The antimicrobial effects of glucosinolates and their respective enzymatic hydrolysis products on bacteria isolated from the human intestinal tract

Aims:  The aim of the study was to evaluate the in vitro antibacterial activity of glucosinolates and their enzymatic hydrolysis product against bacteria isolated from the human intestinal tract.
Methods and results:  Using a disc diffusion bioassay, different doses of intact glucosinolates and their corresponding hydrolysis products were tested. There were clear structure–activity and concentration differences with respect to the in vitro growth inhibition effects as well as differences in the sensitivities of the individual bacteria. The most effective glucosinolate hydrolysis products were the isothiocyanates; sulforaphane and benzyl isothiocyanate were the best inhibitors of growth. Indole-3-carbinol had some inhibitory effects against the Gram-positive bacteria but had no effect, even at the highest dose, against the Gram-negative bacteria. Indole-3-acetonitrile had some inhibitory activity against the Gram-negative bacteria. Glucosinolates, nitriles and amines were ineffective at all the doses used.
Conclusions:  Glucosinolate hydrolysis products and specifically the isothiocyanates SFN and BITC have significant antimicrobial activity against Gram-positive and Gram-negative bacteria, and might be useful in controlling human pathogens through the diet.
Significance and Impact of the Study:  This the first major in vitro study demonstrating the potential of these natural dietary chemicals as an alternative to, or in combination with, current antibiotic-based therapies for treating infectious diseases.     

Humanized Mice Show Clinical Signs of Dengue Fever according to Infecting Virus Genotype

We demonstrated that the infection of humanized NOD-scid IL2rγ ^null mice with different strains (representing the four genotypes) of dengue virus serotype 2 (DEN-2) can induce the development of human-like disease, including fever, viremia, erythema, and thrombocytopenia. Newborn mice were irradiated and received transplants by intrahepatic inoculation of human cord blood-derived hematopoietic progenitor cells (CD34⁺). After 6 weeks, mouse peripheral blood was tested by flow cytometry to determine levels of human lymphocytes (CD45⁺ cells); rates of reconstitution ranged from 16 to 80% (median, 52%). Infection (with approximately 10⁶ PFU, the equivalent of a mosquito bite) of these humanized mice with eight low-passage-number strains produced a high viremia extending to days 12 to 18 postinfection. We observed a significant decrease in platelets at day 10 in most of the mice and an increase in body temperature (fever) and erythema (rash) in comparison with humanized mice inoculated with cell culture medium only. Comparison of Southeast (SE) Asian and other genotype viruses (American, Indian, and West African) in this model showed significant differences in magnitude and duration of viremia and rash, with the SE Asian viruses always being highest. Indian genotype viruses produced lower viremias and less thrombocytopenia than the others, and West African (sylvatic) viruses produced the shortest periods of viremia and the lowest rash measurements. These results correlate with virulence and transmission differences described previously for primary human target cells and whole mosquitoes and may correlate with epidemiologic observations around the world. These characteristics make this mouse model ideal for the study of dengue pathogenesis and the evaluation of vaccine attenuation and antivirals.Dengue viruses, which cause the disease dengue fever (DF) and its more severe form, dengue hemorrhagic fever (DHF), in humans, have been spreading to more areas of the world along with their mosquito (Aedes aegypti and Aedes albopictus) vectors. Now over 100 countries are affected, including some areas of the United States (Texas and Hawaii) (5, 26). Due to the fact that only humans show clinical signs and symptoms of disease, it has been difficult to directly test the mechanisms of pathogenesis of these viruses (4). Through decades of research, including clinical, epidemiologic, and laboratory studies, the factors involved in producing disease, whether it be DF or DHF, have remained unproved. However, there are many indications that both the virus and the host contribute to the occurrence and severity of disease: there are genetic differences in the virus and host immune response that can be measured in vitro, and these factors seem to lead to immunopathology in addition to the damage done by virus replication. Because there are four antigenically distinct dengue viruses (serotypes 1 to 4), humans can theoretically have dengue virus infections leading to clinical disease up to four times, and the immunity to the first virus enhances the probability of developing severe dengue after a subsequent infection. Thus, the development of vaccines has been hampered by the unknown effects of inoculating with a tetravalent preparation that might cause immunopathology or severe disease, and there are no appropriate animal models in which to test vaccine attenuation and efficacy for human applications.In 2005 we reported the development of humanized NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice that produced signs of DF upon infection with one strain of dengue virus (3). The mice were humanized by giving them transplants of purified hematopoietic stem cells from human umbilical cord blood (CB) samples taken from normal births. After subcutaneous infection with a low dose of a Southeast (SE) Asian virus, the viremia, rash, and thrombocytopenia were significantly higher, longer lasting, and more like human disease than in any other animal model described at the time. We concluded that this model could be used to test antiviral treatments, since these mice did not produce measurable human antibodies. Since then, many other immunodeficient mouse strains have been produced that can have enhanced human engraftment levels, and they develop functional human immune system cells, including some level of adaptive immunity (20). It has been reported that some of these mouse strains develop immunoglobulins specific for human immunodeficiency virus and dengue virus, albeit at low levels (14, 25).Here we present results of dengue virus pathogenesis studies in a new mouse strain, NOD-scid IL2rγ ^null, that has a much higher degree of human lymphocyte development (median of 52%, versus 14% previously). The comparison of viruses from different genetic subgroups of dengue serotype 2 has led us to conclude that this model is reflective of actual human dengue pathogenesis, and this development might bring us to a new era in testing the factors that contribute to dengue disease.     

