Manufacturing and preclinical toxicity of GLP grade gene deleted attenuated Leishmania donovani parasite vaccine.

Centrin1 gene deleted Leishmania donovani parasite (LdCen1-/-) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1-/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1-/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1-/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1-/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1-/-, there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1-/- manufactured under cGMP complaint conditions can be suitable for future clinical trials.

Interaction of novel proteins, centrin4 and protein of centriole in Leishmania parasite and their effects on the parasite growth.

Centrins are cytoskeletal proteins associated with the centrosomes or basal bodies in the eukaryotes. We previously reported the involvement of Centrin 1-3 proteins in cell division in the protozoan parasites Leishmania donovani and Trypanosoma brucei. Centrin4 and 5, unique to such parasites, had never been characterized in Leishmania parasite. In the current study, we addressed the function of centrin4 (LdCen4) in Leishmania. By dominant-negative study, the episomal expression of C-terminal truncated LdCen4 in the parasite reduced the parasite growth. LdCen4 double allele gene deletion by either homologous recombination or CRISPR-Cas9 was not successful in L. donovani. However, CRISPR-Cas9-based deletion of the homologous gene was possible in L. mexicana, which attenuated the parasite growth in vitro, but not ex vivo in the macrophages. LdCen4 also interacts with endogenous and overexpressed LdPOC protein, a homolog of centrin reacting human POC (protein of centriole) in a calcium sensitive manner. LdCen4 and LdPOC binding has also been confirmed through in silico analysis by protein structural docking and validated by co-immunoprecipitation. By immunofluorescence studies, we found that both the proteins share a common localization at the basal bodies. Thus, for the first time, this article describes novel centrin4 and its binding protein in the protozoan parasites.

Oxidative status and changes in the adenosine deaminase activity in experimental host infected with tropical liver fluke, Fasciola gigantica.

Fine tuning of the metabolic, physiological and immunological cues along with interplay between the biomolecules of the host and the parasite could be responsible for the successful establishment of parasitic infections. The present investigation was aimed at evaluating the oxidative status and the level of adenosine deaminase (ADA) in the serum and liver of rabbits experimentally infected with Fasciola gigantica. A significant increase in level of ROS, MDA and 4-HNE along with a decline in the SOD, CAT, GR and GST activity was evident in rabbits experimentally infected with Fasciola gigantica. However, there was an increase in the GPX activity in the sera of infected rabbits. The increased GPX activity and decreased GR activity would have resulted in the depletion of GSH, a key non-enzymatic antioxidant, in the infected animals. The level of GSSG was also found to be higher in the sera and liver tissues of the infected rabbits along with a decline in the GSH/GSSG ratio, indicating a high level of oxidative stress in the infected animals, which also showed a significant increase in the activity of the marker enzymes of liver pathology, AST and ALT. Further, a significant inhibition of the adenosine deaminase (ADA) activity in the infected rabbits was accompanied with the reduction in the level of pro-inflammatory cytokine, IL-6 while the anti-inflammatory cytokine, IL-4 level was significantly elevated. In conclusion, the F. gigantica induced significant oxidative stress as evident from the increased levels of ROS and lipid peroxidation along with the disruption of antioxidant and detoxification cascade ultimately lead to pathogenic and inflammatory responses in the experimental host. Whereas, the altered ADA activity could modulate the host's immune responses toward Th-2 type and would facilitate the successful establishment of flukes within their host, thus indicating that ADA could be exploited as a target for the development of novel anthelmintic drugs against fasciolosis.

Complicaciones diabéticas: progresos en el desarrollo del tratamiento / Diabetic complications: progress in the development of treatment

A pesar de los avances en el tratamiento de la diabetes, las complicaciones de la enfermedad a largo plazo, tales como la retinopatía, la nefropatía y la neuropatía, siguen siendo la mayor causa de morbilidad y mortalidad en los pacientes diabéticos. Los estudios clínicos prospectivos con insulinoterapia intensificada han establecido una clara correlación entre la hiperglucemiay el desarrollo de las complicaciones diabéticas estudios en animales han demostrado el vínculo entre el incremento del metabolismo de la glucosa inducido por la hiperglucemia diabética a través de la vía del sorbitol y el desarrollo de lascomplicaciones. Por otra parte, una mejor comprensión de los mecanismos patogénicos de la neuropatía diabética ha promovido la utilización de las mediciones electrofisiológicas como medio confiable de cuantificar la progresión de la neuropatía diabética y también ha redefinido metas realistas de tratamiento para las complicaciones diabéticas, como la detención o atenuación de los procesos de la enfermedad en vez de su reversión. Estudios clínicos recientes con insulinoterapiaintensificada e inhibidores de aldosareductasa, apoyan claramente estos nuevos objetivos de tratamiento. En estudios a largo plazo de 5 a 8 años, la insulinoterapia intensificada no revirtió los signos clínicos y los síntomas de la neuropatía diabética, pero si evitó su aparición en pacientes sin evidencia de neuropatía al comienzo del estudio.La insulinoterapia intensificada tampoco restauró la pérdida preexistente de la función,pero redujo la pérdida acelerada de esa función,característica de la neuropatía diabética

Complicaciones diabéticas: progresos en el desarrollo del tratamiento / Diabetic complications: progress in the development of treatment

A pesar de los avances en el tratamiento de la diabetes, las complicaciones de la enfermedad a largo plazo, tales como la retinopatía, la nefropatía y la neuropatía, siguen siendo la mayor causa de morbilidad y mortalidad en los pacientes diabéticos. Los estudios clínicos prospectivos con insulinoterapia intensificada han establecido una clara correlación entre la hiperglucemiay el desarrollo de las complicaciones diabéticas estudios en animales han demostrado el vínculo entre el incremento del metabolismo de la glucosa inducido por la hiperglucemia diabética a través de la vía del sorbitol y el desarrollo de lascomplicaciones. Por otra parte, una mejor comprensión de los mecanismos patogénicos de la neuropatía diabética ha promovido la utilización de las mediciones electrofisiológicas como medio confiable de cuantificar la progresión de la neuropatía diabética y también ha redefinido metas realistas de tratamiento para las complicaciones diabéticas, como la detención o atenuación de los procesos de la enfermedad en vez de su reversión. Estudios clínicos recientes con insulinoterapiaintensificada e inhibidores de aldosareductasa, apoyan claramente estos nuevos objetivos de tratamiento. En estudios a largo plazo de 5 a 8 años, la insulinoterapia intensificada no revirtió los signos clínicos y los síntomas de la neuropatía diabética, pero si evitó su aparición en pacientes sin evidencia de neuropatía al comienzo del estudio.La insulinoterapia intensificada tampoco restauró la pérdida preexistente de la función,pero redujo la pérdida acelerada de esa función,característica de la neuropatía diabética (AU)