Repurposed Drugs That Activate Autophagy in Filarial Worms Act as Effective Macrofilaricides.

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases caused by filarial nematodes that utilize insect vectors for transmission to their human hosts. Current control strategies are based on annual or biannual mass drug administration (MDA) of the drugs Ivermectin or Ivermectin plus Albendazole, respectively. These drug regimens kill the first-stage larvae of filarial worms (i.e., microfilariae) and interrupt the transmission of infections. MDA programs for these microfilaricidal drugs must be given over the lifetime of the filarial adult worms, which can reach 15 years in the case of Onchocerca volvulus. This is problematic because of suboptimal responses to ivermectin in various endemic regions and inefficient reduction of transmission even after decades of MDA. There is an urgent need for the development of novel alternative treatments to support the 2030 elimination goals of onchocerciasis and lymphatic filariasis. One successful approach has been to target Wolbachia, obligatory endosymbiotic bacteria on which filarial worms are dependent for their survival and reproduction within the human host. A 4-6-week antibiotic therapy with doxycycline, for example, resulted in the loss of Wolbachia that subsequently led to extensive apoptosis of somatic cells, germline, embryos, and microfilariae, as well as inhibition of fourth-stage larval development. However, this long-course regimen has limited use in MDA programs. As an alternative approach to the use of bacteriostatic antibiotics, in this study, we focused on autophagy-inducing compounds, which we hypothesized could disturb various pathways involved in the interdependency between Wolbachia and filarial worms. We demonstrated that several such compounds, including Niclosamide, an FDA-approved drug, Niclosamide ethanolamine (NEN), and Rottlerin, a natural product derived from Kamala trees, significantly reduced the levels of Wolbachia in vitro. Moreover, when these compounds were used in vivo to treat Brugia pahangi-infected gerbils, Niclosamide and NEN significantly decreased adult worm survival, reduced the release of microfilariae, and decreased embryonic development depending on the regimen and dose used. All three drugs given orally significantly reduced Wolbachia loads and induced an increase in levels of lysosome-associated membrane protein in worms from treated animals, suggesting that Niclosamide, NEN, and Rottlerin were effective in causing drug-induced autophagy in these filarial worms. These repurposed drugs provide a new avenue for the clearance of adult worms in filarial infections.

High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics.

Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics. Here we report a high-throughput screening workflow and apply it to screen 61 eukaryotic membrane protein targets. For each target, we tested its expression in lipidic mimetics: two detergents, two liposomes, and two nanodiscs. We show that 35 membrane proteins (57%) can be expressed in a soluble fraction in at least one of the mimetics with the two detergents performing significantly better than nanodiscs and liposomes, in that order. Using the established cell-free workflow, we studied the production and biophysical assays for mitochondrial pyruvate carrier (MPC) complexes. Our studies show that the complexes produced in cell-free are functionally competent in complex formation and substrate binding. Our results highlight the utility of using cell-free systems for screening and production of eukaryotic membrane proteins.

Sporadic inclusion body myositis: clinical, pathological, and genetic analysis of eight Polish patients.

INTRODUCTION: Sporadic inclusion body myositis (sIBM) is one of the most common myopathies in patients above 50 years of age. Its progressive course finally leads to immobilisation, and no effective therapy exists. Its pathogenesis includes both degenerative and inflammatory processes, however, its direct causes remain unknown. Therefore, a possible genetic background of the disease must also be considered. MATERIAL AND METHODS: Here we report on twelve patients: eight with sporadic inclusion body myositis and four with other myopathies with rimmed vacuoles in muscle biopsy. All patients were evaluated clinically, morphologically, radiologically, and genetically. RESULTS: All patients with sIBM presented both shoulder and pelvic girdle muscle involvement. In addition, distal upper and lower limb muscle weakness was noted. Patients with other muscle disorders showed effects mainly in proximal muscles and marked calf muscle hypertrophy. In sIBM cases computed tomography of lower limb muscles revealed atrophy that was most pronounced within the quadriceps femoris and gracilis muscles in the thighs and within the medial head of the gastrocnemius muscle and the tibialis anterior muscle in the lower legs. On light microscopy mononuclear cell invasion of muscle fibres was present in six patients with sIBM. On electron microscopy myofibrillar disorganisation and mitochondrial abnormalities were noted in all sIBM patients, whereas cytoplasmic tubulofilamentous inclusions were seen in three patients and both cytoplasmic and nuclear inclusions in one of them. According to the criteria by Rose et al. (2011) six patients were classified as "clinico-pathologically defined IBM", one as "clinically defined IBM", and one as "probable IBM". Pathological deposits of TDP-43 were found in muscles in all sIBM as well as in control cases. Additionally, accumulation of other proteins thought to be associated with sIBM, like ß-amyloid, -synuclein, and tau protein, was present in the most of examined biopsies. All twelve patients were screened for the presence of causative mutations in TARDBP, VCP, HNRNPA1, and HNRNPA2B1 genes. Additionally, analysis of C9ORF72 hexanucleotide repeat expansion was performed. No causative mutations were found in any of the patients. CONCLUSIONS: Our study provides the first - to our knowledge - comprehensive clinical, pathological, and genetic workup of a group of Polish patients.

The N-terminal pro-brain natriuretic peptide as a marker of mitoxantrone-induced cardiotoxicity in multiple sclerosis patients.

BACKGROUND AND PURPOSE: Mitoxantrone (MTX) has been shown to reduce progression of disability and number of clinical exacerbations in patients with progressive multiple sclerosis (MS). Prolonged administration of MTX, however, is limited by the risk of cardiotoxicity. Cardiac monitoring in MTX-treated patients includes usually measurement of left ventricular ejection fraction (LVEF) by means of echocardiography. The N-terminal pro-brain natriuretic peptide (NT-proBNP) represents a novel diagnostic tool in the assessment of heart failure. This study was aimed to evaluate the usefulness of NT-proBNP for early detection of MTX-induced cardiotoxicity in MS patients. MATERIALS AND METHODS: We measured the NT-proBNP plasma levels in 45 MS patients who completed 24-month MTX therapy and in 37 MS patients of control group. RESULTS: The median NT-proBNP plasma value was 15.12pg/mL. In 12 MTX-treated patients (27%), NT-proBNP plasma values were elevated, though this subgroup of patients neither clinical showed evidence of myocardial damage nor had the LVEF value <50%. In five patients with normal NT-proBNP, we observed LVEF decline >10%. We did not observe correlations between the NT-proBNP levels and patient age, MS duration, relapses index, Extended Disability Status Scale (EDSS), MTX single dose and the total cumulative dose of MTX. In 8 patients (22%) from control group, NT-proBNP plasma levels were also elevated. CONCLUSIONS: The results of our study confirm that MTX therapy is safe for carefully selected and closely monitored MS patients. We believe that serial evaluation of NT-proBNP levels (before, during and after MTX therapy) can identify MS patients at high risk for MTX-induced cardiotoxicity.