SCAMP3 is a component of the Salmonella-induced tubular network and reveals an interaction between bacterial effectors and post-Golgi trafficking

Salmonella enterica are facultative intracellular bacterial pathogens that proliferate within host cells in a membrane-bounded compartment, the Salmonella -containing vacuole (SCV). Intracellular replication of Salmonella is mediated by bacterial effectors translocated on to the cytoplasmic face of the SCV membrane by a type III secretion system. Some of these effectors manipulate the host endocytic pathway, resulting in the formation in epithelial cells of tubules enriched in late endosomal markers, known as Salmonella -induced filaments (SIFs). However, much less is known about possible interference of Salmonella with the secretory pathway. Here, a small-interference RNA screen revealed that secretory carrier membrane proteins (SCAMPs) 2 and 3 contribute to the maintenance of SCVs in the Golgi region of HeLa cells. This is likely to reflect a function of SCAMPs in vacuolar membrane dynamics. Moreover, SCAMP3, which accumulates on the trans -Golgi network in uninfected cells, marked tubules induced by Salmonella effectors that overlapped with SIFs but which also comprised distinct tubules lacking late endosomal proteins. We propose that SCAMP3 tubules reflect a manipulation of specific post-Golgi trafficking that might allow Salmonella to acquire nutrients and membrane, or to control host immune responses.     

One More Piece in the VACV Ecological Puzzle: Could Peridomestic Rodents Be the Link between Wildlife and Bovine Vaccinia Outbreaks in Brazil?

Background

Despite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks – BV), affecting dairy cattle and milkers. Little is known about VACV''s natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated.

Methodology/Principal Findings

In this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks.

Conclusion/Significance

These data provide new biological and epidemiological information on VACV and lead to an interesting question: could peridomestic rodents be the link between wildlife and BV outbreaks?     

Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite''s life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc_con, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.     

Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii

Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.     

