Salmonella enterica Serovar Typhimurium Lacking hfq Gene Confers Protective Immunity against Murine Typhoid

Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generation of live attenuated vaccine strains, focus has gradually shifted towards manipulation of virulence regulator genes. Hfq is a RNA chaperon which mediates the binding of small RNAs to the mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we evaluated the efficacy of the Salmonella Typhimurium Dhfq strain as a candidate for live oral vaccine in murine model of typhoid fever. Salmonella hfq deletion mutant is highly attenuated in cell culture and animal model implying a significant role of Hfq in bacterial virulence. Oral immunization with the Salmonella hfq deletion mutant efficiently protects mice against subsequent oral challenge with virulent strain of Salmonella Typhimurium. Moreover, protection was induced upon both multiple as well as single dose of immunizations. The vaccine strain appears to be safe for use in pregnant mice and the protection is mediated by the increase in the number of CD4(+) T lymphocytes upon vaccination. The levels of serum IgG and secretory-IgA in intestinal washes specific to lipopolysaccharide and outer membrane protein were significantly increased upon vaccination. Furthermore, hfq deletion mutant showed enhanced antigen presentation by dendritic cells compared to the wild type strain. Taken together, the studies in murine immunization model suggest that the Salmonella hfq deletion mutant can be a novel live oral vaccine candidate.

Modulating effect of gelonin gp330 conjugate on Heymann's nephritis induced in rats - A pathomorphological evaluation

Heymann's nephritis (HN) in rats induced by injecting renal proximal tubule brush border protein gp330, is an animal model replicating human autoimmune membranous glomerulonephritis(1). Endogenous IgG gets deposited between the foot processes in the epithelial side of the glomerulus and causes complement-mediated membrane injury, leading to proteinuria and basement membrane thickening. We investigated the effect of a toxin, gelonin conjugated to gp330 and targetted against antigp330-producing cells in ameliorating immune injury and nephrotic state in rats. The groups of animals injected with purified gp330 revealed by immunofluorescence, characteristic granular deposits of IgG along the basement membrane. The rats intravenously injected with gelonin gp330 conjugate, four days after the antigenic challenge with gp330 in two doses, showed amelioration of the nephrotic state and appreciable reduction in glomerular IgG deposits against immune injury. This substantiates our earlier biochemical results and corroborates the possibility of using toxins conjugated to specific antigen in treating antibody-mediated autoimmune diseases.

Heat shock protein 70 as a biomarker of heat stress in a simulated hot cockpit

Background: Fighter pilots are frequently exposed to high temperatures during high-speed low-level flight. Heat strain can result in temporary impairment of cognitive functions and when severe, loss of consciousness and consequent loss of life and equipment. Induction of stress proteins is a highly conserved stress response mechanism from bacteria to humans. induced stress protein levels are known to be cytoprotective and have been correlated with stress tolerance. Although many studies on the heat shock response mechanisms have been performed in cell culture and animal model systems, there is very limited information on stress protein induction in human subjects. Hypothesis: Heat shock proteins (Hsp), especially Hsp70, may be induced in human subjects exposed to high temperatures in a hot cockpit designed to simulate heat stress experienced in low flying sorties. Methods: Six healthy volunteers were subjected to heat stress at 55degreesC in a high temperature cockpit simulator for a period of 1 h at 30% humidity. Physiological parameters such as oral and skin temperatures, heart rate, and sweat rate were monitored regularly during this time. The level of Hsp70 in leukocytes was examined before and after the heat exposure in each subject. Conclusions: Hsp70 was found to be significantly induced in all the six subjects exposed to heat stress. The level of induced Hsp70 appears to correlate with other strain indicators such as accumulative circulatory strain and Craig's modified index. The usefulness of Hsp70 as a molecular marker of heat stress in humans is discussed.

