Multiplex reverse transcription-PCR for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group A rotavirus

A novel multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) that can detect porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (GAR) was developed. The 3 viruses (PEDV, TGEV, and porcine GAR) are major agents in viral enteric diseases of piglets. As the clinical signs of these diseases are similar, including watery diarrhea, differential detection is required for etiologic diagnosis. A mixture of 3 pairs of published primers was used for amplification of viral nucleic acids, yielding 3 different amplicons with sizes of 859 bp, 651 bp, and 309 bp for TGEV, PEDV, and porcine GAR, respectively. A total of 157 specimens (78 fecal and 79 intestinal samples) from piglets with acute gastroenteritis were collected in Korea between January 2004 and May 2005. They were tested for the presence of 3 viruses by multiplex RT-PCR. Coinfections with PEDV and porcine GAR were identified in 16 farms (43.2%). PEDV, porcine GAR, and TGEV infection were 26.3%, 13.2%, and 2.7% respectively. The relative sensitivity and specificity of multiplex RT-PCR were evaluated, with results suggesting that this assay is equal in quality to conventional single-agent RT-PCR assays (sensitivity:100%, 92.9%, 100% for TGEV, PEDV, GARs; specificity: 100% for all 3 viruses). This multiplex RT-PCR is a simple assay and may be a potentially useful for rapid, sensitive, and cost-effective etiological diagnostic tool for acute viral gastroenteritis in piglets.This work was supported by Korea Research Foundation Grants (KRF-2002-070-C00069) and the Brain Korea 21 Project of the Ministry of Education & Human Resources Development, Republic of Korea

c-Jun N-terminal kinase activation has a prognostic implication and is negatively associated with FOXO1 activation in gastric cancer

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Abstract Background Since the biological function of c-Jun N-terminal kinase (JNK) in gastric cancer remains unclear, we investigated the clinical significance of JNK activation and its association with FOXO1 activation. Methods Immunohistochemical tissue array analysis of 483 human gastric cancer specimens was performed, and the results of the immunostaining were quantified. The correlation between JNK activation (nuclear staining for pJNK) and clinicopathological features, the proliferation index, prognosis or FOXO1 inactivation (cytoplasmic staining for pFOXO1) was analyzed. The SNU-638 gastric cancer cell line was used for in vitro analysis. Results Nuclear staining of pJNK was found in 38 % of the gastric carcinomas and was higher in the early stages of pTNM (P < 0.001). pJNK staining negatively correlated with lymphatic invasion (P = 0.034) and positively correlated with intestinal type by Laurens classification (P = 0.037), Ki-67-labeling index (P < 0.001), cyclin D1 (P = 0.045), cyclin E (P < 0.001) and pFOXO1 (P < 0.001). JNK activation correlated with a longer patients survival (P =0.008) and patients with a JNK-active and FOXO1-inactive tumor had a higher survival rate than the remainder of the population (P = 0.004). In vitro analysis showed that JNK inhibition by SP600125 in SNU-638 cells decreased cyclin D1 protein expression and increased FOXO1 activation. Further, JNK inhibition markedly suppressed colony formation, which was partially restored by FOXO1 shRNA expression. Conclusions Our results indicate that JNK activation may serve as a valuable prognostic factor in gastric cancer, and that it is implicated in gastric tumorigenesis, at least in part, through FOXO1 inhibition

Treatment Response and Long Term Follow-up Results of Nonspecific Interstitial Pneumonia

The purpose of this study was to investigate the long-term clinical course of non-specific interstitial pneumonia (NSIP) and to determine which factors are associated with a response to steroid therapy and relapse. Thirty-five patients with pathologically proven NSIP were included. Clinical, radiological, and laboratory data were reviewed retrospectively. The male-to-female ratio was 7:28 (median age, 52 yr). Thirty (86%) patients responded to steroid therapy, and the median follow-up was 55.2 months (range, 15.9-102.0 months). Five patients (14%) showed sustained disease progression and three died despite treatment. In the five with sustained disease progression, NSIP was associated with various systemic conditions, and the seropositivity of fluorescent antinuclear antibody was significantly associated with a poor response to steroids (P = 0.028). The rate of relapse was 25%, but all relapsed patients improved after re-treatment. The initial dose of steroids was significantly low in the relapse group (P = 0.020). In conclusion, progression is associated with various systemic conditions in patients who show progression. A low dose of initial steroids is significantly associated with relapse

c-Jun N-terminal kinase activation has a prognostic implication and is negatively associated with FOXO1 activation in gastric cancer

