Chemotypical variations in Withania somnifera lead to differentially modulated immune response in BALB/c mice

Withania somnifera (Ashwagandha) is a plant with known ethnomedicinal properties and its use in Ayurvedic medicine in India is well documented. The present investigation reports on immunomodulatory efficacy of aqueous-ethanol extracts of roots of three selected Withania somnifera chemotypes designated as NMITLI 101R, NMITLI 118R and NMITLI 128R. Each chemotype was administered 10-100 mg/kg orally to BALB/c mice once daily for 14 days. The immunomodulatory consequences were recorded by determining the humoral immune response with the help of hemagglutination, plaque forming cell assay and cellular response by measuring delayed type hypersensitivity reaction. Additionally, other immune parameters such as proliferation of T and B cells, intracellular and secreted Th1 and Th2 cytokines along with modulation in ROS production by peritoneal macrophages were monitored after feeding with lower doses (3-30 mg/kg/day) of these three chemotypes individually. NMITLI 101R incited both humoral and cellular immune response in terms of higher number of antibody producing cells and enhanced foot pad swelling at the 10 mg dose as also dose dependent B and T cell proliferations. Levels of intracellular and secreted cytokines post-NMITLI 101R treatment illustrated generation of mixed Th1/Th2 response that remained more polarized towards Th1. This chemotype also generated maximum reactive oxygen species. NMITLI 118R provoked comparatively reduced immune response in all humoral and cellular parameters at lower doses but induced highly polarized Th1 cytokine response. In contrast, NMITLI 128R led to enhanced antibody production with minimal cellular response demonstrating marginally Th2 dominance at a lower dose. Taken together, it may therefore be concluded that there were distinct modulation in the immune response exhibited by the three chemotypes of Withania somnifera and NMITLI 101R appeared to possess a better immunostimulatory activity than the other chemotypes at lower doses.     

Clinico‐pathological spectrum of gallbladder disease in children

Aim: Because of wide variation in clinico‐pathological spectrum of gallbladder disease in children the world over, the data of gallbladder disease from this stone belt of India were analysed. Methods: Children who underwent cholecystectomy over a period of 8 years January 2002–December 2009 were reviewed. Results: Out of 7076 cholecystectomies, 56 (0.79%) were in children. Thirty‐nine (69.6%) children were 11–16 years of age. Thirty‐seven (66.07%) children were girls and nineteen (33.9%) were boys. In 12 (21.4%) children, cholecystitis was acalculus. Five (8.9%) children had associated haemolytic disease and 4 (7.1%) children had congenital anomaly in the form of choledochal cyst. Ultrasound findings were available in 44 cases and showed cholelithiasis in 36 cases. Twenty‐two (39.3%) children had mixed cholelithiasis, 8 (14.2%) pigment cholelithiasis, 10 (17.8%) combined cholelithiasis and 4 (7.1%) patients had small concretions. Microscopically, changes of chronic cholecystitis were seen in 98.2% while 1.7% showed acute on chronic cholecystitis. There was single unusual case of cysticercus in the wall of the gallbladder. Conclusions: The frequency of gallstone disease is 0.79%. Nonhaemolytic type of cholelithiasis is more common than haemolytic type in this region. Presence of cysticercus in the gallbladder wall in one case was an unexpected finding.     

Novel RuvB nuclear ATPase is specific to intraerythrocytic mitosis during schizogony of Plasmodium falciparum

RuvB protein belongs to AAA+ family of enzymes involved in diverse cellular activities. In addition to the annotated two RuvB proteins in Plasmodium falciparum database, we report that a third RuvB protein is also present. The amino acid sequence analysis has revealed that P. falciparum RuvB3 (PfRuvB3) possesses Walker motif A, Walker motif B, sensor I and sensor II conserved motifs similar to yeast and human RuvB like proteins. The phylogenetic analysis revealed that PfRuvB3 is closely related to yeast RuvB like proteins which are essential for the survival of yeast. The biochemical characterization of recombinant PfRuvB3 confirms its ssDNA dependent ATPase activity. Using the truncated derivatives we show that Walker motif A is essential for the enzymatic activity of PfRuvB3. Using the immunodepletion assays we further show that the ATPase activity is attributable to PfRuvB3 protein. The endogenous P. falciparum RuvB3 contains the characteristic ATPase and some DNA helicase activities. The confocal microscopy analysis showed that this protein is mainly expressed during intraerythrocytic schizont stages of the parasite and is localized to the nuclear region. Once merozoite comes out from schizont, PfRuvB3 protein distinctly relocalized to the subnuclear region. The co-localization studies with a nucleolar marker PfNop1 further suggest that in P. falciparum RuvB3 localizes into a discrete nuclear compartment. On the basis of these studies it can be speculated that P. falciparum RuvB3 is most likely required for intraerythrocytic schizogony.     