Brazilian Angiostrongylus cantonensis haplotypes,ac8 and ac9, have two different biological and morphological profiles

Angiostrongylus cantonensis is the etiologic agent of eosinophilic meningoencephalitis in humans. Cases have been recorded in many parts of the world, including Brazil. The aim of this study was to compare the differences in the biology and morphology of two different Brazilian haplotypes of A. : ac8 and ac9. A significantly larger number of L1 larvae eliminated in the faeces of rodents at the beginning of the patent period was observed for ac9 haplotype and compared to the total of L1 larvae eliminated, there was a significant difference between the two haplotypes. The ac9 haplotype showed a significant difference in the proportion of female and male specimens (0.6:1), but the same was not observed for ac8 (1.2:1). The morphometric analysis showed that male and female specimens isolated from ac8 haplotype were significantly larger with respect to body length, oesophagus length, spicule length (male) and distance from the anus to the rear end (female) compared to specimens from ac9. The morphological analysis by light microscopy showed little variation in the level of bifurcations at the lateral rays in the right lobe of the copulatory bursa between the two haplotypes. The biological, morphological and morphometric variations observed between the two haplotypes agree with the observed variation at the molecular level using the cytochrome oxidase subunit I marker and reinforce the possible influence of geographical isolation on the development of these haplotypes.     

Evaluation of the WHO classification of dengue disease severity during an epidemic in 2011 in the state of Ceará, Brazil

In 2009, the World Health Organization (WHO) issued a new guideline that stratifies dengue-affected patients into severe (SD) and non-severe dengue (NSD) (with or without warning signs). To evaluate the new recommendations, we completed a retrospective cross-sectional study of the dengue haemorrhagic fever (DHF) cases reported during an outbreak in 2011 in northeastern Brazil. We investigated 84 suspected DHF patients, including 45 (53.6%) males and 39 (46.4%) females. The ages of the patients ranged from five-83 years and the median age was 29. According to the DHF/dengue shock syndrome classification, 53 (63.1%) patients were classified as having dengue fever and 31 (36.9%) as having DHF. According to the 2009 WHO classification, 32 (38.1%) patients were grouped as having NSD [4 (4.8%) without warning signs and 28 (33.3%) with warning signs] and 52 (61.9%) as having SD. A better performance of the revised classification in the detection of severe clinical manifestations allows for an improved detection of patients with SD and may reduce deaths. The revised classification will not only facilitate effective screening and patient management, but will also enable the collection of standardised surveillance data for future epidemiological and clinical studies.     

Effects of adrenal hormones on the expression of adiponectin and adiponectin receptors in adipose tissue, muscle and liver

Background

Adiponectin, an insulin-sensitive hormone that is primarily synthesized in adipose tissue, exerts its effects by binding to two receptors, adipoR1 and adipoR2. Little is known regarding the effects of glucocorticoids on the expression of adiponectin receptors.

Methods

Male Wistar rats were bilaterally adrenalectomized and treated with dexamethasone (0.2 mg/100 g) twice daily for 3 days. To analyze the potential effects of glucocorticoids, rats received two daily injections of the glucocorticoid receptor antagonist (RU-486, 5.0 mg) over the course of 3 days. Additionally, 3T3-L1 adipocytes and C2C12 myotubes were treated with dexamethasone, adrenaline or RU-486. The gene expression of adiponectin, adipoR1 and adipoR2 was determined by real-time PCR, and protein secretion was examined by Western blotting using lysates from retroperitoneal, epididymal and subcutaneous adipose tissue depots, liver and muscle.

Results

In rats, excess glucocorticoids increased the levels of insulin in serum and decreased serum adiponectin concentrations, whereas adrenalectomy decreased the mRNA expression of adiponectin (3-fold) and adipoR2 (7-fold) in epididymal adipose tissue and increased adipoR2 gene expression in muscle (3-fold) compared to control group sham-operated. Dexamethasone treatment did not reverse the effects of adrenalectomy, and glucocorticoid receptor blockade did not reproduce the effects of adrenalectomy. In 3T3-L1 adipocytes, dexamethasone and adrenaline both increased adipoR2 mRNA levels, but RU-486 reduced adipoR2 gene expression in vitro.

Conclusion

Dexamethasone treatment induces a state of insulin resistance but does not affect adiponectin receptor expression in adipose tissue. However, the effects of catecholamines on insulin resistance may be due to their effects on adipoR2.