Insertion Element IS6110 based characterisation of Nepalese tuberculosis strains into different genetic lineages

Nepal is geographically located between India and China, a region containing significant Tuberculosis (TB) and Multi-Drug Resistance (MDR-TB) burdens. However, limited information is available on the phylogenetic diversity of Mycobacterium tuberculosis (Mtb) in Nepal. To gain further insight into the diversity of Mtb in Nepal, consecutive clinical samples from 176 newly diagnosed pulmonary tuberculosis patients were collected from two hospitals in Nepal. Insertion Site IS6110 Fluorescent Amplified Fragment Length Polymorphism (FAFLP) PCR and rpoB sequence analysis were carried out on genomic DNA extracts of cultured strains to assign them to accepted genetic lineages and identify MDR-TB. In this study, the IS6110 based characterisation showed a prevalence of 36.36% Central Asian Strain (CAS), 18.75% Beijing, 7.95 % Haarlem, 3.97% X, 2.2% each of Latin American Mediterranean (LAM), T-Uganda and T, 1.7% S and 24.4% were unassigned. Further, 3.9% of total M. tuberculosis isolates were of rifampicin resistant genotypes thus indicating that the prevalence of MDR could be higher than the country wide prevalence of MDR among new TB cases (2.2%) as reported by the national drug resistance survey carried out in 2011/2012

Magnesium bioavailability after administration of sucrosomial (R) magnesium: results of an ex-vivo study and a comparative, double-blinded, cross-over study in healthy subjects

OBJECTIVE: We conducted an ex-vivo analysis and a study in healthy subjects to compare magnesium bioavailability after administration of Sucrosomial (R) magnesium or commercially available preparations of magnesium citrate, magnesium oxide and magnesium bisglycinate. MATERIALS AND METHODS: In the ex-vivo study we simulated magnesium intestinal absorption after digestion through sections of intestinal mucosa isolated from rats. We compared the absorption of magnesium oxide and Sucrosomial (R) magnesium at two different concentrations: 32.9 mg/ml and 329 mg/ml. The human study was a single day double-blinded repeated crossover study in healthy subjects. Each subject was administered 350 mg magnesium in different formulations (Sucrosomial (R) magnesium, magnesium citrate, magnesium oxide or magnesium bisglycinate) after 1 week of washout. We collected blood and urine samples to measure magnesium concentration in blood, urine and red blood cells. RESULTS: The ex-vivo evaluation showed that magnesium absorption after administration of Sucrosomial (R) magnesium was faster and with higher rates compared to a standard formulation of magnesium oxide. This finding was further confirmed by the results of the study in healthy subjects, that showed a more evident increase in magnesium concentration after administration of Sucrosomial (R) magnesium compared to the other formulations. In particular, the increase in magnesium concentration from baseline to 24 h was statistically higher in blood and in urine for Sucrosomial (R) magnesium compared to magnesium oxide, while in red blood cells Sucrosomial (R) magnesium had a statistically significant advantage compared to magnesium bisglycinate. CONCLUSIONS: Our findings suggest that Sucrosomial (R) magnesium leads to an increased bioavailability of magnesium compared to other formulations. Further studies are needed to investigate if this advantage turns into more evident clinical efficacy

Human beta-defensin 3 is up-regulated in cutaneous leprosy type 1 reactions.

