Simultaneously targeting inflammatory response and parasite sequestration in brain to treat Experimental Cerebral Malaria

Malaria afflicts around 200 million people annually, with a mortality number close to 600,000. The mortality rate in Human Cerebral Malaria (HCM) is unacceptably high (15-20%), despite the availability of artemisinin-based therapy. An effective adjunct therapy is urgently needed. Experimental Cerebral Malaria (ECM) in mice manifests many of the neurological features of HCM. Migration of T cells and parasite-infected RBCs (pRBCs) into the brain are both necessary to precipitate the disease. We have been able to simultaneously target both these parameters of ECM. Curcumin alone was able to reverse all the parameters investigated in this study that govern inflammatory responses, CD8(+) T cell and pRBC sequestration into the brain and blood brain barrier (BBB) breakdown. But the animals eventually died of anemia due to parasite build-up in blood. However, arteether-curcumin (AC) combination therapy even after the onset of symptoms provided complete cure. AC treatment is a promising therapeutic option for HCM.

Fourteen gene GBM prognostic signature identifies association of immune response pathway and mesenchymal subtype with high risk group

Background: Recent research on glioblastoma (GBM) has focused on deducing gene signatures predicting prognosis. The present study evaluated the mRNA expression of selected genes and correlated with outcome to arrive at a prognostic gene signature. Methods: Patients with GBM (n = 123) were prospectively recruited, treated with a uniform protocol and followed up. Expression of 175 genes in GBM tissue was determined using qRT-PCR. A supervised principal component analysis followed by derivation of gene signature was performed. Independent validation of the signature was done using TCGA data. Gene Ontology and KEGG pathway analysis was carried out among patients from TCGA cohort. Results: A 14 gene signature was identified that predicted outcome in GBM. A weighted gene (WG) score was found to be an independent predictor of survival in multivariate analysis in the present cohort (HR = 2.507; B = 0.919; p < 0.001) and in TCGA cohort. Risk stratification by standardized WG score classified patients into low and high risk predicting survival both in our cohort (p = <0.001) and TCGA cohort (p = 0.001). Pathway analysis using the most differentially regulated genes (n = 76) between the low and high risk groups revealed association of activated inflammatory/immune response pathways and mesenchymal subtype in the high risk group. Conclusion: We have identified a 14 gene expression signature that can predict survival in GBM patients. A network analysis revealed activation of inflammatory response pathway specifically in high risk group. These findings may have implications in understanding of gliomagenesis, development of targeted therapies and selection of high risk cancer patients for alternate adjuvant therapies.

Evaluation of benzothiophene carboxamides as analgesics and anti-inflammatory agents

Nonsteroid anti-inflammatory drugs (NSAIDs) represent standard therapy for the alleviation of pain and inflammation. At present various classes of compounds have been reported as selective inhibitors of cyclooxygenase-2 (COX-2). However, they are associated with adverse side effects. To address these issues, we report here a new class of compounds that exhibit potent analgesic and anti-inflammatory response. Substituted bromo-benzothiophene carboxamides (4-11) were examined for their analgesic and anti-inflammatory properties. Our findings demonstrate that newly synthesized bromo-benzothiophene carboxamide derivatives 4, 6, and 8 attenuate nociception and inflammation at lower concentration than classical NSAIDs, such as ibuprofen. These compounds act by selectively inhibiting COX-2 and by disrupting the prostaglandin-E2-dependent positive feedback of COX-2 regulation, which was further substantiated by reduction in the levels of cytokines, chemokines, neutrophil accumulation, synthesis of prostaglandin-E2, expression of COX-2, and neutrophil activation at lower concentration than the classic NSAID ibuprofen. Toxicological study reveals that these compounds are well tolerated and metabolized to avoid any toxicity. Thus, these molecules represent a new class of analgesic and anti-inflammatory agents. (c) 2014 IUBMB Life, 66(3):201-211, 2014

Farnesyltransferase Inhibitors Reduce Ras Activation and Ameliorate Acetaminophen-Induced Liver Injury in Mice

