Characterisation of Colonic Epithelial Stem Cells in Health and Inflammatory Bowel Disease

INTRODUCTION: The epithelium of the large intestine is a very rapidly replicating tissue with an enormous capacity for continuous proliferation, followed by differentiation, senescence and shedding. The proliferative capacity of the large intestinal epithelium is maintained through multipotent stem cells which are thought to be located towards the base of the colonic crypts. As yet poorly defined signals, that may include cytokines and growth factors which are secreted by the lamina propria cells or the intestinal subepithelial myofibroblasts, control the proliferation and differentiation of these epithelial stem cells. OBJECTIVES: 1. To isolate and characterize proliferative cells from the stem cell compartment of the colonic epithelium. 2. To determine whether the above proliferative epithelial cells can ameliorate experimental colitis in mice. 3. To determine whether there is an alteration in epithelial stem cell numbers and distribution in the colonic mucosa of patients with inflammatory bowel disease. METHODS: Human participants were recruited for these studies from those patients who were undergoing colonoscopy in the Department of Gastrointestinal Sciences of this hospital and were recruited into cases or controls depending on their satisfying inclusion criteria. Inclusion criteria: Ulcerative colitis: 1. Patients who were diagnosed to have ulcerative colitis, based on consensus Asian criteria that include clinical information, colonoscopy findings, biopsy findings consistent with idiopathic ulcerative colitis, and exclusion of infections (Ouyang et al, 2006). All patients seen in the Inflammatory Bowel Diseases Clinic and the Gastroenterology Clinic of this hospital underwent investigation according to a defined protocol that included colonoscopy and segmental ileo-colorectal biopsies. Colonoscopy was performed in the Endoscopy Unit and all biopsies were reported under the supervision of specialist gastrointestinal pathologists. 2. Age 18 years or older, of either sex. 3. Ability to give informed consent. Control participants: 1. Patients with irritable bowel syndrome or rectal bleeding undergoing screening colonoscopy to exclude inflammatory bowel disease, colonic polyps or cancer. 2. Normal colonoscopy and rectal biopsy. 3. Age 18 years or older, of either sex. 4. Ability to give informed consent. SUMMARY: In Summary, these studies provide new insight into colonic epithelial stem cell physiology including alterations in ulcerative colitis and in experimental colitis induced by dextran sodium sulphate in mice. Furthermore, alterations in stem cell signalling in the colonic mucosa of ulcerative colitis patients are pinpointed most of which appear to be specific to the disease and only a few secondary to inflammation. The specific Conclusions are as follows: 1. Expression of intracellular stem cell marker Musashi-1 and differentiation marker Hes-1 by immunocytochemistry and PCR revealed that proliferative cells are present in the pool of colonocytes isolated from mouse colon, and their distribution and numbers could be determined. In suspension cultures, we could identify the clones at the end of 14 days which were positive only for Musashi-1 and not Hes-1. So this confirms that zonal proliferative cells of the colon have the clonogenic potential by which it undergoes self-renewal by itself and forms progenitor cells. 2. Luminal infusion of proliferative colonic epithelial cells from donor mice into the colon of mice with dextran sodium sulphate colitis was not an effective method for epithelial stem cell transplantation from donor to recipient mice. 3. The comparison between proximal and distal colon of colonic epithelial stem cells represents that there was no difference observed in the numbers and distribution of these cells between proximal and distal colon of normal mouse as determined by immunostaining using the antibodies to Musashi-1 and Hes-1. 4. In mice with DSS colitis, there was no change in either proximal or distal colon in staining pattern against the antibodies Musashi-1 and Hes-1, even though the distal colon was more inflamed than the proximal colon. 5. There were significant changes observed between the colonic mucosal biopsies of control patients and colitis patients in both the numbers as well as in the location of stem cells in the colon. There was an increase in the numbers of stem cells in colitis patients when compared to control subjects and the stem cell location was also altered with the Musashi-positive cells being seen at the top of crypts in the colitis patients, while they were confined to the bottom of the crypts in normal controls. 6. There was no alteration in mRNA expression of genes related to stem cell multipotency, or in the Sonic and Indian Hedgehog pathways in ulcerative colitis. 7. mRNA expression of Fibroblast Growth Factor Receptor 1 was significantly down-regulated in active ulcerative colitis, while expression of Caudal type homeobox 2 gene was downregulated in UC in remission. The former is involved in tissue repair and the latter in cell differentiation in the crypt. 8. Notch2 and Notch3 expression was down-regulated in inactive ulcerative colitis, reflecting alterations in Notch signalling pathways in the disease. 9. Alterations in transforming growth factor (TGF)-β signalling pathways were reflected as down-regulation of SMAD3 and SMAD9 expression in both active and inactive UC possibly reflecting a reparative response to injury in colitis. Down-regulation of ACVRL1 in inactive UC may be a more specific change in a cell surface receptor for TGF- β. 10. Wnt signalling pathways were affected in inactive UC, including down-regulatino of Frizzled 7 & 8 and nuclear factor of activated T cells 4 (NFATC4), reflecting altered regulation of genes relating to crypt epithelial cell differentiation in the colon. NFACT1 expression was down-regulated in active UC

