Basic fibroblast growth factor: effects on matrix remodeling, receptor expression, and transduction pathway in human periosteal fibroblasts with FGFR2 gene mutation.

The Crouzon syndrome, which is associated with fibroblast growth factor receptor (FGFR2) mutations, is characterized by premature fusion of cranial sutures. We used an in vitro model of cultured periosteal fibroblasts from normal subjects and from Crouzon patients with FGFR2 mutation. We analyzed the matrix turnover rate and the effects of adding FGF2 by evaluating fibronectin synthesis and the activity of some proteolytic enzymes. To assess the role of some FGF signaling molecules involved in FGFR2 regulation, we studied Grb2 tyrosine phosphorylation and the phosphotyrosine proteins associated with Grb2. The iodinate FGF binding assay was performed to quantify FGFR expression. Compared with normal fibroblasts, fibronectin synthesis was decreased in Crouzon fibroblasts, and protease activities in cells and medium were enhanced, suggesting that excess fibronectin catabolism is present. Differences were more marked when FGF2 was added. Very few phosphoproteins were visible in anti-Grb2 immunoprecipitations from Crouzon fibroblasts, which showed a significant increase in the number of high-affinity and low-affinity FGF2 receptors. These results suggest that the abnormal genotype and the Crouzon cellular phenotype are related. To compensate the low levels of tyrosine phosphorylation, Crouzon cells might increase the numbers of FGFR2, thus increasing the cell surface binding sites for FGF2.     

p53 mutation in breast cancer. Correlation with cell kinetics and cell of origin

AIM: Several studies have investigated the expression of the cytokeratins (CKs), vimentin, the epithelial growth factor receptor (EGFR), the oestrogen receptor (ER), and the progesterone receptor (PgR), in breast cancer, but no study has directly compared p53 mutations with these phenotypic and differentiation markers in the same case. The present study was designed to provide some of this information. METHODS: The expression of the p53 and bcl-2 proteins was evaluated by immunohistochemistry in relation to phenotypic characteristics and cellular kinetic parameters (mitotic index and apoptotic index) in 37 cases of ductal carcinoma in situ (DCIS) and 27 cases of infiltrating ductal carcinoma (IDC) of the breast. In addition, p53 gene mutation was examined by polymerase chain reaction single strand conformation polymorphism analysis (SSCP). RESULTS: Thirteen cases (eight DCIS and five IDC) showed expression of CK8, CK14, CK18, vimentin, and EGFR, consistent with a stem cell phenotype, whereas 44 cases (27 DCIS and 17 IDC) showed expression of CK8 and CK1, weak or negative expression of CK18, but were negative for vimentin and EGFR, consistent with a luminal cell phenotype. DCIS and IDC cases with a stem cell phenotype were ER/PgR negative and intermediately or poorly differentiated. In contrast, the cases with luminal cell phenotype were ER/PgR positive and well or intermediately differentiated. In addition, intermediately or poorly differentiated cases with a stem cell phenotype showed higher proliferative activity (per cent of MIB-l positive cells) than did intermediately or well differentiated cases with a luminal cell phenotype. Both DCIS and IDC cases with a stem cell phenotype were p53 positive and bcl-2 negative by immunohistochemistry. In IDC, p53 expression was associated with a reduction of both mitotic index and apoptotic index compared with DCIS. Most of the tumours showing a more differentiated phenotype (luminal) were p53 negative and bcl-2 positive. In these cases, cell kinetic parameters increased from DCIS to IDC. These data suggest the existence of subsets of DCIS and IDC that, because of their phenotypic characteristics, could be derived from subpopulations of normal breast cells having different control mechanisms of cell proliferation and neoplastic progression. CONCLUSIONS: These results are compatible with the hypothesis that the phenotype of the cell of origin constrains both tumour phenotype and the choice of genetic events; however, the occurrence of p53 mutants by chance during neoplastic transformation cannot be excluded.     

Characterization of mycobacterial isolates phylogenetically related to, but different from Mycobacterium simiae.

The use of high-performance liquid chromatography (HPLC) revealed four previously unreported profiles within a group of mycobacteria consisting of 14 clinical isolates. These mycobacteria, whose identification by conventional tests appeared problematic, mostly resembled Mycobacterium avium complex or Mycobacterium simiae. Genetic analysis revealed, within this group, six different nucleic acid sequences in a hypervariable 16S rRNA segment, but all the isolates appeared to be phylogenetically related to M. simiae. Six isolates representing the largest of groups defined by means of genetic sequencing turned out to belong to the newly described species Mycobacterium lentiflavum. Furthermore, three such clusters precisely coincided with three of those defined by HPLC, while the three remaining clusters shared almost identical HPLC profiles. All but one strain (which, although clearly not belonging to the M. avium complex, hybridized with specific commercial DNA probes) showed high-grade resistance to the majority of antimycobacterial drugs. Three of the isolates were clinically significant according to stringent criteria. Sophisticated techniques, like genetic sequencing or HPLC, by now seem indispensable for differentiating unusual and new mycobacteria from well-established ones.     

Drug resistance mutations and newly recognized treatment-related substitutions in the HIV-1 protease gene: prevalence and associations with drug exposure and real or virtual phenotypic resistance to protease inhibitors in two clinical cohorts of antiretroviral experienced patients

This study aimed at identifying HIV-1 protease amino acid changes associated with protease inhibitor (PI) exposure and susceptibility. New amino acid substitutions were correlated with the number of experienced PIs, reaching statistical significance only for those at positions 3, 44, and 74. The correspondence multivariate model demonstrated that > or =3 experienced PIs and substitutions or mutations at positions 3, 46, 54, 73, 74, and 84 were correlated with PI cross-resistance, including resistance for lopinavir and amprenavir in this cohort of patients who were naive for these drugs.     

