Increased production of IL-4 and IL-12p40 from bronchoalveolar lavage cells are biomarkers of Mycobacterium tuberculosis in the sputum

BACKGROUND: Tuberculosis (TB) causes 1.45 million deaths annually world wide, the majority of which occur in the developing world. Active TB disease represents immune failure to control latent infection from airborne spread. Acid-fast bacillus (AFB) seen on sputum smear is a biomarker for contagiousness. METHODS: We enrolled 73 tuberculosis patients with extensive infiltrates into a research study using bronchoalveolar lavage (BAL) to sample lung immune cells and assay BAL cell cytokine production. All patients had sputum culture demonstrating Mycobacterium tuberculosis and 59/73 (81%) had AFB identified by microscopy of the sputum. Compared with smear negative patients, smear positive patients at presentation had a higher proportion with smoking history, a higher proportion with temperature >38.5 0 C, higher BAL cells/ml, lower percent lymphocytes in BAL, higher IL-4 and IL-12p40 in BAL cell supernatants. There was no correlation between AFB smear and other BAL or serum cytokines. Increasing IL-4 was associated with BAL PMN and negatively associated with BAL lymphocytes. Each 10-fold increase in BAL IL-4 and IL-12p40 increased the odds of AFB smear positivity by 7.4 and 2.2-fold, respectively, in a multi-variable logistic model. CONCLUSION: Increasing IL-4 and IL-12p40 production by BAL cells are biomarkers for AFB in sputum of patients who present with radiographically advanced TB. They likely reflect less effective immune control of pathways for controlling TB, leading to patients with increased infectiousness

Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis

BACKGROUND: Hepatocellular carcinoma (HCC) presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes. METHODS: We have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors. RESULTS: While the following sixteen genes remained the unmethylated in all tumor and normal tissues: CDH1, APAF1, hMLH1, BRCA1, hTERC, VHL, RARβ, TIMP3, DAPK1, SURVIVIN, p14(ARF), RB1, p15(INK4b), APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16(INK4a ), RASSF1a, CASP8 and CDH13 genes. For instance, the p16(INK4a )was highly methylated in HCC (17/29, 58.6%) and less significantly methylated in non-cancerous tissue (4/29. 13.79%). The RASSF1a was fully methylated in all tumor tissues (29/29, 100%), and less frequently methylated in corresponding non-cancerous tissue (24/29, 82.75%). CONCLUSIONS: Furthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation) mechanisms may act in concert to inactivate the p16(INK4a )and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16(INK4a )and hypomethylation of the CDH13 genes. For the first time, the survey was carried out on such an extent that it would not only provide new insights into the molecular mechanisms underscoring the aberrant expression of the genes in this study in HCC, but also offer essential information required for a good methylation-based diagnosis of HCC

Nicotinic acetylcholine receptors modulate osteoclastogenesis

Background: Our aim was to investigate the role of nicotinic acetylcholine receptors (nAChRs) in in-vitro osteoclastogenesis and in in-vivo bone homeostasis. Methods: The presence of nAChR subunits as well as the in-vitro effects of nAChR agonists were investigated by ex vivo osteoclastogenesis assays, real-time polymerase chain reaction, Western blot and flow cytometry in murine bone marrow-derived macrophages differentiated in the presence of recombinant receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The bone phenotype of mice lacking various nAChR subunits was investigated by peripheral quantitative computed tomography and histomorphometric analysis. Oscillations in the intracellular calcium concentration were detected by measuring the Fura-2 fluorescence intensity. Results: We could demonstrate the presence of several nAChR subunits in bone marrow-derived macrophages stimulated with RANKL and M-CSF, and showed that they are capable of producing acetylcholine. nAChR ligands reduced the number of osteoclasts as well as the number of tartrate-resistant acidic phosphatase-positive mononuclear cells in a dose-dependent manner. In vitro RANKL-mediated osteoclastogenesis was reduced in mice lacking α7 homomeric nAChR or β2-containing heteromeric nAChRs, while bone histomorphometry revealed increased bone volume as well as impaired osteoclastogenesis in male mice lacking the α7 nAChR. nAChR ligands inhibited RANKL-induced calcium oscillation, a well-established phenomenon of osteoclastogenesis. This inhibitory effect on Ca2+ oscillation subsequently led to the inhibition of RANKL-induced NFATc1 and c-fos expression after long-term treatment with nicotine. Conclusions: We have shown that the activity of nAChRs conveys a marked effect on osteoclastogenesis in mice. Agonists of these receptors inhibited calcium oscillations in osteoclasts and blocked the RANKL-induced activation of c-fos and NFATc1. RANKL-mediated in-vitro osteoclastogenesis was reduced in α7 knockout mice, which was paralleled by increased tibial bone volume in male mice in vivo. © 2016 Mandl et al

