A succinct medical safety: periodic safety update reports

In the rapidly developing era of advanced of medical sciences, pharmacovigilance plays an important role to put necessary brakes and help the medical fraternity to prescribe in a safe manner. Unanimously PSUR contributes in an essential way to regulate the drug marketing agencies to make sure that biological products which are marketed do not harm the general population by its adverse drug reactions. As we all know that the regulatory agencies like EMA, US FDA, CDSCO etc. are very stringent in approving the drugs, they have framed several rules and imposed strict timelines for reporting the necessary documents pertaining to drugs. Inevitably several new drugs like immunomodulators, anticancer drugs are on the raise which further adds a huge responsibility on the regulatory agencies regarding the risk and benefits of such biological products. Thus, the regulatory agencies have added several new implementation and amendments in the existing rules for the marketing of new drugs as well as already prevailing drugs. Hence, it is of paramount importance for the drug companies to follow the guidelines and abide by the rules of regulatory agencies for the benefit of mankind. However, several factors can impede the process of PSUR like improper data retrieval, inadequate training of resource personnel concerned with the development of PSUR. To overcome such difficulties, the field of advanced technology extended its arms for the regulatory authorities to implement electronic documentation and databases for the development of PSURs. In spite of all these efforts several challenges are to be met in the future in the field of pharmacovigilance. In this review several aspects of PSUR in different countries, necessary processes and format in which it needs to be submitted, pitfalls and ways to overcome such situations have been discussed in brief. Therefore, it is the collective responsibility of both Regulatory agencies and the drug companies to ensure a good quality PSUR is produced with its benefits outweighing the risk of the drug for the suffering human population.

Signal thresholds and modular synergy during expression of costimulatory molecules in B lymphocytes

We analyzed intracellular pathways modulating surface densities of CD80 and CD86 in B cells activated through ligation of the Ag receptor, and the adhesion molecule CD54. Whereas B cell Ag receptor (BCR) cross-linking alone stimulated increased expression of CD86, up-regulation of CD80 required dual stimulation with anti-IgM and anti-CD54. The principal downstream component contributed by BCR signaling, toward both CD80 and CD86 induction, was the elevated concentration of free cytoplasmic Ca2+, recruited by way of capacitative influx. This alone was sufficient to generate an increase in CD86 levels. However, CD80 enhancement required the concerted action of both intracellular Ca2+ concentration and CD54-initiated pathways. The nexus between anti-IgM and anti-CD54 stimulation, in the context of CD80 regulation, was identified to involve a self-propagating process of sequential synergy. The first step involved amplified accumulation of intracellular cAMP, as a result of cross-talk between BCR-mobilized Ca2+ and CD54-derived signals. This then facilitated a second synergistic interaction between Ca2+ and cAMP, culminating in CD80 expression. Our findings of distinct signal transducer requirements, with the added consequences of cross-talk, offers an explanation for variable modulation of costimulatory molecule expression in response to diverse physiological stimuli. Importantly, these results also reveal how concentration threshold barriers for recruitment of individual second messengers can be overcome by constructive convergence of signaling modules

Study on the functional outcome of fluoroscopically guided transforaminal epidural steroid injections in patients suffering from lumbar disc herniation

Background: Intervertebral disc herniation of the lumbar region is one of the common causes of acute low back ache and lower extremity pain. While multiple treatment modalities exist, the efficacy of the usage of a transforaminal steroid injection as a tool to either alleviate pain or delay surgery needs to be further evaluated. The aim of this study is to determine the functional outcome of patients suffering from lumbar disc herniation treated with fluroscopically-guided transforaminal epidural steroid injections.Methods: This is a prospective case study in which total of 43 patients were included in the study dating between August 2014 and July 2015. These patients were evaluated and identified with lumbar disc herniation, confirmed with a magnetic resonance imaging prior to the procedure. A pre-injection VAS score was taken. These patients were administered TFESI under fluoroscopic guidance using 2ml of 40mg of Methylprednisolone with 1 ml of 2% xylocaine. They were then evaluated during follow up at 2 weeks, 6 weeks, 12 weeks, and 6 months. Their pain outcome was evaluated using the VAS (visual analog scale) scores and functional outcome was evaluated using Oswestry disability index (ODI).Results: All patients showed significant improvement in the VAS score during their regular follow up when compared to their pre injection levels. Patient satisfaction was the high at 2 weeks post operatively slightly declining over time. 3 patients underwent surgery during the follow up period. The ODI scores also showed significant improvements when compared to the pre injection scores at all follow up periodsConclusions: TFESI provides significant short-term pain relief in patients suffering from a single level lumbar herniated disc and is a viable, effective short-term analgesic tool to address pain and may retard an early surgical intervention

Aedes aegypti lachesin protein binds to the domain III of envelop protein of Dengue virus-2 and inhibits viral replication.

