Oral Therapy Using a Combination of Nanotized Antimalarials and Immunomodulatory Molecules Reduces Inflammation and Prevents Parasite Induced Pathology in the Brain and Spleen of P. berghei ANKA Infected C57BL/6 Mice.

In malaria, anti-parasite immune response of the host may lead to dysregulated inflammation causing severe neuropathology arising from extensive damage to the Blood Brain Barrier (BBB). Use of anti-malarial drugs alone can control parasitemia and reduce inflammation but it cannot reduce pathology if chronic inflammation has already set in. In the present study, we have tested the efficacy of a new oral artemsinin based combination therapy (ACT) regimen using a combination of anti-malarial compounds like nanoartemisinin and nanoallylated-chalcone9 [{1-(4-Chlorophenyl)-3-[3-methoxy-4-(prop-2-en-1-yloxy) phenyl]-prop-2-en-1-one}]given together with anti-inflammatory-cum- anti-malarial compounds like nanoandrographolide and nanocurcumin to C57BL/6 mice infected with P. berghei ANKA. Untreated infected mice developed Experimental Cerebral Malaria (ECM) and died between 10 to 12 days after infection from severe BBB damage. We observed that oral treatments with nanoartemisinin or nano allylated chalcone 9 or nanoandrographolide alone, for 4 days after the onset of ECM, delayed the development of severe neurolopathology but could not prevent it. Nanocurcumin treatment for 4 days on the other hand, prevented damage to the BBB but the mice died because of hyperparasitemia. A single time oral administration of our ACT controlled blood parasitemia and prevented damage to the BBB, but recrudescence occurred due to persistence of parasites in the spleen. However the recrudescent parasites failed to induce ECM and BBB damage, leading to prolonged survival of the animals. A second time treatment at the start of recrudescence led to complete parasite clearance and survival of mice without pathology or parasitemia for 90 days. FACS analysis of spleen cells and gene expression profile in brain and spleen as well as quantitation of serum cytokine by ELISA showed that P. berghei ANKA infection in C57Bl/6 mice leads to a Th1-skewed immune response that result in severe inflammation and early death from ECM. Oral treatment with our ACT prevented a heightened pro-inflammatory response by modulating the Th1, Th2 and Treg immune responses and prevented ECM and death.

Differential Gene Expression in Brain and Liver Tissue of Wistar Rats after Rapid Eye Movement Sleep Deprivation.

Sleep is essential for the survival of most living beings. Numerous researchers have identified a series of genes that are thought to regulate "sleep-state" or the "deprived state". As sleep has a significant effect on physiology, we believe that lack of total sleep, or particularly rapid eye movement (REM) sleep, for a prolonged period would have a profound impact on various body tissues. Therefore, using the microarray method, we sought to determine which genes and processes are affected in the brain and liver of rats following nine days of REM sleep deprivation. Our findings showed that REM sleep deprivation affected a total of 652 genes in the brain and 426 genes in the liver. Only 23 genes were affected commonly, 10 oppositely, and 13 similarly across brain and liver tissue. Our results suggest that nine-day REM sleep deprivation differentially affects genes and processes in the brain and liver of rats.

Andrographolide binds to ATP-binding pocket of VEGFR2 to impede VEGFA-mediated tumor-angiogenesis.

Vasculogenesis and angiogenesis are process of formation of blood vessels. Blood vessels are evolved to distribute nutrients and oxygen to distant organs. These vessels are crucial for growth and repair of wounded tissue. During tumor condition there occurs imbalance in the growth of blood vessels which leads to neo-angiogenesis. Neo-angiogenesis is major perpetrator behind the establishment of tumor. Tumor cells secrete pro-angiogenic factor VEGFA which binds to VEGFR2 present over surface of endothelial cells and triggers formation of new blood vessels. To inhibit tumor-angiogenesis, a physiologically-safe small molecule inhibitor was screened which can potentially interact with kinase domain of VEGFR2 and inhibit its activity. Molecular-docking module and biochemical analysis identified andrographolide as one of the best docking molecules that binds to ATP-binding pocket of VEGFR2 and inhibits its kinase activity. Thus, for a more radical approach towards safe VEGFR2 inhibitor, andrographolide was repurposed to inhibit tumor-angiogenesis and reduce tumor burden.

Rapid Eye Movement sleep deprivation of rat generates ROS in the hepatocytes and makes them more susceptible to oxidative stress.