Effect of Bio sulphur granules (BSG) as fertilizer ingredient on different fractions of sulphur in calcareous soil cultivated with blackgram (Var.VBN-8)

The purpose of this study was to examine the various sulphur (S) fractions in experimental pot calcareous soil treated with Bio sulphur granules (BSG) in order to assess the impact of granular sulphur fertilization in S deficient calcareous soil using blackgram (Var. VBN-8) as a test crop.Factorial randomized block design with ten treatments (T1- Absolute control;T2-Recommended dose of NPK and S (Control);T3-Soil test based NPK; T4-T3 + S as Elemental Sulphur @ 40 kg S/ha; T5-T3 + S as BSGI@ 40 kg S/ha; T6-T3 + S as BSGII@ 40 kg S/ha;T7-T3 + Vermicompost @ 4 t ha-1; T8-T4  + Vermicompost @ 4 t ha-1;T9- T5 + Vermicompost @ 4 t ha-1; T10- T6+ Vermicompost @ 4 t ha-1 ) replicated thrice and 5 pots were maintained for each replication. The results of this study revealed that there was an upward trend in all S fractions in every treatment (T1 to T10), in the following order: organic > inorganic > water soluble > exchangeable S. The pot that received vermicompost coupled with BSG II (T10) (ES@ 40 kg ha-1 and MethylobacteriumthiocyanatumVRI7-A4 as S source) was found to have the greatest S-fraction and was higher than other treatments. Therefore, using BSG II in conjunction with vermicompost is necessary to preserve the availability of S nutrients in calcareous soil and increase the solubility of nutrients through S-oxidation

Defense related enzyme induction in coconut by endophytic bacteria (EPC 5)

Endophytic bacteria Bacillus subtilis (EPC 5) was isolated and tested in vitro along with Pseudomonas fluorescens (Pf1) and the fungus Trichoderma viride (Tv1) against Ganoderma lucidum (Leys) Karst, the causal agent of basal stem rot on coconut palm. The endophytic bacterial strains namely EPC 5 and EPC 8 showed higher vigor index (germination percentage, root and shoot length) and more inhibition against G. lucidum over un-inoculated control. These strains were confirmed as Bacillus subtilis by biochemical tests, cloning and sequencing of internal transcribed spacer (ITS) region. The Bacillus subtilis (EPC 5) along with Pseudomonas fluorescens (Pf1) and Trichoderma viride (Tv1) has been tried as bioconsortia against basal stem rot disease under greenhouse conditions. The soil application of bioconsortia enriched with farm yard manure (FYM) enhanced the coconut saplings growth under greenhouse conditions and showed higher induction of defense related enzymes like peroxidase, polyphenol oxidase, phenylalanine ammonia lyase and phenols when challenged with pathogen

