P2X7 receptor gene polymorphism analysis in rheumatoid arthritis.

The P2X7 receptor, a member of the P2X family of nucleotide-gated channels, is predominantly expressed by monocytic cells. The activation of this receptor has been associated with downstream-signalling cascades, resulting in the release of a number of inflammatory mediators. There are more than 815 single nucleotide polymorphisms (SNPs) that have been described in the human P2X7R gene, but only few have been functionally characterized. The main aim of this study is to determine whether P2X7R gene polymorphisms confer susceptibility to rheumatoid arthritis (RA). A total of 125 patients with RA and 158 healthy volunteers were enrolled in this study. DNA fragment was PCR amplified and sequenced on the AB 3130 Genetic Analyzer. No significant difference in allele frequencies of 489 C→T, 1096 C→G and 1513 A→C polymorphisms, among sporadic cases of RA and healthy controls was found. However, the 1513A/C genotype was significantly associated with the presence of rheumatoid factor and anti-MCV autoantibody in RA patients. Interestingly, the genotype frequency of 1068 A/A was 0.19 in the RA group and 0.09 in control group (P = 0.025). Consequently, this polymorphism (AA) is two folds greater in the RA group compared to controls. Moreover, this polymorphism was significantly associated with mean concentration of C-reactive protein in RA patients. In contrast, 946G→A and 1729 T→A were not detected in both groups. As a result, these two polymorphisms are uncommon in Omani Arab population. Polymorphism at position 1068 and 1513 in the P2X7R gene might contribute to the pathogenesis of RA. Moreover, the loss-of-function SNP at position 1096 C→G or the gain-of-function SNP at position 489 C→T of the P2X7 gene does not appear to be a susceptibility gene locus for the development of RA. Further studies are required to confirm this finding.

Canary in the coliform mine: Exploring the industrial application limits of a microbial respiration alarm system.

Fundamental ecological principles of ecosystem-level respiration are extensively applied in greenhouse gas and elemental cycle studies. A laboratory system termed CEMS (Carbon Dioxide Evolution Measurement System), developed to explore microbial biofilm growth and metabolic responses, was evaluated as an early-warning system for microbial disturbances in industrial settings: in (a) potable water system contamination, and (b) bioreactor inhibition. Respiration was detected as CO2 production, rather than O2 consumption, including aerobic and anaerobic metabolism. Design, thresholds, and benefits of the remote CO2 monitoring technology were described. Headspace CO2 correlated with contamination levels, as well as chemical (R2 > 0.83-0.96) and microbiological water quality indicators (R2 > 0.78-0.88). Detection thresholds were limiting factors in monitoring drinking water to national and international standards (0 CFU/100 mL fecal coliforms) in both open- (>1500 CFU/mL) and closed-loop CO2 measuring regimes (>100 CFU/100 mL). However, closed-loop detection thresholds allow for the detection of significant contamination events, and monitoring less stringent systems such as irrigation water (<100 CFU/mL). Whole-system respiration was effectively harnessed as an early-warning system in bioreactor performance monitoring. Models were used to deconvolute biological CO2 fluctuations from chemical CO2 dynamics, to optimize this real-time, sustainable, low-waste technology, facilitating timeous responses to biological disturbances in bioreactors.

Ameliorative effect of sesamin in cisplatin-induced nephrotoxicity in rats by suppressing inflammation, oxidative/nitrosative stress, and cellular damage.

Nephrotoxicity of cisplatin (CP) involves renal oxidative stress and inflammation, and sesamin (a major liganin in many plants) has strong antioxidant and antiinflammatory actions. Therefore, we investigated here the possible mitigative action of sesamin on CP nephrotoxicity in rats. Sesamin was given orally (5 mg/kg/day, 10 days), and on the 7th day, some of the treated rats were injected intraperitoneally with either saline or CP (5 mg/kg). On the 11th day, rats were sacrificed, and blood and urine samples and kidneys were collected for biochemical estimation of several traditional and novel indices of renal damage in plasma and urine, several oxidative and nitrosative indices in kidneys, and assessment of histopathological renal damage. CP significantly and adversely altered all the physiological, biochemical and histopathological indices of renal function measured. Kidneys of CP-treated rats had a moderate degree of necrosis. This was markedly lessened when CP was given simultaneously with sesamin. Sesamin treatment did not significantly alter the renal CP concentration. The results suggested that sesamin had ameliorated CP nephrotoxicity in rats by reversing the CP-induced oxidative stress and inflammation. Pending further pharmacological and toxicological studies sesamin may be considered a potentially useful nephroprotective agent.

