Improved BM212 MmpL3 Inhibitor Analogue Shows Efficacy in Acute Murine Model of Tuberculosis Infection

1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved in vitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED₉₉ of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery.     

Tamoxifen Ameliorates Peritoneal Membrane Damage by Blocking Mesothelial to Mesenchymal Transition in Peritoneal Dialysis

Mesothelial-to-mesenchymal transition (MMT) is an auto-regulated physiological process of tissue repair that in uncontrolled conditions such as peritoneal dialysis (PD) can lead to peritoneal fibrosis. The maximum expression of peritoneal fibrosis induced by PD fluids and other peritoneal processes is the encapsulating peritoneal sclerosis (EPS) for which no specific treatment exists. Tamoxifen, a synthetic estrogen, has successfully been used to treat retroperitoneal fibrosis and EPS associated with PD. Hence, we used in vitro and animal model approaches to evaluate the efficacy of Tamoxifen to inhibit the MMT as a trigger of peritoneal fibrosis. In vitro studies were carried out using omentum-derived mesothelial cells (MCs) and effluent-derived MCs. Tamoxifen blocked the MMT induced by transforming growth factor (TGF)-β1, as it preserved the expression of E-cadherin and reduced the expression of mesenchymal-associated molecules such as snail, fibronectin, collagen-I, α-smooth muscle actin, and matrix metalloproteinse-2. Tamoxifen-treatment preserved the fibrinolytic capacity of MCs treated with TGF-β1 and decreased their migration capacity. Tamoxifen did not reverse the MMT of non-epitheliod MCs from effluents, but it reduced the expression of some mesenchymal molecules. In mice PD model, we demonstrated that MMT progressed in parallel with peritoneal membrane thickness. In addition, we observed that Tamoxifen significantly reduced peritoneal thickness, angiogenesis, invasion of the compact zone by mesenchymal MCs and improved peritoneal function. Tamoxifen also reduced the effluent levels of vascular endothelial growth factor and leptin. These results demonstrate that Tamoxifen is a therapeutic option to treat peritoneal fibrosis, and that its protective effect is mediated via modulation of the MMT process.     

Antigenic Proteins Involved in Occupational Rhinitis and Asthma Caused by Obeche Wood (Triplochiton Scleroxylon)

Background

Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated.

Objective

To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma

Materials and methods

An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies.

Results

Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA−), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p<0.05). Two new proteins were purified, a 24 kDa protein identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin. Both showed allergenic activity in vitro, with the putative thaumatin being the most active, with 92% recognition by ELISA and 100% by basophil activation test in ORA+ subjects. Cross-reactivity due to CCD was ruled out in 82% of cases.

Conclusions

Two proteins of obeche wood were identified and were recognized by a high percentage of symptomatic subjects and by a small proportion of asymptomatic exposed subjects. Further studies are required to evaluate cross reactivity with other plant allergens.     

Monitoring of Pb Exposure in Waterfowl Ten Years after a Mine Spill through the Use of Noninvasive Sampling

Lead exposure in waterfowl was studied using noninvasive fecal sampling in the Guadalquivir Marshes in Spain, an area affected by the 1998 Aznalcóllar mine disaster. Feces of greylag geese (Anser anser, n = 191) and purple gallinule (Porphyrio porphyrio, n = 91) were collected from three different impacted sites (Entremuros, Caracoles and Cerro de los Ánsares) during the winters of 2004 to 2008. Lead and aluminium (an indicator of sediment ingestion) and Pb isotope signatures (to discriminate between sources of Pb exposure) were analyzed in freeze-dried, acid digested samples. The concentrations of fecal porphyrins and biliverdin were determined as noninvasive biomarkers to study Pb exposure effects. Results showed a decrease in Pb exposure over time in wintering greylag geese. In contrast, for purple gallinule resident in the Entremuros a clear trend was not evident. For both species, sediment ingestion appeared to be the main source of exposure to Pb. In the Entremuros, some samples from purple gallinule were detected with higher Pb levels than expected for simple soil ingestion, and these had Pb isotopic profiles compatible with mining sludge or Pb shot. Whilst fecal Pb isotopic profiles were effective in differentiating between samples from sites with different levels and sources of pollution, the combined use of element ratios (such as Pb/Al) and other non-traditional stable isotope signatures may also prove worthwhile. Overall, the fecal Pb levels detected were below those described in feces for waterfowl from other uncontaminated areas(<10 µg/g d.w.). Despite this, for both species fecal Pb levels were positively correlated with porphyrin excretion, and for purple gallinule, with the coproporphyrin III/I ratio, suggesting some subtle effects on heme synthesis in birds. Ten years after the mine spill, Pb contamination in birds by this pollution source was still detectable and subtlethal effects may persist.     

High Molecular Weight Forms of Mammalian Respiratory Chain Complex II

Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CII_hmw) that can be resolved by clear native electrophoresis. CII_hmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 – over 1000 kDa) and cultured cells (400–670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CII_hmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CII_hmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase.     