Decoding the Distribution of Glycan Receptors for Human-Adapted Influenza A Viruses in Ferret Respiratory Tract

Ferrets are widely used as animal models for studying influenza A viral pathogenesis and transmissibility. Human-adapted influenza A viruses primarily target the upper respiratory tract in humans (infection of the lower respiratory tract is observed less frequently), while in ferrets, upon intranasal inoculation both upper and lower respiratory tract are targeted. Viral tropism is governed by distribution of complex sialylated glycan receptors in various cells/tissues of the host that are specifically recognized by influenza A virus hemagglutinin (HA), a glycoprotein on viral surface. It is generally known that upper respiratory tract of humans and ferrets predominantly express α2→6 sialylated glycan receptors. However much less is known about the fine structure of these glycan receptors and their distribution in different regions of the ferret respiratory tract. In this study, we characterize distribution of glycan receptors going beyond terminal sialic acid linkage in the cranial and caudal regions of the ferret trachea (upper respiratory tract) and lung hilar region (lower respiratory tract) by multiplexing use of various plant lectins and human-adapted HAs to stain these tissue sections. Our findings show that the sialylated glycan receptors recognized by human-adapted HAs are predominantly distributed in submucosal gland of lung hilar region as a part of O-linked glycans. Our study has implications in understanding influenza A viral pathogenesis in ferrets and also in employing ferrets as animal models for developing therapeutic strategies against influenza.Singapore-MIT Alliance for Research and Technolog

Sigma-Metric Analysis to Evaluate Quality Management of Analytical Processes Using RCA and QGI in a Clinical Biochemistry Laboratory, South India

Introduction: This study aimed to identify laboratory errors at the earliest through Sigma-metric analysis and to evaluate quality management of analytical processes. Methods: Sigma-metrics and Quality Goal Indices (QGI) were calculated by harvesting the IQC and EQC data of an accredited laboratory for 31 biochemical parameters run on Roche Cobas6000 and e411. Those with Sigma 2 were further analysed by applying the various Westgard rules, as suggested. Results: Nearly 13 chemistry analytes showed world-class performance with Sigma \u3e 6 and most of the immunoassay parameters showed marginal performance with sigma \u3e 2 6. Sodium, Chloride, Total T4, Beta-HCG and TSH were found to have Sigma \u3c 2 indicating unacceptable performance. A significant improvement was observed in the Sigma-metrics analysis after performing the root cause analysis. Conclusion: Sigma-metric analyses the quality management of various analytical processes in biochemistry. The poor assay performance will be picked up by the Root cause analysis and Quality Goal Indices calculation. With the help of RCA and QGI, we plan to increase the resource management by decreasing the frequency of QC runs

An analysis of stromal expression of CD10 in invasive ductal carcinoma of breast and its correlation with histological grade

Background: Breast cancer is one of the most common cancers among women in India. Stroma has an important role in the pathogenesis of carcinoma of breast. Stromal marker can be novel marker for assessing the prognosis of breast cancer.Methods: With the representative sections of 30 invasive ductal carcinoma of breast NOS type Hematoxylin and eosin staining was done. Immunohistochemistry was done with CD10. CD10 expression in stroma in cases and control slides were studied and statistically analyzed with histopathological grade.Results: 46% (14 out of 30) of cases showed strong positivity for stromal CD10. Only two cases of strong positivity for CD10 were noted in the adjacent normal breast parenchyma. Stromal expression of CD10 had a statistically significant association with breast carcinoma than in control slides, p value is 0.002. 77% (10 out of 13) of CD10 positive cases were high grade carcinomas. Association of CD10 expression with high grade tumour was statistically significant (p value is 0.04 which is less than 0.05). No association was found with mean age.Conclusions: As the grade of breast carcinoma increases the stromal expression of CD10 is increased. Stromal CD10 expression is directly correlated with higher tumour grade. CD10 could be used as novel prognostic marker and used to develop newer drugs.