BACKGROUND: Leprosy, a chronic granulomatous disease affecting the skin and nerves, is caused by Mycobacterium leprae (M. leprae). The type of leprosy developed depends upon the host immune response. Type 1 reactions (T1Rs), that complicate borderline and lepromatous leprosy, are due to an increase in cell-mediated immunity and manifest as nerve damage and skin inflammation. Owing to the increase in inflammation in the skin of patients with T1Rs, we sought to investigate the activation of the innate immune system during reactionary events. Specifically, we investigated the expression levels of human beta-defensins (hBDs) 2 and 3 in the skin of patients with T1Rs, in keratinocytes, and in macrophages stimulated with M. leprae and corticosteroids. RESULTS: Skin biopsies from twenty-three patients with Type 1 reactions were found to have higher transcript levels of hBD3 as compared to fifteen leprosy patients without Type 1 reactions, as measured by qPCR. Moreover, we observed that keratinocytes but not macrophages up-regulated hBD2 and hBD3 in response to M. leprae stimulation in vitro. Corticosteroid treatment of patients with T1Rs caused a suppression of hBD2 and hBD3 in skin biopsies, as measured by qPCR. In vitro, corticosteroids suppressed M. leprae-dependent induction of hBD2 and hBD3 in keratinocytes. CONCLUSIONS: This study demonstrates that hBD3 is induced in leprosy Type 1 Reactions and suppressed by corticosteroids. Furthermore, our findings demonstrate that keratinocytes are responsive to M. leprae and lend support for additional studies on keratinocyte innate immunity in leprosy and T1Rs. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN31894035

The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans.

Exposure to Mycobacterium tuberculosis produces varied early outcomes, ranging from resistance to infection to progressive disease. Here we report results from a forward genetic screen in zebrafish larvae that identify multiple mutant classes with distinct patterns of innate susceptibility to Mycobacterium marinum. A hypersusceptible mutant maps to the lta4h locus encoding leukotriene A(4) hydrolase, which catalyzes the final step in the synthesis of leukotriene B(4) (LTB(4)), a potent chemoattractant and proinflammatory eicosanoid. lta4h mutations confer hypersusceptibility independent of LTB(4) reduction, by redirecting eicosanoid substrates to anti-inflammatory lipoxins. The resultant anti-inflammatory state permits increased mycobacterial proliferation by limiting production of tumor necrosis factor. In humans, we find that protection from both tuberculosis and multibacillary leprosy is associated with heterozygosity for LTA4H polymorphisms that have previously been correlated with differential LTB(4) production. Our results suggest conserved roles for balanced eicosanoid production in vertebrate resistance to mycobacterial infection

T-Cell Regulation in Lepromatous Leprosy

Regulatory T (T(reg)) cells are known for their role in maintaining self-tolerance and balancing immune reactions in autoimmune diseases and chronic infections. However, regulatory mechanisms can also lead to prolonged survival of pathogens in chronic infections like leprosy and tuberculosis (TB). Despite high humoral responses against Mycobacterium leprae (M. leprae), lepromatous leprosy (LL) patients have the characteristic inability to generate T helper 1 (Th1) responses against the bacterium. In this study, we investigated the unresponsiveness to M. leprae in peripheral blood mononuclear cells (PBMC) of LL patients by analysis of IFN-γ responses to M. leprae before and after depletion of CD25(+) cells, by cell subsets analysis of PBMC and by immunohistochemistry of patients' skin lesions. Depletion of CD25(+) cells from total PBMC identified two groups of LL patients: 7/18 (38.8%) gained in vitro responsiveness towards M. leprae after depletion of CD25(+) cells, which was reversed to M. leprae-specific T-cell unresponsiveness by addition of autologous CD25(+) cells. In contrast, 11/18 (61.1%) remained anergic in the absence of CD25(+) T-cells. For both groups mitogen-induced IFN-γ was, however, not affected by depletion of CD25(+) cells. In M. leprae responding healthy controls, treated lepromatous leprosy (LL) and borderline tuberculoid leprosy (BT) patients, depletion of CD25(+) cells only slightly increased the IFN-γ response. Furthermore, cell subset analysis showed significantly higher (p = 0.02) numbers of FoxP3(+) CD8(+)CD25(+) T-cells in LL compared to BT patients, whereas confocal microscopy of skin biopsies revealed increased numbers of CD68(+)CD163(+) as well as FoxP3(+) cells in lesions of LL compared to tuberculoid and borderline tuberculoid leprosy (TT/BT) lesions. Thus, these data show that CD25(+) T(reg) cells play a role in M. leprae-Th1 unresponsiveness in LL