Autoantibodies against the serotoninergic 5-HT4 receptor and congenital heart block: a reassessment

The specificity of autoantibodies against the serotoninergic 5-HT4 receptor in congenital heart block has led to conflicting observations. In order to clarify the situation, a collaborative effort was undertaken to discover the reasons for these discrepancies and to reassess the importance of such autoantibodies by making use of the Research Registry for Neonatal Lupus. Sera from 128 patients (101 anti-SSA/Ro52 positive mothers among which 74 have children with congenital heart block (CHB), 9 anti-SSA/Ro52 negative patients of which 1 had a child with heart block and 18 healthy donors) were assessed in a single blind test using an ELISA coated with a 5-HT4 receptor-derived peptide. Discrepancies between previous observations in our two groups could be ascribed to small differences in the set up of the assay. Of the 75 sera from mothers of children with CHB, 12 were reactive with the 5-HT4 peptide. Four sera among which three were from 35 Ro52 negative mothers without affected children as well as 2 in the 18 controls were positive. Interestingly, in 1 mother with an isolated child with CHB but who had no detectable anti-SSA/Ro52 antibodies and 1 mother with a child with a structural heart block and no detectable antibodies to any component of SSA/Ro, reactivity with the 5-HT4 receptor was noted. While 5-HT4 receptor autoantibodies do not have the predictive value of anti-Ro52 autoantibodies, the presence of these antibodies in a minor subset of mothers whose children have CHB suggests an additional risk factor which may contribute to the pathogenesis of disease.     

Neuroprotective effects of IGF-I against TNFalpha-induced neuronal damage in HIV-associated dementia

Human immunodeficiency virus type 1 (HIV-1) infection often results in disorders of the central nervous system, including HIV-associated dementia (HAD). It is suspected that tumor necrosis factor-alpha (TNFalpha) released by activated and/or infected macrophages/microglia plays a role in the process of neuronal damage seen in AIDS patients. In light of earlier studies showing that the activation of the insulin-like growth factor I receptor (IGF-IR) exerts a strong neuroprotective effect, we investigated the ability of IGF-I to protect neuronal cells from HIV-infected macrophages. Our results demonstrate that the conditioned medium from HIV-1-infected macrophages, HIV/CM, causes loss of neuronal processes in differentiated PC12 and P19 neurons and that these neurodegenerative effects are associated with the presence of TNFalpha. Furthermore, we demonstrate that IGF-I rescues differentiated neurons from both HIV/CM and TNFalpha-induced damage and that IGF-I-mediated neuroprotection is strongly enhanced by overexpression of the wt IGF-IR cDNA and attenuated by the antisense IGF-IR cDNA. Finally, IGF-I-mediated antiapoptotic pathways are continuously functional in differentiated neurons exposed to HIV/CM and are likely supported by TNFalpha-mediated phosphorylation of I(kappa)B. All together these results suggest that the balance between TNFalpha and IGF-IR signaling pathways may control the extent of neuronal injury in this HIV-related experimental setting.     

Tenascin immunoreactivity in normal and pathological bone marrow.

AIMS: To determine the distribution of tenascin in normal and pathological bone marrow. METHODS: 48 different bone marrow lesions were studied immunohistochemically using a monoclonal antibody to tenascin. RESULTS: Tenascin immunoreactivity was found in lesions with increased fibrosis and high numbers of reticular fibres. The strongest immunoreactivity was found in myelofibrosis. Bone marrow from acute and chronic myeloid and lymphatic leukaemias showed weak or moderate immunoreactivity. In hyperplasias inconsistent reticular tenascin immunoreactivity was found; in normal bone marrow, only a few scattered positive fibres were occasionally seen. CONCLUSIONS: Tenascin was generally observed in conditions in which megakaryocytic hyperplasia was a feature. This is in line with the notion that tenascin synthesis in bone marrow fibroblasts is stimulated by TGF-beta which is synthesised by the megakaryocytic lineage. Tenascin also contains EGF-like repeats. It might therefore function as a growth promoter and in this way could also stimulate synthesis of other matrix components. On the other hand, tenascin could function as an adhesive molecule to some cells of the bone marrow. The presence of tenascin in many pathological states of the bone marrow suggests that it may have a role in their pathogenesis and that it also could be a potential marker of disease.     

Involvement of SHV-12 and SHV-2a encoding plasmids in outbreaks of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a Tunisian neonatal ward

Previous genotypic investigations of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae recovered in a Tunisian neonatal ward revealed the spread of two epidemic strains and a high number of genetically unrelated isolates. The aim of the present study was to determine the role of the dissemination of self-transferrable plasmids harboring bla genes in the outbreaks experienced by the ward. The 49 previously identified clinical isolates of ESBL-producing K. pneumoniae were examined for relationships between their enzymes and plasmids. Analysis of crude extracts by isoelectric focusing showed four beta-lactamase-activities at pI 8.2, 7.6, 6, and 5.4. Clinical isolates contained large plasmids that could be transferred by conjugation and transformation conferring resistance to expanded-spectrum cephalosporins. DNA amplification and sequencing were performed to confirm the identities of transferred beta-lactamases. Nucleotide sequence analysis of SHV-specific PCR products from six isolates identified two bla(SHV) genes corresponding to SHV derived ESBLs, SHV-12 and SHV-2a. PstI digestion of plasmid DNA from transformants revealed six restriction patterns. The occurrence of the prevalent plasmid pattern in both epidemic strains and unrelated isolates indicated that diffusion and endemic persistence of the bla(SHV-ESBL) genes in the ward were due to concomitant spread of epidemic strains and plasmid dissemination among unrelated strains.     

