Kalpana Pai

In vitro NADH-oxidase, NADPH-oxidase and myeloperoxidase activity of macrophages after Tinospora cordifolia (guduchi) treatment

It is believed that the enhanced microbicidal and tumoricidal capability of activated macrophages is related to the remarkable increase in the production of oxygen metabolites. Both the production of H2O2 and the oxidation of NAD(P)H are directly dependent upon NAD(P)H-oxidase. It has been established that the respiratory burst is due to activation of NAD(P)H-oxidase localised in the plasmalemma. Myeloperoxidase is believed to be involved in augmenting the cytotoxic activity of H2O2. It was observed that the macrophage cell line J774A.1 when treated with Tinospora cordifolia (guduchi) and LPS showed enhanced NADH-oxidase, NADPH-oxidase and myeloperoxidase production as compared to macrophages treated with medium alone. The direct drug treatment to J774A cells showed activation as assessed by biochemical assays. These results suggest that high NADH-oxidase, NADPH-oxidase and myeloperoxidase activities may account for tumoricidal and microbicidal properties via macrophage activation.

A novel protein coding potential of long intergenic non-coding RNAs (lincRNAs) in the kinetoplastid protozoan parasite Leishmania major

Cutaneous leishmaniasis (CL) is caused by a kinetoplastid protozoan parasite Leishmania major, as a skin ulcer at the site of the sandfly bite. CL is curable and in most cases ulcers heal spontaneously within three to six months leaving a scar and disfiguration. Complete genome of L. major was reported in 2005 at the very initial phase of kinetoplastid parasite genome sequencing project. Presently, L. major genome is most studied and comprehensively annotated genome and therefore, it is being used as a reference genome for annotating recently sequenced Leishmanial genomes. A recent study reporting global transcriptome of L. major promastigotes, identified 1884 uniquely expressed non-coding RNAs (ncRNA) in L. major. In the current study, an in-depth analysis of the 1884 novel ncRNAs was carried out using a proteogenomic approach to identify their protein coding potential. Our analysis resulted in identification of eight novel protein coding genes based on mass spectrometry data. We have analyzed each of these eight novel CDS and in the process have improved the genome annotation of L. major on the basis of mass spectrometry derived peptide data. Although sequenced a decade ago, the improvement in the L. major genome annotation thus is an ongoing process.

Hatching phenology, life history and population dynamics of the Oriental clam shrimp Eulimnadia indocylindrova Durga Prasad and Simhachalam with notes on phenology patterns in the Spinicaudata

ABSTRACT We studied the natural history, hatching phenology and egg bank composition of the Oriental spinicaudatan clam shrimp Eulimnadia indocylindrova Durga Prasad and Simhachalam using both field studies and ex situ sediment rehydration. Field observations revealed that hatching began very early (1–2 days) after inundation, and continued for about 5 days. Mature adults could be observed by 10 days, and they survived up to 16 days. The population showed a largely hermaphrodite-biased sex ratio (male:hermaphrodite 1:3) observed over three years, with a decrease in number of males throughout the hydroperiod. Both amphigenic and monogenic hermaphrodites were observed. The total lifetime fecundity recorded was about 300 eggs laid in multiple clutches. The egg bank composition showed a high proportion of intact eggs, indicative of predictable hydrations and low sediment adversity. Hatching began on the first day post-inundation for all successive cyclical hydration treatments, with peak hatching on days 2 and 3. Hatching rate was highest (57% of total hatching in successive cycles) for the first hydration, decreasing subsequently for the further hydrations. Hatching duration decreased with successive hydrations and was the longest (around 7 days) for the first hydration. Maximum hatching (93%) occurred in the first 10 days for the continuous hydration treatment. Overall, the total emergence of nauplii in successive hydrations was larger than that observed for the continuous hydration treatment, indicative of a risk-spreading strategy across hydroperiods. Early and concentrated naupliar emergence, along with decreased hatching durations for successive cycles, was observed for all the hydrations. A survey of literature revealed a general lack of data on hatching phenology of clam shrimps, particularly from tropical and sub-tropical regions. Based on the available data, it appears that hatching patterns, particularly high, early hatching fractions, are commonly observed in Spinicaudata species, and do not seem to differ much across biogeographical regions.

A proteomic map of the unsequenced kala-azar vector Phlebotomus papatasi using cell line

The debilitating disease kala-azar or visceral leishmaniasis is caused by the kinetoplastid protozoan parasite Leishmania donovani. The parasite is transmitted by the hematophagous sand fly vector of the genus Phlebotomus in the old world and Lutzomyia in the new world. The predominant Phlebotomine species associated with the transmission of kala-azar are Phlebotomus papatasi and Phlebotomus argentipes. Understanding the molecular interaction of the sand fly and Leishmania, during the development of parasite within the sand fly gut is crucial to the understanding of the parasite life cycle. The complete genome sequences of sand flies (Phlebotomus and Lutzomyia) are currently not available and this hinders identification of proteins in the sand fly vector. The current study utilizes a three frame translated transcriptomic data of P. papatasi in the absence of genomic sequences to analyze the mass spectrometry data of P. papatasi cell line using a proteogenomic approach. Additionally, we have carried out the proteogenomic analysis of P. papatasi by comparative homology-based searches using related sequenced dipteran protein data. This study resulted in the identification of 1313 proteins from P. papatasi based on homology. Our study demonstrates the power of proteogenomic approaches in mapping the proteomes of unsequenced organisms.

