Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons

Slit proteins steer the migration of many cell types through their binding to Robo receptors, but how Robo controls cell motility is not clear. We describe the functional analysis of vilse, a Drosophila gene required for Robo repulsion in epithelial cells and axons. Vilse defines a conserved family of RhoGAPs (Rho GTPase-activating proteins), with representatives in flies and vertebrates. The phenotypes of vilse mutants resemble the tracheal and axonal phenotypes of Slit and Robo mutants at the CNS midline. Dosage-sensitive genetic interactions between vilse, slit, and robo mutants suggest that vilse is a component of robo signaling. Moreover, overexpression of Vilse in the trachea of robo mutants ameliorates the phenotypes of robo, indicating that Vilse acts downstream of Robo to mediate midline repulsion. Vilse and its human homolog bind directly to the intracellular domains of the corresponding Robo receptors and promote the hydrolysis of RacGTP and, less efficiently, of Cdc42GTP. These results together with genetic interaction experiments with robo, vilse, and rac mutants suggest a mechanism whereby Robo repulsion is mediated by the localized inactivation of Rac through Vilse.     

Client feedback on physiotherapy counselling in primary health care

The extent of rehabilitation services is adequate, but little feedback is available on these services and service users. This paper is based on a client feedback survey of physiotherapy services conducted in Spring 2000. The purpose of the paper is to compare patients' perceptions of the physiotherapy instruction they received in a private outpatient facility and in a public physiotherapy facility. The results of the study showed that physiotherapy clients were satisfied with the therapy and instruction they received. They felt they received an adequate amount of instructions and advice. The rehabilitee's strong commitment to care, high motivation and a support network are important to independent rehabilitation in the future. As for the point of contact, the majority of clients in the private physiotherapy facility were gainfully employed while the health centre had almost as many retired clients. Client instruction was not dependent on the point of contact.     

Acenocoumarol and heparin compared with acenocoumarol alone in the initial treatment of proximal-vein thrombosis.

BACKGROUND. In most countries, heparin is used in the initial treatment of patients with deep-vein thrombosis. Well-designed studies establishing the efficacy of heparin therapy are lacking, however. Treatment with acenocoumarol alone, according to the hypothesis that high dosages of oral anticoagulants obviate the need for heparin, is considered an effective alternative in some countries. METHODS. In a randomized, double-blind study we compared the efficacy and safety of continuous intravenous heparin plus acenocoumarol with the efficacy and safety of acenocoumarol alone in the initial treatment of outpatients with proximal-vein thrombosis. The principal study end point was a confirmed symptomatic extension or recurrence of venous thromboembolism during six months of follow-up. In addition, we assessed asymptomatic extension or pulmonary embolism by repeating venography and lung scanning after the first week of treatment. The incidence of major bleeding was determined during three months of follow-up. RESULTS. The study was terminated early by the Data Safety and Monitoring Committee because of an excess of symptomatic events in the group that received acenocoumarol alone (in 12 of 60 patients [20 percent], as compared with 4 of 60 patients [6.7 percent] in the combined-therapy group by intention-to-treat analysis; P = 0.058). Asymptomatic extension of venous thrombosis was observed in 39.6 percent of the patients in the acenocoumarol group and in 8.2 percent of patients treated with heparin plus acenocoumarol (P < 0.001). Major bleeding complications were infrequent and comparable in the two groups. CONCLUSIONS. Patients with proximal-vein thrombosis require initial treatment with full-dose heparin, which can safely be combined with acenocoumarol therapy.     

Evaluation of proliferation parameters in in vivo bromodeoxyuridine labelled lung cancers

In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcinoma, the validity of proliferating cell nuclear antigen (PCNA) and Ki67 antigen as proliferative indicators was evaluated in ethanol fixed, paraffin embedded tissue. The percentages of cells positive for these markers were compared to the in vivo bromodeoxyuridine (BrdU) labelling index. A good correlation was found between PCNA immunoreactivity and BrdU labelling index, while Ki67-antigen expression showed a significant relation with BrdU labelling index and with PCNA expression. All three parameters showed a trend towards similar values for the individual cases. Based on the fact that Ki67 antigen is expressed in all cycling cells, whereas replicon-associated PCNA and BrdU only reflect the S-phase fraction, the differences between Ki67-antigen scores on the one hand and BrdU and PCNA scores on the other were smaller than expected. In order to determine the degree of concordance between immunohistochemically and flow cytometrically detected proliferation variables, BrdU incorporation was measured using both methods in duplicate bronchial specimens. Discrepancies in labelling indices were observed predominantly in DNA diploid samples, with consistently lower values in the flow cytometrically analysed specimens. In tumour specimens with an aneuploid DNA content, flow cytometric determination of proliferative activity yielded results similar to those obtained by tissue section examination. We conclude that the scores for PCNA and Ki67 antigen, immunohistochemically detected in ethanol fixed, paraffin embedded tissue reflect functional proliferative activity.     