Hepatotoxicity due to overdose of the analgesic and antipyretic acetaminophen (A-PAIP) is a major cause of liver failure in adults. To better understand the contributions of different signaling pathways, the expression and role of Ras activation was evaluated after oral dosing of mice with APAP (400-500 mg/kg). Ras-guanosine triphosphate (GTP) is induced early and in an oxidative stress-dependent manner. The functional role of Ras activation was studied by a single intraperitoneal injection of the neutral sphingomyelinase and farnesyltransferase inhibitor (FTI) manumycin A (I mg/kg), which lowers induction of Ras-GTP and serum amounts of alanine aminotransferase (ALT). APAP dosing decreases hepatic glutathione amounts, which are not affected by manumycin A treatment. However, APAP-induced activation of c-Jun N-terminal kinase, which plays an important role, is reduced by manumycin A. Also, APAP-induced mitochondrial reactive oxygen species are reduced by manumycin A at a later time point during liver injury. Importantly, the induction of genes involved in the inflammatory response (including iNos, gp91phox, and Fasl) and serum amounts of proinflammatory cytokines interferon-gamma (IFN gamma) and tumor necrosis factor alpha, which increase greatly with APAP challenge, are suppressed with manumycin A. The FTI ctivity of manumycin A is most likely involved in reducing APAP-induced liver injury, because a specific neutral sphingomyelinase inhibitor, GW4869 (I mg/kg), did not show any hepatoprotective effect. Notably, a structurally distinct FTI, gliotoxin (I mg/kg), also inhibits Ras activation and reduces serum amounts of ALT and IFN-gamma after APAP dosing. Finally, histological analysis confirmed the hepatoprotective effect f manumycin A and gliotoxin during APAP-induced liver damage. Conclusion: This study identifies a key role for Ras activation and demonstrates the therapeutic efficacy of FTIs during APAP-induced liver injury.

Alveolar macrophages as accessory cells for human gamma delta T cells activated by Mycobacterium tuberculosis.

Alveolar macrophages form the first line of defense against inhaled droplets containing Mycobacterium tuberculosis by controlling mycobacterial growth and regulating T cell responses. CD4+ and gamma delta T cells, two major T cell subsets activated by M. tuberculosis, require accessory cells for activation. However, the ability of alveolar macrophages to function as accessory cells for T cell activation remains controversial. We sought to determine the ability of alveolar macrophages to serve as accessory cells for resting (HLA-DR-, IL-2R-) and activated (HLA-DR+, IL-2R+) gamma delta T cells in response to M. tuberculosis and its Ag, and to compare accessory cell function for gamma delta T cells of alveolar macrophages and blood monocytes obtained from the same donor. Alveolar macrophages were found to serve as accessory cells for both resting and activated gamma delta T cells in response to M. tuberculosis Ag. At high alveolar macrophage to T cell ratios (> 3:1), however, expansion of resting gamma delta T cells was inhibited by alveolar macrophages. The inhibition of resting gamma delta T cells by alveolar macrophages was dose-dependent, required their presence during the first 24 h, and was partially overcome by IL-2. Alveolar macrophages did not inhibit activated gamma delta T cells even at high accessory cell to T cell ratios, and alveolar macrophages functioned as well as monocytes as accessory cells. Monocytes were not inhibitory for either resting or activated gamma delta T cells. These findings support the following model. In the normal alveolus the alveolar macrophage to T cell ratio is > or = 9:1, and therefore the threshold for resting gamma delta T cell activation is likely to be high. Once a nonspecific inflammatory response occurs, such as after invasion by M. tuberculosis, this ratio is altered, favoring gamma delta T cell activation by alveolar macrophages.

Cytochrome P4504f, a potential therapeutic target limiting neuroinflammation

Inflammatory processes are involved in the pathogenesis and/or progression of acute central nervous system (CNS) infection, traumatic brain injury and neurodegenerative disorders among others indicating the need for novel strategies to limit neuroinflammation. Eicosanoids including leukotrienes, particularly leukotriene B-4 (LTB4) are principle mediator(s) of inflammatory response, initiating and amplifying the generation of cytokines and chemokines. Cytochrome P450 (Cyp), a family of heme proteins mediate metabolism of xenobiotics and endogenous compounds, such as eicosanoids and leukotrienes. Cytochrome P4504F (Cyp4f) subfamily includes five functional enzymes in mouse. We cloned and expressed the mouse Cyp4f enzymes, assayed their relative expression in brain and examined their ability to hydroxylate the inflammatory cascade prompt LTB4 to its inactive 20-hydroxylated product. We then examined the role of Cyp4fs in regulating inflammatory response in vitro, in microglial cells and in vivo, in mouse brain using lipopolysacharide (LPS), as a model compound to generate inflammatory response. We demonstrate that mouse brain Cyp4fs are expressed ubiquitously in several cell types in the brain, including neurons and microglia, and modulate inflammatory response triggered by LPS, in vivo and in microglial cells, in vitro through metabolism of LTB4 to the inactive 20-hydroxy LTB4. Chemical inhibitor or shRNA to Cyp4fs enhance and inducer of Cyp4fs attenuates inflammatory response. Further, induction of Cyp4f expression lowers LTB4 levels and affords neuroprotection in microglial cells or mice exposed to LPS. Thus, catalytic activity of Cyp4fs is a novel target for modulating neuroinflammation through hydroxylation of LTB4. (C) 2011 Elsevier Inc. All rights reserved.

