Monoclonal antibodies to cultured human glomerular mesangial cells. I. Reactivity with haematopoietic cells and normal kidney sections.

The aim of this study was to produce monoclonal antibodies to cultured human glomerular mesangial cells in order to obtain specific markers for these cells and to aid the study of their function. Using standard monoclonal antibody techniques, 29 hybridomas producing antibodies directed to cultured mesangial cells were obtained. Most of these antibodies were not reactive with normal or neoplastic haematopoietic cell lines by flow cytometry. Fourteen of the 29 culture supernatants bound to various components of normal human kidney sections stained by the alkaline phosphatase/anti-alkaline phosphatase (APAAP) method. Ten of these supernatants reacted with components within the glomerulus, with six binding to the mesangium. These studies suggest that (1) mesangial cells in culture may show significant de-differentiation, because most supernatants which reacted with mesangial cells in culture did not do so in tissue sections; (2) antibodies reactive with haematopoietic cells may not detect the majority of immunogenic surface antigens on cells in tissues; and (3) some of the antibodies which we have produced may prove to be useful markers for mesangial cells in glomerular disease.     

The contingent negative variation (CNV) to fear-related stimuli in acquisition and extinction

Contingent Negative Variation (CNV) in anticipation of phobic and neutral slide material was recorded in Acquisition of a learned association between tone pitch and slide type, and in Extinction (following removal of slide presentation). In phobic volunteers, no clear CNV differentiation between 'phobic' and control condition was apparent in either Acquisition or Extinction. For Control subjects, a different pattern entirely has been demonstrated, with anticipation of the neutral condition giving rise to a larger CNV than anticipation of the 'phobic' condition (in Acquisition). In addition, Extinction led to a reversal of this CNV amplitude relationship, with the tone previously associated with the phobic slide producing a DC-shift which was larger than that following the tone previously paired with the neutral slide. The results are compatible with a dynamic model of CNV generation according to which CNV amplitude is positively related to an expectancy for reward or non-punishment but inversely related to an expectancy for punishment or non-reward.     

Specific Interferon-Gamma Producing CD4+ and CD8+ T Cells After Autologous EBV-B Stimulation: The Necessity of Restimulation

The response of T cells to produce interferon-γ, to proliferate and to become cytotoxic after specific stimulation with low dose (2%) autologous EBV-B cells was investigated in 15 EBV seropositive and five seronegative patients. A significantly higher number of interferon-γ producing cells (56 ± 24 per 10⁵ T cells) were found in a spot ELISA in EBV positive than in EBV negative patients (7 ± 2 spots, P<0.01) and it was only found with restimulation after 5–12 days of primary culture. No correlation was found between the extent of interferon-γ production, cytotoxicity or profileration. Specificity of EBV-induced interferon-γ production was demonstrated by comparison of the response to allogeneic EBV-B cells or IL-2 in the reslimulation phase. The response was stronger in CDS⁺ T cells than in CD4⁺ T cells and could be blocked in the restimulation phase with HLA class I and class II antiserum, respectively.     

The prepattern transcription factor Irx2, a target of the FGF8/MAP kinase cascade, is involved in cerebellum formation

The cerebellum develops from the rhombic lip of the rostral hindbrain and is organized by fibroblast growth factor 8 (FGF8) expressed by the isthmus. Here we report characterization of Irx2, a member of the Iroquois (Iro) and Irx class of homeobox genes, that is expressed in the presumptive cerebellum. When Irx2 is misexpressed with Fgf8a in the chick midbrain, the midbrain develops into cerebellum in conjunction with repression of Otx2 and induction of Gbx2. During this event, signaling by the FGF8 and mitogen-activated protein (MAP) kinase cascade modulates the activity of Irx2 by phosphorylation. Our data identify a link between the isthmic organizer and Irx2, thereby shedding light on the roles of Iro and Irx genes, which are conserved in both vertebrates and invertebrates.     

Mutations of the gene encoding the transmembrane transporter protein ABC-C6 cause pseudoxanthoma elasticum

We recently published the precise chromosomal localization on chromosome 16p13.1 of the genetic defect underlying pseudoxanthoma elasticum (PXE), an inherited disorder characterized by progressive calcification of elastic fibers in skin, eye, and the cardiovascular system. Here we report the identification of mutations in the gene encoding the transmembrane transporter protein, ABC-C6 (also known as MRP-6), one of the four genes located in the region of linkage, as cause of the disease. Sequence analysis in four independent consanguineous families from Switzerland, Mexico, and South Africa and in one non-consanguineous family from the United States demonstrated several different mis-sense mutations to cosegregate with the disease phenotype. These findings are consistent with the conclusion that PXE is a recessive disorder that displays allelic heterogeneity, which may explain the considerable phenotypic variance characteristic of the disorder.     

