Only a subset of phosphoantigen-responsive gamma9delta2 T cells mediate protective tuberculosis immunity

Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG) induce potent expansions of human memory Vgamma(9)(+)Vdelta(2)(+) T cells capable of IFN-gamma production, cytolytic activity, and mycobacterial growth inhibition. Certain phosphoantigens expressed by mycobacteria can stimulate gamma(9)delta(2) T cell expansions, suggesting that purified or synthetic forms of these phosphoantigens may be useful alone or as components of new vaccines or immunotherapeutics. However, we show that while mycobacteria-activated gamma(9)delta(2) T cells potently inhibit intracellular mycobacterial growth, phosphoantigen-activated gamma(9)delta(2) T cells fail to inhibit mycobacteria, although both develop similar effector cytokine and cytolytic functional capacities. gamma(9)delta(2) T cells receiving TLR-mediated costimulation during phosphoantigen activation also failed to inhibit mycobacterial growth. We hypothesized that mycobacteria express Ags, other than the previously identified phosphoantigens, that induce protective subsets of gamma(9)delta(2) T cells. Testing this hypothesis, we compared the TCR sequence diversity of gamma(9)delta(2) T cells expanded with BCG-infected vs phosphoantigen-treated dendritic cells. BCG-stimulated gamma(9)delta(2) T cells displayed a more restricted TCR diversity than phosphoantigen-activated gamma(9)delta(2) T cells. In addition, only a subset of phosphoantigen-activated gamma(9)delta(2) T cells functionally responded to mycobacteria-infected dendritic cells. Furthermore, differential inhibitory functions of BCG- and phosphoantigen-activated gamma(9)delta(2) T cells were confirmed at the clonal level and were not due to differences in TCR avidity. Our results demonstrate that BCG infection can activate and expand protective subsets of phosphoantigen-responsive gamma(9)delta(2) T cells, and provide the first indication that gamma(9)delta(2) T cells can develop pathogen specificity similar to alphabeta T cells. Specific targeting of protective gamma(9)delta(2) T cell subsets will be important for future tuberculosis vaccines.     

Expression of NA/1 symporter (NIS) in endometrial mucosa of fertile, sterile and post-menopausal women

The expression of the Na/I Symporter (NIS) in the basolateral cell membrane of the thyroid follicular cells is responsible for the active accumulation of iodide within the thyroid gland and for the subsequent biosynthesis of thyroid hormones. However, several tissues, such as salivary glands, breast, stomach, colon, ovary and endometrium, express NIS even if they are unable to organify iodide. In order to investigate a possible role of NIS in the endometrium, we analyzed, by immunochemistry, the expression of NIS in 44 endometrial samples of 20 patients with primary unexplained infertility, 14 fertile women and 10 in postmenopausal. NIS immunostaining was detected in endometrial cells belonging to the majority of sterile, post-menopausal and fertile women. However, the sterile and post-menopausal patients showed a higher percentage of NIS reactive cells compared to the fertile women (60+/-21% and 57+/-18% vs 19+/-9%; p=0.0001). NIS immunostaining was localized on the membrane and cytoplasm of the endometrial cells. We could not find any correlation between endometrial thickness and NIS immunoexpression. Our results indicate that, in the absence of histological markers, a sterile endometrium can be recognized because of the high expressions of NIS. Moreover, NIS expressions, elevated in both sterile and menopause women, is not related to the estrogen levels, but it could be modulated by factors common to the two conditions. In conclusion, we speculate that NIS may play a role in the development of female sterility.     

A framework for scabies control

Scabies is a neglected tropical disease (NTD) that causes a significant health burden, particularly in disadvantaged communities and where there is overcrowding. There is emerging evidence that ivermectin-based mass drug administration (MDA) can reduce the prevalence of scabies in some settings, but evidence remains limited, and there are no formal guidelines to inform control efforts. An informal World Health Organization (WHO) consultation was organized to find agreement on strategies for global control. The consultation resulted in a framework for scabies control and recommendations for mapping of disease burden, delivery of interventions, and establishing monitoring and evaluation. Key operational research priorities were identified. This framework will allow countries to set control targets for scabies as part of national NTD strategic plans and develop control strategies using MDA for high-prevalence regions and outbreak situations. As further evidence and experience are collected and strategies are refined over time, formal guidelines can be developed. The control of scabies and the reduction of the health burden of scabies and associated conditions will be vital to achieving the targets set in WHO Roadmap for NTDs for 2021 to 2030 and the Sustainable Development Goals.     

Gene-linked shift in ganglioside distribution influences growth and vascularity in a mouse astrocytoma

Brain tumor growth and progression is dependent upon vascularity, and is associated with altered ganglioside composition and distribution. In this study, we examined the influence of gangliosides on growth and vascularity in a malignant mouse astrocytoma, CT-2A. Ganglioside distribution was altered in CT-2A tumor cells using an antisense construct to beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T), a key enzyme that uses the simple ganglioside GM3 as a substrate for the synthesis of the more complex gangliosides, GM2, GM1 and GD1a. GalNAc-T gene expression was significantly lower in CT-2A cells stably transfected with the antisense GalNAc-T plasmid, pcDNA3.1/TNG (CT-2A/TNG) than in either non-transfected CT-2A or mock-transfected (CT-2A/V) control tumor cells. GM3 was elevated from 16% to 58% of the total ganglioside distribution, whereas GM1 and GD1a were reduced from 17% and 49% to 10% and 17%, respectively, in CT-2A/TNG tumor cells. Growth, vascularity (blood vessel density and Matrigel assay) and vascular endothelial growth factor (VEGF) expression was significantly less in CT-2A/TNG tumors than in control CT-2A brain tumors. In addition, the expression of VEGF, hypoxia-inducible factor 1alpha (HIF-1alpha) and neuropilin-1 (NP-1) was significantly lower in CT-2A/TNG tumor cells than in control CT-2A tumor cells. These data suggest that gene-linked changes in ganglioside composition influence the growth and angiogenic properties of the CT-2A astrocytoma.     

