Ras-transfection up-regulated HaCaT cell migration: Inhibition by Marimastat

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-ras clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines. This revised version was published online in July 2006 with corrections to the Cover Date.     

MUC1 expression is correlated with nuclear grade and tumor progression in pT1 renal clear cell carcinoma

We studied, by immunohistochemical analysis, the expression of MUC1 and epithelial membrane antigen in 44 stage pT1 renal cell carcinomas (RCCs). Six patients had a metastatic evolution. The percentage of stained cells was determined for each tumor. All tumors and normal adjacent renal parenchyma were stained. In normal kidney, distal convoluted tubules and collecting ducts stained strongly with an apical distribution. In tumors, there was a significant statistical correlation of the MUC1 expression level with the nuclear grade and with tumor progression. High-grade tumors had more stained cells than did low-grade tumors. Metastatic tumors also were more stained than nonmetastatic lesions. By using the Kaplan-Meier method and the log-rank test, we observed that patients with fewer than 10% of stained cells had no metastatic evolution. In contrast, patients with 70% or more stained cells had significantly lower metastasis-free survival rates. We conclude that MUC1 is expressed in RCC and is associated with tumor progression in pT1 RCC.     

Electronic implementation of guidelines in the EsPeR system: a knowledge specification method

Despite initiatives to standardize methods for the development of clinical guidelines, several barriers hinder their integration in daily clinical practice: failure to fulfil quality criteria, poor effectiveness of their dissemination. Computerization of guidelines can favor their dissemination. The initial step of computerization is the knowledge specification from the text of the guideline. We describe the method of knowledge specification, which is used in EsPeR (Personalized Estimate of Risks), a web-based decision support system in preventive medicine, which allows, for a given person, to estimate risks and access recommendations, based on clinical profile. This method is based on a structured and systematic analysis of text allowing detailed specification of a decision tree. We use decision tables to validate the decision algorithm and decision trees to specify this algorithm, along with elementary messages of recommendation. Editing tools are used to facilitate the process of validation and the workflow between expert physicians and computer scientists. Applied to eleven different guidelines, the method allows a quick and valid computerization and integration in the EsPeR system. The method used for computerization could help to define a framework usable at the initial step of guideline development in order to produce guidelines ready for electronic implementation.     

Origin and filiation of human plasmacytoid dendritic cells

Human plasmacytoid dendritic cells represent a rare population of leukocytes which produce high amounts of type I interferon in response to certain viruses. Although those cells were first described in 1958, there are still unsolved issues related to their origin and function. Recently, a leukemic counterpart of plasmacytoid dendritic cells was identified. Molecular approaches using either normal or leukemic plasmacytoid dendritic cells provide some new insights into the controversial lymphoid origin of those cells. The need for specific markers is still a critical aspect for the identification of plasmacytoid dendritic cells, whatever stage of differentiation, in normal as well as in pathological conditions. Hopefully, novel markers will allow delineation of the relationships between dendritic cells at different stages of differentiation/maturation along the myeloid and lymphoid lineages.     

Formalin-fixed and paraffin-embedded nodal non-Hodgkin's lymphomas demonstrate the same chromosome changes as those found in frozen samples: a comparative study using interphase fluorescence in situ hybridization.

Cytogenetic studies in lymphomas classically require fresh or frozen tissue, whereas in many instances only paraffin-embedded biopsies are available. We applied an interphase FISH assay on nuclei extracted from thick paraffin sections to determine accuracy of molecular cytogenetics in such samples. Twenty-three lymphoma samples and 4 reactive lymph nodes were tested with various commercially available DNA probes, and hybridization patterns were compared with those obtained on frozen nuclei counterparts. Successful hybridization with all probes tested was observed for 23/27 (85%) paraffin-embedded tissues and for all (100%) frozen samples, and cut-off levels defining positivity were superimposable for both situations. Chromosome changes were detected in the same way, without any false-positive or false-negative cases. Hybridization signals observed on dewaxed samples were either those classically expected to define the relevant chromosome change or were atypical: all atypical changes could be demonstrated also into nuclei from the frozen counterpart. Moreover, all typical and atypical chromosome changes observed on frozen nuclei were also detected in paraffin-embedded tissues. Our study shows that our interphase FISH assay performed on paraffin-embedded samples is a valuable alternate to conventional methods to ascertain diagnosis of lymphomas as to include patients into therapeutic trials.     

Management of non-palpable breast lesions with vacuum-assisted large core needle biopsies (Mammotome). Experience with 560 procedures at the Val d'Aurelle Center

Percutaneous vacuum-assisted large core needle biopsy of breast microcalcifications is now commonly performed as the initial approach to nonpalpable breast lesions. It can obviate the need for surgery in women with benign lesions and often lead to a one-stage surgical procedure when malignant lesions are diagnosed. To illustrate this strategy, we describe our experience based on 560 procedures performed within a 36 Month-period. Sixty percent of the lesions were benign, mostly fibrocystic changes. Thirty percent of the specimens were malignant, almost exclusively intraductal carcinomas, sometimes associated with an invasive component. This component must be identified by the pathologist in order to avoid incomplete treatment and to plan lymph node excision. Finally, 10% of the specimens were boderline including lobular neoplasia, atypical ductal hyperplasia and columnar cell lesions with atypia. Surgical excision is recommended for atypical ductal hyperplasia, columnar cell lesions with atypia and lobular neoplasia with particular features, pleomorphic or comedo-like, to avoid missing more aggressive associated lesions. A strict procedure is required for the analysis of needle core biopsies and the subsequent surgical specimens, to accurately classify breast lesions provided by a mammographic screening program. This procedure should be based on a multidisciplinary approach and dialog.     

