Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

Type I osteogenesis imperfecta and multiple osteochondromas in the same child.

A male infant showed a humeral diaphysis fracture at 5 months of age and a distal tibial physis fracture at 2 years of age. A specialized consultant ruled out child abuse. This child had the characteristic features of type I osteogenesis imperfecta: blue sclerae, osseous fragility, and presumably autosomal dominant inheritance, as his father suffered from similar disorders. Later on, multiple painful osteochondromas were also found and some of these were surgically treated. The child's mother showed several peripheral osteochondromas. We describe the follow-up of this patient up to the age of 18 years. To our knowledge, the fortuitous association of these two inherited conditions has not been reported in medical literature.

Apparent stable-isotope heterogeneities in gangue carbonates of the Mississippi Valley-type Zn-Pb deposits of San-Vicente, Central Peru

The aim of the present communication is to emphasize that some variations of the measured delta(13)C and delta(18)O values are apparent, and due to analytical interferences caused by the presence of sulfur and organosulfur compounds in the analyzed carbonates. This is particularly relevant for isotopic studies on carbonate-hosted mineral deposits, where the nearly ubiquitous association of the host carbonates with organic matter and sulfides can certainly affect the metallogenetic interpretations. In this work two methods were used to overcome the disturbing effects of sulfides and organic matter: (1) sample pretreatment following the method proposed by Charef and Sheppard (1984), combining the oxidation of organic matter with sodium hypochlorite and trapping of the sulfur species with silver phosphate; and (2) laser-based microprobe extraction. Apparent isotopic variations in sparry dolomite from a single hand sample of zebra ore from the MVT Zn-Pb deposit, San Vicente, central Peru, are as large as 6 parts per thousand delta(13)C and 4 parts per thousand delta(18)O. These variations are reduced to several tenths of a per mil when the samples are pretreated. A careful examination of the effects of treatment with NaOCl and/or Ag3PO4 in relation to the concentration of sulfide inclusions indicates that the main disturbing effects for delta(13)C values are the presence of sulfur species and organic matter, whereas the delta(18)O values are mainly affected by the presence of sulfides. Fine- and medium-grained replacement carbonates from MVT and other sediment-hosted base metal deposits are potentially the most affected during isotope analysis, due to the common presence of organic matter and sulfides. Using in situ laser microprobe techniques, it is possible to determine isotopic variations at a sub-millimeter scale. Our results show that laser extraction analysis allows a more precise sampling of the carbonate minerals, and minimizes contamination of the sample with sulfides and to some extent with intergrown organic matter. However, there is an isotopic shift associated with the laser extraction technique, of the order of 0.5-1 parts per thousand for delta(13)C and delta(18)O values.

Surveillance of HIV type 1 drug resistance among naive patients from Venezuela.

We have studied 65 HIV-1-infected untreated patients recruited in Caracas, Venezuela with TCD4 counts > or =350/microl. The reverse transcriptase and protease sequences of the virus were sequenced, aligned with reference HIV-1 group M strains, and analyzed for drug resistance mutations. Most of the viruses were subtype B genotype in both the protease and RT genomic regions. Five of the 62 virus isolates successfully amplified showed evidence of recombination between protease and RT, with their protease region being non-B while their RT region was derived from subtype B. Four strains were found bearing resistance mutations either to NRTIs, NNRTIs, or PIs. The prevalence of HIV-1 isolates bearing resistance mutations was therefore above the 5% threshold of WHO.

Subclinical coronary and aortic atherosclerosis detected by magnetic resonance imaging in type 1 diabetes with and without diabetic nephropathy.