Copper removal ability by Streptomyces strains with dissimilar growth patterns and endowed with cupric reductase activity

Morphological, physiological and molecular characterization of three copper-resistant actinobacterial strains (AB2A, AB3 and AB5A) isolated from copper-polluted sediments of a drainage channel showed that they belonged to the genus Streptomyces. These characteristics plus their distinctive copper resistance phenotypes revealed considerable divergence among the isolates. Highly dissimilar growth patterns and copper removal efficiency were observed for the selected Streptomyces strains grown on minimal medium (MM) added with 0.5 mM of copper sulfate (MM(Cu)). Strain AB2A showed an early mechanism of copper uptake/retention (80% until day 3), followed by a drastic metal efflux process (days 5-7). In contrast, Streptomyces sp. AB3 and AB5A showed only copper retention phenotypes under the same culture conditions. Particularly, Streptomyces sp. AB5A showed a better efficiency in copper removal (94%), although a longer lag phase was observed for this microorganism grown for 7 days in MM(Cu). Cupric reductase activity was detected in both copper-adapted cells and nonadapted cells of all three strains but this activity was up to 100-fold higher in preadapted cells of Streptomyces sp. AB2A. To our knowledge, this is the first time that cupric reductase activity was demonstrated in Streptomyces strains.     

PB183, a sigma receptor ligand, as a potential PET probe for the imaging of prostate adenocarcinoma

PB183, a non-selective sigma receptor ligand displaying high sigma-1 and sigma-2 receptor affinity, was evaluated in prostate tumour cell lines for its suitability as PET radiotracer. The pharmacodynamic and pharmacokinetic properties suggested PB183 as a potential PET radiotracer to visualize prostate adenocarcinoma.     

Picoinjection of Microfluidic Drops Without Metal Electrodes

Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of these electrodes adds cumbersome and error-prone steps to the device fabrication process. We have developed a technique that obviates the needs for metal electrodes during picoinjection. Instead, it uses the injection fluid itself as an electrode, since most biological reagents contain dissolved electrolytes and are conductive. By eliminating the electrodes, we reduce device fabrication time and complexity, and make the devices more robust. In addition, with our approach, the injection volume depends on the voltage applied to the picoinjection solution; this allows us to rapidly adjust the volume injected by modulating the applied voltage. We demonstrate that our technique is compatible with reagents incorporating common biological compounds, including buffers, enzymes, and nucleic acids.     

Nitric oxide production is required for murine resident peritoneal macrophages to suppress mitogen-stimulated T cell proliferation. Role of IFN-gamma in the induction of the nitric oxide-synthesizing pathway

Lymphocyte proliferation in Con A- or LPS-stimulated murine splenic cell (SC) cultures was suppressed by the addition of excess macrophages. In Con A-stimulated cultures, suppression was associated with the expression of nitric oxide-synthesizing pathway (NOSP) activity as demonstrated by the accumulation of nitrite, a degradation product of nitric oxide (NO), in the culture supernatants. That NO, a cytotoxic and anti-proliferative metabolite of l-arginine, or other reactive nitrogen intermediates generated through the NOSP mediated the suppressive effect was suggested by the reversal of suppression brought about by the addition of a specific inhibitor of the NOSP (NG-monomethyl-l-arginine acetate) to the culture media. No NOSP activity was detectable in LPS-stimulated SC/macrophage cocultures. The role of T cell-derived IFN-gamma in the induction of the NOSP was investigated by the use of anti-IFN-gamma-mAb. Antibody-treated Con A supernatants failed to induce the NOSP in macrophages, and the addition of the mAb to Con A-stimulated SC/macrophage cocultures obviated the suppressive effects. Indomethacin and catalase only partially restored proliferation in Con A-stimulated SC/macrophage cocultures but were remarkably efficient in preventing macrophage-dependent suppression when LPS was used as the mitogenic stimulus. These results demonstrate a regulatory system of potential relevance in sites of predominant macrophage infiltration by which T cell-derived IFN-gamma activates the production of the mediator, NO, that suppresses T cell proliferation. In addition, these data demonstrate that, although the suppressive effects of excess macrophages appear to be expressed nonspecifically toward both T and B cells, suppression is mediated through a different mechanism in each case.     

Simul taneous production of alpha and beta amylases byBacillus subtilis MIR-5 in batch and continuous culture

Summary Simultaneous production of - and -amylase was studied in batch and continuous culture using starch as substrate inB. subtilis MIR-5. By mainipulating the cultural condition, both enzymes could then be produced by the same strain.Footnote: dedicated to 75^th aniverssary of Dr. Raul E. Trucco.