Immunomodulator Effect of Picroliv and its Potential in Treatment Against Resistant Plasmodium yoelii (MDR) Infection in Mice

PURPOSE: The present study was envisaged to evaluate potential of combination therapy comprising of immunomodulator picroliv and antimalarial chloroquine against drug resistant Plasmodium yoelii (P. yoelii) infection in BALB/c mice. METHODS: The immunomodulatory potential of picroliv was established by immunizing animals with model antigen along with picroliv. Immune response was assessed using T-cell proliferation assay and also by determining the antibody isotype-profile induced in the immunized mice. In the next set of experiment, prophylactic potential of picroliv to strengthen antimalarial properties of chloroquine against P. yoelii (MDR) infection in BALB/c mice was assessed. RESULTS: T-cell proliferation as well as antibody production study reveals that picroliv helps in evoking strong immuno-potentiating response against model antigen in the immunized mice. Co-administration of picroliv enhances efficacy of CHQ against experimental murine malaria. CONCLUSION: The activation of host immune system can increase the efficacy of chloroquine for suppression of drug resistant malaria infection in BALB/c mice.     

BRCA1/Trp53 heterozygosity and replication stress drive esophageal cancer development in a mouse model

BRCA1 germline mutations are associated with an increased risk of breast and ovarian cancer. Recent findings of others suggest that BRCA1 mutation carriers also bear an increased risk of esophageal and gastric cancer. Here, we employ a Brca1/Trp53 mouse model to show that unresolved replication stress (RS) in BRCA1 heterozygous cells drives esophageal tumorigenesis in a model of the human equivalent. This model employs 4-nitroquinoline-1-oxide (4NQO) as an RS-inducing agent. Upon drinking 4NQO-containing water, Brca1 heterozygous mice formed squamous cell carcinomas of the distal esophagus and forestomach at a much higher frequency and speed (∼90 to 120 d) than did wild-type (WT) mice, which remained largely tumor free. Their esophageal tissue, but not that of WT control mice, revealed evidence of overt RS as reflected by intracellular CHK1 phosphorylation and 53BP1 staining. These Brca1 mutant tumors also revealed higher genome mutation rates than those of control animals; the mutational signature SBS4, which is associated with tobacco-induced tumorigenesis; and a loss of Brca1 heterozygosity (LOH). This uniquely accelerated Brca1 tumor model is also relevant to human esophageal squamous cell carcinoma, an often lethal tumor.