BACKGROUND: Patients with type 1 diabetes and nephropathy maintain an excess cardiovascular mortality compared with diabetic patients with normoalbuminuria. We sought to evaluate coronary and aortic atherosclerosis in a cohort of asymptomatic type 1 diabetic patients with and without diabetic nephropathy using cardiovascular magnetic resonance imaging. METHODS AND RESULTS: In a cross-sectional study, 136 subjects with long-standing type 1 diabetes without symptoms or history of cardiovascular disease, including 63 patients (46%) with nephropathy and 73 patients with normoalbuminuria, underwent cardiovascular magnetic resonance imaging. All subjects underwent cardiac exercise testing and noninvasive tests for peripheral artery disease and autonomic neuropathy. Coronary artery stenoses were identified in 10% of subjects with nephropathy (versus 0% with normoalbuminuria; P=0.007). Coronary plaque burden, expressed as right coronary artery mean wall thickness (1.7+/-0.3 versus 1.3+/-0.2 mm; P<0.001) and maximum right coronary artery wall thickness (2.2+/-0.5 versus 1.6+/-0.3 mm; P<0.001), was greater in subjects with nephropathy. The prevalence of thoracic (3% versus 0%; P=0.28) and abdominal aortic plaque (22% versus 16%; P=0.7) was similar in both groups. Subjects with and without abdominal aortic plaques had similar coronary plaque burden. CONCLUSIONS: In asymptomatic type 1 diabetes, cardiovascular magnetic resonance imaging reveals greater coronary plaque burden in subjects with nephropathy compared with those with normoalbuminuria.

Gene transfer of a soluble IL-1 type 2 receptor-Ig fusion protein improves cardiac allograft survival in rats.

OBJECTIVE: Interleukin-1 (IL-1) mediates ischemia-reperfusion injury and graft inflammation after heart transplantation. IL-1 affects target cells through two distinct types of transmembrane receptors, type-1 receptor (IL-1R1), which transduces the signal, and the non-signaling type-2 receptor (IL-1R2), which acts as a ligand sink that subtracts IL-1beta from IL-1R1. We analyzed the efficacy of adenovirus (Ad)-mediated gene transfer of a soluble IL-1R2-Ig fusion protein in delaying cardiac allograft rejection and the mechanisms underlying the protective effect. METHODS: IL-1 inhibition by IL-1R2-Ig was tested using an in vitro functional assay whereby endothelial cells preincubated with AdIL-1R2-Ig or control virus were stimulated with recombinant IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and urokinase-type plasminogen activator (u-PA) induction was measured by zymography. AdIL-1R2-Ig was delivered to F344 rat donor hearts ex vivo, which were placed in the abdominal position in LEW hosts. Intragraft inflammatory cell infiltrates and proinflammatory cytokine expression were analyzed by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: IL-1R2-Ig specifically inhibited IL-1beta-induced u-PA responses in vitro. IL-1R2-Ig gene transfer reduced intragraft monocytes/macrophages and CD4(+) cell infiltrates (p<0.05), TNF-alpha and transforming growth factor-beta (TGF-beta) expression (p<0.05), and prolonged graft survival (15.6+/-5.7 vs 10.3+/-2.5 days with control vector and 10.1+/-2.1 days with buffer alone; p<0.01). AdIL-1R2-Ig combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone (19.4+/-3.0 vs 15.9+/-1.8 days; p<0.05). CONCLUSIONS: Soluble IL-1 type-2 receptor gene transfer attenuates cardiac allograft rejection in a rat model. IL-1 inhibition may be useful as an adjuvant therapy in heart transplantation.

Eosinophilic oesophagitis and proton pump inhibitor-responsive oesophageal eosinophilia have similar clinical, endoscopic and histological findings.

