Testosterone does not affect lower urinary tract symptoms while improving markers of prostatitis in men with benign prostatic hyperplasia: a randomized clinical trial.

PURPOSE: Benign Prostatic Hyperplasia (BPH) is a result of prostate inflammation, frequently occurring in metabolic syndrome (MetS). Low testosterone is common in MetS. A randomized clinical trial was designed to evaluate if 24 weeks of testosterone therapy (TTh) in BPH men with MetS and low testosterone improve urinary symptoms and prostate inflammation. METHODS: One-hundred-twenty men with MetS waitlisted for BPH surgery were enrolled. They were categorized into normal testosterone (TT ≥ 12 nmol/L and cFT ≥ 225 pmol/L; n = 48) and testosterone deficient (TD) (TT < 12 nmol/L and/or cFT < 225 pmol/L; n = 72) then randomized to testosterone gel 2% (5 g/daily) or placebo for 24 weeks. At baseline and follow-up, questionnaires for urinary symptoms and trans-rectal ultrasound were performed. Prostate tissue was collected for molecular and histopathological analyses. RESULTS: No differences in the improvement of urinary symptoms were found between TTh and placebo (OR [95% CI] 0.96 [0.39; 2.37]). In TD + TTh, increase in prostate but not adenoma volume was observed (2.64 mL [0.07; 5.20] and 1.82 mL [- 0.46; 0.41], respectively). Ultrasound markers of inflammation were improved. In a subset of 61 men, a hyper-expression of several pro-inflammatory genes was found in TD + placebo when compared with normal testosterone. TTh was able to counteract this effect. For 80 men, the inflammatory infiltrate was higher in TD + placebo than in normal testosterone (0.8 points [0.2; 1.4]) and TD + TTh men (0.9 points [0.2; 1.5]). CONCLUSIONS: Twenty-four weeks of TTh in TD men with BPH and MetS improves ultrasound, molecular and histological proxies of prostate inflammation. This does not result in symptom improvement.

Testosterone positively regulates vagina NO-induced relaxation: an experimental study in rats.

PURPOSE: Female sexual response involves a complex interplay between neurophysiological mechanisms and the nitric oxide (NO)-mediated relaxation of clitoris and vagina. The aim of this study was to evaluate sex steroids regulation of the relaxant pathway in vagina, using a validated animal model. METHODS: Subgroups of OVX Sprague-Dawley rats were treated with 17ß-estradiol, testosterone, or testosterone and letrozole, and compared with a group of intact animals. Masson's trichrome staining was performed for morphological evaluation of the distal vaginal wall, in vitro contractility studies investigated the effect of OVX and in vivo treatments on vaginal smooth muscle activity. RNA from vaginal tissue was analyzed by semi-quantitative RT-PCR. RESULTS: Immunohistochemical analysis showed that OVX induced epithelial and smooth muscle structural atrophy, testosterone and testo + letrozole increased the muscle bundles content and organization without affecting the epithelium while 17ß-estradiol mediated the opposite effects. In vitro contractility studies were performed on noradrenaline pre-contracted vaginal strips from each experimental group. Acetylcholine (0.001-10 µM) stimulation induced a concentration-dependent relaxation, significantly reduced by NO-synthase inhibitor L-NAME and by guanylate cyclase inhibitor ODQ. OVX resulted in a decreased responsiveness to acetylcholine, restored by testosterone, with or without letrozole, but not by 17ß-estradiol. OVX sensitivity to the NO-donor SNP was higher than in the control. Vardenafil, a PDE5 inhibitor, enhanced SNP effect in OVX + testosterone as well as in control, as supported by RNA expression analysis. CONCLUSIONS: Our study demonstrates that testosterone improves the NO-mediated smooth muscle vaginal cells relaxation confirming its role in maintaining the integrity of muscular relaxant machinery.

Treatment potential of LPCN 1144 on liver health and metabolic regulation in a non-genomic, high fat diet induced NASH rabbit model.

