Interleukin-17 regulates matrix metalloproteinase activity in human pulmonary tuberculosis.

Tuberculosis (TB) is characterized by extensive pulmonary matrix breakdown. Interleukin-17 (IL-17) is key in host defence in TB but its role in TB-driven tissue damage is unknown. We investigated the hypothesis that respiratory stromal cell matrix metalloproteinase (MMP) production in TB is regulated by T-helper 17 (TH -17) cytokines. Biopsies of patients with pulmonary TB were analysed by immunohistochemistry (IHC), and patient bronchoalveolar lavage fluid (BALF) MMP and cytokine concentrations were measured by Luminex assays. Primary human airway epithelial cells were stimulated with conditioned medium from human monocytes infected with Mycobacterium tuberculosis (Mtb) and TH -17 cytokines. MMP secretion, activity, and gene expression were determined by ELISA, Luminex assay, zymography, RT-qPCR, and dual luciferase reporter assays. Signalling pathways were examined using phospho-western analysis and siRNA. IL-17 is expressed in TB patient granulomas and MMP-3 is expressed in adjacent pulmonary epithelial cells. IL-17 had a divergent, concentration-dependent effect on MMP secretion, increasing epithelial secretion of MMP-3 (p < 0.001) over 72 h, whilst decreasing that of MMP-9 (p < 0.0001); mRNA levels were similarly affected. Both IL-17 and IL-22 increased fibroblast Mtb-dependent MMP-3 secretion but IL-22 did not modulate epithelial MMP-3 expression. Both IL-17 and IL-22, but not IL-23, were significantly up-regulated in BALF from TB patients. IL-17-driven MMP-3 was dependent on p38 MAP kinase and the PI3K p110α subunit. In summary, IL-17 drives airway stromal cell-derived MMP-3, a mediator of tissue destruction in TB, alone and with monocyte-dependent networks in TB. This is regulated by p38 MAP kinase and PI3K pathways. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Molecular Epidemiology of Extended-Spectrum ß-Lactamase-Producing Escherichia coli Pathotypes in Diarrheal Children from Low Socioeconomic Status Communities in Bihar, India: Emergence of the CTX-M Type.

BACKGROUND: Childhood diarrheal diseases remain highly endemic in India, but the emergence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli among children with diarrhea in Bihar remains elusive. In this study, we determine and characterize ESBL-producing E coli pathotypes among hospitalized diarrheal preschool children living in low socioeconomic level communities in Bihar, India. MATERIALS AND METHODS: The stool samples were collected everyday throughout the year for 2 consecutive years. In our study, we collected stool samples randomly from every fifth patient. Stool samples were collected from a total of 633 randomly selected diarrheal children (age: 0-60 months) belonging to 17 communities and screened for identification of virulent diarrheagenic E coli (DEC) pathotype (viz, enteropathogenic E coli [EPEC], enteroaggregative E coli [EAEC], enterotoxigenic E coli [ETEC], enteroinvasive E coli [EIEC], and enterohemorrhagic E coli [EHEC]) by a multiplex polymerase chain reaction (PCR) assay. Furthermore, ESBLs were screened by conventional antibiotic resistance pattern testing and later characterized for the presence of ß-lactamase (bla) genes by PCR and DNA sequencing. RESULTS: Diarrheagenic E coli was detected in 191 cases (30.2%) of the total 633 diarrheic children. Maximum occurrence of DEC was found in ≤12 months age group (72.7%) with prevalence of the EAEC pathotype. Most isolates were resistant to ampicillin, ciprofloxacin, piperacillin, levofloxacin, ceftazidime, cefotaxime, ceftriaxone, and gentamicin, whereas over 96% of them were sensitive to amikacin. About 37.6% of total 191 DEC isolates were ESBL producers (n = 72), being prevalent among ETEC (n = 35; 18.32%), followed by EPEC (n = 21; 10.9%), EAEC (n = 13; 6.8%), and EIEC (n = 3; 1.57%). Interestingly, the commonest ß-lactamase was CTX-M type (blaCTX-M) in 86.1% (n = 62) of the ESBL isolates, followed by blaSHV (n = 49; 68%), blaTEM (n = 37; 51.8%), and blaOXA (n = 21; 29.1%) determinants. Resistance of ESBL isolates was mostly related to ampicillin (100%), ceftriaxone (98.1%), cefotaxime (92.4%), gentamicin (74.1%), and levofloxacin (73.2%), whereas best antimicrobial activities were observed for piperacillin-tazobactam, amikacin, meropenem, and imipenem. CONCLUSIONS: This study revealed that EAEC (72.1%) is the predominant pathotype in Bihar, significantly high in ≤12 months age group children (P = .04). Moreover, the widespread prevalence of ESBLs in children, especially the CTX-M type, is of great concern, which requires monitoring of infection control measures through efficient antimicrobial management and detection of ESBL-producing isolates.

Antimycobacterial drugs modulate immunopathogenic matrix metalloproteinases in a cellular model of pulmonary tuberculosis.

Tuberculosis is characterized by extensive destruction and remodelling of the pulmonary extracellular matrix. Stromal cell-derived matrix metalloproteinases (MMPs) are implicated in this process and may be a target for adjunctive immunotherapy. We hypothesized that MMPs are elevated in bronchoalveolar lavage fluid of tuberculosis patients and that antimycobacterial agents may have a modulatory effect on MMP secretion. Concentrations of MMP-1, -2, -3, -7, -8, and -9 were elevated in the bronchoalveolar lavage fluid from tuberculosis patients compared to those in bronchoalveolar lavage fluid from patients with other pulmonary conditions. There was a positive correlation between MMP-3, MMP-7, and MMP-8 and a chest radiological score of cavitation and parenchymal damage. Respiratory epithelial cell-derived MMP-3 was suppressed by moxifloxacin, rifampicin, and azithromycin in a dose-dependent manner. Respiratory epithelial cell-derived MMP-1 was suppressed by moxifloxacin and azithromycin, whereas MMP-9 secretion was only decreased by moxifloxacin. In contrast, moxifloxacin and azithromycin both increased MMP-1 and -3 secretion from MRC-5 fibroblasts, demonstrating that the effects of these drugs are cell specific. Isoniazid did not affect MMP secretion. In conclusion, MMPs are elevated in bronchoalveolar lavage fluid from tuberculosis patients and correlate with parameters of tissue destruction. Antimycobacterial agents have a hitherto-undescribed immunomodulatory effect on MMP release by stromal cells.

Telescoped approach to aryl hydroxymethylation in the synthesis of a key pharmaceutical intermediate.

An efficient synthetic approach leading to introduction of the hydroxymethyl group to an aryl moiety via combination of the Bouveault formylation and hydride reduction has been optimized using a rational, mechanistic-based approach. This approach enabled telescoping of the two steps into a single efficient process, readily amenable to scaleup.

Stem cell through present and future.

Stem cell transplantation (SCT) has the potential to transform the lives of children with a wide variety of genetic diseases, ranging from inherent defects of hemopoietic cell production or function through to metabolic diseases mostly affecting solid organs. For these children life expectancy or quality of life would otherwise be very poor. It ranks as one of the most remarkable therapeutic advances of the past 40 years. Despite rapid technological improvements, however, there are still many short term risks and potential long term toxicities. Consequently, the rapid emergence of alternative therapies (including new drugs, enzyme and gene therapies), necessitate constant re-evaluation of the risk/benefit ratio for each disease and hence the appropriateness of SCT. This review describes the major aspects of the transplant process, indications for transplantation, outcome statistics, and areas where alternative therapies are becoming available. SCT remains a highly experimental therapy. Due to the relatively short history of the discipline no data exists on truly long term follow up. This is important as some organs benefit relatively poorly or problems may emerge which were never apparent as part of the untreated disease. The speed of technological change makes randomised trials on these diseases, which are individually quite rare, almost impossible to perform.

Mechanistic insights into the Pd(BINAP)-catalyzed amination of aryl bromides: kinetic studies under synthetically relevant conditions.

Kinetic studies using reaction calorimetry were carried out under synthetically relevant conditions to study the mechanism of the amination of bromobenzene with primary and secondary amines using Pd(2)(dba)(3)/BINAP mixtures as well as preformed (dba)Pd(BINAP), (p-tolyl)(Br)Pd(BINAP), and Pd(BINAP)(2) complexes. The presence of a significant induction period in the reaction was attributed to the slow activation of the catalytic precursor, resulting in an increase in the concentration of active species within the catalytic cycle. The induction period can mask the true kinetics of the reaction, which exhibits positive order dependences on aryl bromide and amine and zero-order dependence on base. It is also determined that the bis-ligand complex Pd(BINAP)(2) does not play a role directly on the catalytic cycle. In addition to the conventionally accepted pathway involving oxidative addition of the aryl halide to (BINAP)Pd as the first step, a pathway initiated by addition of the amine to the catalyst is proposed and supported by kinetic modeling of sequential reaction experiments. A subtle dependence of the reaction mechanism on the relative concentrations of substrates is revealed in these studies. The dependence of the catalyst resting state on reaction conditions is also discussed. This work suggests that conclusions from kinetic studies may be meaningful only for the conditions under which they are carried out, calling into question the use of conventional kinetic methods in this system.