BACKGROUND: Some patients with a phenotypic appearance of eosinophilic oesophagitis (EoE) respond histologically to PPI, and are described as having PPI-responsive oesophageal eosinophilia (PPI-REE). It is unclear if PPI-REE is a GERD-related phenomenon, a subtype of EoE, or a completely unique entity. AIM: To compare demographic, clinical and histological features of EoE and PPI-REE. METHODS: Two databases were reviewed from the Walter Reed and Swiss EoE databases. Patients were stratified into two groups, EoE and PPI-REE, based on recent EoE consensus guidelines. Response to PPI was defined as achieving less than 15 eos/hpf and a 50% decrease from baseline following at least a 6-week course of treatment. RESULTS: One hundred and three patients were identified (63 EoE and 40 PPI-REE; mean age 40.2 years, 75% male and 89% Caucasian). The two cohorts had similar dysphagia (97% vs. 100%, P = 0.520), food impaction (43% vs. 35%, P = 0.536), and heartburn (33% vs. 32%, P = 1.000) and a similar duration of symptoms (6.0 years vs. 5.8 years, P = 0.850). Endoscopic features were also similar between EoE and PPI-REE; rings (68% vs. 68%, P = 1.000), furrows (70% vs. 70%, P = 1.000), plaques (19% vs. 10%, P = 0.272), strictures (49% vs. 30%, P = 0.066). EoE and PPI-REE were similar in the number of proximal (39 eos/hpf vs. 38 eos/hpf, P = 0.919) and distal eosinophils (50 vs. 43 eos/hpf, P = 0.285). CONCLUSIONS: EoE and PPI-responsive oesophageal eosinophilia are similar in clinical, histological and endoscopic features and therefore are indistinguishable without a PPI trial. Further studies are needed to determine why a subset of patients with oesophageal eosinophilia respond to PPI.

Quette place pour l'automesure glycémique dans la prise en charge du diabète de type 2 [Which role for self-monitoring of blood glucose in type 2 diabetes?]

There is a sustained controversy in the literature about the role and utility of self-monitoring of blood glucose (SMBG) in type 2 diabetes. The study results in this field do not provide really useful clues for the integration of SMBG in the follow-up of the individual patient, because they are based on a misconception of SMBG. It is studied as if it was a medical treatment whose effect on glycemic control is to be isolated. However, SMBG has no such intrinsic effect. It gains its purpose only as an inseparable component of a comprehensive and structured educational strategy. To be appropriate this strategy cannot be based on the health care professionals' view on diabetes only. It rather has to be tailored to the individual patient's needs through an ongoing process of shared reflection with him.

Hemodynamic and humoral effects of the new renin inhibitor enalkiren in normal humans.

The effect of the renin inhibitor enalkiren (Abbott-64662) was evaluated in eight normal volunteer subjects on a standardized sodium diet (100 mmol/day) by measurement of various components of the renin-angiotensin system and drug levels in plasma. On day 1, vehicle and doses of 0.001, 0.003, and 0.01 mg/kg i.v. were administered within 2 minutes at 90-minute intervals. On day 2, vehicle and doses of 0.01, 0.03, and 0.1 mg/kg i.v. were given. With the higher doses, blood pressure tended to decrease slightly with no change in heart rate. Plasma renin activity and plasma angiotensin-(1-8)octapeptide (angiotensin II) fell markedly in a dose-dependent manner. Inhibition of plasma renin activity was maximal 5 minutes after administration of the drug and persisted 90 minutes after the doses of 0.03 and 0.1 mg/kg. Not surprisingly, there was a close correlation between plasma renin activity and plasma angiotensin II levels (r = 0.81, n = 28, p less than 0.001). In contrast, active and total renin measured directly by monoclonal antibodies rose in dose-related fashion in response to renin inhibition. Pharmacokinetic parameters were calculated using the plasma drug concentrations obtained up to 6 hours after the 0.1 mg/kg dose. By means of a two-compartment model, plasma mean half-life of the drug was estimated at 1.60 +/- 0.43 hours.

Seroepidemiology of herpes simplex virus type 1 and 2 in pregnant women in Switzerland: an obstetric clinic based study.