Artemisinin-based combination with curcumin adds a new dimension to malaria therapy

Malaria afflicts 300 million people worldwide, with over a million deaths every year. With no immediate prospect of a vaccine against the disease, drugs are the only choice to treat it. Unfortunately, the parasite has become resistant to most antimalarials, restricting the option to use artemisinins (ARTs) for effective cure. With the use of ARTs as the front-line antimalarials, reports are already available on the possible resistance development to these drugs as well. Therefore, it has become necessary to use ART-based combination therapies to delay emergence of resistance. It is also necessary to discover new pharmacophores to eventually replace ART. Studies in our laboratory have shown that curcumin not only synergizes with ART as an antimalarial to kill the parasite, but is also uniquely able to prime the immune system to protect against parasite recrudescence in the animal model. The results indicate a potential for the use of ART curcumin combination against recrudescence/relapse in falciparum and vivax malaria. In addition, studies have also suggested the use of curcumin as an adjunct therapy against cerebral malaria. In this review we have attempted to highlight these aspects as well as the studies directed to discover new pharmacophores as potential replacements for ART.

Osteoprotective effect of propranolol in ovariectomized rats: a comparison with zoledronic acid and alfacalcidol

Currently beta-adrenergic receptor blockers are considered to be potential drugs under investigation for preventive or therapeutic effect in osteoporosis. However, there is no published data showing the comparative study of beta-blockers with well accepted agents for the treatment of osteoporosis. To address this question, we compared the effects of propranolol with well accepted treatments like zoledronic acid and alfacalcidol in an animal model of postmenopausal osteoporosis. Five days after ovariectomy, 36 ovariectomized (OVX) rats were divided into 6 equal groups, randomized to treatments zoledronic acid (100 mu g/kg, intravenous single dose); alfacalcidol (0.5 mu g/kg, oral gauge daily); propranolol (0.1 mg/kg, subcutaneously 5 days per week) for 12 weeks. Untreated OVX and sham OVX were used as controls. At the end of treatment serum calcium and alkaline phosphatase were assayed. Femurs were removed and tested for bone density, bone porosity, bone mechanical properties and trabecular micro-architecture. Propranolol showed a significant decrease in alkaline phosphatase levels and bone porosity in comparison to OVX control. Moreover, propranolol significantly improved bone density, bone mechanical properties and inhibited the deterioration of trabecular microarchitecture when compared with OVX control. The osteoprotective effect of propranolol was comparable with zoledronic acid and alfacalcidol. Based on this comparative study, the results strongly suggest that propranolol can be a candidate therapeutic drug for the management of postmenopausal osteoporosis.

Comparative Evaluation of Zoledronic Acid, Alfacalcidol and Propranolol in Pharmacological Correction of Experimental Osteoporosis

Propranolol, a beta-adrenergic receptor blocker, is presently considered to be a potential therapeutic intervention under investigation for its role in prevention and treatment of osteoporosis. However, no studies have compared the osteoprotective properties of propranolol with well accepted therapeu-tic interventions for the treatment of osteoporosis. To address this question, this study was designed to evaluate the bone protective effects of zoledronic acid, alfacalcidol and propranolol in an animal model of postmenopausal osteoporosis. Five days after ovariectomy, 36 ovariectomized (OVX) rats were divided in- to 6 equal groups, randomized to treatments zoledronic acid (100 μg/kg, intravenous single dose); alfacal-cidol (0.5 μg/kg, oral gauge daily); propranolol (0.1mg/kg, subcutaneously 5 days per week) for 12 weeks. Untreated OVX and sham OVX were used as controls. At the end of the study, rats were killed under anesthesia. For bone porosity evaluation, whole fourth lumbar vertebrae (LV4) were removed. LV4 were also used to measure bone mechanical propeties. Left femurs were used for bone histology. Propranolol showed a significant decrease in bone porosity in comparison to OVX control. Moreover, propranolol sig- nificantly improved bone mechanical properties and bone quality when compared with OVX control. The osteoprotective effect of propranolol was comparable with zoledronic acid and alfacalcidol. Based on this comparative study, the results strongly suggest that propranolol might be new therapeutic intervention for the management of postmenopausal osteoporosis in humans.