BACKGROUND: Since the biological function of c-Jun N-terminal kinase (JNK) in gastric cancer remains unclear, we investigated the clinical significance of JNK activation and its association with FOXO1 activation. METHODS: Immunohistochemical tissue array analysis of 483 human gastric cancer specimens was performed, and the results of the immunostaining were quantified. The correlation between JNK activation (nuclear staining for pJNK) and clinicopathological features, the proliferation index, prognosis or FOXO1 inactivation (cytoplasmic staining for pFOXO1) was analyzed. The SNU-638 gastric cancer cell line was used for in vitro analysis. RESULTS: Nuclear staining of pJNK was found in 38 % of the gastric carcinomas and was higher in the early stages of pTNM (P < 0.001). pJNK staining negatively correlated with lymphatic invasion (P = 0.034) and positively correlated with intestinal type by Lauren’s classification (P = 0.037), Ki-67-labeling index (P < 0.001), cyclin D1 (P = 0.045), cyclin E (P < 0.001) and pFOXO1 (P < 0.001). JNK activation correlated with a longer patients survival (P =0.008) and patients with a JNK-active and FOXO1-inactive tumor had a higher survival rate than the remainder of the population (P = 0.004). In vitro analysis showed that JNK inhibition by SP600125 in SNU-638 cells decreased cyclin D1 protein expression and increased FOXO1 activation. Further, JNK inhibition markedly suppressed colony formation, which was partially restored by FOXO1 shRNA expression. CONCLUSIONS: Our results indicate that JNK activation may serve as a valuable prognostic factor in gastric cancer, and that it is implicated in gastric tumorigenesis, at least in part, through FOXO1 inhibition

Single cell RNA-seq reveals profound transcriptional similarity between Barrett's oesophagus and oesophageal submucosal glands

Barrett’s oesophagus is a precursor of oesophageal adenocarcinoma. In this common condition, squamous epithelium in the oesophagus is replaced by columnar epithelium in response to acid reflux. Barrett’s oesophagus is highly heterogeneous and its relationships to normal tissues are unclear. Here we investigate the cellular complexity of Barrett’s oesophagus and the upper gastrointestinal tract using RNA-sequencing of single cells from multiple biopsies from six patients with Barrett’s oesophagus and two patients without oesophageal pathology. We find that cell populations in Barrett’s oesophagus, marked by LEFTY1 and OLFM4, exhibit a profound transcriptional overlap with oesophageal submucosal gland cells, but not with gastric or duodenal cells. Additionally, SPINK4 and ITLN1 mark cells that precede morphologically identifiable goblet cells in colon and Barrett’s oesophagus, potentially aiding the identification of metaplasia. Our findings reveal striking transcriptional relationships between normal tissue populations and cells in a premalignant condition, with implications for clinical practice

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Multiple pulmonary atypical carcinoids presenting with long-standing Cushing syndrome masked by pulmonary tuberculosis

Pulmonary neuroendocrine tumors, especially small cell carcinoma and typical carcinoids, are the most common cause of ectopic Cushing syndrome (CS). Sometimes these adrenocorticotropic hormone (ACTH)-producing tumors are too small to localize, and it may take a long time to identify them. This report describes the case of a 27-year-old-man with CS. This CS was due to the ectopic ACTH produced by the pulmonary atypical carcinoids, which were recognized at the disseminated stage 5 years after the initial presentation of CS. Microscopically, multiple whitish nodules (up to 1.0 cm in diameter) of the wedge-resected lung were composed of small round cells appearing to be typical neuroendocrine but were diagnosed as atypical carcinoid, based on the findings of focal necrosis, high mitotic rates and multiple endolymphatic tumor emboli. Immunohistochemically, tumor cells robustly stained for ACTH. The delay of diagnosis is, in part, attributable to the pulmonary tuberculosis (one of the complications of hypercortisolism), because the pulmonary tuberculosis presented in the form of multiple nodules throughout the lung. In addition, this patient had a Crooke`s cell hyperplasia of the pituitary gland, which represents the reactive change of corticotrophs exposed to the excess cortisol rather than neoplastic change leading to CS.Aubry MC, 2007, CHEST, V131, P1635, DOI 10.1378/chest.06-2788Davies SJ, 2007, THORAX, V62, P248, DOI 10.1136/thx.2006.063065WONG M, 2007, MED J MALAYSIA, V62, P168Salgado LR, 2006, EUR J ENDOCRINOL, V155, P725, DOI 10.1530/eje.1.02278Isidori AM, 2006, J CLIN ENDOCR METAB, V91, P371, DOI 10.1210/jc.2005-1542Asamura H, 2006, J CLIN ONCOL, V24, P70, DOI 10.1200/JCO.2005.04.1202Isidori AM, 2006, FRONT HORM RES, V35, P143Meinardi JR, 2006, NETH J MED, V64, P23Ilias I, 2005, J CLIN ENDOCR METAB, V90, P4955Deb SJ, 2005, ANN THORAC SURG, V79, P1132, DOI 10.1016/j.arthoracsur.2004.07.021Kaltsas GA, 2004, ENDOCR REV, V25, P458, DOI 10.1210/er.2003-0014PAPLA B, 2004, POL J PATHOL, V55, P31ISOWA N, 2003, JPN J THORAC CARDIOV, V51, P427de Perrot M, 2002, ANN THORAC SURG, V73, P675Cooper WA, 2001, CHEST, V119, P14Granberg D, 2000, J CLIN ENDOCR METAB, V85, P3425Ozbey N, 2000, J ENDOCRINOL INVEST, V23, P536Arioglu E, 1998, NEW ENGL J MED, V339, P883Travis WD, 1998, AM J SURG PATHOL, V22, P934Shrager JB, 1997, J THORAC CARDIOV SUR, V114, P367LOH KC, 1997, SINGAPORE MED J, V38, P29Rusch VW, 1996, ANN THORAC SURG, V62, P798Dusmet ME, 1996, WORLD J SURG, V20, P189deMontpreville VT, 1996, J THORAC CARDIOV SUR, V111, P134AMANN ST, 1994, SOUTHERN MED J, V87, P855SAKAMOTO K, 1992, NIPPON NAIBUNPI GAKK, V68, P743