Up-regulated expression of zonula occludens protein-1 in human melanoma associates with N-cadherin and contributes to invasion and adhesion

During the process of malignant transformation, nascent melanoma cells escape keratinocyte control through down-regulation of E-cadherin and instead communicate among themselves and with fibroblasts via N-cadherin-based cell-cell contacts. The zonula occludens (ZO) protein-1 is a membrane-associated component of both the tight and adherens junctions found at sites of cell-cell contact. In most cancers, levels of ZO-1 are typically down-regulated, leading to increased motility. Here we report the novel observation that ZO-1 expression is up-regulated in melanoma cells and is located at adherens junctions between melanoma cells and fibroblasts. Immunofluorescence and co-immunoprecipitation studies showed co-localization of ZO-1 with N-cadherin. Down-regulation of ZO-1 in melanoma cells through RNA interference produced marked changes in cell morphology--leading to a less-dendritic, more rounded phenotype. Consistent with a role in N-cadherin-based adhesion, RNAi-treated melanoma cells were less adherent and invasive when grown in a collagen gel. These data provide the first evidence that increased ZO-1 expression in melanoma contributes to the oncogenic behavior of this tumor and further illustrate that protein products of genes, such as ZO-1, can function in either a pro- or anti-oncogenic manner when expressed in different cellular contexts.     

Identification of a gene from Xp21 with similarity to the tctex-1 gene of the murine t complex

Long range physical mapping within the p21 region of the X chromosomeIdentified a CpG rich Island approximately 180 kb centromericto the chronic granulomatous disease (CGD) locus. The segmentsadjacent to the CpG Island hybridized to discrete bands In DNAsof several species and when used to screen retinal cDNA librariesled to the Identification of cDNAs that detected a mRNA of 2.1kb in many tissues. Molecular characterization of correspondinggenomic clones of this novel human gene confirmed the originof the cDNA clones and Indicated a genomic structure with fiveexons spanning a total of 9 kb. The complete cDNA sequence revealedthat this gene contained a putative open reading frame of 116amino acids with a 3' untranslated region of 1.74 kb. The aminoacid sequence shows a high degree of similarity to the predictedproduct of the tctex-1 gene of the mouse t complex. As linkagestudies and patients with deletions have Implicated the Xp21region as containing the retInltls plgmentosa defect (RP3),the gene was assessed as a candidate disease gene In RP3 families.A single base pair polymorphism was Identified within the codingregion but no disease associated changes were found by singlestrand conformational polymorphism and sequencing analysis ofamplified exons of 20 RP patients. Analysis of a dinucleotiderepeat polymorphism within this gene In families affected withRP3 suggested refinement of the RP3 region.     

Expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in ischemic skeletal muscle and its regeneration

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible endothelial cell mitogen and survival factor. Its receptor VEGFR-2 (KDR/Flk-1) mediates these effects. We studied the expression of VEGF and VEGFR-2 in ischemic human and rabbit skeletal muscle by immunohistochemistry and in situ hybridization. Human samples were obtained from eight lower limb amputations because of acute or chronic critical ischemia. In chronically ischemic human skeletal muscle VEGF and VEGFR-2 expression was restricted to atrophic and regenerating skeletal myocytes, whereas in acutely ischemic limbs VEGF and VEGFR-2 were expressed diffusely in the affected muscle. Hypoxia-inducible factor-1alpha was associated with VEGF and VEGFR-2 expression both in acute and chronic ischemia but not in regeneration. Hindlimb ischemia was induced in 20 New Zealand White rabbits by excising the femoral artery. Magnetic resonance imaging and histological sections revealed extensive ischemic damage in the thigh and leg muscles of ischemic rabbit hindlimbs with VEGF expression similar to acute human lower limb ischemia. After 1 and 3 weeks of ischemia VEGF expression was restricted to regenerating myotubes and by 6 weeks regeneration and expression of VEGF was diminished. VEGFR-2 expression was co-localized with VEGF expression in regenerating myotubes. Macrophages and an increased number of capillaries were associated with areas of ischemic muscle expressing VEGF and VEGFR-2. In conclusion, two patterns of VEGF and VEGFR-2 expression in human and rabbit ischemic skeletal muscle are demonstrated. In acute skeletal muscle ischemia VEGF and VEGFR-2 are expressed diffusely in the affected muscle. In chronic skeletal muscle ischemia and in skeletal muscle recovering from ischemia VEGF and VEGFR-2 expression are restricted to atrophic and regenerating muscle cells suggesting the operation of an autocrine pathway that may promote survival and regeneration of myocytes.     