Dengue virus (DENV) comprises of four serotypes (DENV-1 to -4) and is medically one of the most important arboviruses (arthropod-borne virus). DENV infection is a major human health burden and is transmitted between humans by the insect vector, Aedes aegypti. Ae. aegypti ingests DENV while feeding on infected humans, which traverses through its gut, haemolymph and salivary glands of the mosquito before being injected into a healthy human. During this process of transmission, DENV must interact with many proteins of the insect vector, which are important for its successful transmission. Our study focused on the identification and characterisation of interacting protein partners in Ae. aegypti to DENV. Since domain III (DIII) of envelope protein (E) is exposed on the virion surface and is involved in virus entry into various cells, we performed phage display library screening against domain III of the envelope protein (EDIII) of DENV-2. A peptide sequence showing similarity to lachesin protein was found interacting with EDIII. The lachesin protein was cloned, heterologously expressed, purified and used for in vitro interaction studies. Lachesin protein interacted with EDIII and also with DENV. Further, lachesin protein was localised in neuronal cells of different organs of Ae. aegypti by confocal microscopy. Blocking of lachesin protein in Ae. aegypti with anti-lachesin antibody resulted in a significant reduction in DENV replication

Protective Immunity to Mycobacterium tuberculosis Infection by Chemokine and Cytokine Conditioned CFP-10 Differentiated Dendritic Cells

BACKGROUND: Dendritic cells (DCs) play major roles in mediating immune responses to mycobacteria. A crucial aspect of this is the priming of T cells via chemokines and cytokines. In this study we investigated the roles of chemokines RANTES and IP-10 in regulating protective responses from Mycobacterium tuberculosis (M. tb) 10 kDa Culture Filtrate Protein-10 (CFP-10) differentiated DCs (CFP10-DCs). METHODS AND FINDINGS: Infection of CFP10-DCs with mycobacteria down-modulated RANTES and IP-10 levels. Pathway specific microarray analyses showed that in addition to RANTES and IP-10, mycobacteria infected CFP10-DCs showed reduced expression of many Th1 promoting chemokines and chemokine receptors. Importantly, T cells co-cultured with RANTES and IP-10 conditioned CFP10-DCs mediated killing of mycobacteria from infected macrophages. Similarly, T cells recruited by RANTES and IP-10 conditioned CFP10-DCs mediated significant killing of mycobacteria from infected macrophages. IFN-gamma treatment of CFP10-DCs restored RANTES and IP-10 levels and T cells activated by these DCs mediated significant killing of virulent M. tb inside macrophages. Adoptive transfer of either RANTES and IP-10 or IL-12 and IFN-gamma conditioned CFP10-DCs cleared an established M. tb infection in mice. The extent of clearance was similar to that obtained with drug treatment. CONCLUSIONS: These results indicate that chemokine and cytokine secretion by DCs differentiated by M. tb antigens such as CFP-10 play major roles in regulating protective immune responses at sites of infection

Voltage Gated Calcium Channels Negatively Regulate Protective Immunity to Mycobacterium tuberculosis

Mycobacterium tuberculosis modulates levels and activity of key intracellular second messengers to evade protective immune responses. Calcium release from voltage gated calcium channels (VGCC) regulates immune responses to pathogens. In this study, we investigated the roles of VGCC in regulating protective immunity to mycobacteria in vitro and in vivo. Inhibiting L-type or R-type VGCC in dendritic cells (DCs) either using antibodies or by siRNA increased calcium influx in an inositol 1,4,5-phosphate and calcium release calcium activated channel dependent mechanism that resulted in increased expression of genes favoring pro-inflammatory responses. Further, VGCC-blocked DCs activated T cells that in turn mediated killing of M. tuberculosis inside macrophages. Likewise, inhibiting VGCC in infected macrophages and PBMCs induced calcium influx, upregulated the expression of pro-inflammatory genes and resulted in enhanced killing of intracellular M. tuberculosis. Importantly, compared to healthy controls, PBMCs of tuberculosis patients expressed higher levels of both VGCC, which were significantly reduced following chemotherapy. Finally, blocking VGCC in vivo in M. tuberculosis infected mice using specific antibodies increased intracellular calcium and significantly reduced bacterial loads. These results indicate that L-type and R-type VGCC play a negative role in M. tuberculosis infection by regulating calcium mobilization in cells that determine protective immunity

Phenylalanine-Rich Peptides Potently Bind ESAT6, a Virulence Determinant of Mycobacterium tuberculosis, and Concurrently Affect the Pathogen's Growth

BACKGROUND:The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung. METHODOLOGY/PRINCIPAL FINDINGS:During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage. CONCLUSIONS/SIGNIFICANCE:While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide's binding to Esat6-as the latter is not an essential protein of M. tuberculosis-nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen

Characterization of Leishmania donovani Aquaporins Shows Presence of Subcellular Aquaporins Similar to Tonoplast Intrinsic Proteins of Plants

Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field