Quantification of Enteric Dysfunction in Cystic Fibrosis: Inter- and Intraindividual Variability.

OBJECTIVES: To test the utility of various biomarkers as indicators of gut dysfunction in cystic fibrosis (CF) and determine whether intraindividual variations in these measures are repeatable over short intervals and whether interindividual variations correlate with clinical outcomes. STUDY DESIGN: We performed a cross-sectional, limited longitudinal study of children with CF aged 1-21 years who provided blood and stool samples at 2 or 3 visits, 2 weeks and 3 months apart, which were assayed for markers of intestinal inflammation (fecal calprotectin [fCal], lipocalin-2 [fLcn2], neopterin), and permeability (plasma lipopolysaccharide [LPS] antibodies, LPS-binding protein) by enzyme immunoassays. Control specimens were obtained from children without CF who had undergone esophagogastroduodenoscopy and had no evidence of gut inflammation. RESULTS: Twenty-six of 29 participants with CF completed the study. Sixty-nine stools (57 case/12 control) and 76 plasmas (60 case/16 control) were analyzed. LPS antibody had reliable intraindividual stability. fCal, fLcn2, and neopterin were significantly greater in CF than in control samples. fCal was negatively correlated with 3-month interval change (Δ) in weight-for-age z-score, body mass index/weight-for-length z-score, and forced expiratory volume in 1 second. fLcn2 was negatively correlated with FEV1 but not with anthropometrics. No marker correlated with Δbody mass index/weight-for-length z-score or ΔFEV1. CONCLUSIONS: fLcn2 is elevated in people with CF and might predict worse interval pulmonary function. Expanded studies are warranted to test if fLcn2 correlates with changes in additional outcomes.

Very-Early Onset Chronic Active Colitis with Heterozygous Variants in LRBA1 and CARD11, a Case of "Immune TOR-Opathies".

BACKGROUND: A small subset of cases of inflammatory bowel disease (IBD) occurs as a result of single gene defects, and typically occurs in young or very young pediatric patients, referred to as "monogenic very-early onset IBD (VEO-IBD)". The gene variants leading to monogenic VEO-IBD are often associated with primary immunodeficiency syndromes. CASE REPORT: A six year-old girl presented to our gastroenterology clinic with LRBA deficiency with a heterozygous mutation at c.1399 A > G, p Met467Val, histopathologic chronic active colitis without granulomas and clinical chronic colitis. Her gastrointestinal symptoms began at age 5 with bloody diarrhea, abdominal pain and weight loss. Whole exome sequencing revealed a CARD11 heterozygous de novo mutation (c.220 + 1G > A). She was in clinical remission on only abatacept. DISCUSSION: We present a case of monogenic VEO-IBD associated with two heterozygous variants in LRBA1 and CARD11, both considered as key players in the newly proposed "immune TOR-opathies".

Histological Evidence of Chronic Mycoplasma genitalium-Induced Cervicitis in HIV-Infected Women: A Retrospective Cohort Study.

BACKGROUND: Mycoplasma genitalium is an emerging sexually transmitted pathogen implicated in inflammatory syndromes of the female reproductive tract. The objective of this study was to investigate human immunodeficiency virus (HIV)-infected women for an association between M. genitalium and cervicitis, a putative mechanism for enhanced HIV transmission efficiency to an uninfected partner. METHODS: Using a longitudinal cohort of antiretroviral therapy-adherent New Orleans women, we retrospectively screened for M. genitalium and quantitatively characterized several markers of cervical inflammation, including secreted cytokines and cytological and histological signs of leukocyte infiltration. RESULTS: We observed a high prevalence of M. genitalium (7.4%) among HIV-infected New Orleans women. Chronic M. genitalium infection was associated with increased secretion of proinflammatory cytokines, including interleukin 1ß, interleukin 6, and interleukin 8, and marked inflammatory cervical infiltrates in the cervix with enrichment of HIV target cells. Cure of M. genitalium infection resulted in ablation of all signs of inflammation. CONCLUSIONS: These findings implicate M. genitalium as an etiologic agent of cervicitis in HIV-infected women, providing a potential mechanism for enhanced HIV transmission to an uninfected partner. Screening and treatment of M. genitalium among HIV-infected individuals may be warranted to further understand this coinfection scenario, improve cervical health, and reduce the spread of HIV.

