Virulence potential of group A streptococci isolated from throat cultures of children from north India

Background & objectives: Rheumatic fever (RF)/rheumatic heart disease (RHD) caused by Group A streptococcus (GAS) are more prevalent in north India as compared to the western world, where invasive diseases are common. This could be due to variation in the virulence of GAS in different geographic locations. Hence, we studied the virulence potential of GAS isolated from the throat of children from north India Methods: Fifty GAS isolated consecutively, from children with mild pharyngitis (20), severe pharyngitis (24) and asymptomatic pharyngeal carriers (6), were characterized by emm typing and opacity factor (OF). Adherence and internalization of GAS in HEp-2 cells and opsonophagocytosis in convalescent serum samples were studied. Results: Twenty emm types, six sequence types, and one non-typeable GAS were circulating in the community. emm type 74, 11, 68, StI129 and NS292 were most prevalent. Twenty seven (54%) GAS isolates were OF negative. Sixty five per cent of the most prevalent emm types were OF negative indicating their rheumatogenic potential. Adhesion of GAS ranged from 0.1 to 100 per cent. Forty eight per cent of GAS were highly adherent. Invasion of GAS in HEp-2 cells ranged between 0 to 30 per cent. Only 20 per cent isolates exhibited highest invasion. GAS were opsonophagocytosed with highly divergent efficiency ranging from 0 to 91.7 per cent. Nineteen GAS were not opsonophagocytosed and 15 multiplied during the assay. Isolates of the same emm type also varied in their virulence potential. Interpretation & conclusions: GAS isolates from the throat of children from north India belonged to several emm types, majority were OF negative, excellent adherents but poor invaders. This explains why throat infections in these children tend to lead to ARF/RHD rather than invasive diseases. A few isolates exhibiting high invasion efficiency indicate that GAS throat cultures can also lead to invasive diseases

Partial characterization of a 36-kDa antigen of Entamoeba histolytica and its recognition by sera from patients with amoebiasis

A 36-kDa antigen of axenically grown pathogenic Entamoeba histolytica (HM1-IMSS) was eluted from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved crude amoebic extract antigens. The immunoreactivity of this partially purified 36-kDa antigen with monoclonal antibody (MoAb) 3D10 altered significantly (P<0.01) after heat and trypsin treatment but remained unaltered after treatment with sodium metaperiodate (P>0.5), thereby indicating the protein nature of the epitope recognized by MoAb 3D10. The epitope was found to be localized on the surface as well as in the cytoplasm of the E. histolytica trophozoites with the majority of it in the cytoplasm. In addition, this epitope was also found to be present on the cyst form of the parasite. The 36-kDa molecule was recognized by the sera from 29 (85%) of the 34 patients with amoebic liver abscess and five (83%) of the six patients with amoebic colitis. No serum samples from asymptomatic cyst passers, from patients with non-amoebic hepatic or intestinal disorders and apparently healthy subjects had antibodies that reacted with this 36-kDa molecule. The immune responses in man to this 36-kDa amoebic molecule indicate a potential specific role for this molecule in invasive amoebiasis

Distinct role of CD80 and CD86 in the regulation of the activation of B cell and B cell lymphoma

To date, not much has been known regarding the role of CD80 and CD86 molecules in signaling of B cells. The CD28/CTLA4 ligands, CD80 (B7-1) and CD86 (B7-2), are expressed on the surface of freshly isolated splenic B cells and their expression is up-regulated by lipopolysaccharides. In the present study, we have investigated whether signaling via CD80/CD86 could alter the proliferation and immunoglobulin synthesis of B cells. Splenic B cells were stimulated with lipopolysaccharides in the presence of anti-B7-1 (16-10A1) and anti-B7-2 (GL1) monoclonal antibodies (mAbs). Exciting features observed during the study were that cross-linking of CD86 with GL1 enhanced the proliferation and production of IgG1 and IgG2a isotypes. In contrast, anti-B7-1 (16-10A1) mAb could efficiently block the proliferation and production of IgG1 and IgG2a. Furthermore, GL1 mAb could also induce the secretion of IgG isotypes from B cell lymphomas. Importantly, 16-10A1 could retard the growth of lymphomas and favored the up-regulation of pro-apoptotic molecules caspase-3, caspase-8, Fas, FasL, Bak and Bax and down-regulation of anti-apoptotic molecule Bcl-x(L). In contrast, GL1 augmented the level of anti-apoptotic molecules Bcl-w and Bcl-x(L) and decreased the levels of pro-apoptotic molecule caspase-8, thereby providing a novel insight into the mechanism whereby triggering through CD80 and CD86 could deliver regulatory signals. Thus, this study is the first demonstration of a distinct signaling event induced by CD80 and CD86 molecules in B cell lymphoma. Finally, the significance of the finding is that CD80 provided negative signal for the proliferation and IgG secretion of normal B cells and B cell lymphomas. In contrast, CD86 encouraged the activity of B cells

A Versatile High Throughput Screening Platform for Plant Metabolic Engineering Highlights the Major Role of ABI3 in Lipid Metabolism Regulation

Traditional functional genetic studies in crops are time consuming, complicated andcannot be readily scaled up. The reason is that mutant or transformed crops need tobe generated to study the effect of gene modifications on specific traits of interest.However, many crop species have a complex genome and a long generation time. Asa result, it usually takes several months to over a year to obtain desired mutants ortransgenic plants, which represents a significant bottleneck in the development of newcrop varieties. To overcome this major issue, we are currently establishing a versatileplant genetic screening platform, amenable to high throughput screening in almost anycrop species, with a unique workflow. This platform combines protoplast transformationand fluorescence activated cell sorting. Here we show that tobacco protoplasts canaccumulate high levels of lipid if transiently transformed with genes involved in lipidbiosynthesis and can be sorted based on lipid content. Hence, protoplasts can be usedas a predictive tool for plant lipid engineering. Using this newly established strategy, wedemonstrate the major role ofABI3in plant lipid accumulation. We anticipate that thisworkflow can be applied to numerous highly valuable metabolic traits other than storagelipid accumulation. This new strategy represents a significant step toward screeningcomplex genetic libraries, in a single experiment and in a matter of days, as opposed toyears by conventional means.This work was partly funded through the CSIRO Synthetic Biology Future Science Platform and the CSIRO Research Office CERC Postdoctoral Fellowship schem

Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization

The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they ‘chaperone’ p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes

Induced sputum nitrites correlate with FEV1 in children with cystic fibrosis

Aim: To determine the difference in the levels of nitrites in induced sputum of children with cystic fibrosis (CF) and controls. Furthermore, to evaluate the association between induced sputum nitrites and lung function in children with CF. Methods: Nitrites, cell differentials, white blood cell count, were estimated in induced sputum of 20 children with CF and 10 age‐matched healthy controls. Nitrites in induced sputum samples were measured using the Greiss assay. Lung function was ascertained by spirometry. Results: We observed high levels of nitrites in CF (184.8 ± 11.07 lM ⁄ L) versus controls (56.4 ± 5.7 lM ⁄ L) (p < 0.01). A positive correlation between neturophil percent and nitrites, white blood cell count and nitrites (p < 0.05) in children with CF was observed. Sputum nitrites correlated negatively with FEV1 (p < 0.05) in children with CF. Conclusion: Induced sputum nitrite could serve as a useful non invasive marker for assessing the degree of inflammation in the airways of children with CF