PURPOSE: Low free testosterone (T) level in men is independently associated with presence and severity of Non-Alcoholic Steatohepatitis (NASH). The histological and molecular effects of oral testosterone prodrug LPCN 1144 treatment on hepatic fibrosis and NASH features are unknown. A metabolic syndrome-induced NASH model in rabbits consuming high fat diet (HFD) has been previously used to assess treatment effects of injectable T on hepatic fibrosis and NASH features. Here we present results on LPCN 1144 in this HFD-induced, NASH preclinical model. METHODS: Male rabbits were randomly assigned to five groups: regular diet (RD), HFD, HFD + 1144 vehicle (HFD + Veh), HFD + 1144 (1144), and HFD + 1144 + α-tocopherol (1144 + ALPHA). Rabbits were sacrificed after 12 weeks for liver histological, biochemical and genetic analyses. Histological scores were obtained through Giemsa (inflammation), Masson's trichrome (steatosis and ballooning), and Picrosirius Red (fibrosis) staining. RESULTS: Compared to RD, HFD and HFD + Veh significantly worsened NASH features and hepatic fibrosis. Considering HFD and HFD + Veh arms, histological and biomarker features were not significantly different. Both 1144 and 1144 + ALPHA arms improved mean histological scores of NASH as compared to HFD arm. Importantly, percentage of fibrosis was improved in both 1144 (p < 0.05) and 1144 + ALPHA (p = 0.05) treatment arms vs. HFD. Both treatment arms also reduced HFD-induced inflammation and fibrosis mRNA markers. Furthermore, 1144 treatments significantly improved HFD-induced metabolic dysfunctions. CONCLUSIONS: Histological and biomarker analyses demonstrate that LPCN 1144 improved HFD-induced hepatic fibrosis and NASH biochemical, biomolecular and histochemical features. These preclinical findings support a therapeutic potential of LPCN 1144 in the treatment of NASH and of hepatic fibrosis.

Testosterone treatment is associated with reduced adipose tissue dysfunction and nonalcoholic fatty liver disease in obese hypogonadal men.

PURPOSE: In both preclinical and clinical settings, testosterone treatment (TTh) of hypogonadism has shown beneficial effects on insulin sensitivity and visceral and liver fat accumulation. This prospective, observational study was aimed at assessing the change in markers of fat and liver functioning in obese men scheduled for bariatric surgery. METHODS: Hypogonadal patients with consistent symptoms (n = 15) undergoing 27.63 ± 3.64 weeks of TTh were compared to untreated eugonadal (n = 17) or asymptomatic hypogonadal (n = 46) men. A cross-sectional analysis among the different groups was also performed, especially for data derived from liver and fat biopsies. Preadipocytes isolated from adipose tissue biopsies were used to evaluate insulin sensitivity, adipogenic potential and mitochondrial function. NAFLD was evaluated by triglyceride assay and by calculating NAFLD activity score in liver biopsies. RESULTS: In TTh-hypogonadal men, histopathological NAFLD activity and steatosis scores, as well as liver triglyceride content were lower than in untreated-hypogonadal men and comparable to eugonadal ones. TTh was also associated with a favorable hepatic expression of lipid handling-related genes. In visceral adipose tissue and preadipocytes, TTh was associated with an increased expression of lipid catabolism and mitochondrial bio-functionality markers. Preadipocytes from TTh men also exhibited a healthier morpho-functional phenotype of mitochondria and higher insulin-sensitivity compared to untreated-hypogonadal ones. CONCLUSIONS: The present data suggest that TTh in severely obese, hypogonadal individuals induces metabolically healthier preadipocytes, improving insulin sensitivity, mitochondrial functioning and lipid handling. A potentially protective role for testosterone on the progression of NAFLD, improving hepatic steatosis and reducing intrahepatic triglyceride content, was also envisaged. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02248467, September 25th 2014.

Therapeutic effects of the selective farnesoid X receptor agonist obeticholic acid in a monocrotaline-induced pulmonary hypertension rat model.