Expression of Bioactive Callithrix jacchus Follicle-Stimulating Hormone in Pichia pastoris

Callithrix jacchus (common marmoset) is a New World primate monkey, used as an animal model in biomedical research. Marmoset-specific follicle-stimulating hormone (FSH) preparation is required to improve superovulation protocols and to develop homologous FSH monitoring assays in these monkeys. In this study, we document the large-scale expression of recombinant marmoset FSH in methylotropic yeast, Pichia pastoris. The recombinant preparation was found to be immunologically active in Western blotting and radioimmunoassay. The preparation displayed receptor binding ability in radioreceptor assay. Based on the receptor binding ability, the yield of fermentation was estimated to be 7.2 mg/L. FSH-induced cAMP assay and estradiol assay revealed that the recombinant hormone is able to induce signal transduction. Both immunological and in vitro biological activity of marmoset FSH was found to be comparable to purified human pituitary FSH, which served as reference hormone for these assays. Thus, the study suggests that a Pichia expression system can be used for large-scale expression of bioactive recombinant marmoset FSH.

Increased expression of cystine/glutamate antiporter in multiple sclerosis

Background: Glutamate excitotoxicity contributes to oligodendrocyte and tissue damage in multiple sclerosis (MS). Intriguingly, glutamate level in plasma and cerebrospinal fluid of MS patients is elevated, a feature which may be related to the pathophysiology of this disease. In addition to glutamate transporters, levels of extracellular glutamate are controlled by cystine/glutamate antiporter x(c)(-), an exchanger that provides intracellular cystine for production of glutathione, the major cellular antioxidant. The objective of this study was to analyze the role of the system x(c)(-) in glutamate homeostasis alterations in MS pathology. -- Methods: Primary cultures of human monocytes and the cell line U-937 were used to investigate the mechanism of glutamate release. Expression of cystine glutamate exchanger (xCT) was quantified by quantitative PCR, Western blot, flow cytometry and immunohistochemistry in monocytes in vitro, in animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, and in samples of MS patients. -- Results and discussion: We show here that human activated monocytes release glutamate through cystine/glutamate antiporter x(c)(-) and that the expression of the catalytic subunit xCT is upregulated as a consequence of monocyte activation. In addition, xCT expression is also increased in EAE and in the disease proper. In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells. In particular, cells from monocyte-macrophage-microglia lineage have higher xCT expression in MS and in EAE, indicating that immune activation upregulates xCT levels, which may result in higher glutamate release and contribution to excitotoxic damage to oligodendrocytes. -- Conclusions: Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)(-) in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

Pravastatin inhibits cell proliferation and increased MAT1A expression in hepatocarcinoma cells and in vivo models

Background: Statins may have therapeutic effects on hepatocarcinoma (HCC). This type of disorder is the most common malignant primary tumour in the liver. Our objective was to determine whether pravastatin had a therapeutic effect in vitro and in vivo models. Method: We design in vitro and in vivo model. In vitro we used PLC and determine cell proliferation. In vivo, we used and animal model to determined, PCNA and MAT1A expression and transaminases levels. Results: We found that pravastatin decreases cell proliferation in vitro (cell proliferation in pravastatin group was 82%, in sorafenib group 51% and in combined group 40%) and in vivo (in pravastatin group 80%, in sorafenib group 76.4% and in combined group 72.72%). The MAT1A levels, was significantly higher in Pravastatin group (D 62%, P 94%, S 71%, P + S 91%). The transaminases levels, decreased significantly in Pravastatin group (GOT and GPT levels D 619.5 U/L; 271 U/L) (P 117.5 U/L; 43.5 U/L) (S 147 U/L; 59 U/L) (P + S 142 U/L; 59 U/L). Conclusion: The combination of pravastatin + sorafenib were more effective than Sorafenib alone.

Contribution of Pannexin1 to Experimental Autoimmune Encephalomyelitis

Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.