Cytochrome P450-mediated metabolism in brain: functional roles and their implications

Introduction: Cytochromes P450 (P450) and associated monooxygenases are a family of heme proteins involved in metabolism of endogenous compounds (arachidonic acid, eicosanoids and prostaglandins) as also xenobiotics including drugs and environmental chemicals. Liver is the major organ involved in P450-mediated metabolism and hepatic enzymes have been characterized. Extrahepatic organs, such as lung, kidney and brain have the capability for biotransformation through P450 enzymes. Brain, including human brain, expresses P450 enzymes that metabolize xenobiotics and endogenous compounds. Areas covered: An overview of P450-mediated metabolism in brain is presented focusing on distinct differences seen in expression of P450 enzymes, generation of unique P450 enzymes in brain through alternate splicing and their consequences in terms of metabolism of psychoactive drugs and inflammatory prompts, such as leukotrienes, thus modulating inflammatory response. Expert opinion: The brain possesses unique P450s that metabolize drugs and endogenous compounds through pathways that are markedly different from that seen in liver indicating that extrapolation directly from liver to brain is not appropriate. It is therefore necessary to characterize the unique brain P450s and their ability to metabolize xenobiotics and endogenous compounds to better understand the functions of this important class of enzymes in brain, especially human brain.

Absence of systemic toxicity in mouse model towards BaTiO3 nanoparticulate based eluate treatment

One of the existing issues in implant failure of orthopedic biomaterials is the toxicity induced by the fine particles released during long term use in vivo, leading to acute inflammatory response. In developing a new class of piezobiocomposite to mimic the integrated electrical and mechanical properties of bone, bone-mimicking physical properties as well as in vitro cytocompatibility properties have been achieved with spark plasma sintered hydroxyapatite (HA)-barium titanate (BaTiO3) composites. However, the presence of BaTiO3 remains a concern towards the potential toxicity effect. To address this issue, present work reports the first result to conclusively confirm the non-toxic effect of HA-BaTiO3 piezobiocomposite nanoparticulates, in vivo. Twenty BALB/c mice were intraarticularly injected at their right knee joints with different concentrations of HA-BaTiO3 composite of up to 25 mg/ml. The histopathological examination confirmed the absence of any trace of injected particles or any sign of inflammatory reaction in the vital organs, such as heart, spleen, kidney and liver at 7 days post-exposure period. Rather, the injected nanoparticulates were found to be agglomerated in the vicinity of the knee joint, surrounded by macrophages. Importantly, the absence of any systemic toxicity response in any of the vital organs in the treated mouse model, other than a mild local response at the site of delivery, was recorded. The serum biochemical analyses using proinflammatory cytokines (TNF-alpha and IL-1 beta) also complimented to the non-immunogenic response to injected particulates. Altogether, the absence of any inflammatory/ adverse reaction will open up myriad of opportunities for BaTiO3 based piezoelectric implantable devices in biomedical applications.

Flow Cytometry Analysis of Cytotoxicity In Vitro and Long-Term Toxicity of HA-40wt% BaTiO3 Nanoparticles In Vivo

The development of new implantable biomaterials requires bone-mimicking physical properties together with desired biocompatible property. In continuation to our earlier published research to establish compositional dependent multifunctional bone-like properties and cytocompatibility response of hydroxyapatite (HA)-BaTiO3 composites, the toxicological property evaluation, both invitro and invivo, were conducted on HA-40wt% BaTiO3 and reported in this work. In particular, this work reports invitro cytotoxicity of mouse myoblast cells as well as invivo long-term tissue and nanoparticles interaction of intra-articularly injected HA-40wt% BaTiO3 and BaTiO3 up to the concentration of 25mg/mL in physiological saline over 12weeks in mouse model. The careful analysis of flow cytometry results could not reveal any statistically significant difference in terms of early/late apoptotic cells or necrotic cells over 8d in culture. Extensive histological analysis could not record any signature of cellular level toxicity or pronounced inflammatory response in vital organs as well as at knee joints of Balb/c mice after 12weeks. Taken together, this study establishes nontoxic nature of HA-40wt% BaTiO3 and therefore, HA-40wt% BaTiO3 can be used safely for various biomedical applications.