Intestinal Stem Cell Markers in the Intestinal Metaplasia of Stomach and Barrett's Esophagus.

Gastric intestinal metaplasia (IM) is a highly prevalent preneoplastic lesion; however, the molecular mechanisms regulating its development remain unclear. We have previously shown that a population of cells expressing the intestinal stem cell (ISC) marker LGR5 increases remarkably in IM. In this study, we further investigated the molecular characteristics of these LGR5+ cells in IM by examining the expression profile of several ISC markers. Notably, we found that ISC markers-including OLFM4 and EPHB2-are positively associated with the CDX2 expression in non-tumorous gastric tissues. This finding was confirmed in stomach lesions with or without metaplasia, which demonstrated that OLFM4 and EPHB2 expression gradually increased with metaplastic progression. Moreover, RNA in situ hybridization revealed that LGR5+ cells coexpress several ISC markers and remained confined to the base of metaplastic glands, reminiscent to that of normal intestinal crypts, whereas those in normal antral glands expressed none of these markers. Furthermore, a large number of ISC marker-expressing cells were diffusely distributed in gastric adenomas, suggesting that these markers may facilitate gastric tumorigenesis. In addition, Barrett's esophagus (BE)-which is histologically similar to intestinal metaplasia-exhibited a similar distribution of ISC markers, indicating the presence of a stem cell population with intestinal differentiation potential. In conclusion, we identified that LGR5+ cells in gastric IM and BE coexpress ISC markers, and exhibit the same expression profile as those found in normal intestinal crypts. Taken together, these results implicate an intestinal-like stem cell population in the pathogenesis of IM, and provide an important basis for understanding the development and maintenance of this disease

Distribution of<i>LGR5</i>⁺ Cells and Associated Implications during the Early Stage of Gastric Tumorigenesis

<div><p><i>Lgr5</i> was identified as a promising gastrointestinal tract stem cell marker in mice. Lineage tracing indicates that <i>Lgr5</i><b>⁺</b> cells may not only be the cells responsible for the origin of tumors; they may also be the so-called cancer stem cells. In the present study, we investigated the presence of <i>Lgr5</i><b>⁺</b> cells and their biological significance in normal human gastric mucosa and gastric tumors. RNAscope, a newly developed RNA <i>in situ</i> hybridization technique, specifically labeled <i>Lgr5</i><b>⁺</b> cells at the basal glands of the gastric antrum. Notably, the number of <i>Lgr5</i><b>⁺</b> cells was remarkably increased in intestinal metaplasia. In total, 76% of gastric adenomas and 43% of early gastric carcinomas were positive for <i>LGR5</i>. <i>Lgr5</i><b>⁺</b> cells were found more frequently in low-grade tumors with active Wnt signaling and an intestinal gland type, suggesting that <i>LGR5</i> is likely involved in the very early stages of Wnt-driven tumorigenesis in the stomach. Interestingly, similar to stem cells in normal tissues, <i>Lgr5</i><b>⁺</b> cells were often restricted to the base of the tumor glands, and such <i>Lgr5</i><b>⁺</b> restriction was associated with high levels of intestinal stem cell markers such as <i>EPHB2</i>, <i>OLFM4</i>, and <i>ASCL2</i>. Thus, our findings show that <i>Lgr5</i><b>⁺</b> cells are present at the base of the antral glands in the human stomach and that this cell population significantly expands in intestinal metaplasias. Furthermore, <i>Lgr5</i><b>⁺</b> cells are seen in a large number of gastric tumors ; their frequent basal arrangements and coexpression of ISC markers support the idea that <i>Lgr5</i><b>⁺</b> cells act as stem cells during the early stage of intestinal-type gastric tumorigenesis.</p></div