OBJECTIVE: To assess the seroprevalence of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) IgG antibodies and the seroincidence of HSV-1 and HSV-2 infections in pregnant women attending the maternity clinic of the University Hospital Lausanne. STUDY DESIGN: Blood samples from 1030 women were taken at the usual pregnancy visit in the first trimester to assess the prevalence rate of IgG antibodies against HSV-1 and HSV-2 using a type-specific assay. A second blood sample was taken 6-8 weeks postpartum from returning women who were seronegative for HSV-2 or HSV-1 to assess the incidence of seroconversion (primary infection). RESULTS: The seroprevalence rates were 79.4% (95% CI: 76.9-81.9) for HSV-1 and 21.2% (18.7-23.7) for HSV-2 in women 14-46 years old. Type-specific serostatus patterns were as follows: 17.3% HSV-1/-2: +/+, 62.1% HSV-1/-2: +/-, 3.9% HSV-1/-2: -/+, 16.7% HSV-1/-2: -/-. Two hundred and sixty five women (59 of the 212 seronegative for HSV-1 (27.8%) and 265 of the 812 seronegative for HSV-2 (32.6%)) returned to the outpatient clinic for the post-delivery check and a second blood sample was obtained. One HSV-1 seroconversion was detected (HSV-1 seroconversion rate 2.4%/100 patient×year (95% CI: 0.06-13.4)) in a patient who had symptoms compatible with primary genital herpes. No HSV-2 seroconversion was detected (HSV-2 seroconversion rate: 0/100 patient×year (97.5% one-sided CI: 0-2)). CONCLUSION: Compared to a previous population-based study, our study results suggest a rise in the prevalence of HSV-2 among pregnant women in Switzerland. The low incidence of seroconversion detected during pregnancy is consistent with the very low reported incidence of neonatal herpes in Switzerland. CONDENSATION: This study in a public hospital in Western Switzerland suggests an increasing prevalence of HSV-2, but a low incidence of primary infections in women of childbearing age.

Diabetic neuropathy is a more important determinant of baroreflex sensitivity than carotid elasticity in type 2 diabetes.

The object of this study was to evaluate the contribution of carotid distensibilty on baroreflex sensitivity in patients with type 2 diabetes mellitus with at least 2 additional cardiovascular risk factors. Carotid distensibility was measured bilaterally at the common carotid artery in 79 consecutive diabetic patients and 60 matched subjects without diabetes. Spontaneous baroreflex sensitivity assessment was obtained using time and frequency methods. Baroreflex sensitivity was lower in diabetic subjects as compared with nondiabetic control subjects (5.25+/-2.80 ms/mm Hg versus 7.55+/-3.79 ms/mm Hg; P<0.01, respectively). Contrary to nondiabetic subjects, diabetic subjects showed no significant correlation between carotid distensibility and baroreflex sensitivity (r2=0.08, P=0.04 and r2=0.04, P=0.13, respectively). In diabetic subjects, baroreflex sensitivity was significantly lower in subjects with peripheral neuropathy than in those with preserved vibration sensation (4.1+/-0.5 versus 6.1+/-0.4 ms/mm Hg, respectively; P=0.005). Age in nondiabetic subjects, diabetes duration, systolic blood pressure, peripheral or sensitive neuropathy, and carotid distensibility were introduced in a stepwise multivariate analysis to identify the determinants of baroreflex sensitivity. In diabetic patients, neuropathy is a more sensitive determinant of baroreflex sensitivity than the reduced carotid distensibility (stepwise analysis; F ratio=5.1, P=0.028 versus F ratio=1.9, P=0.16, respectively). In diabetic subjects with 2 additional cardiovascular risk factors, spontaneous baroreflex sensitivity is not related to carotid distensibility. Diabetic subjects represent a particular population within the spectrum of cardiovascular risk situations because of the marked neuropathy associated with their metabolic disorder. Therefore, neuropathy is a more significant determinant of baroreflex sensitivity than carotid artery elasticity in patients with type 2 diabetes.

