Apoptosis incidence and protein expression of p53, TGF-beta receptor II, p27Kip1, and Smad4 in benign, premalignant, and malignant human prostate

Deregulation of apoptosis is involved in prostate cancer development and progression. This study involved an immunohistochemical "profiling" of prostate tissue specimens from patients who underwent prostatectomy for localized prostate cancer, to identify apoptosis-specific alterations associated with premalignant precursor lesions. Prostate tissue was pathologically evaluated, and areas of benign acini, high-grade prostate intraepithelial neoplasia (HGPIN), and prostate cancer were identified. Immunohistochemical analysis was performed to determine the expression of p27Kip1, a key cell cycle regulator, transforming growth factor (TGF)-beta receptor II (TbetaRII), a critical signaling effector of TGF-beta; Smad4, a downstream intracellular effector of TGF-beta signaling; p53, a key apoptosis regulator; and prostate-specific antigen (PSA), a clinical marker of prostate cancer. The apoptotic index of the same cell populations was determined using the transferase-mediated digoxigenin-tagged 16-desoxy-uridine-triphosphate nick end labeling assay. Our findings indicate a significant reduction in p27Kip1 immunoreactivity in HGPIN (P<0.0001) and prostate cancer (P<0.0001) compared with the benign tissue. A significant down-regulation was detected in TbetaRII expression in HGPIN and prostate cancer compared with benign prostatic hyperplasia (BPH)(P<0.001). A significant decrease was also observed in Smad4 levels in HGPIN and prostate cancer compared with BPH (P<0.001). Evaluation of the incidence of apoptosis revealed a significant decrease in the apoptotic index among the epithelial cell populations in HGPIN and a further decrease in prostate carcinoma (P<0.01). This reduced apoptotic index correlated with a significant increase in p53 immunoreactivity in the prostatic carcinoma foci. Prostate cancer cells exhibited strong nuclear staining for p53 compared with adjacent HGPIN (P<0.05) and the benign lesions of the same prostate specimens (P<0.05). A significant reduction in PSA immunostaining was detected in HGPIN and prostate carcinoma foci compared with the benign glandular epithelia (P<0.001). These results further define deregulation of TGF-beta signaling effectors as a molecular basis for loss of apoptotic control contributing to the development of prostate tumors. Identification of apoptotic regulators in precursor premalignant lesions may have prognostic significance in disease progression as well as therapeutic value for targeting prostate cancer.     

Immunity in HIV-1-Infected Adults with a Previous State of Moderate-Severe Immune-Suppression and More Than 500 CD4+ T Cell After Highly Active Antiretroviral Therapy

We evaluated phenotypic and functional parameters of immune restoration of 27 HIV-infected patients on highly active antiretroviral therapy (HAART) (HIV-cases) with HIV-RNA levels below detectable limits at least during 18 months, and CD4+ cell per microliter higher than 500 at the moment of the study and lower than 300 anytime before. These patients were compared with 11 HIV-controls that never had less than 500 CD4+ cell per microliter and 20 healthy-controls (HIV seronegative subjects) in a cross-sectional study. HIV-cases had lower counts of naïve CD4+ than HIV-controls and healthy-controls. HIV-patients (both HIV-cases and HIV-controls) showed higher values of naïve and memory CD8+ counts than healthy-controls. TREC-bearing cell levels were significantly lower in HIV-cases than in healthy-controls. Peripheral blood mononuclear cells (PBMC) cultures, HIV-cases had lower values in proliferation to streptokinase (SK) and tetanus toxin (TT) than in healthy-controls. HIV-cases had lower IFN-γ and higher IL-5 production with pokeweed than healthy-controls (P < 0.01). However, IL-5 production of HIV-cases after TT stimulation was lower than in HIV-controls and healthy-controls. Total IgG and IgG1 levels were significantly higher in HIV-cases than in HIV-controls and healthy-controls. Also, IgM levels were significantly higher in HIV-cases than in healthy-controls. Nevertheless, IgG2 levels were significantly lower in HIV-cases and HIV-controls than in healthy-controls. The levels of specific Igs antipneumococcal capsular polysaccharide and TT were significantly lower in HIV-cases than in healthy-controls. HIV-patients with a previous state of severe-moderate immunosuppression normalizing their CD4+ counts have a incomplete immune reconstitution after HAART. Long-term consequences of this subclinical immune deficiency remain to be determined.     