BACKGROUND: Activation of the farnesoid X receptor (FXR), a member of the nuclear receptor steroid superfamily, leads to anti-inflammatory and anti-fibrotic effects in several tissues, including the lung. We have recently demonstrated a protective effect of the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) in rat models of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and bleomycin-induced pulmonary fibrosis. The aim of the present study was to investigate whether the positive effects of OCA treatment could be exerted also in established MCT-induced PAH, i.e., starting treatment 2 weeks after MCT administration. METHODS: Rats with MCT-induced PAH were treated, 2 weeks after MCT administration, with OCA or tadalafil for two additional weeks. Pulmonary functional tests were performed at week 2 (before treatment) and four (end of treatment). At the same time points, lung morphological features and expression profile of genes related to smooth muscle relaxation/contraction and tissue remodeling were also assessed. RESULTS: 2 weeks after MCT-induced injury, the treadmill resistance (a functional parameter related to pulmonary hypertension) was significantly decreased. At the same time point, we observed right ventricular hypertrophy and vascular remodeling, with upregulation of genes related to inflammation. At week 4, we observed a further worsening of the functional and morphological parameters, accompanied by dysregulation of inflammatory and extracellular matrix markers mRNA expression. Administration of OCA (3 or 10 mg/kg/day), starting 2 weeks after MCT-induced injury, significantly improved pulmonary function, effectively normalizing the exercise capacity. OCA also reverted most of the lung alterations, with a significant reduction of lung vascular wall thickness, right ventricular hypertrophy, and restoration of the local balance between relaxant and contractile pathways. Markers of remodeling pathways were also normalized by OCA treatment. Notably, results with OCA treatment were similar, or even superior, to those obtained with tadalafil, a recently approved treatment for pulmonary hypertension. CONCLUSIONS: The results of this study demonstrate a significant therapeutic effect of OCA in established MCT-induced PAH, improving exercise capacity associated with reduction of right ventricular hypertrophy and lung vascular remodeling. Thus, OCA dosing in a therapeutic protocol restores the balance between relaxant and contractile pathways in the lung, promoting cardiopulmonary protective actions in MCT-induced PAH.

Therapeutic effects of obeticholic acid (OCA) treatment in a bleomycin-induced pulmonary fibrosis rat model.

PURPOSE: We recently demonstrated a protective effect of the farnesoid X receptor agonist obeticholic acid (OCA) in rat models of bleomycin-induced pulmonary fibrosis (PF). Aim of the present study was to investigate whether the positive effects of OCA treatment are apparent also on ongoing bleomycin-induced PF, i.e., after 2 weeks of bleomycin administration. METHODS: Bleomycin-induced PF rats were treated 2 weeks after bleomycin administration with OCA or pirfenidone for two additional weeks. Pulmonary function test was performed at 2 and 4 weeks in all experimental groups. At the same time points, lung morphological features and mRNA expression profile of genes related to fibrosis, inflammation and epithelial-mesenchymal transition were also assessed. RESULTS: After 2 weeks, bleomycin significantly increased the pressure at the airway opening (PAO), a functional parameter related to fibrosis-induced lung stiffness, and induced diffuse lung interstitium fibrosis, with upregulation of inflammation (IL1ß, MCP1) and tissue remodeling (COL1A1, COL3A1, ET1, MMP7, PDGFa, αSMA, SNAI1) markers. At week four, a further increase of lung fibrosis and PAO was observed, accompanied by upregulation of extracellular matrix-related mRNA expression. OCA administration, even after the establishment of PF, significantly improved pulmonary function, normalizing PAO, and reverted the bleomycin-induced lung alterations, with significant reduction of markers of inflammation (CD206, COX2, HIF1, IL1ß, MCP1), epithelial proliferation (CTGF, PDGFa) and fibrosis (COL1A1, COL3A1, ET1, FN1, MMPs, αSMA, SNAIs, TGFß1, TIMPs). Results with OCA were similar or superior to those obtained with pirfenidone. CONCLUSIONS: In conclusion, our results demonstrate a significant therapeutic effect of OCA in already established PF.

The revised ghent nosology; reclassifying isolated ectopia lentis.

