Padmanabhan P. Nair

Understanding the nature of cell surface markers on exfoliated colonic cells is a crucial step in establishing criteria for a normally functioning mucosa. We have found that colonic cells isolated from stool samples (SCSR-010 Fecal Cell Isolation Kit, NonInvasive Technologies, Elkridge, MD), preserved at room temperature for up to one week, with viability of >85% and low levels of apoptosis (8% - 10%) exhibit two distinct cell size subpopulations, in the 2.5μM– 5.0 μM and 5.0μM-8.0μM range. In addition to IgA, about 60% of the cells expressed a novel heterodimeric IgA/IgG immunoglobulin that conferred a broad-spectrum cell mediated cytotoxicity against tumor cells. In a cohort of 58 subjects the exclusive absence of this immunoglobulin in two African-Americans was suggestive of a germline deletion. Serial cultures in stem cell medium retained the expression of this heterodimer. Since a majority of the cystic cells expressed the stem cell markers Lgr5 and Musashi-1 we termed these cells as gastrointestinal progenitor stem cells (GIP-C**). CXCR-4, the cytokine co-receptor for HIV was markedly expressed. These cells also expressed CD20, IgA, IgG, CD45, and COX-2. We assume that they originated from mature columnar epithelium by dedifferentiation. Our observations indicate that we have a robust noninvasive method to study mucosal pathophysiology and a direct method to create a database for applications in regenerative medicine.

Evidence of Multimeric Forms of HSP70 with Phosphorylation on Serine and Tyrosine Residues - Implications for Roles of HSP70 in Detection of GI Cancers HSP70;multimeric forms;phosphorylation;serine;tyrosine;GI cancer detection; Background: Heat-shock protein70 (HSP70) are intracellular protein chaperones, with emerging evidence of their association with various diseases. We have previously reported significantly elevated plasma-HSP70 (pHSP70) in pancreatic cancer. Current methods of pHSP70 isolation are ELISA-based which lack specificity due to cross-reactivity by similarities in the amino-acid sequence in regions of the protein backbone resulting in overestimated HSP70 value. Materials and Methods: This study was undertaken to develop a methodology to capture all isoforms of pHSP70, while further defining their tyrosine and serine phosphorylation status. Results: The methodology included gel electrophoresis on centrifuged supernatant obtained from plasma incubated with HSP70 antibody-coupled beads. After blocking non-specific binding sites, blots were immunostained with monoclonal-antibody specific for human-HSP70, phosphoserine and phosphotyrosine. Conclusions: Our novel immunocapture approach has distinct advantages over the commercially available methods of pHSP70 quantification by allowing isolation of molecular aggregates of HSP70 with additional ability to precisely distinguish phosphorylation state of HSP70 molecules at serine and tyrosine residues.

Epidemiological studies suggest a protective role for β-carotene with several malignancies. Esophageal adenocarcinoma frequently arises from Barrett's esophagus (BE). We postulated that β-carotene therapy maybe protective in BE. We conducted a prospective study in which 25 mg of β-carotene was administered daily for six-months to six patients. Each patient underwent upper endoscopy before and after therapy and multiple mucosal biopsies were obtained. Additionally, patients completed a gastroesophageal reflux disease (GERD) symptoms questionnaire before and after therapy and severity score was calculated. To study the effect of β-carotene at molecular level, tissue extracts of the esophageal mucosal biopsy were subjected to assessment of heat-shock protein 70 (HSP70). A significant (p<0.05) reduction in mean GERD symptoms severity score from 7.0±2.4 to 2.7±1.7 following β-carotene therapy was noted. Measurement of Barrett's segment also revealed a significant reduction in mean length after therapy. In fact, two patients had complete disappearance of intestinal metaplasia. Furthermore, marked enhancement of HSP70 expression was demonstrated in biopsy specimens from Barrett's epithelium in four cases that were tested. Long- term β-carotene therapy realizes amelioration of GERD symptoms along with restitution of the histological and molecular changes in esophageal mucosa of patients with BE, associated with concurrent increase in mucosal HSP70 expression.