Hapten-induced colitis associated with maintained Th1 and inflammatory responses in IFN-gamma receptor-deficient mice

IFN-gamma is a potent pro-inflammatory cytokine thought to be involved in the pathogenesis of Crohn's disease. To further define the role of IFN-gamma in intestinal inflammation, we studied the effects of intra-colonic 2,4,6-trinitrobenzene sulfonic acid (TNBS) instillation in mice with a functionally inactivated IFN-gamma receptor 1 (IFN-gammaR1(- / -)). Our results indicate that IFN-gamma is not necessary for the induction of hapten-induced colitis: after TNBS administration both wild-type and IFN-gammaR1(- / -) mice lost body weight, and the histological features of TNBS-induced colitis were comparable. Colons of IFN-gammaR1(- / -) mice contained a greater number of cells, represented by macrophages and CD4(+) T cells; caudal lymph node cells produced more IFN-gamma and TNF-alpha upon stimulation in vitro. Moreover, IL-18 and IL-12 p40 RNA levels were comparably up-regulated after TNBS treatment in IFN-gammaR1(- / -) wild-type mice. These findings demonstrate that IFN-gamma is dispensable for the development of TNBS-induced colitis. Importantly, the production of Th1 cytokines (e. g. IFN-gamma and TNF-alpha) by caudal lymph node T lymphocytes was enhanced rather than decreased in IFNgammaR1(- / -) mice with no evidence for default Th2 development.     

Localization of a novel X-linked congenital stationary night blindness locus: close linkage to the RP3 type retinitis pigmentosa gene region

X-linked congenital stationary night blindness (CSNBX) is anon-progressive retinal disorder characterized by decreasedvisual acuity and loss of night vision. CSNBX Is clinicallyheterogeneous with respect to the involvement of retinal rodsand/or cones in the disease. in this study, we localize a newlocus for CSNBX to Xp21.1, thus providing evidence that CSNBXis also genetically heterogeneous. A clear correlation betweendifferent genotypes and phenotypes cannot be found yet. Thenew CSNBX gene described here is closely linked to the X-linkedretinitis pigmentosa type 3 gene region, which supports thehypothesis that there may be a functional relationship betweencongenital stationary night blindness and retinitis pigmentosa.     

Outcome of surgical treatment for early adenocarcinoma of the esophagus or gastro-esophageal junction

Adenocarcinoma of the esophagus, or GEJ, has a poor prognosis. Early lesions [i.e. high grade dysplasia (HGD) or T1-carcinoma] are potentially curable. Local endoscopic therapies are promising treatment options for superficial lesions; however, for deeper lesions, surgical resection is considered to be the treatment of choice. To contribute to therapeutic decision-making, we retrospectively analysed the outcome of transhiatal esophagectomy in 120 patients with pathologically proven HGD (n=13) or T1-adenocarcinoma (n=107) of the distal esophagus or gastro-esophageal junction (GEJ). Tumors were subdivided into six different depths of invasion (T1-mucosal m1-m3, T1-submucosal sm1-sm3), and the frequency of lymphatic dissemination and time to locoregional and/or distant recurrence were analysed. Only one of the 79 T1m1-3/sm1 tumors (1%) showed lymph node metastases as compared with 18 out of 41 T1sm2-3 tumors (44%). There was a significant difference in recurrence-free period between T1m1-m3/sm1 versus T1sm2-sm3 tumor patients (P log rank <0.0001), with 5-year recurrence-free percentages of 97% and 57%, respectively. In multivariate analysis including age, gender, tumor differentiation grade, N-stage and depth of invasion, only N-stage was an independent prognostic factor for recurrence-free period (hazard rate=5.9, 95% CI 1.7–20.7). However, if N-stage was excluded from analysis, only depth of invasion (T1sm2-3 versus T1m1-m3/sm1) was an independent prognostic factor for recurrence-free period (hazard rate=7.5, 95% CI 2.0–27.7). These data indicate that T1m1-m3/sm1 adenocarcinomas of esophagus or GEJ show a very low risk of lymphatic dissemination and are therefore eligible for local endoscopic therapy. After transhiatal surgical resection, almost half of the patients with T1sm2-sm3 lesions develop recurrent disease within 5 years, and therefore need additional therapy to improve survival.