Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As₂O₃) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.     

Effects of X-rays and Colchicine on Pollen of Trichosanthes anguina L. (Cucurbitaceae)

Pollen grains of M, and C, generation plants of Trichosanthes anguina L. were studied after treatment of seeds with X-rays and colchicine respectively. Pollen sterility increased with increase in X-ray dose and colchicine concentration. The polar axis of X-irradiated populations was shorter than that of controls. But colchicine treatment resulted in larger pollen grains than the controls, although the exine did not increase as much. Ex-perimentally-produced tetraploids had larger pollen grains than diploids and triploids. The triploids were characterised by changes in pore morphology. Of 293 treated plants examined, 11 aberrant plants were isolated in the M, and C, generations. These plants had large and small fertile pollen grains, and also showed treatment effects in external morphology and chromosomal aberration during meiosis. The changes in polar diameter were not associated with variation in chromosomal material.     

Hypoparathyroidism-retardation-dysmorphism syndrome

Congenital hypoparathyroidism, growth retardation and facial dysmorphism is a rare autosomal recessive disorder seen among children born to consanguineous couple of Arab ethnicity. This syndrome is commonly known as Sanjad-Sakati or hypoparathyroidism-retardation-dysmorphism syndrome (HRD). We report 13-year-old Hindu boy with hypoparathyroidism, tetany, facial dysmorphism and developmental delay, compatible with HRD syndrome.     

Study of Surface Enhanced Raman Scattering of Single Molecule Adsorbed on the Surface of Metal Nanogeometries: Electrostatic Approach

Plasmonics - In this paper, we have studied the surface enhanced raman scattering (SERS) from a molecule adsorbed on coated and non-coated spherical shape metallic nanoparticles. We have accounted...     

Optofluidic single‐cell absorption flow analyzer for point‐of‐care diagnosis of malaria

In this work, an optofluidic flow analyzer, which can be used to perform malaria diagnosis at the point‐of‐care is demonstrated. The presented technique is based on quantitative optical absorption measurements carried out on a single cell level for a given population of Human Red Blood Cells (RBCs). By measuring the optical absorption of each RBC, the decrease in the Hemoglobin (Hb) concentration in the cytoplasm of the cell due to the invasion of malarial parasite is detected. Cells are assessed on a single cell basis, as they pass through a microfluidic channel. The proposed technique has been implemented with inexpensive off‐the‐shelf components like laser diode, photo‐detector and a micro‐controller. The ability of the optofluidic flow analyzer to asses about 308,049 cells within 3 minutes has been demonstrated. The presented technique is capable of detecting very low parasitemia levels with high sensitivity.

     

Contrasting Roles of Islet Resident Immunoregulatory Macrophages and Dendritic Cells in Experimental Autoimmune Type 1 Diabetes

The innate immune system critically shapes diabetogenic adaptive immunity during type 1 diabetes (T1D) pathogenesis. While the role of tissue-infiltrating monocyte-derived macrophages in T1D is well established, the role of their tissue-resident counterparts remains undefined. We now demonstrate that islet resident macrophages (IRMs) from non-autoimmune mice have an immunoregulatory phenotype and powerfully induce FoxP3+ Tregs in vitro. The immunoregulatory phenotype and function of IRMs is compromised by TLR4 activation in vitro. Moreover, as T1D approaches in NOD mice, the immunoregulatory phenotype of IRMs is diminished as is their relative abundance compared to immunostimulatory DCs. Our findings suggest that maintenance of IRM abundance and their immunoregulatory phenotype may constitute a novel therapeutic strategy to prevent and/or cure T1D.     

Induction of IL-10 and TGFβ from CD4+CD25+FoxP3+ T Cells Correlates with Parasite Load in Indian Kala-azar Patients Infected with Leishmania donovani

Background

Visceral leishmaniasis (VL) is distinguished by a complex interplay of immune response and parasite multiplication inside host cells. However, the direct association between different immunological correlates and parasite numbers remains largely unknown.

Methodology/Principal Findings

We examined the plasma levels of different disease promoting/protective as well as Th17 cytokines and found IL-10, TGFβ and IL-17 to be significantly correlated with parasite load in VL patients (r = 0.52, 0.53 and 0.51 for IL-10, TGFβ and IL-17, respectively). We then extended our investigation to a more antigen-specific response and found leishmanial antigen stimulated levels of both IL-10 and TGFβ to be significantly associated with parasite load (r = 0.71 and 0.72 for IL-10 and TGFβ respectively). In addition to cytokines we also looked for different cellular subtypes that could contribute to cytokine secretion and parasite persistence. Our observations manifested an association between different Treg cell markers and disease progression as absolute numbers of CD4⁺CD25⁺ (r = 0.55), CD4⁺CD25^hi (r = 0.61) as well as percentages of CD4⁺CD25⁺FoxP3⁺ T cells (r = 0.68) all correlated with parasite load. Encouraged by these results, we investigated a link between these immunological components and interestingly found both CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ Treg cells to secrete significantly (p<0.05) higher amounts of not only IL-10 but also TGFβ in comparison to corresponding CD25^- T cells.

Conclusions/Significance

Our findings shed some light on source(s) of TGFβ and suggest an association between these disease promoting cytokines and Treg cells with parasite load during active disease. Moreover, the direct evidence of CD4⁺CD25⁺FoxP3⁺ Treg cells as a source of IL-10 and TGFβ during active VL could open new avenues for immunotherapy towards cure of this potentially fatal disease.     

Postprandial Hypertriglyceridemia Predicts Development of Insulin Resistance Glucose Intolerance and Type 2 Diabetes

Insulin resistance (IR) and type 2 diabetes mellitus (T2DM) have been found to be associated with postprandial hypertriglyceridemia (PPHTg). However, whether PPHTg can cause IR and diabetes is not clear. We therefore investigated the role of PPHTg in development of T2DM in rat model of T2DM. 96 male Wistar rats were randomized into four groups (24 rats each). Control Group A, high sucrose diet (HSD) Group B, HSD+Pioglitazone (10mg/kg/day) Group C and HSD+Atorvastatin (20mg/kg/day) Group D. Fat and glucose tolerance tests were done at regular intervals in all groups besides insulin and body weight measurement. At 26 weeks, low dose streptozotocin (15mg/kg,i.p.) was given to half of the rats. All rats were followed up till 48 weeks. PPHTg developed as early as week 2 in Group B and stabilized by week 14. Group B displayed highest PPHTg compared to other groups. Atorvastatin treatment (Group D) abolished PPHTg which became comparable to controls, pioglitazone treatment partially blunted PPHTg resulting in intermediate PPHTg. Group B with highest PPHTg showed highest subsequent IR, glucose intolerance (GI) and highest incidence of prediabetes at week 26 and diabetes at week 34 and 46 compared to other groups. Group D rats displayed lower IR, GI, low incidence of prediabetes and diabetes at these time points compared to Groups B and C. ROC analysis showed that triglyceride area under the curve of each time point significantly predicts the risk of diabetes. Present study provides the evidence that PPHTg predicts the development of IR, GI and T2DM in rat model of diet induced T2DM.