Inoculation of Triatoma Virus (Dicistroviridae: Cripavirus) elicits a non-infective immune response in mice

Background: Dicistroviridae is a new family of small, non-enveloped, +ssRNA viruses pathogenic to both beneficial arthropods and insect pests. Little is known about the dicistrovirus replication mechanism or gene function, and any knowledge on these subjects comes mainly from comparisons with mammalian viruses from the Picornaviridae family. Due to its peculiar genome organization and characteristics of the per os viral transmission route, dicistroviruses make good candidates for use as biopesticides. Triatoma virus (TrV) is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of the human trypanosomiasis disease called Chagas disease. TrV was postulated as a potential control agent against Chagas' vectors. Although there is no evidence that TrV nor other dicistroviruses replicate in species outside the Insecta class, the innocuousness of these viruses in humans and animals needs to be ascertained. Methods: In this study, RT-PCR and ELISA were used to detect the infectivity of this virus in Mus musculus BALB/c mice. Results: In this study we have observed that there is no significant difference in the ratio IgG2a/IgG1 in sera from animals inoculated with TrV when compared with non-inoculated animals or mice inoculated only with non-infective TrV protein capsids. Conclusions: We conclude that, under our experimental conditions, TrV is unable to replicate inmice. This study constitutes the first test to evaluate the infectivity of a dicistrovirus in a vertebrate animal model.

Behavioral, neurochemical and morphological changes induced by the overexpression of munc18-1a in brain of mice: relevance to schizophrenia

Overexpression of the mammalian homolog of the unc-18 gene (munc18-1) has been described in the brain of subjects with schizophrenia. Munc18-1 protein is involved in membrane fusion processes, exocytosis and neurotransmitter release. A transgenic mouse strain that overexpresses the protein isoform munc18-1a in the brain was characterized. This animal displays several schizophrenia-related behaviors, supersensitivity to hallucinogenic drugs and deficits in prepulse inhibition that reverse after antipsychotic treatment. Relevant brain areas (that is, cortex and striatum) exhibit reduced expression of dopamine D-1 receptors and dopamine transporters together with enhanced amphetamine-induced in vivo dopamine release. Magnetic resonance imaging demonstrates decreased gray matter volume in the transgenic animal. In conclusion, the mouse overexpressing brain munc18-1a represents a new valid animal model that resembles functional and structural abnormalities in patients with schizophrenia.

A via L-arginina-óxido nítrico no transtorno depressivo e sua associação com a função plaquetária e o estresse oxidativo

O transtorno depressivo (TD) é um fator de risco cardiovascular independente que apresenta elevada morbi-mortalidade. Recentes evidências sugerem a participação do óxido nítrico (NO), potente vasodilatador e anti-agregante plaquetário, na patogênese de doenças cardiovasculares e psiquiátricas. A síntese do NO ocorre através da conversão do aminoácido L-arginina em L-citrulina e NO, pela ação da enzima NO sintase (NOS). Esta tese aborda o papel da via L-arginina-NO em plaquetas de pacientes com TD e sua associação com a função plaquetária e estresse oxidativo. Para análise comportamental da depressão em modelo animal, foi utilizado o modelo de estresse pós-natal de separação única (SMU). Os animais foram divididos em quatro grupos para a realização do estudo: Grupo Controle Sedentário (GCS), Grupo Controle Exercício (GCE), Grupo SMU Sedentário (SMUS) e Grupo SMU Exercício (SMUE). O treinamento físico (TF) dos animais englobou 8 semanas, com duração de 30 minutos e uma velocidade de treinamento estabelecida pelo teste máximo (TE). Para o estudo em humanos, 10 pacientes com TD com score Hamilton: 201, (média de idade: 384anos), foram pareados com 10 indivíduos saudáveis (média de idade: 383anos). Os estudos em humanos e animais foram aprovados pelos Comitês de Ética: 1436 - CEP/HUPE e CEUA/047/2010, respectivamente. Foi mensurado em humanos e em animais: transporte de L-arginina, concentração GMPc, atividade das enzimas NOS e superóxido dismutase (SOD) em plaquetas e cortisol sistêmico. Experimentos realizados somente em humanos: expressão das enzimas NOS, arginase e guanilato ciclase através de Western Blotting. A agregação plaquetária foi induzida por colágeno e foi realizada análise sistêmica de proteína C-reativa, fibrinogênio e L-arginina. Para o tratamento estatístico utilizou-se três testes estatísticos para avaliar as diferenças das curvas de sobrevida: Kaplan-Meier, e os testes de Tarone-Ware e Peto-Prentice. Em humanos, houve uma redução do transporte de L-arginina, da atividade das enzimas NOS e SOD, e da concentração de GMPc em plaquetas, e nas concentrações plasmáticas de L-arginina no grupo com TD em relação ao grupo controle. Foi observado um aumento dos níveis plasmáticos de fibrinogênio no TD. Esses resultados demonstram uma inibição da via L-arginina-NO-GMPc e da enzima anti-oxidante SOD em pacientes com TD sem afetar a função plaquetária. Em relação ao TF, para o modelo animal, foram encontradas alterações iniciais quanto à distância percorrida e tempo de execução do TE entre os grupos controles e o grupos SMUs, apresentando estes últimos menores valores para o TE. Após 8 semanas de TF, verificou-se um maior influxo no transporte de L-arginina para o SMUE em comparação ao grupo SMUS. As diferenças observadas para o tempo e a distância percorrida no TE inicial entre os grupos controle e no modelo de estresse foram revertidas após as 8 semanas de TF, demonstrando o efeito benéfico do exercício físico na capacidade cardiorespiratória em modelos de depressão.