Production and characterization of murine monoclonal antibodies to Histoplasma capsulatum yeast cell antigens

Four monoclonal antibodies (MAbs) were produced by immunizing mice with a disrupted yeast cell homogenate of Histoplasma capsulatum. MAbs 1 and 2 reacted only with the yeast cell antigens of H. capsulatum and Blastomyces dermatitidis, whereas MAbs 3 and 4 showed broader cross-reactivity. MAb 3 cross-reacted with B. dermatitidis, Paracoccidioides brasiliensis, Sporothrix schenckii, and Candida albicans, and MAb 4 cross-reacted with B. dermatitidis, C. albicans, Coccidioides immitis, Aspergillus fumigatus, and Mycobacterium tuberculosis. All four MAbs exhibited unique specificity when reacted with three different strains of H. capsulatum (G217B, A811, and P-IN). MAb 1 belonged to the IgG2b subclass, MAb 3 belonged to the IgG1 subclass, and MAbs 2 and 4 belonged to the IgG3 subclass. MAbs 1, 2, and 3 formed bands in the Western immunoblot assay; the two dominant distinct bands had apparent molecular masses of 72 and 62 kilodaltons.     

Quantitative comparison between two phase contrast techniques: diffraction enhanced imaging and phase propagation imaging

Two x-ray phase contrast imaging techniques are compared in a quantitative way for future mammographic applications: diffraction enhanced imaging (DEI) and phase propagation imaging (PPI). DEI involves, downstream of the sample, an analyser crystal acting as an angular filter for x-rays refracted by the sample. PPI simply uses the propagation (Fresnel diffraction) of the monochromatic and partially coherent x-ray beam over large distances. The information given by the two techniques is assessed by theoretical simulations and compared at the level of the experimental results for different kinds of samples (phantoms and real tissues). The imaging parameters such as the energy, the angular position of the analyser crystal in the DEI case or the sample to detector distance in the PPI case were varied in order to optimize the image quality in terms of contrast, visibility and figure of merit.     

Thymocyte expression of cathepsin L is essential for NKT cell development

CD1d antigen presentation to natural killer T (NKT) cells expressing the semi-invariant T cell receptor V(alpha)14J(alpha)18 requires CD1d trafficking through endosomal compartments; however, the endosomal events remain undefined. We show that mice lacking the endosomal protease cathepsin L (catL) have greatly reduced numbers of V(alpha)14(+)NK1.1(+) T cells. In addition, catL expression in thymocytes is critical not only for selection of these cells in vivo but also for stimulation of V(alpha)14(+)NK1.1(+) T cells in vitro. CD1d cell-surface expression and intracellular localization appear normal in catL-deficient thymocytes, as does the lysosomal morphology; this implies a specific role for catL in regulating presentation of natural CD1d ligands mediating V(alpha)14(+)NK1.1(+) T cell selection. These data implicate lysosomal proteases as key regulators of not only classical major histocompatibility complex class II antigen presentation but also nonclassical CD1d presentation.     

Biochemical characterization of a novel extended-spectrum beta-lactamase from Pseudomonas aeruginosa 802

Pseudomonas aeruginosa 802 was isolated at Rabta hospital in Tunis and was resistant to extended-spectrum cephalosporins and aztreonam. It produced a pI 7.6 extended-spectrum beta-lactamase (ESBL). The ESBL, named LBT 802, was purified to homogeneity by filtration on Sephadex G-75 followed by CM-Sepharose chromatography and high-performance liquid chromatography (HPLC) on a TSK-gel SP-5PW column. The LBT 802 enzyme had a molecular mass of 30 kDa. It showed a broad-substrate profile by hydrolyzing benzylpenicillin, ampicillin, cephalothin, cephaloridine, cefotaxime, ceftriaxone, and cefpirome but not ceftazidime, cefoxitin, imipenem, or aztreonam. The highest hydrolytic efficiency (Vmax/Km) was obtained for ampicillin, cephalothin, cephaloridine, and benzylpenicillin. Among extended-spectrum cephalosporins the best substrate was ceftriaxone followed by cefotaxime and cefpirome. LBT 802 activity was inhibited by clavulanic acid, sulbactam, imipenem, cefoxitin, and aztreonam. It showed its lowest Ki values for clavulanic acid, imipenem and sulbactam.