Prolyl-leucyl-glycinamide shares some effects with oxytocin but decreases oxytocin levels

Oxytocin treatment in rats induces long-lasting antistress and growth promoting effects. This study investigated whether prolyl-leucyl-glycinamide (PLG) (the c-terminal tripeptide of oxytocin) or tocinoic acid (the ring structure of oxytocin) could induce some of these effects in male rats. For this purpose, PLG (2 or 10 mg/kg, s.c.) or tocinoic acid (1 mg/kg, s.c.) was administered to rats once a day for 3 or 5 days. Blood pressure, heart rate, spontaneous motor activity, nociceptive thresholds, and the survival of ischaemic musculocutaneous flaps were measured. In addition, endogenous oxytocin levels and plasma levels of some hormones known to be influenced by oxytocin were determined. PLG (2 mg/kg, s.c., but not 10 mg/kg, s.c.) decreased diastolic blood pressure (p<0.05) and locomotor activity (p<0.05). PLG (10 mg/kg, s.c.) decreased gastrin (p<0.05) and endogenous oxytocin levels in plasma (p<0.01). Tocinoic acid decreased locomotor activity (p<0.05), but did not affect any of the other parameters measured. In conclusion, this study showed that both PLG and tocinoic acid decrease locomotor activity. In addition, PLG also induced some other effects similar to those induced by oxytocin treatment but when administered in high doses it decreased oxytocin levels.     

Novel mutations in the EXT1 gene in two consanguineous families affected with multiple hereditary exostoses (familial osteochondromatosis)

Multiple hereditary exostoses (HME) is an autosomal dominant developmental disorder exhibiting multiple osteocartilaginous bone tumors that generally arise near the ends of growing long bones. Here, we report two large consanguineous families from Pakistan, who display the typical features of HME. Affected individuals also show a previously unreported feature--bilateral overriding of single toes. Analysis using microsatellite markers for each of the known EXT loci, EXT1, EXT2, and EXT3 showed linkage to EXT1. In the first family, mutation analysis of the EXT1 gene revealed that affected individuals were heterozygous for an in-frame G-to-C transversion at the conserved splice donor site in intron 1. This mutation is predicted to disrupt splicing of the first intron and produce a frameshift that leads to a premature termination codon. In the second family, an insertion of an A in exon 8 is predicted to produce a frameshift at codon 555 followed by a premature termination, a further 10 codons downstream. In both families, an increased number of affected male subjects were observed. In affected females in family 2, phenotypic variability and incomplete penetrance were noted.     

The effect of inflammatory cytokines on secretion of macrophage colony-stimulating factor and monocyte chemoattractant protein-1 in human granulosa cells

PROBLEM: In order to investigate the role of macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein -1 (MCP-1) in human ovulation, we studied the regulation of M-CSF and MCP-1 in cultured human granulosa cells. METHOD OF STUDY: Immortalized granulosa cells (GC1a) were cultured in serum-free medium, and incubated with interleukin (IL)-1alpha, IL-1 receptor antagonist (ra) and tumor necrosis factor (TNF)-alpha. The supernatants were collected, and M-CSF and MCP-1 were measured by ELISA. RESULTS: The levels of M-CSF and MCP-1 were increased after treatment with IL-1alpha (1 nm) and TNF-alpha (1 nm) in a time-dependent manner. The levels of M-CSF and MCP-1 were significantly increased after treatment with IL-1alpha and TNF-alpha in a dose-dependent manner. However, the levels of M-CSF and MCP-1 were significantly decreased by treatment with IL-1alpha (1 nm) and/or increasing concentrations of IL-1 ra. CONCLUSIONS: Our data indicated that M-CSF and MCP-1 were regulated by IL-1alpha and TNF-alpha. It was suggested that M-CSF and MCP-1 may play an important role in human preovulatory processes.     