Studies on the arginase, 5'-nucleotidase and lysozyme activity by monocytes from visceral leishmaniasis patients.

Intracellular pathogenic protozoan infection like visceral leishmaniasis is considered in terms of the overall inflammatory response and the complex cellular interactions leading to formation of the activated macrophage. Analysis of the development of activation is facilitated when operationally defined stage of activation are characterized using a library of objective markers. There is a role of arginase in the immune response supporting its involvement in macrophage effector mechanism in vitro and in vivo. 5′-Nucleotidase a plasma membrane component has been cited as a biochemical correlate of macrophage function in an altered morphological and biochemical state of activation and stimulation. Depression in 5′-nucleotidase activity has been generally referred to as a characteristic marker of activated macrophages. Lysozyme or lysosomal enzymes are released into the endocytic or autophagic vacuole macrophage where they serve the purpose of intracellular digestion of engulfed or segregated materials. In the present study, we have studied levels of arginase and 5′-nucleotidase (marker for macrophage activation) in monocytes of active VL patients and healthy controls. Lysozyme a secretary product of macrophages was also measured in supernatants collected from monocytes of active VL patients and healthy controls. Elevated levels of 5′-nucleotidase were observed in supernatants of monocytes from active VL patients as compared to healthy controls. Low levels of arginase and lysozyme production by monocytes isolated from VL patients were observed as compared to healthy controls. Our studies suggest that low levels of arginase and elevated 5′-nucleotidase activity could be one of the mechanisms in the pathology of VL infection. Low lysozyme activity in patients may account for persistence of Leishmania parasites in VL infections.

Preparation and characterization of microencapsulated DwPT trivalent vaccine using water soluble chitosan and its in-vitro and in-vivo immunological properties

The paper explained the microencapsulation of three different antigenic materials viz. Diphtheria toxoid (DT), whole cell pertussis antigens (PT and FHA) and tetanus toxoid (TT) by coacervation method using water soluble chitosan as a polymer crosslinked by vanillin/TPP co-crosslinkers for the development of oral trivalent DwPT vaccine. Instrumental characterization of chitosan microspheres suggested specific interaction with vanillin/TPP, higher thermal stability, amorphous nature, spherical morphology with size less than 2μm along with positive charge density offering mucoadhesive properties. Furthermore, PT and FHA showed higher encapsulation up to 94% followed by TT and DT. Cumulative release rate of DT was (68.47%), TT (73.67%), PT (43%) and FHA (53%). Release kinetics interpreted using DD solver program, indicated protein release followed first order kinetics and obeyed Korsmeyer-peppas model, stating fickian diffusion relates to diffusion, erosion and controlled release rate of the encapsulated toxoids. Application of formulations on caco-2 cell line showed negligible cytotoxic effect and efficient uptake of FITC labelled microspheres. The obtained in-vivo results suggests that the final trivalent DwPT formulation were having successful elicitation of both systemic (IgG) and mucosal (sIgA) immune response in balb/c mice. Overall studies indicated that DwPT formulation could be a suitable alternative to available injectable DaPT vaccine.

Involvement of tyrosine-specific protein kinase and protein kinase C in J774A.1 macrophage functions activated by Tinospora cordifolia

Background Macrophages are the first line of defense and constitute important participant in the bi-directional interaction between innate and specific immunity. Macrophages are in a quiescent form and get activated when given a stimulus. In our previous studies we have reported that guduchi or LPS treatment of macrophages enhanced production of nitric oxide (NO) and increased tumoricidal activity against L929 fibroblast cells. Objective In the present study effect of Tinospora cordifolia commonly known as guduchi on macrophage activation and the mechanism of action i.e. involvement of protein kinase C inhibitor and tyrosine-specific protein kinase inhibitor was investigated. Materials and Methods The present study was undertaken to determine whether H-7 (inhibitor of protein kinase C) and/or genistein (inhibitor of tyrosine-specific protein kinase) could inhibit guduchi or LPS-induced macrophage NO and TNF-α production or reduce the cytolysis of L929 fibroblast cells. Results It was observed that in vitro incubation with H-7 and/or genistein completely inhibited guduchi or LPS-induced NO and TNF-α production by macrophages (J774A.1). Conclusion The inhibitory effects of H-7 and/or genistein, suggest that phosphorylation via these kinases may upregulate the NO synthase activity in macrophages.

Role of Cytokines in the Pathogenesis of Visceral Leishmaniasis

Infection in humans with Leishmania manifests into a spectrum of diseases. The manifestations of the disease depend on the resultant evasion of the parasite to immune responses namely macrophages, which is an exclusive host of leishmania. The B cells valiantly mount antibody responses, however to no avail as the Leishmania parasites occupy the intracellular niches of the macrophages. Extensive studies have been documented on the role of cell-mediated immunity (CMI) in protection and counter survival strategies of the parasites leading to down-regulation of CMI. The present review attempts to discuss the cytokines in progression or resolution of visceral form of leishmaniasis or kala-azar, predominantly affecting the Indian subcontinent. The components/cytokine(s) responsible for the regulation of the critical balance of Th1/Th2/Th9/Th17/Treg cells has been discussed in the perspective. Therefore, any strategy involving the treatment of VL needs to consider the balance and regulation of CD4+ T cell function.