Inherited ectopia lentis (EL) is most commonly caused by Marfan syndrome (MFS), a multisystemic disorder caused by mutations in FBN1. Historically the diagnosis for patients with EL who have no systemic features of MFS is isolated EL (IEL). However, the Ghent nosology for MFS was updated in 2010 and made some important alterations. In particular, patients with EL and a FBN1 mutation are now categorically diagnosed with MFS, if their mutation has previously been described with aortic dilation/dissection. This carries significant systemic implications, as many patients previously diagnosed with IEL are now reclassified. We provide a review of all published cases of IEL caused by FBN1 mutations over the last 20 years to assess what impact the new Ghent nosology has on these. Indeed, 57/123 probands (46.3%) are now classified as MFS according to the revised Ghent nosology and 37/96 mutations (38.5%) reported to cause isolated EL have also been found in patients with aortic dilation/dissection. These findings suggest that EL caused by mutations in FBN1 is actually part of a spectrum of fibrillinopathies with MFS, and the term 'IEL' should be avoided in such cases.

Opposite effects of tamoxifen on metabolic syndrome-induced bladder and prostate alterations: a role for GPR30/GPER?

BACKGROUND: BPH and LUTS have been associated to obesity, hypogonadism, and metabolic syndrome (MetS). MetS-induced prostate and bladder alterations, including inflammation and tissue remodeling, have been related to a low-testosterone and high-estrogen milieu. In addition to ERs, GPR30/GPER is able to mediate several estrogenic non-genomic actions. METHODS: Supplementing a subgroup of MetS rabbits with tamoxifen, we analyzed the in vivo effects on MetS-induced prostate and bladder alterations. The effects of selective ER/GPER ligands and GPER silencing on prostate inflammation were also studied in vitro using hBPH cells. RESULTS: ERα, ERß, and PR expression was upregulated in MetS bladder, where tamoxifen decreased ERα and PR expression, further stimulating ERß. In addition, tamoxifen-dosing decreased MetS-induced overexpression of inflammatory and tissue remodeling genes. In prostate, sex steroid receptors, pro-inflammatory and pro-fibrotic genes were upregulated in MetS. However, tamoxifen did not affect them and even increased COX-2. In hBPH cells, 17ß-estradiol increased IL-8 secretion, an effect blunted by co-treatment with GPER antagonist G15 but not by ER antagonist ICI 182,780, which further increased it. GPER agonist G1 dose-dependently (IC50 = 1.6 nM) induced IL-8 secretion. In vitro analysis demonstrated that GPER silencing reverted these stimulatory effects. CONCLUSIONS: GPER can be considered the main mediator of estrogen action in prostate, whereas in bladder the mechanism appears to rely on ERα, as indicated by in vivo experiments with tamoxifen dosing. Limiting the effects of the MetS-induced estrogen action via GPER could offer new perspectives in the management of BPH/LUTS, whereas tamoxifen dosing showed potential benefits in bladder.

Human prostatic urethra expresses vitamin D receptor and responds to vitamin D receptor ligation.

BACKGROUND: Chronic inflammation is now considered a determinant of benign prostatic hyperplasia (BPH), promoting, together with the hormonal milieu, prostate overgrowth and lower urinary tract symptoms (LUTS). Prostatic urethra actively participates in determining progression of LUTS associated with BPH. AIM: To investigate the expression of the vitamin D receptor (VDR) and the ability of the VDR agonist elocalcitol to reduce inflammatory responses in human prostatic urethra (hPU) cells. MATERIALS AND METHODS: Human prostatic urethra, prostate and bladder neck were obtained from patients affected by BPH. Immunohistochemical studies for VDR expression were performed in tissue samples, from which primary cell cultures were also derived. In hPU cells, proliferation and chemiotaxis were studied, along with Rho kinase (ROCK) activity (MYPT-1 phosphorylation) by western blot. Quantitative RT-PCR was performed for VDR, cyclooxygenase (COX-2), and interleukin (IL)-8 expression. RESULTS: Urethra displays higher VDR expression compared to prostate and bladder neck tissues. The VDR agonist elocalcitol partially reverts COX-2 and IL-8 mRNA upregulation induced by a pro-inflammatory cytokine mixture (IL-17, interferon-γ, tumor necrosis factor-α) and inhibits cell migration in urethral cells. Elocalcitol prevents activation of ROCK, as previously demonstrated in bladder and prostate cell cultures. CONCLUSIONS: Our results suggest that prostatic urethra is, within the lower urinary tract, a novel target for VDR agonists, as shown by the capacity of elocalcitol to inhibit ROCK activity and to limit inflammatory responses in human primary urethra cells.