Objectives We examined the results of pancreatic juice cytodiagnosis using the method of endoscopic nasopancreatic drainage (ENPD) to identify pancreatic carcinoma in situ and compared the images and pathologic diagnosis of pancreatic carcinoma in situ as well as clinicopathologic characteristics. Methods In patients who underwent endoscopic retrograde cholangiopancreatography and had ENPD place, only patients presenting with focal stenosis and distal dilatation of the main pancreatic duct were included in the ENPD placement group. Endoscopic nasopancreatic drainage was conducted 27 times in 20 patients in the ENPD placement group. In an average session, cytodiagnosis of the pancreatic juice was conducted 5.3 times (range, 2–11 times). Results Results of cytodiagnosis were positive in 15 of 20 patients. Results of ENPD cytodiagnosis and diagnosis of pancreatic cancer showed sensitivity of 100%, specificity of 83.3%, and accuracy of 95%. Seven of 15 patients were diagnosed with carcinoma in situ. In these 7 patients, tumor markers (carcinoembryonic antigen, CA-19-9) were within reference limits, and the tumors were not visible on imaging tests. Pathologic histology revealed a propensity for the cancer to proliferate around the stenosis of the pancreatic duct. Conclusions Cytodiagnosis of pancreatic juice using ENPD multiple times proved to be useful in the diagnosis of pancreatic carcinoma in situ.

There is significant evidence supporting the hypotheses that lifestyle, diet, and bioactive components in foods are important modifiers of cancer risk. However, our ability to assess host response noninvasively is limited. To overcome this, we have developed a technology to isolate several million viable exfoliated somatic colonic cells from a small sample of stool (0.5–1.0 g) by a procedure known as somatic cell sampling and recovery (SCSR). Orally administered carotenoids appear in these cells several days after consuming the supplement, usually showing a peak concentration between 5–7 d after their ingestion. The time lag observed for the appearance of orally administered carotenoids in SCSR cells corresponds to the turnover rate of the colonic mucosa. This is an example of how changes in cell turnover rates can be carefully assessed using the SCSR system. The specific mechanisms by which individual constituents of diet affect the cancer process are not fully understood. However, host response to dietary constituents may be investigated, noninvasively, by following the modulation of tumor-associated molecular markers in these exfoliated SCSR cells. We have demonstrated the feasibility of using SCSR cells to detect the expression of carcinoembryonic antigen, CD44, and its splice variants, c-myc, c-erbb2, and mutations in the p53 gene. In this regard, SCSR cells are a readily available surrogate cellular target that may serve to monitor changes in cell turnover, differentiation, and expression of cancer-associated biomarkers that are likely to be modulated by bioactive food components.

Objective. Colorectal cancer is a major cause of cancer mortality in the industrialized nations in the West. Because mortality is closely related to the stage of the disease at the time of diagnosis, detection at an early stage is likely to result in improved recovery rates. Since current diagnostic procedures such as colonoscopy are invasive and the fecal occult blood test (FOBT) lacks sensitivity and specificity for the detection of early lesions, the development of non-invasive methods based on molecular markers of neoplasia can lead to earlier diagnosis and more favorable outcomes for patients with colorectal cancer. Recent advances in the technology for isolating colonocytes from stool (SCSR (somatic cell sampling and recovery)) provide a non-invasive tool for the study of biomarkers expressed in colorectal cancer. The aim of this study was to detect mRNA expression of three biomarkers: (c-erbb-2 and two forms of c-myc: p64 and p67) in fecal colonocytes and to evaluate its use in diagnosing colorectal cancer. Material and methods. Colonocytes (SCSR cells) were isolated from stools from 30 subjects: 15 colorectal cancer patients and 15 normal controls. One cancer patient was excluded from the final data analysis because the tumor was a gastrointestinal lymphoma. Each sample yielded two fractions: a pellet and an interphase. Expression of c-myc p64, c-myc p67 and c-erbb-2 mRNA was evaluated in each of the fractions by reverse transcriptase-polymerase chain reaction (RT-PCR). A marker was considered positive upon detecting an amplicon of the expected size in agarose gel electrophoresis. Results. c-myc p64 mRNA expression was observed in both fractions in 78.5% of colorectal cancer patients, compared with 13.3% in the control group (p=0.009). For c-myc p67, 78.6% of the colorectal cancer patients showed mRNA expression in both fractions in comparison with only 13.3% of the controls (p=0.003). C-erbb-2 showed no significant difference in mRNA expression between colorectal cancer and controls. When the data were analyzed for co-expression of c-myc p64 and c-myc p67, in both pellet and interphase, sensitivity was 64% and specificity was 100%. Conclusions. Fecal colonocytes isolated by somatic cell sampling and recovery (SCSR) technology could be used for the non-invasive assessment of the expression of biomarkers of colon cancer such as c-myc p64, c-myc p67 and c-erbb-2. The expression of c-myc p64 and c-myc p67 in colonocytes showed a significant association with colorectal cancer and may be helpful as a biomarker for the non-invasive detection of colorectal cancer.