BACKGROUND: Rapid Eye Movement sleep deprivation (REMSD) of rats causes inflammation of the liver and apoptotic cell death of neurons and hepatocytes. Studies also suggest that REM sleep deprivation can cause muscle as well as cardiac injury and neurodegenerative diseases. OBJECTIVE AND METHODS: The aim of this research was to determine whether REM sleep deprivation of rats would increase the levels of reactive oxygen species (ROS) in the hepatocytes and create oxidative stress in them. We selectively deprived the rats for REM sleep using the standard flower pot method. RESULTS: We observed that when rats were subjected to REM sleep deprivation, the levels of ROS in their hepatocytes increased ~184.33% compared to large platform control (LPC) group by day 9 of deprivation, but it returned towards normal level (~49.27%) after recovery sleep for 5 days. Nitric oxide synthase (iNOS) gene expression and protein levels as determined by real-time PCR and western blot analysis respectively were found to be elevated in hepatocytes of REM sleep deprived rats as compared to the LPC group. The level of nitric oxide (NO) in the hepatocytes of REMSD rats also increased by ~404.40% as compared to the LPC group but sleep recovery for 5 days normalized the effect (~135.35% compared to LPC group). We used a large platform control group as a reference group to compare with the REM sleep deprived group as the effect on the hepatocytes of both LPC group and cage control groups were not significantly different. DISCUSSION: We have analyzed the oxidative stress generated in the hepatocytes of rats due to REM sleep deprivation and further consequences of it. REMS deprivation not only increased the levels of ROS in the hepatocytes but also induced iNOS and NO in them. REM sleep deprived hepatocytes became more susceptible to oxidative stresses on further exposures. Furthermore, our study has great pathological and physiological.

Nanoparticle-Formulated Curcumin Prevents Posttherapeutic Disease Reactivation and Reinfection with Mycobacterium tuberculosis following Isoniazid Therapy.

Curcumin, the bioactive component of turmeric also known as "Indian Yellow Gold," exhibits therapeutic efficacy against several chronic inflammatory and infectious diseases. Even though considered as a wonder drug pertaining to a myriad of reported benefits, the translational potential of curcumin is limited by its low systemic bioavailability due to its poor intestinal absorption, rapid metabolism, and rapid systemic elimination. Therefore, the translational potential of this compound is specifically challenged by bioavailability issues, and several laboratories are making efforts to improve its bioavailability. We developed a simple one-step process to generate curcumin nanoparticles of ~200 nm in size, which yielded a fivefold enhanced bioavailability in mice over regular curcumin. Curcumin nanoparticles drastically reduced hepatotoxicity induced by antitubercular antibiotics during treatment in mice. Most interestingly, co-treatment of nanoparticle-formulated curcumin along with antitubercular antibiotics dramatically reduced the risk for disease reactivation and reinfection, which is the major shortfall of current antibiotic treatment adopted by Directly Observed Treatment Short-course. Furthermore, nanoparticle-formulated curcumin significantly reduced the time needed for antibiotic therapy to obtain sterile immunity, thereby reducing the possibility of generating drug-resistant variants of the organisms. Therefore, adjunct therapy of nano-formulated curcumin with enhanced bioavailability may be beneficial to treatment of tuberculosis and possibly other diseases.

Heightened inflammation in severe malaria is associated with decreased IL-10 expression levels and neutrophils.

Dysregulation of the cytokine network in severe malaria owing to variations in factors like parasite load, strains and host factors is well documented but the key cytokines that are dysregulated remain poorly elucidated. Longitudinal changes in cytokine levels in an individual with parasitemia and disease resolution is likely to identify the key cytokines. We have analyzed the mRNA expression of cytokines over a 7-d period in severe (SM) and uncomplicated (UM) Plasmodium falciparum malaria. We found up-regulated expression of TNF-α, IL-1ß, IFN-γ and TGF-ß in SM, with decreased expression of IL-10 on d 0. Further, we observed a negative correlation of IL-10 expression with parasitemia and pro-inflammatory cytokines, suggesting IL-10 to be the key cytokine in tilting the balance to an inflammatory response. Longitudinal analysis revealed that the key cytokines associated with disease were TNF-α, IL-1ß, IFN-γ, IL-12α, RANTES and TGF-ß, while TNF-α, IL-10 and TGF-ß discriminated between SM and UM. A higher neutrophil count in SM and its positive association with parasite density and IL-1ß and IL-8 provides support for neutrophils in inflammation in malaria. Our findings suggest subversion of anti-inflammatory response in SM by parasite factors towards an exaggerated pro-inflammatory response with involvement of neutrophils, the classical inflammatory cells.

MEK inhibition prevents tumour-shed transforming growth factor-ß-induced T-regulatory cell augmentation in tumour milieu.

Tumour progression is associated with immune-suppressive conditions that facilitate the escape of tumour cells from the regimen of immune cells, subsequently paralysing the host defence mechanisms. Induction of CD4(+)  CD25(+)  FoxP3(+) T regulatory (Treg) cells has been implicated in the tumour immune escape mechanism, although the novel anti-cancer treatment strategies targeting Treg cells remain unknown. The focus of this study is to define the interaction between tumour and immune system, i.e. how immune tolerance starts and gradually leads to the induction of adaptive Treg cells in the tumour microenvironment. Our study identified hyperactivated mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) -signalling as a potential target for reversing Treg cell augmentation in breast cancer patients. In more mechanistic detail, pharmacological inhibitors of MEK/ERK signalling inhibited transforming growth factor-ß (TGF-ß) production in tumour cells that essentially blocked TGF-ß-SMAD3/SMAD4-mediated induction of CD25/interleukin-2 receptor α on CD4(+) T-cell surface. As a result high-affinity binding of interleukin-2 on those cells was prohibited, causing lack of Janus kinase 1 (JAK1)/JAK3-mediated signal transducer and activator of transcription 3 (STAT3)/STAT5 activation required for FoxP3 expression. Finally, for a more radical approach towards a safe MEK inhibitor, we validate the potential of multi-kinase inhibitor curcumin, especially the nano-curcumin made out of pure curcumin with greater bioavailability; in repealing tumour-shed TGF-ß-induced Treg cell augmentation.