Sonic Hedgehog Carried by Microparticles Corrects Angiotensin II-Induced Hypertension and Endothelial Dysfunction in Mice

Microparticles are small fragments of the plasma membrane generated after cell stimulation. We recently showed that Sonic hedgehog (Shh) is present in microparticles generated from activated/apoptotic human T lymphocytes and corrects endothelial injury through nitric oxide (NO) release. This study investigates whether microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in mice. Male Swiss mice were implanted with osmotic minipumps delivering angiotensin II (0.5 mg/kg/day) or NaCl (0.9%). Systolic blood pressure and heart rate were measured daily during 21 days. After 7 day of minipump implantation, mice received i.v. injections of microparticles (10 µg/ml) or i.p. Shh receptor antagonist cyclopamine (10 mg/kg/2 days) during one week. Angiotensin II induced a significant rise in systolic blood pressure without affecting heart rate. Microparticles reversed angiotensin II-induced hypertension, and cyclopamine prevented the effects of microparticles. Microparticles completely corrected the impairment of acetylcholine- and flow-induced relaxation in vessels from angiotensin II-infused mice. The improvement of endothelial function induced by microparticles was completely prevented by cyclopamine treatment. Moreover, microparticles alone did not modify NO and O₂ ^{. -} production in aorta, but significantly increased NO and reduced O₂ ^{. -} productions in aorta from angiotensin II-treated mice, and these effects were blocked by cyclopamine. Altogether, these results show that microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in aorta through a mechanism associated with Shh-induced NO production and reduction of oxidative stress. These microparticles may represent a new therapeutic approach in cardiovascular diseases associated with decreased NO production.     

BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

TGF-β family members play a relevant role in tumorigenic processes, including hepatocellular carcinoma (HCC), but a specific implication of the Bone Morphogenetic Protein (BMP) subfamily is still unknown. Although originally isolated from fetal liver, little is known about BMP9, a BMP family member, and its role in liver physiology and pathology. Our results show that BMP9 promotes growth in HCC cells, but not in immortalized human hepatocytes. In the liver cancer cell line HepG2, BMP9 triggers Smad1,5,8 phosphorylation and inhibitor of DNA binding 1 (Id1) expression up- regulation. Importantly, by using chemical inhibitors, ligand trap and gene silencing approaches we demonstrate that HepG2 cells autocrinely produce BMP9 that supports their proliferation and anchorage independent growth. Additionally, our data reveal that in HepG2 cells BMP9 triggers cell cycle progression, and strikingly, completely abolishes the increase in the percentage of apoptotic cells induced by long-term incubation in low serum. Collectively, our data unveil a dual role for BMP9, both promoting a proliferative response and exerting a remarkable anti-apoptotic function in HepG2 cells, which result in a robust BMP9 effect on liver cancer cell growth. Finally, we show that BMP9 expression is increased in 40% of human HCC tissues compared with normal human liver as revealed by immunohistochemistry analysis, suggesting that BMP9 signaling may be relevant during hepatocarcinogenesis in vivo. Our findings provide new clues for a better understanding of BMPs contribution, and in particular BMP9, in HCC pathogenesis that may result in the development of effective and targeted therapeutic interventions.     

Risks and Benefits of Early Antithrombotic Therapy after Thrombolytic Treatment in Patients with Acute Stroke

Background

Current guidelines recommend withholding antithrombotic therapy (ATT) for at least 24 h in patients with acute ischemic stroke treated with thrombolytic therapy. Herein, we report a retrospective analysis of a single-centre experience on the safety and efficacy of antithrombotic therapy (ATT) started before or after 24 h of intravenous thrombolysis in a cohort of acute ischemic stroke patients.

Methods

A total of 139 patients (Rapid ATT group) received antithrombotic therapy before 24 h of thrombolysis, and 33 patients (Standard ATT group) after 24 h. The brain parenchyma and vessel status were assessed using simple CT scan on admission, multimodal CT scan at the end of thrombolysis, and angio-CT/MRI scan at day 3. Functional outcome was scored using the modified Rankin Scale (mRS) at day 90.

Results

The two ATT groups had similar demographics, stroke subtypes, baseline NIHSS, thrombolytic strategies, vessel-patency rates at the end of thrombolysis, and incidence of bleeding complications at follow up. At day 3, the Rapid ATT group had a non-significant improved vessel-patency rate than the Standard ATT group. At day 90, a greater proportion of patients in the rapid ATT group had shifted down the mRS, and had improved in the NIHSS score.

Conclusions

ATT initiated before 24 h of intravenous thrombolytic therapy in acute stroke patients disclosed no safety concerns compared with a conventional antithrombotic therapy delay of 24 h and showed better functional outcome at follow up. The value of early initiation of ATT after thrombolysis deserves further assessment in randomized controlled trials.