The risk of lymphedema after postoperative radiation therapy in endometrial cancer

Objective: Lower extremity lymphedema adversely affects quality of life by causing discomfort, impaired mobility and increased risk of infection. The goal of this study is to investigate factors that influence the likelihood of lymphedema in patients with endometrial cancer who undergo adjuvant radiation with or without chemotherapy. Methods: A retrospective chart review identified all stage I–III endometrial cancer patients who had a hysterectomy with or without complete staging lymphadenectomy and adjuvant radiation therapy between January 2006 and February 2013. Patients with new-onset lymphedema after treatment were identified. Logistic regression was used to find factors that influenced lymphedema risk. Results: Of 212 patients who met inclusion criteria, 15 patients (7.1%) developed new-onset lymphedema. Lymphedema was associated with lymph-node dissection (odds ratio [OR], 5.6; 95% CI, 1.01 to 105.5; p=0.048) and with the presence of pathologically positive lymph nodes (OR, 4.1; 95% CI, 1.4 to 12.3; p=0.01). Multivariate logistic regression confirmed the association with lymph-node positivity (OR, 3.2; 95% CI, 1.0007 to 10.7; p=0.0499) when controlled for lymph-node dissection. Median time to lymphedema onset was 8 months (range, 1 to 58 months) with resolution or improvement in eight patients (53.3%) after a median of 10 months. Conclusion: Lymph-node positivity was associated with an increased risk of lymphedema in endometrial cancer patients who received adjuvant radiation. Future studies are needed to explore whether node-positive patients may benefit from early lymphedema-controlling interventions

Effect of phenolic compounds released during degradation of Coir pith by Oscillatoria annae on Albino rat ( Rattus norvegicus )

This study was carried out to investigate the effect of degraded coir pith based cyanobacterial culture filtrate on Rattus norvegicus . The culture filtrate containing phenolic compound was administered at a rate of 42 mg/Kg for 15 days and its effect on serum glucose, protein, creatinine, urea, uric acid, alkaline phosphate (ALP), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and sperm analysis was studied. The results showed a significant reduction in the levels of serum glucose and liver marker enzymes while serum protein and renal function test showed normal level in both experimental and control rats. Also, a significant reduction in semen count was reported. The results suggested that the phenolic compound of the culture filtrate appears to be non toxic in the tested animals @ JASE

Quantitative Description of Glycan-Receptor Binding of Influenza A Virus H7 Hemagglutinin

In the context of recently emerged novel influenza strains through reassortment, avian influenza subtypes such as H5N1, H7N7, H7N2, H7N3 and H9N2 pose a constant threat in terms of their adaptation to the human host. Among these subtypes, it was recently demonstrated that mutations in H5 and H9 hemagglutinin (HA) in the context of lab-generated reassorted viruses conferred aerosol transmissibility in ferrets (a property shared by human adapted viruses). We previously demonstrated that the quantitative binding affinity of HA to α2→6 sialylated glycans (human receptors) is one of the important factors governing human adaptation of HA. Although the H7 subtype has infected humans causing varied clinical outcomes from mild conjunctivitis to severe respiratory illnesses, it is not clear where the HA of these subtypes stand in regard to human adaptation since its binding affinity to glycan receptors has not yet been quantified. In this study, we have quantitatively characterized the glycan receptor-binding specificity of HAs from representative strains of Eurasian (H7N7) and North American (H7N2) lineages that have caused human infection. Furthermore, we have demonstrated for the first time that two specific mutations; Gln226→Leu and Gly228→Ser in glycan receptor-binding site of H7 HA substantially increase its binding affinity to human receptor. Our findings contribute to a framework for monitoring the evolution of H7 HA to be able to adapt to human host.National Institutes of Health (U.S.) (GM R37 GM057073-13)Singapore-MIT Alliance for Research and Technolog