Comprehensive molecular screening of the FBN1 gene favors locus homogeneity of classical Marfan syndrome

In order to estimate the contribution of mutations at the fibrillin-1 locus (FBN1) to classical Marfan syndrome (MFS) and to study possible phenotypic differences between patients with an FBN1 mutation vs. without, a comprehensive molecular study of the FBN1 gene in a cohort of 93 MFS patients fulfilling the clinical diagnosis of MFS according to the Ghent nosology was performed. The initial mutation screening by CSGE/SSCP allowed identification of an FBN1-mutation in 73 patients. Next, sequencing of all FBN1-exons was performed in 11 mutation-negative patients, while in nine others, DHPLC was used. This allowed identification of seven and five additional mutations, respectively. Southern blot analysis revealed an abnormal hybridization pattern in one more patient. A total of 23 out of the 85 mutations identified here are reported for the first time. Phenotypic comparison of MFS patients with cysteine-involving mutations vs. premature termination mutations revealed significant differences in ocular and skeletal involvement. The phenotype of the eight patients without proven FBN1 mutation did not differ from the others with respect to the presence of major cardiac, ocular, and skeletal manifestations or positive familial history. Most likely, a portion of FBN1-mutations remains undetected because of technical limitations. In conclusion, the involvement of the FBN1-gene could be demonstrated in at least 91% of all MFS patients (85/93), which strongly suggests that this gene is the predominant, if not the sole, locus for MFS.     

肿瘤转移抑制基因nm23—H1 mRNA在乳腺癌中的转录表达

目的：探讨nm23—H1基因mRNA在乳腺患组织中的表达及其与临床的关系．方法:采用半定量RT—PCR方法，检测30例乳腺患组织nm23—H1的表达．结果:①淋巴结阳性的原发灶组织nm23—H1 mRNAA表达明显低于淋巴结阴性的原发灶组织，Ⅲ期乳腺患组织nm23—H1 mRNA水平较Ⅰ、Ⅱ期的明显低．②多因素分析发现淋巴结转移与nm23—H1 mRNA的表达有显著性相关．结论nm23—H1基因mRNA表达强度与淋巴结转移呈负相关．在乳胰患转移过程中，nm23—H1 mRNA起重要的作用。     

A mutation analysis of the<Emphasis Type="Italic"> BRCA1</Emphasis> gene in 140 families from southeast France with a history of breast and/or ovarian cancer

A mutation analysis of the BRCA1 gene in 140 French families with a history of breast cancer or breast-ovarian cancer revealed several deleterious germline mutations, as well as rare sequence variants. The 19 genetics variants were of 15 different types, two of which had not been reported in the Breast cancer Information Core (BIC) database. Five distinct truncating mutations, leading to putative nonfunctional proteins, were identified out of 140 index cases (3.5%). One novel nonsense mutation, C4491T, was reported, whereas the four other BRCA1 deleterious mutations identified consisted of frequent frameshifts in the nucleotide sequence. One splice variant (331+3A>G) and thirteen missense variations leading to amino acid substitutions of unknown structural and functional importance were identified. Among these, two BRCA1 missense mutations, A120G and T243C could be considered as suspected deleterious. The first missense mutation modified the initiation codon (M1V) and the second (C39R) may have consequences on the structure and functioning of the BRCA1 protein by modifying cysteine ligands from the RING finger domain. As expected BRCA1 gene alteration, including missense mutations of unknown biological significance, were more frequent in families with a history of breast-ovarian-cancer (32%) than in breast-cancer-only families (12%).     

Nitric Oxide Modulates MCP-1 Expression in Endothelial Cells: Implications for the Pathogenesis of Pulmonary Granulomatous Vasculitis

Monocyte chemoattractant protein-1 (MCP-1) is a pivotal mediator of angiocentric granuloma formation in glucan-induced pulmonary granulomatous vasculitis. Based on the rationale that mononuclear phagocytes retrieved from granulomas are rich sources of nitric oxide (NO) and that the recruitment of mononuclear phagocytes into lesions abates as granuloma formation slows, we tested the hypothesis that MCP-1 gene expression is regulated by a NO-sensitive mechanism. Preexposure of endothelial cell (EC) monolayers to NO donor compounds markedly reduced cytokine-induced MCP-1 expression and cytosolic-to-nuclear translocation of nuclear factor-kappa B (NF-B), reversed fluctuations in endothelial reduced glutathione (GSH) pools but did not affect cGMP concentrations. The lungs of mice bearing targeted disruptions of the inducible nitric oxide synthase (iNOS) gene exhibited significantly higher concentrations of MCP-1 following glucan infusion than did those of wild-type mice. Cumulatively, these data suggest that NO suppresses MCP-1 expression by blunting the redox changes associated with cytokine-induced EC activation.