistoquímica de la densidad microvascular y de la anhidrasa carbónica IX en carcinoma renal. Relación con el tipo histológico y con la progresión tumoral.

PURPOSE: to correlate the immunohistochemical expression of microvascular density (MVD) and the carbonic anhydrase IX (CAIX) with the different histological subtypes of renal carcinoma and its progression. MATERIAL AND METHODS: we studied 93 patients with renal cell carcinoma operated between 1990 and 2008. Antibodies employed for immunohistochemistry (IHC); CD31 (1: 40, Dako) and CD34 (1: 50, Dako) for MVD and CAIX (1: 100, Santa Cruz). CAIX was validated semiquantitatively as: strongly positive (>85%); weakly positive (10% -85%); and negative (< 10%), independently of the intensity of the stain. MVD was validated with both anti-CD31 and anti-CD34 by means of a whole section, to select the microscopic field (x100) with highest density of stained vessels, counting the number of vessels in a photographic field of 0.53 mm(2). Results are expressed as the maximal number of vessels by mm(2) of tumour tissue. RESULTS: median follow up was 40 months (1-160). We found no differences of expression with any of the 3 IHC markers between tumours that progressed (49) and tumours that did not progress (44). The IHC expression of CAIX was strongly related to MVD, measured for both CD31 and CD34 (p<0.0001). MVD with both antibodies was inversely related to tumour size and Fuhrman grade and was also stronger in clear cell carcinomas compared to the rest of histological subtypes, measured by CD31 (p = 0.001) and CD34 (p = 0.003). CONCLUSIONS: neither MVD nor CAIX expressions were related to tumour progression, but were related to histological subtypes. This fact, added to their co-expression, could prompt the use of the CAIX expression, which is far more reproducible, as a quick and easy approximation to MVD. More research should be done to use it as marker for targeted therapy.

Analysis of the human G protein-coupled receptor kinase 2 (GRK2) gene promoter: regulation by signal transduction systems in aortic smooth muscle cells.

BACKGROUND: Desensitization of G protein-coupled receptors (GPCR) is emerging as an important feature of several cardiovascular diseases. G protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of a variety of these receptors, and its cardiac expression levels are altered in pathological situations such as chronic heart failure. However, very little is known about the signals and mechanisms that modulate GRK2 expression in cardiovascular cells. METHODS AND RESULTS: We have studied the transcriptional activity of the 1.6-kb-long proximal genomic region of the human GRK2 gene. In an aortic smooth muscle cell line, agents that lead to physiological vasoconstriction and hypertrophy, such as phorbol esters, increased GRK2 promoter activity. Activation of signaling pathways by cotransfected G(alphaq) subunits or alpha(1)-adrenergic receptors also markedly enhanced the expression of the GRK2 promoter constructs. Conversely, proinflammatory cytokines, such as interleukin-1beta, tumor necrosis factor-alpha, or interferon-gamma, led to the opposite effect, decreasing the activity of the GRK2 promoter. CONCLUSIONS: Our results suggest that the expression of GRK2 in vascular cells is tightly controlled at the transcriptional level by the interplay between several extracellular messengers, which may trigger alterations of normal GRK2 levels in some physiopathological circumstances, thus promoting changes in the efficacy of the GPCR signal transduction.

The gene coding for the beta-chain of C4b-binding protein (C4BPB) has become a pseudogene in the mouse.

C4BP beta is one of the two polypeptides that in humans compose the plasma glycoprotein C4b-binding protein (C4BP). C4BP beta binds the anticoagulant vitamin K-dependent protein S. Two, nonmutually exclusive, roles have been proposed for the C4BP-protein S interaction. It has been suggested to play a role in the control of the protein C anticoagulatory pathway. In addition, it may serve an important role in localizing C4BP to the surface of injured or activated cells. While the physiological significance of C4BP-protein S interaction is unclear, it has clinical relevance because elevated plasma levels of C4BP are associated with increased risk for thromboembolic disorders in humans, due to an inactivation of the protein C anticoagulatory pathway. Using a human C4BP beta cDNA probe, we have isolated and characterized a genomic DNA fragment that includes the murine C4BPB gene. Murine C4BPB is a single-copy gene that maps close to the C4BPA gene in chromosome 1. It contains two exons homologous to the exons coding for the SCR-1 and SCR-2 repeats of the human C4BP beta polypeptide chain. Sequence analysis of the C4BPB exons in the Mus musculus inbred strains CBA, Balb/c, and C57BL/6, in pen-bred Swiss mice, and in Mus spretus demonstrated the presence of two in-phase stop codons that are incompatible with the expression of a functional C4BP beta polypeptide. Thus, the characterization of the murine C4BPB gene documents the peculiar situation of a single-copy gene that is functional in humans but has become a pseudogene in the mouse.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of T-cell proliferation by rat synoviocytes.

Antigen presentation by synoviocytes to arthritis-related T-cell clones was studied in Lewis rats. Freshly isolated synoviocytes may be able to take up and process mycobacterial antigens but they seem inefficient as antigen presenting cells. Furthermore, synovial cells inhibited the specific proliferative responses of T lymphocytes by a mechanism which apparently was not antigen-specific or mediated by secreted cytokines. Synovial cells isolated from rats at the period of developing adjuvant arthritis showed a lower inhibitory capacity associated with a lower degree of antigen clearance. These results suggest that synoviocytes might inhibit T-cell responses in normal joints and that this negative control diminishes following arthritis induction.

Aggregated human immunoglobulins bind to modified proteins.

Human immunoglobulins treated at 55 degrees C in vitro are able to interact with maleylated bovine serum albumin (mBSA), but not with unmodified BSA. Gel filtration experiments demonstrated that the mBSA binding is associated with a high molecular weight complex of aggregated IgG. This aggregated IgG with binding capacity for mBSA could also be generated in vitro by treatment of human IgG at 37 degrees C or 40 degrees C and by incubation with human neutrophils. Furthermore, IgG aggregates with binding activity for mBSA could be detected in untreated synovial fluids from rheumatoid arthritis patients, indicating that these complexes occur in vivo. The phenomenon of binding to aggregated IgG was extended to other modified proteins such as maleylated human serum albumin (mHSA), acetyl low density lipoprotein (Ac-LDL) and BSA reacted with oxidized linolenic acid. Soluble forms of these modified proteins were able to compete for the interaction between aggregated IgG and surface-bound mBSA. We also found that aggregated IgG enhanced the Ac-LDL-dependent foam cell formation. These findings suggest a role for aggregated IgG in the metabolism of oxidized proteins.

Decreased tubulin synthesis in synoviocytes from adjuvant-induced arthritic rats.

The first microscopical alterations along adjuvant arthritis induction in rats seem to appear in the synovium. We have studied the protein synthesis pattern of the cells constitutively present in synovial membrane (synoviocytes) and have found an impairment of synthesis of some proteins when synoviocytes are derived from adjuvant arthritic rats. One of these polypeptides was identified as beta tubulin by two-dimensional gel electrophoresis, a membrane transfer assay using a specific monoclonal antibody and peptide mapping. We postulate that a repressed synthesis of tubulin may be an initial step in the triggering of the disease, since the effect was evident at pre-arthritic stages, when infiltration by inflammatory cells had not yet occurred.

Arthritis transferred by cells derived from pre-inflammatory rat synovium.

Different cell populations isolated from rats during the period of latency of adjuvant arthritis were injected into the bloodstream of naive rats to test their ability to transfer articular disorders. Synovium-derived cells (synoviocytes) were able to induce arthritis in 3 out of 4 recipient animals, whereas peripheral blood leukocytes, peritoneal exudate macrophages, lymph node cells, synoviocyte lysates and synoviocytes from control animals were not able to do so. This model of cellular transferred arthritis is associated with antibody titres to hsp65 in rat sera. Our findings suggest a crucial role for synovial cells in the pathogenesis of adjuvant disease, which might be linked to their function as accessory cells.