Production of positive controls for calcivirus-specific PCR using recombinant baculiovirus technology

Recent advances in our knowledge of the genetic structure of human caliciviruses (HuCVs) and small round-structured viruses (SRSVs) have led to the development of polymerase chain reaction (PCR)-based molecular tests specific for these viruses. These methods have been developed to detect a number of human pathogenic viruses in environmental samples including water, sewage and shellfish. HuCVs and SRSVs are not culturable, and no animal model is currently available. Therefore there is no convenient method of preparing viruses for study or for reagent production. One problem facing those attempting to use PCR-based methods for the detection of HuCVs and SRSVs is the lack of a suitable positive control substrate. This is particularly important when screening complex samples in which the levels of inhibitors present may significantly interfere with amplificiation. Regions within the RNA polymerase regions of two genetically distinct human caliciviruses have been amplified and used to produce recombinant baculoviruses which express RNA corresponding to the calicivirus polymerase. This RNA is being investigated as a positive control substrate for PCR testing, using current diagnostic primer sets. Recombinant baculovirus technology will enable efficient and cost-effective production of large quantities of positive control RNA with a specific known genotype. We consider the development of these systems as essential for successful screening and monitoring applications.

Alterações estruturais pancreáticas e metabólicas em camundongos C57BL/6 alimentados com dieta de alta densidade energética

Camundongos C57BL/6 machos com oito semanas de idade alimentados com diferentes dietas durante 16 semanas: de alta densidade energética (ADE, 26% das calorias de carboidrato, 60% de gordura e 14% de proteína) ou dieta padrão (CO, 76% das calorias de carboidrato, 10% de gordura e 14% de proteína). Comparado ao grupo CO, o grupo ADE apresentou maior ganho de massa e maior depósito de tecido adiposo, bem como maiores níveis plasmáticos de triglicerídeos, LDL-c, ALT, AST e fosfatase alcalina e com maiores níveis de corticosterona plasmática, glicose de jejum e insulina com uma consequente resistência à insulina (avaliado pelo HOMA-IR). No TOTG, a glicose plasmática aumentou ao máximo após 15 min. da administração de glicose oral em ambos os grupos. Entretanto os níveis de glicose foram maiores no grupo ADE que no grupo CO (P<0.0001). O clearance de glicose no grupo ADE foi reduzido, permanecendo aumentado após 120 min. (P<0.001), caracterizando intolerância a glicose no grupo ADE. O teste intraperitoneal de tolerância à insulina mostrou uma rápida redução na glicose plasmática após 15 minutos da administração de insulina em ambos os grupos, mas significativamente aumentada no grupo ADE (P<0.0001), permanecendo desta forma até os 120 min. após a administração. Concluindo, camundongos C57BL/6 respondem a dieta ADE desenvolvimento os sinais e sintomas associados à síndrome metabólica observada em humanos. Por conseguinte, este modelo animal poderá ajudar-nos a compreender melhor as alterações em órgãos alvos associadas com a síndrome metabólica, assim como a possibilidade de tratamentos diferentes.