Germline BRCA1 mutations predispose humans to an elevated cancer risk and especially that of the breast and ovary (1, 2). In recent times the suggestion of an increased risk of esophageal cancer in BRCA1 mutation carriers has also been reported, e.g., in an individual with a germline BRCA1 mutation (3). Also, a complete clinical response to platinum treatment was observed in a patient with BRCA1 mutant esophageal cancer (4). Furthermore, the overall esophageal squamous cell carcinoma (ESCC) risk in BRCA1 carriers is significant (relative risk [RR] of 2.9 [95% CI 1.1 to 6.0]) (5, 6). This is in keeping with the observation that a relatively frequent loss of heterozygosity (LOH) is detected in the BRCA1-containing region of chromosome 17 in squamous cell carcinoma of the esophagus (7–9).BRCA1 maintains genome integrity by engaging in multiple cellular processes, including the repair of DNA damage (10, 11), including double strand breaks (DSBs), stalled replication forks, and other abnormalities. Stalled forks, when not resolved, can lead to mutations or can collapse into DSBs (12–15). Both outcomes are components of what is commonly referred to as replication stress (RS), which, when chronic, can serve as a cancer driving force (16–18).Loss of certain DNA damage repair functions in BRCA1 mutant tumor cells also renders these cells sensitive to platinum-based derivatives and PARP inhibitors (19, 20). Success of these agents in suppressing BRCA1 mutant tumor growth has made them therapeutic agents of choice for treating BRCA1 mutant cancer (21, 22). Loss of BRCA1 function either by germline deletion and/or promoter hypermethylation is now a predictive classifier of response to these agents (23).Currently, multiple Brca1 mouse models facilitate the study of BRCA1 loss-associated tumorigenesis. Complete loss of BRCA1 is embryonically lethal (24); thus, successful, tumorigenic models either conditionally delete both alleles of Brca1 in a tissue of interest or express a hypomorphic mutant version of Brca1 (25–27). For example, conditional Brca1 loss can be driven by Cre-mediated deletion of two Brca1 floxed alleles in a tissue of choice. And mice bearing hypomorphic Brca1 mutant alleles, like delta 11, and those that express an incompletely functioning or a truncated version of the Brca1 protein instead of the full-length polypeptide also develop Brca1 tumors (26, 28, 29).Often, BRCA1 knockout (KO) mice also incur a loss of p53 function, which results in accelerated tumor formation, and, in the case of hypomorphic Brca1 mutant mice, their survival as well (25, 27, 30). Mice in both models develop tumors, including mammary and ovarian tumors, which, on average, take ∼1 y to develop. Brca1 delta 11 (Brca1^Δ11/Δ11) mice, which synthesize a markedly deleted but still partially functional Brca1 allele, also form esophageal tumors that can be accelerated by addition of the oxidative stress–inducing agent methyl-N-amylnitrosamine (MNAN) to their drinking water (31).However, the role of BRCA1 haploinsufficiency has not been extensively evaluated in mouse models. One reason is that Brca1 heterozygous mice did not generate tumors more often or faster than their wild-type (WT) counterparts (27, 32). This is unlike the increased predisposition to cancer observed in human BRCA1 mutation carriers (who carry a germline loss-of-function mutation in a single BRCA1 allele). They manifest a significantly greater than normal breast and/or ovarian cancer incidence by age 70 (1, 2).BRCA1 haploinsufficiency can be linked to increased genomic instability, in part, because of its defective participation in stalled fork repair and replication stress suppression (33) and, possibly, because of its role in regulating SIRT1 levels and affecting pRb pathway activation (34). Given the importance of RS development in tumorigenesis (16, 18, 35), this effect would be a logical contributor to BRCA1 mutant cancer development. Of note, BRCA1 heterozygous human cells are haploinsufficient for RS suppression (33), raising the possibility that this defect operates as a general contributor to the increased tumorigenicity observed in many germline BRCA1 heterozygous families.To test this hypothesis, we have established a Brca1 mutant esophageal mouse cancer model that is capable of addressing the role of replication stress accumulation in BRCA1 mutant cancer. Here one allele of Brca1 and one of Trp53 were deleted through the action of Meox2Cre, which acts very early during embryogenesis (embryonic day 5 [E5]) (36) and results in the development of Brca1 and p53 heterozygosity in all tissues. Using this mouse model, we have found that BRCA1 deficiency in replication stress suppression is enhanced by exposure to 4-nitroquinoline-1-oxide (4NQO) in BRCA1 heterozygous tissue where it serves as an efficient and abnormally rapid driver of tumor formation.     

Gel core liposomes: an advanced carrier for improved vaccine delivery

Many sub-unit vaccines are successful in preventing the occurrence of disease, but their use is largely restrained due to low immunogenicity. Novel carrier-based vaccine could serve as a vaccine adjuvant to overcome low immunogenicity of sub-unit vaccines. The use of liposomes as a delivery system for antigen is well recognized but they are unstable and release of antigen from them cannot be controlled over a prolonged period of time. To overcome the limitation of liposomes, this study has developed gel core liposomes in which a core of polymer was incorporated inside the liposomal vesicles, which serve the function of skeleton and provide mechanical strength to vesicles. In the present investigation BSA-loaded gel core liposomes were prepared by reverse phase evaporation method and characterized for vesicles size, shape, entrapment efficiency, in vitro release and stability studies. The in vivo studies to evaluate antigen presenting potential of the gel-core liposomes was performed in Balb/c mice by measuring the immune response elicited by intramuscular administration of BSA-loaded gel core liposomes and compared with intramuscularly administered BSA-loaded conventional liposomes, alum adsorbed BSA and plain antigen. Results indicate that intramuscular immunization with gel core liposomes induces efficient systemic antibody responses against BSA as compared to other formulations. The gel core liposomal formulation provides good entrapment efficiency, enhanced in vitro stability, prolonged antigen release and effective immunoadjuvant property, justifying its potential for improved vaccine delivery.     

Scrub typhus causing neonatal hepatitis with acute liver failure—A case series

Neonatal hepatitis with acute liver failure due to varied etiology including various infections is reported in the past. Scrub typhus as a cause of neonatal hepatitis has rarely been reported in literature. A high index of clinical suspicion is required for early diagnosis and timely treatment. Severity and prognosis of the disease varies widely because several different strains of Orientia tsutsugamushi exist with different virulence. Delayed diagnosis can result in complication and significant morbidity and mortality. Here, we report three cases of neonatal hepatitis with acute liver failure caused by scrub typhus to increase awareness.