CDK9/CYCLIN T1 expression during normal lymphoid differentiation and malignant transformation

CDK9 is a member of the CDC2-like family of kinases. Its cyclin partners are members of the CYCLIN T family (T1, T2a, and T2b) and CYCLIN K. The CDK9/CYCLIN T1 complex is very important in the differentiation programme of several cell types, controlling specific differentiation pathways. Limited data are available regarding the expression of CDK9/CYCLIN T1 in haematopoietic and lymphoid tissues. The aim of this study was to analyse the expression of the CDK9/CYCLIN T1 complex in lymphoid tissue, in order to assess its role in B- and T-cell differentiation and lymphomagenesis. CDK9/CYCLIN T1 expression was found by immunohistochemistry in precursor B and T cells. In peripheral lymphoid tissues, germinal centre cells and scattered B- and T-cell blasts in interfollicular areas expressed CDK9/CYCLIN T1, while mantle cells, plasma cells, and small resting T-lymphocytes displayed no expression of either molecule. CDK9/CYCLIN T1 expression therefore appears to be related to particular stages of lymphoid differentiation/activation. CDK9 and CYCLIN T1 were highly expressed in lymphomas derived from precursor B and T cells, from germinal centre cells, such as follicular lymphomas, and from activated T cells (ie anaplastic large cell lymphomas). Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma also showed strong nuclear staining. Diffuse large B-cell, Burkitt's lymphomas, and peripheral T-cell lymphomas, among T-cell lymphoproliferative disorders, showed a wide range of values. No expression of CDK9 or CYCLIN T1 was detected in mantle cell and marginal zone lymphomas. However, at the mRNA level, an imbalance in the CDK9/CYCLIN T1 ratio was found in follicular lymphoma and diffuse large B-cell lymphomas with germinal centre phenotype, and in the cell lines of classical Hodgkin's lymphomas, Burkitt's lymphomas, and anaplastic large cell lymphoma, in comparison with reactive lymph nodes. These results suggest that the CDK9/CYCLIN T1 complex may affect the activation and differentiation programme of lymphoid cells. The molecular mechanism through which the CDK9/CYCLIN T1 complex is altered in malignant transformation needs to be elucidated.     

CD1a and antitumour immune response

Primary immune response is based on the capacity of local professional antigen-presenting cells (whose prototype is represented by dendritic cells, DCs) to take up and present antigens to selected clones of T cells, but also to non-specific effector cells such as macrophages or natural killer cells. The four CD1 proteins, all of which share a limited homology to class I MHC proteins, are differently expressed in various cell types, of both mesenchymal and, as recently described, epithelial lineage. Regarding the role of CD1 molecules in the anti-tumour response, it has been reported that CD1+ dendritic cells are involved in the first steps of the primary immune response in a number of malignancies. Moreover, the presence of a high number of DCs in the tumoral or peritumoral area, as well as in the draining lymph nodes, has been shown to correlate with a better prognosis. A recent report on the presence of CD1a in metaplastic epithelial cells of Barrett esophagus introduced new questions about CD1a expression patterns. Moreover, the strong correlation between the lack of CD1a+ cells and the malignant evolution of the lesion may indicate a possible role of non-professional APCs in mediating and/or potentiating immune responses to tumours.     

The CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Belgian Diabetes Registry

Susceptibility to autoimmune insulin-dependent (type 1) diabetes mellitus is determined by a combination of environmental and genetic factors, which include variation in MHC genes on chromosome 6p21 (IDDM1) and the insulin gene on chromosome 11p15 (IDDM2). However, linkage to IDDM1 and IDDM2 cannot explain the clustering of type 1 diabetes in families, and a role for other genes is inferred. In the present report we describe linkage and association of type 1 diabetes to the CTLA-4 gene (cytotoxic T lymphocyte associated-4) on chromosome 2q33 (designated IDDM12). CTLA-4 is a strong candidate gene for T cell- mediated autoimmune disease because it encodes a T cell receptor that mediates T cell apoptosis and is a vital negative regulator of T cell activation. In addition, we provide supporting evidence that CTLA-4 is associated with susceptibility to Graves' disease, another organ- specific autoimmune disease.      

Toll-like receptors (TLRs) in innate immune defense against Staphylococcus aureus

Toll-like receptors (TLRs) are the most important class of innate pattern recognition receptors (PRRs) by which host immune and non-immune cells are able to recognize pathogen-associated molecular patterns (PAMPs). Most mammalian species have 10 to 15 types of TLRs. TLRs are believed to function as homo- or hetero-dimers. TLR2, which plays a crucial role in recognizing PAMPs from Staphylococcus aureus, forms heterodimers with TLR1 or TLR6 and each dimer has a different ligand specificity. Staphylococcal lipoproteins, Panton-Valentine toxin and Phenol Soluble Modulins have been identified as potent TLR2 ligands. Conversely, the ligand function attributed to peptidoglycan and LTA remains controversial. TLR2 uses a MyD88-dependent signaling pathway that results in NF-kB translocation into the nucleus and activation of the expression of pro-inflammatory cytokine genes. Recognition rouses both an inflammatory response, culminating in the phagocytosis of bacteria, and an adaptive immune response, with the presentation of resulting bacterial compounds to T cells. Here, recent advances on the recognition of S. aureus by TLRs are presented and discussed, as well as the new therapeutic opportunities deriving from this new knowledge.