Pathogenic FBN1 mutations in 146 adults not meeting clinical diagnostic criteria for Marfan syndrome: further delineation of type 1 fibrillinopathies and focus on patients with an isolated major criterion.

Mutations in the FBN1 gene cause Marfan syndrome (MFS) and have been associated with a wide range of milder overlapping phenotypes. A proportion of patients carrying a FBN1 mutation does not meet diagnostic criteria for MFS, and are diagnosed with "other type I fibrillinopathy." In order to better describe this entity, we analyzed a subgroup of 146 out of 689 adult propositi with incomplete "clinical" international criteria (Ghent nosology) from a large collaborative international study including 1,009 propositi with a pathogenic FBN1 mutation. We focused on patients with only one major clinical criterion, [including isolated ectopia lentis (EL; 12 patients), isolated ascending aortic dilatation (17 patients), and isolated major skeletal manifestations (1 patient)] or with no major criterion but only minor criteria in 1 or more organ systems (16 patients). At least one component of the Ghent nosology, insufficient alone to make a minor criterion, was found in the majority of patients with isolated ascending aortic dilatation and isolated EL. In patients with isolated EL, missense mutations involving a cysteine were predominant, mutations in exons 24-32 were underrepresented, and no mutations leading to a premature truncation were found. Studies of recurrent mutations and affected family members of propositi with only one major clinical criterion argue for a clinical continuum between such phenotypes and classical MFS. Using strict definitions, we conclude that patients with FBN1 mutation and only one major clinical criterion or with only minor clinical criteria of one or more organ system do exist but represent only 5% of the adult cohort.

Contribution of molecular analyses in diagnosing Marfan syndrome and type I fibrillinopathies: an international study of 1009 probands.

BACKGROUND: The diagnosis of Marfan syndrome (MFS) is usually initially based on clinical criteria according to the number of major and minor systems affected following international nosology. The number of FBN1 mutation carriers, at risk of aortic complications who would not be properly diagnosed based only on clinical grounds, is of growing importance owing to the increased availability of molecular screening. The aim of the study was to identify patients who should be considered for FBN1 mutation screening. METHODS: Our international series included 1009 probands with a known FBN1 mutation. Patients were classified as either fulfilling or not fulfilling "clinical" criteria. In patients with unfulfilled "clinical" criteria, we evaluated the percentage of additional patients who became positive for international criteria when the FBN1 mutation was considered. The aortic risk was evaluated and compared in patients fulfilling or not fulfilling the "clinical" international criteria. RESULTS: Diagnosis of MFS was possible on clinical grounds in 79% of the adults, whereas 90% fulfilled the international criteria when including the FBN1 mutation. Corresponding figures for children were 56% and 85%, respectively. Aortic dilatation occurred later in adults with unfulfilled "clinical criteria" when compared to the Marfan syndrome group (44% vs 73% at 40 years, p<0.001), but the lifelong risk for ascending aortic dissection or surgery was not significantly different in both groups. CONCLUSIONS: Because of its implications for aortic follow-up, FBN1 molecular analysis is recommended in newly suspected MFS when two systems are involved with at least one major system affected. This is of utmost importance in patients without aortic dilatation and in children.

Scoliosis, blindness and arachnodactyly in a large Turkish family: is it a new syndrome?