Thymoquinone, a bioactive component of black caraway seeds, causes G1 phase cell cycle arrest and apoptosis in triple-negative breast cancer cells with mutant p53.

Thymoquinone (TQ) from black caraway seeds has several anticancer activities; however, its effect on triple-negative breast cancer (TNBC) cells that lack functional tumor suppressor p53 is not known. Here, we explored the growth inhibitory effect of TQ on 2 TNBC cell lines with mutant p53. Cell metabolism assays showed that TQ inhibited TNBC cell growth without affecting normal cell growth. Flow cytometric analyses of TQ-treated TNBC cells showed G1 phase cell cycle arrest and apoptosis characterized by the loss of mitochondrial membrane integrity. Western blots of lysates from TQ-treated TNBC cells showed cytochrome c and apoptosis-inducing factor in the cytoplasm, as well as caspase-9 activation consistent with the mitochondrial pathway of apoptosis. Caspase-8 was also activated in TQ-treated TNBC cells, although the mechanism of activation is not clear at this time. Importantly, TQ-induced apoptosis was only partially inhibited by zVAD-fmk, indicating a role for caspase-independent effector molecules. Poly(ADP-ribose) polymerase cleavage and increased γH2AX, as well as reduced Akt phosphorylation and decreased expression of X-linked inhibitor of apoptosis, were evident in TQ-treated cells. Finally, TQ enhanced cisplatin- and docetaxel-induced cytotoxicity. These findings suggest that TQ could be useful in the management of TNBC, even when functional p53 is absent.

Noninvasive Markers of Inflammation and Protein Loss Augment Diagnosis of Pediatric Celiac Disease.

INTRODUCTION: Circulating tissue transglutaminase immunoglobulin A concentration is a sensitive and specific indicator of celiac disease, but discrepancies between serologic and histologic findings occur. We hypothesized that fecal markers of inflammation and protein loss would be greater in patients with untreated celiac disease than in healthy controls. Our study aims to evaluate multiple fecal and plasma markers in celiac disease and correlate these findings with serologic and histologic findings as noninvasive means of evaluating disease activity. METHODS: Participants with positive celiac serologies and controls with negative celiac serologies were prospectively enrolled before upper endoscopy. Blood, stool, and duodenal biopsies were collected. Concentrations of fecal lipocalin-2, calprotectin, and alpha-1-antitrypsin and plasma lipocalin-2 were determined. Biopsies underwent modified Marsh scoring. Significance was tested between cases and controls, modified Marsh score and tissue transglutaminase immunoglobulin A concentration. RESULTS: Lipocalin-2 was significantly elevated in the stool ( P = 0.006) but not the plasma of participants with positive celiac serologies. There was no significant difference in fecal calprotectin or alpha-1 antitrypsin between participants with positive celiac serologies and controls. Fecal alpha-1 antitrypsin >100 mg/dL was specific, but not sensitive for biopsy-proven celiac disease. DISCUSSION: Lipocalin-2 is elevated in the stool but not the plasma of patients with celiac disease suggesting a role of local inflammatory response. Calprotectin was not a useful marker in the diagnosis of celiac disease. While random fecal alpha-1 antitrypsin was not significantly elevated in cases compared with controls, an elevation of greater than 100 mg/dL was 90% specific for biopsy-proven celiac disease.

Non-Invasive markers of inflammation and protein loss augment diagnosis of celiac disease.

Background: Circulating tissue transglutaminase IgA (TTG IgA) concentrations are sensitive and specific indicators of celiac disease, but discrepancies between serologic and histologic findings still occur. We hypothesized that fecal markers of inflammation and protein loss would be greater in patients with untreated celiac disease than in healthy controls. Our study aims to evaluate multiple fecal and plasma markers in celiac disease and correlate these findings with serologic and histologic findings as non-invasive means of evaluating disease activity. Methods: Participants with positive celiac serologies and controls with negative celiac serologies were enrolled at the time of upper endoscopy. Blood, stool and duodenal biopsies were collected. Concentrations of fecal lipocalin-2, calprotectin and alpha-1-antitrypsin and plasma lipcalin-2 were determined. Biopsies underwent modified Marsh scoring. Significance was tested between cases and controls, modified Marsh score and TTG IgA concentration. Results: Lipocalin-2 was significantly elevated in the stool ( p =0.007) but not the plasma of participants with positive celiac serologies compared to controls. There was no significant difference in fecal calprotectin or alpha-1 antitrypsin between participants with positive celiac serologies and controls. Fecal alpha-1 antitrypsin >100mg/dL was specific, but not sensitive for biopsy proven celiac disease. Conclusions: Lipocalin-2 is elevated in the stool but not the plasma of patients with celiac disease suggesting a role in the local inflammatory response. Calprotectin was not a useful marker in the diagnosis of celiac disease and did not correlate with degree of histologic changes on biopsy. While random fecal alpha-1 antitrypsin was not significantly elevated in cases compared to controls, an elevation of greater than 100mg/dL was 90% specific for biopsy proven celiac disease.

NADPH quinone oxidoreductase 1 mediates breast cancer cell resistance to thymoquinone-induced apoptosis.

Thymoquinone (TQ), a bioactive component of black caraway seed (Nigella sativa) oil, is reported to have antineoplastic properties. In this study we investigated the effect of TQ on a panel of human breast cancer cell lines. Cell viability assays showed that TQ killed T-47D, MDA-MB-231, and MDA-MB-468 cells via p53-independent induction of apoptosis; however, MCF-7 cells were refractory to the cytotoxic action of TQ. Western Blot analysis showed that MCF-7 cells expressed high levels of cytoprotective NADPH quinone oxidoreductase 1 (NQO1), which was responsible for TQ-resistance since inhibition of NQO1 with dicoumarol rendered MCF-7 cells TQ-sensitive. These findings may be clinically important when considering TQ as a possible adjunct treatment for breast cancer since a high percentage of breast tumors express NQO1.

Curcumin-induced apoptosis in PC3 prostate carcinoma cells is caspase-independent and involves cellular ceramide accumulation and damage to mitochondria.

Curcumin, the principal curcuminoid of tumeric, has potent anticancer activity. To determine the mechanism of curcumin-induced cytotoxicity in prostate cancer cells, we exposed PC3 prostate carcinoma cells to 25 to 100 microM curcumin for 24 to 72 h. Curcumin treatment of PC3 cells caused time- and dose-dependent induction of apoptosis and depletion of cellular reduced glutathione (GSH). Exogenous GSH and its precursor N-acetyl-cysteine, but not ascorbic acid (AA) or ebselen, decreased curcumin accumulation in PC3 cells and also prevented curcumin-induced DNA fragmentation. The failure of AA and ebselen to protect PC3 cells from curcumin-induced apoptosis argued against the involvement of reactive oxygen species; rather, GSH-mediated inhibition of curcumin-induced cytotoxicity was due to reduced curcumin accumulation in PC3 cells. Curcumin-treated PC3 cells showed apoptosis-inducing cellular ceramide accumulation and activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Caspase-3, caspase-8, and caspase-9 were activated, and cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria following curcumin treatment. Interestingly, curcumin-induced apoptosis was not prevented by p38 MAPK, JNK, or caspase inhibition. We conclude that curcumin-induced cytotoxicity was due to cellular ceramide accumulation and damage to mitochondria that resulted in apoptosis mediated by AIF and other caspase-independent processes.

Analysis of parental and nurse weight estimates of children in the pediatric emergency department.

OBJECTIVE: To evaluate the accuracy of parent and triage nurse estimates of children's weights in the pediatric emergency department. METHODS: A convenience sample of parents was surveyed before triage regarding their child's chief complaint, when and where the child was last weighed, and an estimate of their child's weight. Children unable to be weighed or with possible dehydration or external orthopedic devices were excluded. Nurses also estimated children's weights before weighing them and were blinded to parent estimates. All nurses were experienced in the department, had participated in a triage course, and had completed a background survey. RESULTS: Nine hundred forty-two patients presenting to pediatric emergency department triage were enrolled, resulting in 812 surveys. Parents were more accurate at weight estimation than nurses, with 79% of parents and 83% of legal guardians estimating weights within 10%. Only 55% of nurse estimates were within 10%. Parents and nurses tended to underestimate patients' weights (P < 0.0001). Parent estimates were not affected by a child's age or sex or the location the child was last weighed (P > 0.05). Accuracy of nurse estimates was not related to nurse education or experience (P > 0.05). Parent estimates of weight were more accurate than nurse estimates 74.6% of the time. CONCLUSIONS: Parents were more accurate at estimating children's weights than triage nurses but were within 10% of the children's actual weights only 79% of the time. Nurse estimates were highly inaccurate. Other methods to estimate patient weights should be used when actual patient weights are unobtainable.

Immigrant Youth Have Significantly Lower Rates of Externalizing Behavior than Native-Born Americans: Differences by Region of Birth.

A large proportion of Americans have the opinion that immigrants increase crime. Although past research has not found immigrant status to be associated with criminal behavior, American immigration policy has historically discriminated against certain groups based on their region of birth due to safety concerns. The purpose of the present study was to examine differences in externalizing behavior by immigrant's region of birth. Data was used from the Early Childhood Longitudinal Study-Kindergarten Cohort (ECLS-K), a nationally representative and longitudinal study of 21,260 kindergarteners. A series of Poisson regression models were used to predict externalizing behavior of fifth grade students from immigrant status and parent's region of birth. Analyses controlled for demographic characteristics of the child and family and were adjusted by probability weights and primary sampling unit provided by the ECLS-K. After controlling for family income and parents' educational status, immigrant youth had 0.04 lower externalizing behavior scores compared to native-born American youth (B = - 0.04, 95% CI - 0.06 to - 0.01). When considering differences by region of origin, youth from Asia (B = - 0.12, 95% CI - 0.17 to - 0.07) and Central America (B = - 0.10, 95% CI - 0.14 to - 0.05) had significantly lower externalizing behavior compared to native-born American youth, after controlling for covariates. In fifth grade, immigrant youth have significantly lower rates of externalizing behavior than native-born Americans. In particular, immigrant youth from Asia and Central America engaged in significantly less externalizing behavior than native-born Americans. No region of origin engaged in significantly more externalizing behavior than native-born youth.

Nkx2-5 Second Heart Field Target Gene Ccdc117 Regulates DNA Metabolism and Proliferation.

The cardiac transcription factor Nkx2-5 is essential for normal outflow tract (OFT) and right ventricle (RV) development. Nkx2-5-/- null mouse embryos display severe OFT and RV hypoplasia and a single ventricle phenotype due to decreased proliferation of Second Heart Field (SHF) cells, a pool of cardiac progenitors present in anterior pharyngeal arch mesoderm at mid-gestation. However, definition of the precise role of Nkx2-5 in facilitating SHF expansion is incomplete. We have found that Nkx2-5 positively and directly regulates a novel target gene, Ccdc117, in cells of the SHF at these stages. The nuclear/mitotic spindle associated protein Ccdc117 interacts with the MIP18/MMS19 cytoplasmic iron-sulfur (FeS) cluster assembly (CIA) complex, which transfers critical FeS clusters to several key enzymes with functions in DNA repair and replication. Loss of cellular Ccdc117 expression results in reduced proliferation rates associated with a delay at the G1-S transition, decreased rates of DNA synthesis, and unresolved DNA damage. These results implicate a novel role for Nkx2-5 in the regulation of cell cycle events in the developing heart, through Ccdc117's interaction with elements of the CIA pathway and the facilitation of DNA replication during SHF expansion.

Piperine inhibits the growth and motility of triple-negative breast cancer cells.

Piperine, an alkaloid from black pepper, is reported to have anticancer activities. In this study, we investigated the effect of piperine on the growth and motility of triple-negative breast cancer (TNBC) cells. Piperine inhibited the in vitro growth of TNBC cells, as well as hormone-dependent breast cancer cells, without affecting normal mammary epithelial cell growth. Exposure to piperine decreased the percentage of TNBC cells in the G2 phase of the cell cycle. In addition, G1- and G2-associated protein expression was decreased and p21(Waf1/Cip1) expression was increased in piperine-treated TNBC cells. Piperine also inhibited survival-promoting Akt activation in TNBC cells and caused caspase-dependent apoptosis via the mitochondrial pathway. Interestingly, combined treatment with piperine and γ radiation was more cytotoxic for TNBC cells than γ radiation alone. The in vitro migration of piperine-treated TNBC cells was impaired and expression of matrix metalloproteinase-2 and -9 mRNA was decreased, suggesting an antimetastatic effect by piperine. Finally, intratumoral administration of piperine inhibited the growth of TNBC xenografts in immune-deficient mice. Taken together, these findings suggest that piperine may be useful in the treatment of TNBC.