Cognitive control in childhood-onset obsessive-compulsive disorder: a functional MRI study

BACKGROUND: Failure to resist chronic obsessive-compulsive symptoms may denote an altered state of cognitive control. We searched for the cerebral regions engaged in this dysfunction. METHOD: Differences in brain regional activity were examined by event-related functional magnetic regional imaging (fMRI) in a group of adolescents or young adults (n = 12) with childhood-onset obsessive-compulsive disorder (OCD), relative to healthy subjects. Subjects performed a conflict task involving the presentation of two consecutive and possibly conflicting prime and target numbers. Patients' image dataset was further analysed according to resistance or non-resistance to symptoms during the scans. RESULTS: Using volume correction based on a priori hypotheses, an exploratory analysis revealed that, within the prime-target repetition condition, the OCD subjects activated more than healthy subjects a subregion of the anterior cingulate gyrus and the left parietal lobe. Furthermore, compared with 'resistant' patients, the 'non-resistant' OCD subjects activated a bilateral network including the precuneus, pulvinar and paracentral lobules. CONCLUSIONS: Higher regional activations suggest an abnormal amplification process in OCD subjects during the discrimination of repetitive visual stimuli. The regional distribution of functional changes may vary with the patients' ability to resist obsessions.     

Monoclonal antibodies to a specific 54-kilodalton antigen of Nocardia spp.

Two monoclonal antibodies (MAbs) of the immunoglobulin G2A isotype, reacting with a Nocardia-specific 54-kDa antigen, were generated. As determined by Western blot (immunoblot), both MAbs reacted only with the 54-kDa band. As determined by indirect immunofluorescence or enzyme immunoassay with whole microorganisms, the MAbs did not react with Nocardia cells. One of the MAbs showed weak cross-reactivity with mycobacterial antigens, while the other showed no cross-reactivity.     

Virus overrides the propensity of human CD40L-activated plasmacytoid dendritic cells to produce Th2 mediators through synergistic induction of IFN-{gamma} and Th1 chemokine production

Depending on the activation status, plasmacytoid dendritic cells (PDC) and myeloid DC have the ability to induce CD4 T cell development toward T helper cell type 1 (Th1) or Th2 pathways. Thus, we tested whether different activation signals could also have an impact on the profile of chemokines produced by human PDC. Signals that induce human PDC to promote a type 1 response (i.e., viruses) and a type 2 response [i.e., CD40 ligand (CD40L)] also induced PDC isolated from tonsils to secrete chemokines preferentially attracting Th1 cells [such as interferon-gamma (IFN-gamma)-inducible protein (IP)-10/CXC chemokine ligand 10 (CXCL10) and macrophage inflammatory protein-1beta/CC chemokine ligand 4 (CCL4)] or Th2 cells (such as thymus and activation-regulated chemokine/CCL17 and monocyte-derived chemokine/CCL22), respectively. Activated natural killer cells were preferentially recruited by supernatants of virus-activated PDC, and supernatants of CD40L-activated PDC attracted memory CD4(+) T cells, particularly the CD4(+)CD45RO(+)CD25(+) T cells described for their regulatory activities. It is striking that CD40L and virus synergized to trigger the production of IFN-gamma by PDC, which induces another Th1-attracting chemokine monokine-induced by IFN-gamma/CXCL9 and cooperates with endogenous type I IFN for IP-10/CXCL10 production. In conclusion, our studies reveal that PDC participate in the selective recruitment of effector cells of innate and adaptive immune responses and that virus converts the CD40L-induced Th2 chemokine patterns of PDC into a potent Th1 mediator profile through an autocrine loop of IFN-gamma.     

Genetic heterogeneity of the NS3 protease gene in hepatitis C virus genotype 1 from untreated infected patients

NS3 protease is essential for hepatitis C Virus (HCV) replication, and is one of the most promising targets for specific anti-HCV therapy. Its natural polymorphism has not been studied at the quasispecies level. In the present work, the genetic heterogeneity of the NS3 protease gene was analyzed in 17 HCV genotype 1 (5 subtypes 1a and 12 subtypes 1b) samples collected from infected patients before anti-viral therapy. A total of 294 clones were sequenced. Although the protease NS3 is considered to be one of the less variable genes in the HCV genome, variability of both nucleotide and amino acid sequences was found. In variants belonging to 1a and 1b subtypes, 224 and 267 of 543 positions showed one or more nucleotide substitutions, respectively. Forty and 74 of the 181 NS3 amino acid positions showed at least one mutation in HCV-1a and HCV-1b isolates, respectively. Most substitutions were conservative. This substantial polymorphism of the NS3 protease produced by HCV-1a and HCV-1b suggests that, despite the numerous functional and structural constraints, the enzyme is sufficiently flexible to tolerate substitutions.