In this report we have described an affected sib in a large Turkish family who appears to have a new distinct dominantly-inherited blindness, scoliosis and arachnodactyly syndrome. The combination of clinical abnormalities in these patients did not initially suggest Marfan syndrome or other connective tissue disorders associated with ectopia lentis. The proband was a 16-year-old boy who was referred to our clinics for scoliosis. He had arachnodactyly of both fingers and toes. He had been suffering from progressive visual loss and strabismus since he was eight-years-old. His 20-year-old brother had severe kyphoscoliosis, and arachnodactyly of fingers and toes. He was 130 cm tall and was bilaterally blind. His 23-year-old sister had only eye findings but no arachnodactyly or scoliosis. His 60-year-old father had mild scoliosis, blindness and arachnodactyly and mother was normal. We performed routine mutation analyses in the genes FBN1, TGFBR1 and TGFBR2, but no mutation has been detected. Our Turkish patients are most likely affected by a hitherto unrecorded condition which is caused by an autosomal dominant gene defect with variable expression but we can not exclude multigenic inheritance. Further studies are needed to assess the contribution of sex influence to the syndrome because the female relative is less affected.

Fibroblast growth factor and endothelin-1 receptors mediate the response of human striatal precursor cells to hypoxia.

Fetal striatal transplantation has emerged as a new therapeutic strategy in Huntington's disease (HD). Hypoxia is one of the microenvironmental stress conditions to which fetal tissue is exposed as soon as it is isolated and transplanted into the diseased host brain. Mechanisms that support neuroblast survival and replenishment of damaged cells within the HD brain in the hypoxic condition have yet to be fully elucidated. This study is aimed at investigating the molecular pathways associated with the hypoxic condition in human fetal striatal neuroblasts (human striatal precursor (HSP) cells), using the hypoxia-mimetic agent cobalt chloride (CoCl2). We analyzed the effect of CoCl2 on HSP cell proliferation and on the expression of hypoxia-related proteins, such as hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Moreover, we evaluated fibroblast growth factor 2 (FGF2; 50ng/ml) and endothelin-1 (ET-1; 100nM) proliferative/survival effects in HSP cells in normoxic and hypoxic conditions. Dose-response experiments using increasing concentrations of CoCl2 (50-750µM) showed that the HSP cell growth was unaffected after 24h, while it increased at 48h, with the maximal effect observed at 400µM. In contrast, cell survival was impaired at 72h. Hypoxic conditions determined HIF-1α protein accumulation and increased gene and protein expression of VEGF, while FGF2 and ET-1 significantly stimulated HSP cell proliferation both in normoxic and hypoxic conditions, thus counteracting the apoptotic CoCl2 effect at 72h. The incubation with selective receptor (FGFR1, endothelin receptor A (ETA) and endothelin receptor B (ETB)) inhibitors abolished the FGF2 and ET-1 neuroprotective effect. In particular, ET-1 stimulated HSP cell survival through ETA in normoxic conditions and through ETB during hypoxia. Accordingly, ETA expression was down-regulated, while ETB expression was up-regulated by CoCl2 treatment. Overall, our results support the idea that HSP cells possess the machinery for their adaptation to hypoxic conditions and that neurotrophic factors, such as FGF2 and ET-1, may sustain neurogenesis and long-term survival through complex receptor-mediated mechanisms.

Detection of six novel FBN1 mutations in British patients affected by Marfan syndrome.

Marfan syndrome (MFS), an autosomal dominant disorder of the extracellular matrix, is due to mutations in fibrillin-1 (FBN1) gene. Investigations carried out in the last decade, unveiled the unpredictability of the site of the mutation, which could be anywhere in the gene. FBN1 mutations have been reported in a spectrum of diseases related to MFS, with no clear evidence for a phenotype-genotype correlation. In this paper we analysed 10 British patients affected by MFS and we were able to characterise five novel missense mutations (C474W, C1402Y, G1987R, C2153Y, G2536R), one novel frameshift mutation (7926delC), one already described mutation (P1424A) and one FBN1 variant (P1148A) classified as a polymorphism in the Asian population. Four out of the five novel missense mutations involved either cysteines or an amino acid conserved in the domain structure. The mutation yield in this study is calculated at 80.0% (8/10), thus indicating that SSCA is a reliable and cost-effective technique for the screening of such a large gene. Our results suggest that this method is reliable to search for FBN1 mutations and that FBN1 screening could be a helpful tool to confirm and possibly anticipate the clinical diagnosis in familial cases. Hum Mutat 18:251, 2001.