Mutations in subunit interface and B-cell epitopes improve antileukemic activities of Escherichia coli asparaginase-II: evaluation of immunogenicity in mice.

L-Asparaginase-II from Escherichia coli (EcA) is a central component in the treatment of acute lymphoblastic leukemia (ALL). However, the therapeutic efficacy of EcA is limited due to immunogenicity and a short half-life in the patient. Here, we performed rational mutagenesis to obtain EcA variants with a potential to improve ALL treatment. Several variants, especially W66Y and Y176F, killed the ALL cells more efficiently than did wild-type EcA (WT-EcA), although nonleukemic peripheral blood monocytes were not affected. Several assays, including Western blotting, annexin-V/propidium iodide binding, comet, and micronuclei assays, showed that the reduction in viability of leukemic cells is due to the increase in caspase-3, cytochrome c release, poly(ADP-ribose) polymerase activation, down-regulation of anti-apoptotic protein Bcl-XL, an arrest of the cell cycle at the G0/G1 phase, and eventually apoptosis. Both W66Y and Y176F induced significantly more apoptosis in lymphocytes derived from ALL patients. In addition, Y176F and Y176S exhibited greatly decreased glutaminase activity, whereas K288S/Y176F, a variant mutated in one of the immunodominant epitopes, showed reduced antigenicity. Further in vivo immunogenicity studies in mice showed that K288S/Y176F was 10-fold less immunogenic as compared with WT-EcA. Moreover, sera obtained from WT-EcA immunized mice and ALL patients who were given asparaginase therapy for several weeks recognized the K288S/Y176F mutant significantly less than the WT-EcA. Further mechanistic studies revealed that W66Y, Y176F, and K288S/Y176F rapidly depleted asparagine and also down-regulated the transcription of asparagine synthetase as compared with WT-EcA. These highly desirable attributes of these variants could significantly advance asparaginase therapy of leukemia in the future.

Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency.

A non-toxic, direct-acting fibrinolytic serine protease (Bafibrinase) demonstrating thrombolytic and anticoagulant properties was purified from Bacillus sp. strain AS-S20-I. Bafibrinase was monomeric, with a molecular mass of 32.3 kDa. The peptide mass fingerprinting of Bafibrinase revealed only 8.3% sequence coverage, suggesting it was a novel fibrinolytic enzyme. However, two of the tryptic digested de novo peptide sequences of Bafibrinase demonstrated good similarity with endopeptidases possessing serine in their catalytic triad. Further, catalytic activity of Bafibrinase was inhibited by serine protease inhibitor reinforcing this is a subtilisin-like serine protease. The apparent K(m) and V(max) values of Bafibrinase towards fibrin were determined as 0.24 µM and 2.8 µmol/min, respectively. It showed a K(m) value of 0.139 mM towards a chromogenic substrate for plasmin (D-Val-Leu-Lys-p-Nitroanilide dihydrochloride) and optimum activity at physiological conditions (37 °C and pH 7.4). Based on the cleavage pattern of fibrin and fibrinogen, Bafibrinase may be classified as an α,ß-fibrinogenase. Bafibrinase could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, Bafibrinase at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug. Bafibrinase was also superior to human plasmin in degrading in vitro thrombus. The in vivo anticoagulant nature of Bafibrinase is being explored for the treatment and prevention of thrombosis and other cardiovascular diseases.

Brugia malayi adult low molecular weight IgG4-reactive antigens induce differential cytokine response in lymphocytes of endemic normal and asymptomatic microfilariae carriers in vitro.

To characterize putatively protective immune response in bancroftian filariasis, Th1/Th2 cytokine profile induced in peripheral blood mononuclear cells (PBMCs) of endemic normal (EN) and asymptomatic microfilaremic (ASM) individuals were studied using different molecular weight fractions of Brugia malayi adult soluble antigens (BmA), which are differentially recognized by IgG4 antibodies present in their sera. Infection free and putatively immune individuals living in a filaria endemic area were identified and included in the present study as EN only after careful longitudinal follow up for three years. It was observed that the low molecular weight antigens present in Fr4 and Fr5 induced differential cytokine response; EN individuals showed a strong Th1 bias whereas ASM individuals showed a strong Th2 bias even though both the groups produced Th1 cytokines, albeit of different quantity, when a nonhelminthic antigen like H37Rv whole cell lysate was used. Since antigens present in Fr5 induced a highly polarized response, they should be examined for their diagnostic potential in lymphatic filariasis.