Dramatic response of vemurafenib-induced cutaneous lesions upon switch to dual BRAF/MEK inhibition in a metastatic melanoma patient.

BRAF inhibitory therapy is the mainstream treatment for BRAF mutant advanced melanoma. However vemurafenib, a type I mutant BRAF V600 inhibitor, induces an array of proliferative skin disorders from keratosis pilaris-like and keratoacanthoma-like lesions to locally aggressive cutaneous squamous cell carcinoma (cuSCC). Dual BRAF/MEK inhibition is known to lower the incidence of such manifestations, but it is not known whether it can counteract established lesions. Here we show, for the first time, a dramatic response and a restitution ad integro upon dual inhibition of a widespread proliferative affection induced by BRAF monotherapy. A 75-year-old woman was diagnosed with a BRAF V600E mutated metastatic melanoma. Following dacarbazine (DTIC) and ipilimumab, the patient was started on 960 mg twice daily vemurafenib (Zelboraf), which resulted in complete response, but the patient also developed grade IV skin toxicity. Despite dose-reduction to 720 mg twice daily the side effects persisted. We hypothesized that a switch to double inhibition of the mitogen-activated protein kinase pathway with dabrafenib and trametinib could lead to improvement of the skin lesions, while preserving tumor control. The patient was closely followed for changes in skin lesions. We witnessed a rapid regression followed by complete disappearance of all side effects of vemurafenib except for grade I fatigue. The biopsied skin lesions show regression of established keratoacanthoma-like lesions with signs of apoptosis. Switching from the current standard of care vemurafenib therapy to the double BRAF/MEK inhibition in BRAF mutant melanoma patients results in rapid disappearance of established proliferative skin disorders.

Fructose overconsumption causes dyslipidemia and ectopic lipid deposition in healthy subjects with and without a family history of type 2 diabetes.

BACKGROUND: Both nutritional and genetic factors are involved in the pathogenesis of nonalcoholic fatty liver disease and insulin resistance. OBJECTIVE: The aim was to assess the effects of fructose, a potent stimulator of hepatic de novo lipogenesis, on intrahepatocellular lipids (IHCLs) and insulin sensitivity in healthy offspring of patients with type 2 diabetes (OffT2D)--a subgroup of individuals prone to metabolic disorders. DESIGN: Sixteen male OffT2D and 8 control subjects were studied in a crossover design after either a 7-d isocaloric diet or a hypercaloric high-fructose diet (3.5 g x kg FFM(-1) x d(-1), +35% energy intake). Hepatic and whole-body insulin sensitivity were assessed with a 2-step hyperinsulinemic euglycemic clamp (0.3 and 1.0 mU x kg(-1) x min(-1)), together with 6,6-[2H2]glucose. IHCLs and intramyocellular lipids (IMCLs) were measured by 1H-magnetic resonance spectroscopy. RESULTS: The OffT2D group had significantly (P < 0.05) higher IHCLs (+94%), total triacylglycerols (+35%), and lower whole-body insulin sensitivity (-27%) than did the control group. The high-fructose diet significantly increased IHCLs (control: +76%; OffT2D: +79%), IMCLs (control: +47%; OffT2D: +24%), VLDL-triacylglycerols (control: +51%; OffT2D: +110%), and fasting hepatic glucose output (control: +4%; OffT2D: +5%). Furthermore, the effects of fructose on VLDL-triacylglycerols were higher in the OffT2D group (group x diet interaction: P < 0.05). CONCLUSIONS: A 7-d high-fructose diet increased ectopic lipid deposition in liver and muscle and fasting VLDL-triacylglycerols and decreased hepatic insulin sensitivity. Fructose-induced alterations in VLDL-triacylglycerols appeared to be of greater magnitude in the OffT2D group, which suggests that these individuals may be more prone to developing dyslipidemia when challenged by high fructose intakes. This trial was registered at clinicaltrials.gov as NCT00523562.

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h(2) of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRES.