Rapid detection of 17p11.2 rearrangements by FISH without cell culture (direct FISH,DFISH): a prospective study of 130 patients with inherited peripheral neuropathies

Charcot-Marie-Tooth (CMT) disease and hereditary neuropathy with pressure palsies (HNPP) are two frequent hereditary motor and sensory neuropathies. CMT is characterized by slowly progressive weakness and atrophy, primarily in peroneal and distal leg muscles. The most frequent form, CMT1A, is due, in most cases, to the duplication of a 1.5 Mb region on chromosome 17p11.2 containing the peripheral myelin protein 22 gene (PMP22). The phenotype seems to result from dosage of the PMP22 gene. This hypothesis is reinforced by the existence of HNPP, which is clinically characterized by various recurrent truncular palsies or sensory loss precipitated by minor trauma, which is caused by deletion of the same 1.5 Mb region in 17p11.2. In clinical practice, the detection of the duplication or the deletion in 17p11.2, which permits a positive diagnosis, is still performed by time consuming methods (Southern blot or various combinations of molecular tools). We developed a method for the rapid detection of 17p11.2 rearrangements, using "direct FISH" and PRINS analyses, which does not require cell culture. In a prospective study of 92 patients with CMT and 38 with suspected HNPP, we compared this new technique to classical strategies like Southern blot. The results demonstrate the high sensitivity and specificity of the new FISH technique for the diagnosis of CMT1A and HNPP. Moreover, because of its simplicity and rapidity, this technique provides a useful alternative to the molecular approaches that have been used to diagnose segmental aneusomies, especially in the case of duplications that often go undetected.     

Human eosinophils express and release IL-13 following CD28-dependent activation

Human eosinophils produce a large number of cytokines, including immunoregulatory cytokines. Given that eosinophils store and release interleukin (IL)-4, a key cytokine in the pathogenesis of allergic inflammation, and that IL-4 and IL-13 share common biological functions, we investigated the possibility that IL-13 may be synthesized by these cells. Using flow cytometry and immunocytochemistry, we show that eosinophils synthesize and store IL-13. Granule localization was demonstrated after subcellular fractionation, and IL-13 immunoreactivity was localized to crystalloid, granule-enriched fractions. Furthermore, electron microscopic analyses specifically localized IL-13 to the dense cores of bicompartmental secondary granules. Upon CD28 ligation, IL-13 was released by eosinophils, whereas a combination of CD28 and immunoglobulin A complexes resulted in decreased IL-13 secretion. Furthermore, eosinophil-derived IL-13 exerts a biological effect, inducing CD23 expression on B cells. By having the capacity to synthesize and release IL-13, eosinophils may participate in the development and maintenance of the T helper cell type 2 response, a prominent feature of allergic diseases.     

Quantitative measurement of regional lung gas volume by synchrotron radiation computed tomography

The aim of this study was to assess the feasibility of a novel respiration-gated spiral synchrotron radiation computed tomography (SRCT) technique for direct quantification of absolute regional lung volumes, using stable xenon (Xe) gas as an inhaled indicator. Spiral SRCT with K-edge subtraction using two monochromatic x-ray beams was used to visualize and directly quantify inhaled Xe concentrations and airspace volumes in three-dimensional (3D) reconstructed lung images. Volume measurements were validated using a hollow Xe-filled phantom. Spiral images spanning 49 mm in lung height were acquired following 60 breaths of an 80% Xe-20% O2 gas mixture, in two anaesthetized and mechanically ventilated rabbits at baseline and after histamine aerosol inhalation. Volumetric images of 20 mm lung sections were obtained at functional residual capacity (FRC) and at end-inspiration. 3D images showed large patchy filling defects in peripheral airways and alveoli following histamine provocation. Local specific lung compliance was calculated based on FRC/end-inspiration images in normal lung. This study demonstrates spiral SRCT as a new technique for direct determination of regional lung volume, offering possibilities for non-invasive investigation of regional lung function and mechanics, with a uniquely high spatial resolution. An example of non-uniform volume distribution in rabbit lung following histamine inhalation is presented.     

Distribution of TT virus genomic groups 1-5 in Brazilian blood donors, HBV carriers, and HIV-1-infected patients

Human isolates of the highly prevalent TT virus (TTV) have been classified into five major genomic groups (1-5). The geographical distribution of the groups throughout the world is not well known. Five different PCR assays were developed in an attempt to amplify specifically TTV DNAs of each genomic group. Serum samples collected from 72 Brazilian adults (24 voluntary blood donors, 24 hepatitis B virus (HBV) carriers, and 24 human immunodeficiency virus type 1 (HIV-1)-infected patients) were tested. TTV DNA from at least one genomic group was detected in 11 (46%) blood donors, 13 (54%) HBV carriers, and 24 (100%) HIV-1 patients. All five genomic groups were detected in the three populations, with the exception of group 2 in blood donors. Some samples, negative with all five specific assays, were positive with the commonly used untranslated region (UTR) PCR system. On the other hand, TTV DNA was detected in some samples by using specific assays but not with the UTR PCR. Mixed infections with 2-5 TTV isolates from different groups were detected in 21% blood donors, 29% HBV carriers, and 71% HIV-1 patients. Fifteen PCR products (three obtained with each assay) were sequenced. Most sequences showed high (>86%) homology with those of TTV isolates belonging to their presumed groups. However, three sequences had low homology with all TTV sequences available from the DNA databanks. In conclusion, TTV isolates belonging to all five known genomic groups circulate in Brazil, and the results suggest the existence of new and as yet uncharacterised major genomic groups.     