Inhibition of cell movement and associated changes by hexachlorocyclohexanes due to unregulated intracellular calcium increases

Hexachlorocyclohexanes (HCCHs) are potent stimulators of polymorphonuclear leukocyte (PMN) oxidative metabolism and of mobilization of calcium from intracellular stores. It was of interest, therefore, to evaluate the effect of HCCHs on PMN orientation and chemotaxis and to determine their effectiveness as chemotaxins. Chemotaxis was evaluated using micro-Boyden chambers, f-actin was quantitated by nitrobenzoxadiazole (NBD)-phallacidin fluorescence, and microtubules were quantitated by observing the concanavalin A (Con A) capping phenomenon. We also evaluated changes in intracellular calcium [Ca2+]i using quin 2 fluorescence. We found that the HCCH isomers were not chemotaxins and that the HCCH isomers that stimulated O2- formation (delta and gamma HCCH) inhibited chemotaxis. This effect was associated with inhibition of orientation. In addition, we found extensive inhibition of both f-actin and Con A cap formation. These effects of HCCH on cell function were associated with marked increases of [Ca2+]i. This work suggests that non-receptor-mediated increases of [Ca2+]i associated with HCCH have divergent effects on cell function and suggests that physiologic responses of PMNs requiring cytoskeletal alterations, such as chemotaxis, depend on the controlled responses of receptor-mediated stimulation.     

Pseudocyst or cystic neoplasm? Differential diagnosis and initial management of cystic pancreatic lesions

Owing to advances in pancreatic imaging, cystic lesions of the pancreas are being recognized with increasing frequency. A presumptive diagnosis of "pseudocyst", based upon CT appearance alone, will prove to be in error in as many as one-third of patients. Neoplastic cysts of the pancreas are particularly susceptible to this misdiagnosis, which can result in inappropriate drainage rather than resection. Using a combination of historical features and computed tomography, the differential diagnosis between pseudocyst and cystic neoplasm can usually be made. In borderline patients transcutaneous aspiration for cytology, and analysis of the cyst fluid for neoplastic markers may prove helpful. Final resolution of doubtful cases is achieved by biopsy of the cyst wall.     

Basic fibroblast growth factor expression in human bone marrow and peripheral blood cells

We have shown previously that basic fibroblast growth factor (bFGF) is a mitogen for human bone marrow (BM) stromal cells and that bFGF stimulates myelopoiesis in primary BM cultures. In this article, we demonstrate the presence of bFGF in two cell lineages in human BM and peripheral blood as well as the deposition of bFGF into the extracellular matrix of BM stromal cell cultures. In immunofluorescence experiments on BM and peripheral blood smears, megakaryocytes and platelets stained strongly for bFGF, whereas weaker staining was observed in immature and mature cells of the granulocyte series. The presence of bFGF in platelets was confirmed by enzyme-linked immunosorbent assay as well as by immunoprecipitation followed by immunoblotting. bFGF was synthesized by BM stromal cell cultures and was found either cell associated or localized in the nucleus and the nucleoli, and its location was dependent on the fixation procedure used. Addition of exogenous bFGF to stromal cells showed the presence of extracellular binding molecules for this cytokine. bFGF could be released from these sites by soluble heparin or phosphatidylinositol- specific phospholipase C. This study supports the role of bFGF as a stromal cell mitogen and stimulator of myelopoiesis. The data indicate that the stromal cells produce bFGF and that their extracellular matrix can serve as a reservoir for this growth factor. In addition, the results suggest a possible involvement of bFGF in platelet function as well as in megakaryocytopoiesis.     

Interaction of single-chain urokinase with its receptor induces the appearance and disappearance of binding epitopes within the resultant complex for other cell surface proteins

Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI- 1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell- bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.     

Rechargeable deep brain stimulators in the management of paediatric dystonia: well tolerated with a low complication rate

Background: Deep brain stimulation (DBS) is a recognised method of treatment for primary and secondary dystonia. The size of non-rechargeable batteries has limited their use in small children. Our severe dystonia patients have required battery replacement every 20-24 months. Objectives: To evaluate reliability, care burden, patients' satisfaction and complications related to the rechargeable neurostimulator Activa? RC (launched by Medtronic in Europe in autumn 2008). Methods: Complications were recorded prospectively, and a questionnaire on neurostimulator maintenance, care burden and parental satisfaction was applied to all patients with at least 3 months of follow-up. Results: 30 Activa RCs were implanted between December 2008 and June 2010, 25 with a follow-up of 3-17 months (mean 10); the mean patient's age at surgery was 11.1 years; 22/25 questionnaires were completed. All families achieved good standards of recharging. Caregivers were responsible for recharging in 82% of cases. With higher parameters of stimulation, recharging time was longer than initially recommended by the manufacturer. All but one family would recommend Activa RC to other patients. Transient recharging problems were the most common complication (36% of cases). Infection/skin erosion occurred in 8% of cases, self-resolving early seroma in 20%. Conclusions: Activa was found to offer reliable stimulation with a low rate of significant complications and a suitable treatment option for children with dystonia.