Erratum: Detection of six novel FBN1 mutations in British patients affected by Marfan syndrome.

Marfan syndrome (MFS), an autosomal dominant disorder of the extracellular matrix, is due to mutations in fibrillin-1 (FBN1) gene. Investigations carried out in the last decade, unveiled the unpredictability of the site of the mutation, which could be anywhere in the gene. FBN1 mutations have been reported in a spectrum of diseases related to MFS, with no clear evidence for a phenotype-genotype correlation. In this paper we analysed 10 British patients affected by MFS and we were able to characterise five novel missense mutations (C474W, C1402Y, G1987R, C2153Y, G2536R), one novel frameshift mutation (7926delC), one already described mutation (P1424A) and one FBN1 variant (P1148A) classified as a polymorphism in the Asian population. Four out of the five novel missense mutations involved either cysteines or an amino acid conserved in the domain structure. The mutation yield in this study is calculated at 80.0% (8/10), thus indicating that SSCA is a reliable and cost-effective technique for the screening of such a large gene. Our results suggest that this method is reliable to search for FBN1 mutations and that FBN1 screening could be a helpful tool to confirm and possibly anticipate the clinical diagnosis in familial cases.

A novel de novo mutation in the triple helix of the COL6A3 gene in a two-generation Italian family affected by Bethlem myopathy. A diagnostic approach in the mutations' screening of type VI collagen.

Bethlem myopathy is an autosomal dominant inherited disease producing a mild neuromuscular disorder, characterized mainly by muscular weakness and multiple joint contractures. Bethlem myopathy is caused by mutations in one of the three chains of collagen type VI. Here we report the clinical description and the molecular characterization of the defect in a two-generation Italian family in which a Gly-->Arg substitution disrupts the triple helix structure of the alpha 3 chain of collagen type VI, an ubiquitous glycoprotein of the extracellular matrix. In this family the identification of the mutation also allowed one to exclude the disease in the grandfather. It is noteworthy that the father of the proband carries a de novo mutation, the first described for Bethlem myopathy.

Cytokine gene expression in human LPS- and IFNgamma-stimulated mononuclear cells is inhibited by heparin.

In addition to its well-understood anticoagulant activity, heparin is known to modulate a variety of biological functions including immunologic responses. In order to investigate whether heparin influences the humoral immunity by interacting with cellular elements and affecting gene expression in blood circulating cells. we studied the effect of heparin on IL-1beta, IL-6 and TNFalpha mRNAs in human lipopolysaccharide-(LPS)- or interferon-gamma(IFN-gamma)-stimulated mononuclear cells. The study of mRNA was carried out by an initial PCR screening followed by a Northern blot quantitative analysis. Heparin (0.5 U/ml) turned out to inhibit all three cytokine gene expressions. The mRNA decrease was 37 +/- 6% for IL-1beta, 53 +/- 3% for IL-6 and 47 +/- 4% for TNFalpha with LPS stimulus. No differences could be observed in the inhibitory effect of heparin on IFNgamma-stimulated cells. This effect of heparin was confirmed in a subset of experiments performed on purified monocytes. These results suggest an important immunosuppressive effect of heparin on cell-mediated immune responses.

Tissue factor reduction and tissue factor pathway inhibitor release after heparin administration.