Outer membrane protein A renders dendritic cells and macrophages responsive to CCL21 and triggers dendritic cell migration to secondary lymphoid organs

Outer membrane protein A (OmpA) is a class of bacterial cell wall protein that is immunogenic without adjuvant. As specific immune responses are initiated in the lymph nodes (LN, we analyzed the effect of the OmpA from Klebsiella pneumoniae (KpOmpA) onchemokine/ chemokine receptor expression by APC and on cell migration to the LN. Upon contact with KpOmpA, human immature DC and macrophages acquire CCR7 expression and responsiveness to CCL21. In parallel, CCR1 and CCR5 expression is down-regulated and CXCL8, CCL2, CCL3 and CCL5 production is up-regulated. Mice injected subcutaneously with KpOmpA present a transient inflammatory reaction at the site of injection accompanied by an enlargement of the draining LN with a higher proportion of DC and macrophages. Lastly, when exposed to KpOmpA prior injection, DC but not macrophages migrate to the draining LN. In conclusion, KpOmpA confers a migratory phenotype to DC and triggers their migration to the regional LN. This property contributes to explain how innate cells initiate adaptive immune response upon recognition of conserved bacterial components and also why OmpA is immunogenic in the absence of adjuvant.     

Heparin inhibits lipopolysaccharide (LPS) binding to leukocytes and LPS-induced cytokine production

The glycosaminoglycan heparin is known to exhibit anti-inflammatory properties unrelated to its anticoagulant activity. However, in a generalized inflammatory response with implanted or extracorporeal devices, the beneficial effect of heparin coating and/or systemic administration is still unclear as well as the precise mechanisms of action. In the present study, we have first studied the effect of heparin on lipopolysaccharide (LPS)-induced cytokine production by human blood monocytes. Our results indicated that the production of interleukin-1alpha, tumor necrosis factor-alpha, and interleukin-8 was significantly decreased when heparin was simultaneously incubated with Escherichia coli LPS. Because the modulation of heparin on monocyte activation could be mediated by its binding via CD14, the main LPS receptor on monocytes, we then studied the binding of LPS and heparin to leukocytes from human blood and to Chinese hamster ovary cells transfected with the human CD14 gene. The data by flow cytometry showed the binding of biotinylated heparin to leukocytes. Moreover, the experiments performed on leukocytes and on CD14-positive Chinese hamster ovary cells indicated that heparin inhibited LPS binding. From our results, we conclude that: 1. heparin is an effective inhibitor of LPS-induced monocyte activation, and 2. heparin inhibits the binding of LPS to cells via a CD14-independent pathway. This study suggests a potentially important therapeutic application for heparin or heparin analogs to prevent inflammation with biomaterials.     

Antigenic and immunogenic mimicry of the HER2/neu oncoprotein by phage-displayed peptides

To recover peptides that antigenically and immunogenically mimic the p185^HER2 oncoprotein, we selected the phage-peptide libraries pVIII-9aa and pVIII-9aa. Cys using murine monoclonal antibodies (mAb) MGr2 and MGr6, directed against two distinct epitopes of the p185^HER2 extracellular domain. Phagedisplayed peptides containing consensus amino acid motifs were recovered and shown to compete specifically for mAb binding on tumor cells that overexpress p185^HER2. The deduced amino acid sequence of the peptides suggests that both epitopes defined by the mAb on p185^HER2 are discontinuous and that hydrophobic interactions are involved in binding with the mAb. A phage clone displaying the GPLDSLFAQ peptide elicited a specific immune response against the p185^HER2 in BALB/c mice, demonstrating that this phage-displayed peptide represents an immunological equivalent of the MGr2 epitope on p185^HER2 and might be used as a substitute for this oncoprotein in in vitro and in vivo immunological studies.     

WNT10B variants in split hand/foot malformation: Report of three novel families and review of the literature

Split‐hand/foot malformation (SHFM) is a genetically heterogeneous congenital limb malformation typically limited to a defect of the central rays of the autopod, presenting as a median cleft of hands and feet. It can be associated with long bone deficiency or included in more complex syndromes. Among the numerous genetic causes, WNT10B homozygous variants have been recently identified in consanguineous families, but remain still rarely described (SHFM6; MIM225300). We report on three novel SHFM families harboring WNT10B variants and review the literature, allowing us to highlight some clinical findings. The feet are more severely affected than the hands and there is a frequent asymmetry without obvious side‐bias. Syndactyly of third–fourth fingers was a frequent finding (62%). Polydactyly, which was classically described in SHFM6, was only present in 27% of patients. No genotype–phenotype correlation is delineated but heterozygous individuals might have mild features of SHFM, suggesting a dose‐effect of the WNT10B loss‐of‐function.     

Expression of voltage-dependent sodium and transient potassium currents in an identified sub-population of dorsal root ganglion cells acutely isolated from 12-day-old mouse embryos

The electrophysiological properties of a subset of dorsal root ganglion (DRG) neurons microdissected from 12-day-old (E12) mouse embryos and acutely isolated were analyzed as soon as 3 after their isolation. Two classes of neurons were defined according to their mean diameter. The larger diameter class was examined in this study. They display uniform cytoskeletal properties with co-expression of vimentin and neurofilament triplet proteins. Patch-clamp methods also revealed a homogeneous and limited repertoire of ionic channels that included (1) a TTX-sensitive Na⁺ current whose properties are similar to that reported in mature mammalian neurons, and (2) two types of K⁺ currents that can be compared with the delayed rectifier (I _k ) and the transient (I _a) potassium currents found in other mammalian preparations. It may be possible to use this in vitro model to examine the development of new types of currents, such as Ca²⁺ currents during neuronal growth and differentiation.