The possibility of using exfoliated colonic epithelial cells for assessing the bioavailability of β-carotene was examined. Analysis of exfoliated colonic epithelial cells showed the presence of β-carotene and vitamin A. The β-carotene content was significantly lower in cells from stool samples of subjects on a β-carotene-poor diet than those receiving a single dose of a β-carotene supplement. Colonic epithelial cells isolated from stool samples collected daily during a wash-out period while the subjects were on a β-carotene-poor diet showed a steady decrease in β-carotene content, reaching the lowest value on day 7. Kinetic analysis showed that a single dose of a β-carotene supplement in the form of spirulina (Spirulina platensis) or agathi (Sesbania grandiflora) after the wash-out period caused an increase in the β-carotene content after a lag period of 5–7 d, but the vitamin A levels during these periods were not significantly affected. Analysis of plasma β-carotene concentration also showed similar changes, which correlated with those of exfoliated colonic cells. A relationship between the β-carotene content of the diet and that of the colonic epithelial cells suggests that analysis of the β-carotene content in exfoliated human colonic epithelial cells is a useful non-invasive method to assess the bioavailability of provitamin A β-carotene.

The gastrointestinal epithelium is known to undergo constant and rapid renewal resulting in millions of cells being shed into the fecal stream every day. The conventional wisdom was that these cells disintegrate upon exfoliation and will not survive the transit through the intestinal tract. In 1990, we (P.N.) made the discovery that a significant number of these cells remain intact and viable and that they can be isolated. The implications of this important discovery became apparent when we demonstrated that these cells are exclusively of colonic origin, are anatomically representative of the entire colon, and can be used for clinical investigations of disease processes. The term coprocytobiology (CCB) was coined to encompass the broad range of applications of this new technology. The somatic cell sampling and recovery (SCSR) process involves the isolation of exfoliated colonocytes from a small sample of stool (approximate to 1 g) collected and transported in a unique medium at ambient temperature, providing cells for the detection of a number of biomarkers of disease propensity. These exfoliated colonocytes express cytokeratins indicating epithelial lineage as well as colon-specific antigen. Over the years, the study of exfoliated colonocytes has provided striking new insights into the biology of colon cancer and inflammatory bowel disease, including detection of p53 gene mutations, reverse transcriptase polymerase chain reaction amplification, and identification of CD44 splice variants, neoplasia-associated specific binding of plant lectins, and expression of COX-2, the inducible form of cyclooxygenase. The functional diversity of cells isolated by SCSR is revealed by the demonstration of cell surface markers such as secretory component, IgA, and IgG on the one hand and the amplification and cloning of the human insulin receptor and the expression of the multidrug resistance gene mdr-1 on the other hand. This review portrays the immense potential of CCB as a powerful tool for investigating the pathophysiology of disease, identifying genetic variants in pharmacogenetics, assessment of mucosal immunity, and several other applications that use somatic cells.