Pregnant Behind Bars: Meeting the Nutrition Needs of Incarcerated Pregnant Women

The number of women involved in the criminal justice system has increased dramatically over the past 20 years. Due to their marginalized background, incarcerated women have a complex set of health-related needs. This is especially true of those who are pregnant, a particularly vulnerable, high-risk group. Although guidelines have been developed that recommend pregnancy screening, provision of dietary supplements, regular nutritious meals, and nutritional counseling for incarcerated pregnant women, jail policies and health care protocols often fail to heed these recommendations. In this chapter, we discuss the nutritional needs of pregnant incarcerated women as well as breastfeeding in the context of the criminal justice system and consider some of the challenges in developing programming and policies to address these health-related needs. We also present findings from the William & Mary Healthy Beginnings Project, a nutrition intervention program developed for pregnant incarcerated women in Southeastern Virginia. Assessment of this program suggests that through the development of protocols and polices that consider the health-related needs of pregnant women, correctional facilities could play a pivotal role in helping incarcerated women develop healthier habits to better care for themselves and their newborns.https://scholarworks.wm.edu/asbookchapters/1106/thumbnail.jp

Effects of long-term weekly iron and folic acid supplementation on lower genital tract infection - a double blind, randomised controlled trial in Burkina Faso

BACKGROUND: Provision of routine iron supplements to prevent anaemia could increase the risk for lower genital tract infections as virulence of some pathogens depends on iron availability. This trial in Burkina Faso assessed whether weekly periconceptional iron supplementation increased the risk of lower genital tract infection in young non-pregnant and pregnant women. METHODS: Genital tract infections were assessed within a double blind, controlled, non-inferiority trial of malaria risk among nulliparous women, randomised to receive either iron and folic acid or folic acid alone, weekly, under direct observation for 18 months. Women conceiving during this period entered the pregnancy cohort. End assessment (FIN) for women remaining non-pregnant was at 18 months. For the pregnancy cohort, end assessment was at the first scheduled antenatal visit (ANC1). Infection markers included Nugent scores for abnormal flora and bacterial vaginosis (BV), T. vaginalis PCR, vaginal microbiota, reported signs and symptoms, and antibiotic and anti-fungal prescriptions. Iron biomarkers were assessed at baseline, FIN and ANC1. Analysis compared outcomes by intention to treat and in iron replete/deficient categories. RESULTS: A total of 1954 women (mean 16.8 years) were followed and 478 (24.5%) became pregnant. Median supplement adherence was 79% (IQR 59-90%). Baseline BV prevalence was 12.3%. At FIN and ANC1 prevalence was 12.8% and 7.0%, respectively (P < 0.011). T. vaginalis prevalence was 4.9% at FIN and 12.9% at ANC1 (P < 0.001). BV and T. vaginalis prevalence and microbiota profiles did not differ at trial end-points. Iron-supplemented non-pregnant women received more antibiotic treatments for non-genital infections (P = 0.014; mainly gastrointestinal infections (P = 0.005), anti-fungal treatments for genital infections (P = 0.014) and analgesics (P = 0.008). Weekly iron did not significantly reduce iron deficiency prevalence. At baseline, iron-deficient women were more likely to have normal vaginal flora (P = 0.016). CONCLUSIONS: Periconceptional weekly iron supplementation of young women did not increase the risk of lower genital tract infections but did increase general morbidity in the non-pregnant cohort. Unabsorbed gut iron due to malaria could induce enteric infections, accounting for the increased administration of antibiotics and antifungals in the iron-supplemented arm. This finding reinforces concerns about routine iron supplementation in highly malarious areas

Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis

Prokaryotic ubiquitin-like protein (Pup) in Mycobacterium tuberculosis (Mtb) is the first known post-translational small protein modifier in prokaryotes, and targets several proteins for degradation by a bacterial proteasome in a manner akin to ubiquitin (Ub) mediated proteolysis in eukaryotes. To determine the extent of pupylation in Mtb, we used tandem affinity purification to identify its “pupylome”. Mass spectrometry identified 55 out of 604 purified proteins with confirmed pupylation sites. Forty-four proteins, including those with and without identified pupylation sites, were tested as substrates of proteolysis in Mtb. Under steady state conditions, the majority of the test proteins did not accumulate in degradation mutants, suggesting not all targets of pupylation are necessarily substrates of the proteasome under steady state conditions. Four proteins implicated in Mtb pathogenesis, Icl (isocitrate lyase), Ino1 (inositol-1-phosphate synthase), MtrA (Mtb response regulator A) and PhoP (phosphate response regulator P), showed altered levels in degradation defective Mtb. Icl, Ino1 and MtrA accumulated in Mtb degradation mutants, suggesting these proteins are targeted to the proteasome. Unexpectedly, PhoP was present in wild type Mtb but undetectable in the degradation mutants. Taken together, these data demonstrate that pupylation regulates numerous proteins in Mtb and may not always lead to degradation

First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

Background: in a previous study, we detected the presence of a Mycobacterium avium species-specific insertion sequence, IS1245, in Mycobacterium kansasii. Both species were isolated from a mixed M. avium-M. kansasii bone marrow culture from an HIV-positive patient. the transfer mechanism of this insertion sequence to M. kansasii was investigated here.Methodology/Principal Findings: A linear plasmid (pMA100) was identified in all colonies isolated from the M. avium-M. kansasii mixed culture carrying the IS1245 element. the linearity of pMA100 was confirmed. Other analyses suggested that pMA100 contained a covalently bound protein in the terminal regions, a characteristic of invertron linear replicons. Partial sequencing of pMA100 showed that it bears one intact copy of IS1245 inserted in a region rich in transposase-related sequences. These types of sequences have been described in other linear mycobacterial plasmids. Mating experiments were performed to confirm that pMA100 could be transferred in vitro from M. avium to M. kansasii. pMA100 was transferred by in vitro conjugation not only to the M. kansasii strain from the mixed culture, but also to two other unrelated M. kansasii clinical isolates, as well as to Mycobacterium bovis BCG Moreau.Conclusions/Significance: Horizontal gene transfer (HGT) is one of most important mechanisms leading to the evolution and diversity of bacteria. This work provides evidence for the first time on the natural occurrence of HGT between different species of mycobacteria. Gene transfer, mediated by a novel conjugative plasmid, was detected and experimentally reproduced.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Cooperacion Interuniversitaria UAM-Banco Santander con America Latina (CEAL), UAM, SpainConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilLab Nacl Comp Cient, Petropolis, BrazilUniv Autonoma Madrid, Fac Med, Dept Prevent Med, Madrid, SpainInst Adolfo Lutz Registro, Nucleo TB & Micobacterioses, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilFAPESP: FAPESP - 06/01533-9Web of Scienc

High Content Phenotypic Cell-Based Visual Screen Identifies Mycobacterium tuberculosis Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials