[Clinical application of expanded noninvasive prenatal testing for fetal chromosome abnormalities].

To assess the positive predictive value (PPV) of extended noninvasive prenatal testing (NIPT-plus) for fetal chromosomal abnormalities. This retrospective research enrolled 511 cases of pregnant women with positive NIPT-plus results at the Obstetrics and Gynecology Hospital of Fudan University from May 2017 to January 2021. Karyotype analysis and chromosome microarray analysis (CMA) techniques was applied for verification. All cases were followed to determine their pregnancy outcome. The Chi-square test was used in PPV. 63 out of 511 refused prenatal diagnosis after counseling, 448 pregnant women with prenatal diagnosis showed that the PPVs of NIPT-plus test for fetal trisomy 21, 18 and 13 (T21, T18, T13), sex chromosome aneuploidy (SCAs) and chromosome microdeletion/microduplication syndrome (MMS) were 86.0% (92/107), 79.5% (35/44), 54.5% (12/22), 39.5% (75/190), and 41.7% (30/72), respectively. The results revealed that the PPV was higher among older pregnant women compared to young pregnant women (77.8% vs. 51.9%,P<0.01). With increasing maternal age, the PPV of NIPT-plus presented increasing trends for T21, T13, and composite PPV except for T18 or SCAs. In addition, the termination rates for confirmed SCAs fetal karyotypes 45, X; 47, XXX; 47, XXY and 47, XYY were 11/11, 3/15, 91.7% (22/24) and 1/14, respectively. NIPT-plus can safely and effectively detect fetal chromosomal abnormalities and can be extended to MMS screening, significantly reducing the proportion of interventional prenatal diagnoses, and those with positive screening still require further confirmation.

Prognostic impact of prognostic nutritional index in advanced (stage IIIB/IV) non-small cell lung cancer patients.

Prognostic nutritional index (PNI) is a parameter reflecting prognosis for various cancers, including resected lung cancer. However, there were few reports to study the relationship between the PNI and overall survival (OS) in patients with advanced (stage IIIB/IV) non-small lung cancer (NSCLC). In this study, we collected the clinical data of 315 patients with advanced (stage IIIB/IV) NSCLC who had received chemotherapy or epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) between January 2010 and June 2011. Survival curves were plotted using the Kaplan-Meier method. Multivariate analyses were used to evaluate prognostic significance of PNI in patients with advanced (stage IIIB/IV) NSCLC. In our analysis, we found that PNI (p=0.001) was significantly associated with OS in patients with advanced (stage IIIB/IV) NSCLC, so was smoking (p<0.001) and disease stage (p=0.005). We demonstrated that PNI could be utilized to predict survival outcomes in patients with advanced (stage IIIB/IV) NSCLC. Patients with a lower PNI may have worse prognosis.

[The clinical characteristics of patients with monomorphic epitheliotropic intestinal T-cell lymphoma characterized by minor endoscopic abnormalities].

Objective: To clarify the clinical features of monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) with minor endoscopic abnormalities. Methods: The clinical data of 6 patients with MEITL characterized by minor endoscopic abnormalities in Peking Union Medical College Hospital from 2012 to 2016 were retrospectively analyzed, including clinical manifestations, endoscopic, pathological features, medications and prognosis. Results: Five out of 6 patients were male, with an average age of 61.2 years old. The median disease duration was 4.5 months. All patients initially presented with diarrhea without specific findings for serologic testing. CT enterography showed continuous intestinal lesions, including symmetric thickening of the bowel wall, abnormal hyperenhancement of mucosal surface and lymphadenopathy. Endoscopic appearances were only mildly abnormal, including mucosal swelling, atrophy of villus, mosaic sign and shallow ulcers. Histopathologic findings revealed massive small to medium sized T lymphocytes infiltration with positive expression of CD(3) and CD(8). Chemotherapy and palliative treatment were administrated after diagnosis. Conclusions: Clinical presentations of MEITL are non-specific with minor endoscopic abnormalities. Therefore, biopsy is indispensable for patients with a relatively normal endoscopic result.

[The 462nd case: chronic watery diarrhea and acute kidney injury].

A 60-year-old man presented with severe watery diarrhea for 2 months complicated with weight loss and acute kidney injury. He did not respond well to antidiarrheal medicines, empirical antibiotics and dietary exclusion of gluten or even complete bowel rest. The final diagnosis of autoimmune enteropathy (AIE) was made based on histopathologic findings of endoscopic biopsy from duodenal mucosa after excluding neoplastic disease, inflammatory bowel disease, and infectious diarrhea, etc. Chronic diarrhea and oliguria alleviated after the administration of corticosteroids.

Chronologic changes in serum hepatitis B virus DNA, genotypes, surface antigen mutants and reverse transcriptase mutants during 25-year nationwide immunization in Taiwan.

We investigated breakthrough infection and hepatitis B virus (HBV) genetic changes in immunized subjects after 25 years of a universal infant immunization. Specifically, serum HBV DNA, genotypes, surface antigen mutants and nucleoside analog-resistant (NAr) mutants were assessed in 2853 subjects (<25 years old) surveyed in 2009, and these data were compared with the data from previous serosurveys. A comparison across different age-stratified groups using the 2009 data revealed a significant increase in the seropositive rate of anti-HBc (5.51% vs 12.38%, P=.001) and HBV DNA (1.13% vs 3.96%, P=.007) between those 17-22 and 23-24 years of age, possibly due to selective infant immunization in 1984-1986. Well-characterized NAr mutants, potential NAr mutants and surface "a" determinant mutants were detected in none, 15 (45.5%) and nine (27.3%) of 33 HBV DNA-positive subjects, respectively. Of 15 immunized, HBV DNA-positive young adults (18-24 years), three (20%) carried "a" determinant mutants. Amongst 1176 HBsAg-negative subjects evaluated for occult HBV infection, those seropositive for anti-HBc had a higher seropositive rate for HBV DNA (10/110, 9.1% vs 7/1066, 0.66%; P<.001) and "a" determinant mutants (4/110, 3.6% vs 0/1066; P<.001) than those seronegative for anti-HBc. Overall, the HBsAg-positive subjects in six serosurveys showed no significant increase in genotype C frequency in the comparison between the vaccinated and unvaccinated cohorts (25/98, 25.5% versus 14/79, 17.7%, P=.188). Over the 25-year programme, there was no increase in the prevalence of genotype C in HBsAg carriers and no increase in breakthrough HBV infection or surface mutant prevalence beyond adolescence. Nucleic acid amplification should still be considered the primary screening method for occult hepatitis B detection in high-risk recipients.

Knockdown of human serine/threonine kinase 33 suppresses human small cell lung carcinoma by blocking RPS6/BAD signaling transduction.

Small cell lung cancer (SCLC) is characterized by rapid growth rate and a tendency to metastasize to distinct sites of patients' bodies. The human serine/threonine kinase 33 (STK33) gene has shown its potency as a therapeutic target for prevention of lung carcinomas including non-small cell lung cancer (NSCLC), but its function in the oncogenesis and development of SCLC remains unrevealed. In the current study, it was hypothesized that STK33 played a key role in the proliferation, survival, and invasion of SCLC cells. The expression of STK33 in human SCLC cell lines NCI-H466 and DMS153 was inhibited by specific shRNA. The cell proliferation, cell apoptosis, and cell invasion of the cells were assessed with a series of in vitro assays. To explore the mechanism through which STK33 gene exerted its function in the carcinogenesis of SCLC cells, the effect of STK33 knockdown on the activity of S6K1/RPS6/BAD signaling was detected. Then the results were further confirmed with STK33 inhibitor ML281 and in vivo assays. The results demonstrated that inhibition of STK33 in SCLC cells suppressed the cell proliferation and invasion while induced cell apoptosis. Associated with the change in the phenotypic features, knockdown of STK33 also decreased the phosphorylation of RPS6 and BAD while increased the expression of cleaved caspase 9, indicating that apoptosis induced by STK33 suppression was mediated via mitochondrial pathway. Similar to the results of STK33 knockdown, incubating NCI-H466 cells with STK33 inhibitor also reduced the cell viability by suppressing RPS6/BAD pathways. Additionally, STK33 knockdown also inhibited tumor growth and RPS6/BAD activity in mice models. Findings outlined in our study were different from that in NSCLC to some extent: knockdown of STK33 in SCLC cells induced the apoptosis through mitochondrial pathway but independent of S6K1 function, inferring that the function of STK33 might be cancer type specific.

[Effect of interleukin-22 on proliferation and activation of hepatic stellate cells induced by acetaldehyde and related mechanism].

Objective: To investigate the effect of interleukin-22 (IL-22) on the activation and proliferation of hepatic stellate cells (HSCs) induced by acetaldehyde, as well as the role of the antioxidant axis Nrf2-keap1-ARE. Methods: Hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and after 24 and 48 hours of acetaldehyde stimulation at various concentrations (25, 50, 100, 200, and 400 µmol/L), MTT assay was used to measure cell proliferation rate to screen out the optimal conditions for model establishment. HSC-T6 cells were treated first with the optimal concentration of acetaldehyde (200 µmol/L) for 24 hours and then with different concentrations of IL-22 (10, 20, and 50 ng/ml) for 24 hours. MTT assay was used to measure cell proliferation, Western blot and cell immunohistochemistry were used to measure the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and α-smooth muscle actin (α-SMA), and spectrophotometry was used to measure the changes in the content of malondialdehyde (MDA) and reduced glutathione (GSH) in culture supernatant. SPSS 17.0 was used for statistical analysis and data were expressed as mean±SD. P < 0.05 was considered statistically significant. A one-way analysis of variance was used for comparison of means between any two groups. Results: HSCs had significantly enhanced proliferation and activation after being treated with acetaldehyde, especially at 200 µmol/L for 48 hours. After the intervention with gradient concentrations of IL-22, the proliferation and activation of HSCs were inhibited in a dose-dependent manner, and the proliferation and migration rates in the 10, 20, and 50 ng/ml IL-22 groups were 14%, 25%, and 35%, respectively (all P < 0.05). The results of Western blot and immunohistochemistry showed that there was no significant difference in the expression of Nrf2 total protein in HSCs between groups, while there was extremely low expression of Nrf2 nucleoprotein in the blank control group. There was increased expression of Nrf2 nucleoprotein after acetaldehyde stimulation (compared with the blank control group, P < 0.05), and after the intervention with gradient concentrations of IL-22, the expression of Nrf2 nucleoprotein was further increased (all P < 0.05). The results of spectrophotometry showed that compared with the blank control group, the model group had increased levels of MDA and GSH in culture supernatant after acetaldehyde stimulation; after the intervention with gradient concentrations of IL-22, there was a significant reduction in the MDA level and a significant increase in the GSH level in a dose-dependent manner (all P < 0.05). Conclusion: The activation and proliferation of HSCs induced by acetaldehyde helps with the successful establishment of an in vitro model of alcoholic liver fibrosis. IL-22 effectively inhibits the activation and proliferation of HSCs induced by acetaldehyde, and its mechanism may be related to promoting Nrf2 nuclear translocation in HSCs and expression of the downstream target gene GSH and increasing the activity of the antioxidant axis Nrf2-keap1-ARE.

Heme oxygenase-1 gene promoter polymorphism and the risk of pediatric nonalcoholic fatty liver disease.

BACKGROUND AND OBJECTIVES: Oxidative stress and the insulin-resistant state are thought to be key components in the pathogenesis of pediatric nonalcoholic fatty liver disease (NAFLD). Heme oxygenase (HO) is important in the defense against oxidative stress. This study aimed to assess the association of HO-1 gene promoter polymorphism and insulin resistance with NAFLD among obese children. METHODS: A total of 101 obese children aged 6-17 years were recruited. Anthropometric, serum biochemical variables and biomarkers for glucose and insulin metabolism were measured. We screened the allelic frequencies of (GT)n repeats in the HO-1 gene promoter among these obese children. NAFLD was determined through liver ultrasonography. Because the distribution of numbers of (GT)n repeats was bimodal, we divided the alleles into two classes: class S included shorter (27) repeats, and class L included longer (⩾27) repeats. We assessed the effects of the length of (GT)n repeats in HO-1 gene promoter on pediatric NAFLD. RESULTS: Of the 101 obese subjects, 27 (26.7%) had NAFLD. The alanine aminotransferase level was higher in patients carrying L alleles (L/L and L/S) than patients with S alleles (S/S) (46.2±49.3 IU|(-1) versus 30.2±20.1 IU|(-1); P=0.027). The significant risk factors for pediatric NAFLD were patients carrying L alleles (L/L and L/S) (odds ratio (OR)=18.84; 95% confidence interval (CI): 1.45-245.22; P=0.025), homeostasis model assessment of insulin resistance (OR=1.40; 95% CI: 1.07-1.83; P=0.014) and age (OR=1.24; 95% CI: 1.03-1.50; P=0.025). CONCLUSION: In this hospital-based study, the obese children with longer GT repeats in the HO-1 gene promoter and insulin resistance were susceptible to NAFLD.

Genome-wide normalized score: a novel algorithm to detect fetal trisomy 21 during non-invasive prenatal testing.

OBJECTIVES: Non-invasive prenatal testing for fetal trisomy 21 (T21) by massively parallel shotgun sequencing (MPSS) is available for clinical use but its efficacy is limited by several factors, e.g. the proportion of cell-free fetal DNA in maternal plasma and sequencing depth. Existing algorithms discard DNA reads from the chromosomes for which testing is not being performed (i.e. those other than chromosome 21) and are thus more susceptible to diluted fetal DNA and limited sequencing depth. We aimed to describe and evaluate a novel algorithm for aneuploidy detection (genome-wide normalized score (GWNS)), which normalizes read counts by the proportions of DNA fragments from chromosome 21 in normal controls. METHODS: We assessed the GWNS approach by comparison with two existing algorithms, i.e. Z-score and normalized chromosome value (NCV), using theoretical approximations and computer simulations in a set of 86 cases (64 euploid and 22 T21 cases). We then validated GWNS by studying an expanded set of clinical samples (n = 208). Finally, dilution experiments were undertaken to compare performance of the three algorithms (Z-score, NCV, GWNS) when fetal DNA concentration was low. RESULTS: At fixed levels of significance and power, GWNS required a smaller fetal DNA proportion and fewer total MPSS reads compared to Z-score or NCV. In dilution experiments, GWNS also outperformed the other two methods by reaching the correct diagnosis with the lowest range of fetal DNA concentrations (GWNS, 3.83-4.75%; Z-score, 4.75-5.22%; NCV, 6.47-8.58%). CONCLUSION: Our results demonstrate that GWNS is comparable to Z-score and NCV methods regarding the performance of detecting fetal T21. Dilution experiments suggest that GWNS may perform better than the other methods when fetal fraction is low.

Analysis and prediction of risk factors of ovarian hyperstimulation caused by Long-acting GnRH agonist protocol in follicular phase.

OBJECTIVE: The aim of the study was to explore the risk factors of ovarian hyperstimulation in patients undergoing long-acting gonadotropin-releasing hormone (GnRH) agonist protocol in follicular phase of ovulation induction therapy and to establish a predictive model. PATIENTS AND METHODS: A total of 1289 patients who received Long-acting GnRH agonist protocol in follicular phase for ovulation induction in the Fujian Provincial Maternity and Child Health Hospital from July 1, 2018, to July 31, 2019, were selected. Among them, 33 patients developed moderate/severe ovarian hyperstimulation syndrome. The relevant indicators of the two groups were followed up for comparison, and Lasso regression was used to screen independent risk factors and construct a nomogram prediction model.  A receiver operating characteristic (ROC) curve and calibration curve were used to evaluate the discrimination and calibration of the prediction model. RESULTS: Univariate analysis suggested that the woman's age, basal antral follicle number (AFC), total gonadotropin (Gn) dose, Gn starting dose, basal estradiol (E2) level, basal anti-Müllerian hormone (AMH) value, number of follicles obtained, Gn start day E2, the difference in follicle-stimulating hormone (FSH) value and Gn starting day were statistically significant. Significant indicators of univariate analysis and clinical significance were included in the Lasso regression model, and AFC, woman's age, polycystic ovary syndrome, Gn starting dose and number of follicles obtained were finally screened as final predictors. The ROC curve indicated that the area under the curve (AUC) was 0.812. CONCLUSIONS: Ovarian hyperstimulation caused by long-acting GnRH agonist protocol in follicular phase for ovulation stimulation has a certain predictability. Paying attention to the patient's age, AFC, Gn starting dose, number of follicles obtained, and whether PCOS is evident may lead to early detection of ovarian hyperstimulation syndrome, which has clinical guiding significance.

[Outcomes of surgical management of typeâ ¢ laryngotracheal clefts: anterior laryngofissure approach and posterior cartilage graft laryngotracheoplasty].

Objective: Our aim of this study is to describe the outcomes of a series of patients who underwent cleft repair and posterior cartilage grafts laryngotracheoplasty (LTP) from anterior midline cervical approach for type Ⅲ laryngotracheoesophageal clefts (LETC). Methods: A review of patients with type Ⅲ LETC between May 2017 and December 2021 was performed. Demographic features including gender, age at surgery, weight, airway support, feeding status, and airway and other comorbidities were collected preoperatively. Patients were evaluated in breathing, swallowing and phonation postoperatively. The developmental status and morbidities were recorded. Results: Five patients who underwent cleft repair and posterior cartilage grafts LTP from anterior midline cervical approach were included. All patients survived and thrived postoperatively. At last follow-up, 3 patients were able to successfully extubate with acceptable voice, and 2 patients were tracheostomied. Four patients were able to be fed orally without aspiration, and one patient needed to be fed by thick food. Conclusion: The combination of cleft repair and posterior cartilage grafts LTP from anterior midline cervical approach is an effective and safe treatment for type Ⅲ LETC.

Stabilization of blood methylmalonic acid level in methylmalonic acidemia after liver transplantation.

Methylmalonic acidemia with complete mutase deficiency (mut(0) type) is an inborn error of metabolism with high mortality and morbidity. LT has been suggested to be a solution to this disease, but elevation of urinary and blood MMA was still observed after LT. In this study, we measured dry blood spot MMA and its precursor propionyl-carnitine (C3-carnitine) for mut(0) patients. The results revealed that when C3-carnitine rose during metabolic stress, MMA rose exponentially (up to 1000 micromol/L) in patients who did not undergo LT. In patients who underwent LT, MMA rose to 100-200 micromol/L when C3-carnitine reached 10-20 micromol/L. However, when C3-carnitine rose further to 40-50 micromol/L, MMA levels just stayed put. Therefore, LT stabilized blood MMA level, though there might be a threshold for blood MMA clearance by the donor liver. This finding should be critical to understand the long-term outcome for LT in methylmalonic acidemia.

Hepatitis B vaccination in children: the Taiwan experience.

The world's first nationwide hepatitis B virus (HBV) universal vaccination program for infants was launched in Taiwan in July, 1984. All infants received three to four doses plasma or recombinant HBV vaccines. In addition, infants of HBeAg-positive mothers received 0.5ml of hepatitis B immunoglobulin within 24hours after birth. The vaccination coverage rate is as high as 97%. Seroprevalence of hepatitis B surface antigen (HBsAg) declined from 9.8% (prevaccination period) to 0.6% in children in Taipei City after 20years of mass vaccination. The seropositive rates for HBsAg, antibody to HBsAg, and antibody to hepatitis B core antigen were 1.2%, 50.5%, and 3.7%, respectively, in those born after the vaccination program (<20years old) in 2004. In line with the decrease of chronic HBV infection, the incidence of hepatocellular carcinoma (HCC) also decreased in children in Taiwan. From 1981 to 1994, the incidence of HCC in 6- to 9-year-olds declined from 0.52/100,000 for those born between 1974 and 1984 to 0.13 for those born between 1984 and 1986 (p<0.001). We extended the observation to 2000, the incidence of HCC per 100,000 children declined from 0.54 to 0.20. The prevalence of a determinant mutants (amino acids 121-149 of HBsAg) in Taiwanese carrier children was 7.8% (eight out of 103) in 1984, increased to 19.6% (10 out of 51) in 1989, peaked at 28.1% (nince out of 32) in 1994, and remained stationary at 23.1% (three out of 13) and about 25% in 1999 and 2004, respectively; it was higher in those fully vaccinated compared with those not vaccinated. The other group of subjects who are susceptible to vaccine failure is the immunocompromized hosts. We observed some de novo HBV infection in children after liver transplantation. Despite of the success of hepatitis B immunization, childhood chronic HBV infection and HCC were not eliminated by the universal vaccination program. Among those HBsAg carriers born after the vaccination program, 89% of their mothers were found to be positive for HBsAg, indicating the importance of maternal transmission. This was also true in the mothers of children with HCC, of them 96% were HBsAg positive. After two decades of universal infant HBV vaccination, we found this program provides long-term protection for up to more than 20years, and a universal booster is not required for the primary HBV vaccinees before adulthood. Mother-to-child transmission, although largely diminished, is still the main cause for immunoprophylaxis failure. The emergence of escape mutant did not impose increased risk of chronic infection at present. Nevertheless, development of new vaccines may overcome the vaccine failure.

Low viraemia at enrollment in children with chronic hepatitis C favours spontaneous viral clearance.

The significance of hepatitis C viral (HCV)-RNA levels in long-term clinical outcomes of children with chronic HCV infection is not well understood. We conducted a long-term follow-up study of 42 children with chronic HCV infection that included clinical evaluation, biochemical tests, HCV genotyping and repeated quantitative HCV-RNA detection. Patients were divided into low and high viraemia groups according to RNA levels at enrollment (below/above 4.5 x 10(4) IU/mL), and clinical, biochemical and virological factors were evaluated. Overall, 14.3% (6/42) of patients developed spontaneous viral clearance during a median 10.1 years of follow-up. HCV-RNA levels at enrollment and mean RNA levels during follow-up for each patient were significantly correlated (R = 0.9018, 95% CI: 0.6637-0.9038, P < or = 0.001). HCV-RNA level fluctuation was within two log units in 76% of patients. Cumulative viraemia probability during follow-up could be predicted by viraemia levels at enrollment (P = 0.0092). Chronic HCV-infected children, with an RNA level below 4.5 x 10(4) IU/mL at enrollment, have a higher spontaneous viral clearance rate.

[Clinical evaluation of vocal fold paralysis in 207 children].

Objective: To investigate the etiology and clinical characteristics of vocal fold paralysis in children. To provide useful information for diagnosis, management and prognosis in the clinical work. Methods: Two hundred and seven children with vocal fold paralysis in Children's Hospital of Fudan University were retrospectively studied, and followed-up. Results: All the patients had hoarseness.151 cases had vocal paralysis in the left side and the main etiology was pulmonary arterial hypertension.43 cases had bilateral vocal paralysis and all of them had respiratory problems.The main etiology were congenital tracheoesophageal malformations.13 cases had vocal paralysis in the right side.In terms of etiology, 8 cases were related to intracranial lesions, 2 cases were idiopathic. Conclusions: The main etiologies of left vocal fold paralysis were cardiovascular diseases, and bilateral vocal paralysis were congenital tracheoesophageal malformations.The main etiologies of right vocal fold paralysis were neoplastic and central lesion.The prognosis of bilateral vocal fold paralysis and right vocal fold paralysis was poor.

Postoperative radiotherapy is dispensable for OSCC patients with micrometastases in lymph nodes.

Lymph node metastasis is a decisive factor for performing postoperative radiotherapy for oral squamous cell carcinoma (OSCC). However, whether OSCC patients with only micrometastasis need postoperative radiotherapy is unclear. In this study, OSCC patients (n = 311) with negative (n = 247), only micrometastasis (n = 44) and macrometastasis (n = 20) were detected and selected by HE staining. Micrometastasis was re-assessed using immunohistochemical staining of cytokeratin (CK) in HE-negative patients to find out the false negative cases. The results indicated that, among the negative lymph node cases (n = 247), the positive rate of CK was 4.94% (n = 12). Besides, the clinical features of the primary tumor in relation to the only micrometastatic status and the value of the postoperative radiotherapy on the only micrometastasis patients were evaluated. Patients with only micrometastasis had higher T stage and inferior worst pattern of invasion (WPOI) than patients without micrometastasis, but they had longer overall survival (OS), metastasis-free survival (MFS), and disease-free survival (DFS) than macrometastasis patients. However, the survival time of only micrometastasis patients with or without postoperative radiotherapy was comparable, even in patients with inferior WPOI. Radiotherapy, however, may only benefit patients with IV/V levels of micrometastasis. These data indicated that postoperative radiotherapy is dispensable for only micrometastasis OSCC patients.

LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis.

Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14(+)TLR4(-), whereas cancerous tissues were CD14(+)TLR4(+), by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14(+)TLR4(+) colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14(+)TLR4(-)) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.

Nephromegaly and elevated plasma hepatocyte growth factor-transforming growth factor-beta1 ratio in infants with fulminant hepatitis or biliary atresia.

Nephromegaly, assessed by calculating kidney volume using renal ultrasound, was studied in infants with biliary atresia, neonatal hepatitis, or fulminant hepatitis. We evaluated kidney volume in 29 patients with biliary atresia, 17 patients with neonatal hepatitis, and 10 patients with fulminant hepatitis, as well as 32 healthy infants. Levels of plasma hepatocyte growth factor (HGF) were measured in all infants. Levels of plasma transforming growth factor-beta1 (TGF-beta1) were also measured in diseased infants and 20 healthy infants. Significant nephromegaly was found in infants with biliary atresia compared with healthy infants (P < 0.001 by analysis of covariance). Marked nephromegaly was also noted in all infants with fulminant hepatitis and 35% of infants with neonatal hepatitis. No nephromegaly was found in infants at 2 months of age with biliary atresia or neonatal hepatitis despite mildly elevated plasma HGF levels. Regardless of the duration of HGF exposure and healthy renal growth by a certain age, a positive correlation existed between plasma HGF level and kidney volume (r = 0.529; P < 0.001), but an inverse correlation was found between plasma TGF-beta1 level and nephromegaly (r = -0.505; P < 0.001) in all diseased infants. There was a stronger positive correlation between plasma HGF-TGF-beta1 ratio and kidney volume (r = 0.666; P < 0.001) and degree of nephromegaly (r = 0.717; P < 0.001). These results confirm the presence of large kidneys not only in patients with biliary atresia but also in patients with fulminant hepatitis, which suggests the possible pathogenic role of HGF and manifests as elevated HGF-TGF-beta1 ratios in patients with such conditions. Nephromegaly in patients with severe or chronic liver dysfunction may provide a new in vivo model to study the mechanisms of renal growth.

Prevalence and rapid identification of clarithromycin-resistant Helicobacter pylori isolates in children.

BACKGROUND: Little is known about the prevalence of antibiotic-resistant Helicobacter pylori infection in children. Culture and antimicrobial susceptibility testing are generally time-consuming and not a routine in many hospitals. OBJECTIVE: To investigate the prevalence of clarithromycin-resistant H. pylori strains in children, to identify those isolates via rapid methodology and to examine the severity of gastritis caused by the antibiotic-resistant H. pylori isolates. METHODS: Enrolled were 245 children investigated for H. pylori infection by endoscopic examination. The gastric antral specimens were subjected to DNA extraction and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with primers specific to the H. pylori 23S rRNA gene. Conventional bacterial cultures were performed simultaneously as the diagnostic standard. Minimal inhibitory concentrations of clarithromycin and metronidazole were determined by E test. This was used as a standard to determine the sensitivity and specificity of the above PCR-RFLP assay. The specimens were processed for histologic examination and evaluated by the updated Sydney system. RESULTS: H. pylori was isolated in 67 of the 245 children; 12 (18%) of them were clarithromycin-resistant and 6 (9%) were metronidazole-resistant. No difference in histologic examinations was noted between the antibiotic-resistant and -susceptible strains. We performed PCR-RFLP with all 12 clarithromycin-resistant isolates: 10 had a 23S ribosomal RNA A2144G point mutation; 1 had a mixture of an A2143G point mutant and susceptible strains; and 1 had neither of the 2 mutations. CONCLUSIONS: The prevalence of clarithromycin-resistant H. pylori isolates in Taiwanese children is 18%. PCR-RFLP had a high sensitivity (92%) and specificity (100%) for the clarithromycin resistance gene mutation determination. The dominant mutation is A2144G. PCR-RFLP provides a rapid and accurate approach to detect clarithromycin-resistant strains within 24 h.