Elevated plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) and large amounts of monocyte procoagulant activity (PCA) have been documented in unstable angina (UA) patients. In in vitro experiments heparin is able to blunt monocyte TF production by inhibiting TF and cytokine gene expression by stimulated cells and after in vivo administration it reduces adverse ischemic outcomes in UA patients. TF and TFPI plasma levels and monocyte PCA have been investigated in 28 refractory UA patients before and during anticoagulant subcutaneous heparin administration (thrice daily weight- and PTT-adjusted for 3 days) followed by 5000 IU X 3 for 5 days. After 2-day treatment, immediately prior to the heparin injection, TF and TFPI plasma levels [(median and range): 239 pg/ml, 130-385 pg/ ml and 120 ng/ml, 80-287 ng/ml] were lower in comparison to baseline samples (254.5 pg/ml, 134.6-380 pg/ml and 135.5 ng/ml, 74-306 ng/ml). Four h after the heparin injection TF furtherly decreased (176.5 pg/ml, 87.5-321 pg/ml; -32.5%. p<0.001) and TFPI increased (240.5 ng/ml, 140-450 ng/ml; +67%, p<0.0001). After 7-day treatment, before the injection of heparin, TF and TFPI plasma levels (200 pg/ml, 128-325 pg/ml and 115 ng/ml, 70-252 ng/ml) significantly decreased (p<0.05) in comparison to the pre-treatment values. On the morning of the 8th day, 4 h after the injection of heparin TF plasma levels and monocytes PCA significantly decreased (156.5 pg/ml, 74-259 pg/ml and from 180 U/105 monocytes, 109-582 U/10(5) monocytes to 86.1 U/10(5) monocytes, 28-320 U/10(5) monocytes; - 38% and -55% respectively) and TFPI increased (235.6 ng/ml, 152-423 ng/ ml; +70%, p<0.001). In conclusion, heparin treatment is associated with a decrease of high TF plasma levels and monocyte procoagulant activity in UA patients. These actions of heparin may play a role in determining the antithrombotic and antiinflammatory properties of this drug.

Platelet activation and platelet lipid composition in pulmonary cancer.

In order to investigate the possible mechanisms underlying platelet functional changes in patients affected by neoplasms, platelet lipid composition, plasma beta-thromboglobulin (Beta-TG) and serum thromboxane B2 (TXB2) were investigated in 16 male patients affected by pulmonary carcinoma and in 16 comparable control subjects. In patients high levels of plasma Beta-TG (67 +/- 9 versus controls 14 +/- 4 ng/ml, p < 0.001) and serum TXB2 (434 +/- 56 versus 223 +/- 48 ng/ml, p < 0.001) were observed. Also platelet lipid composition was found altered in patients with respect to controls (lower percent levels in n-3 fatty acids and in linoleic acid esterified in the main platelet phospholipid fractions: at least p < 0.05). These results indicate that in vivo platelet activation is detectable in neoplastic patients and it is associated with alterations in platelet lipid composition. In the light of the important role played by membrane lipids in platelet functions related to thrombosis and haemostasis we conclude that platelet lipid changes could cooperate in platelet activation and increased thrombotic risk so frequently observed in neoplastic disease.

D-dimer concentrations during normal pregnancy, as measured by ELISA.

In pregnant women a number of changes in blood clotting and fibrinolysis proteins have been reported so indicating the existence of a state of hypercoagulability. In addition to fibrinogen and antithrombin III (AT), D-dimer is frequently checked during pregnancy, in particular during at risk pregnancy, but the exact pattern of D-dimer modifications during uncomplicated pregnancy is not definitively described. The aim of this study was to establish the range values in three different periods of uncomplicated pregnancy (A: 1-20 wks; B: 21-30 wks; C: 31-40 wks). We measured plasma levels of D-dimer, clottable fibrinogen and AT in 108 consecutive normal pregnant women aged 16 to 42 years. In period A, the range of D-dimer values was 43-211 ng/mL, not different from controls, while fibrinogen levels were significantly higher (p < 0.05) than in matched non pregnant women. Mean D-dimer levels were higher in periods B (p < 0.05) and C (p < 0.05) vs period A. Similarly, mean fibrinogen levels were found more elevated in periods B and C vs period A (p < 0.05). A significant correlation was found between fibrinogen and D-dimer levels (p < 0.001). No differences in AT levels were found among the three periods of pregnancy. The results of this study indicate that levels of D-dimer up to 685 micrograms/L may be reached at the end of physiological pregnancy. This fact should be taken into account in the evaluation of hemostatic studies performed in uncomplicated and complicated pregnant women.