Detection of K-ras mutation in colonic effluent samples from patients without evidence of colorectal carcinoma.

BACKGROUND: K-ras gene mutation appears in more than 50% of patients with colon tumors. Both its frequency and early appearance may qualify this mutation as a potential biomarker. To enable early detection of mutant K-ras alleles, we had previously developed a sensitive polymerase chain reaction (PCR)-based assay, i.e., enriched PCR, which enables detection of one mutant K-ras allele present within 10,000 normal alleles. Using the enriched PCR, we were able to detect mutant K-ras alleles in "normal-appearing" colonic mucosa in patients with colorectal cancer. PURPOSE: A study was initiated to determine whether mutant K-ras alleles could be identified in colonic effluent samples of patients who may be at risk to develop colorectal cancer. METHODS: Over 9 years, colonic effluent samples were collected prior to routine colonoscopy from 39 patients who were apparently free of colorectal cancer. These samples were collected from patients with a family history of colorectal cancer (n = 7), adenomatous polyps (n = 7), previously resected colorectal cancer (n = 5), inflammatory bowel disorders (n = 13), normal colonoscopic examination (n = 6), and familial adenomatous polyposis (n = 1). All of the samples were double coded and analyzed for K-ras gene mutation. RESULTS: Of the 39 patients, seven were found to harbor mutant K-ras codon 12 alleles. Mutations were found in patients with a family history of colorectal cancer (three of seven), adenomatous polyps (one of seven), previously resected colorectal carcinoma (two of five), and familial adenomatous polyposis (one of one). In one case, effluent was found to harbor a mutant K-ras allele 4 years before the patient was diagnosed with colorectal cancer. CONCLUSIONS: (a) Effluent samples contain enough DNA to be detected with enriched PCR. Such samples may well be representative of the entire colon in general as opposed to a localized area such as that usually analyzed during colonoscopy. (b) K-ras gene mutation can be identified in routinely obtained colonic washings of patients who are at risk of developing colorectal cancer. Such mutations were absent in patients with inflammatory bowel disorders and in those who had undergone normal colonoscopic examinations. Detection of K-ras mutation in colonic washings may assist in identifying patients who may be at high risk for developing adenocarcinoma of the colon. IMPLICATIONS: The ability to examine colonic effluents provides a powerful and convenient source of sampling and may be adapted for future large-scale screening.

Polyps as biomarkers for colorectal neoplasia.

Current understanding of colorectal carcinogenesis suggests a series of genetic changes occurring pari passu with morphological changes ultimately resulting in a cancerous lesion. The adenomatous polyp was originally the prototype of the preneoplastic lesion but recently, other colonic polyps, primarily the hamartomas, have been clinically characterized as colorectal cancer biomarkers with genetic changes found mainly in the mesenchymal component as opposed to the ectodermal, or epithelial element. This, with the current interest in angiogenesis playing a role in the propagation of neoplastic lesions, has now encompassed every tissue element and opened the way for an understanding of the oncogenic process. This has suggested that considerable interaction occurs between all tissue elements, including what was previously described as epithelial-matrix interactions. While hyperplastic polyps are thought not to confer risk for cancer, they may offer clues as to the first steps of the overall process. Microadenomas have introduced new clinical as well as biological considerations, as unique risk factors. Investigation of these lesions has moved from purely morphological correlations to mechanistic dissections of important biological pathways using both genetic and protein chemistry tools. This review explores the microcosm of the colonic polyp and its relation to cancer as the quintessential premalignant biomarker.

Cancer associated antigen CA19-9 in colonic effluent of patients with neoplasia of the colon and inflammatory bowel disease.

We measured colonic effluent samples from 10 patients with colorectal cancer, 13 with adenomatous polyps, 14 with normal colons and compared them to 10 patients with inflammatory bowel disease by measuring this CA19-9 content. Results showed considerable overlap between the different pathologic categories, making differentiation impossible. A lower level of CA19-9 in the effluent samples from patients with adenomas was noted. These differences were reproducible for assays performed several months apart. CA19-9 may originate from the upper gastrointestinal tract since large amounts are present in pancreatico-biliary secretions. This antigen is therefore not useful in the diagnosis of neoplasia or inflammatory bowel disease using colonic effluent samples as the test material.

A putative CEA moiety is shared by the cotton-top tamarin (Saguinus oedipus) and humans.

CEA-like molecules immunologically distinct from those in humans have been described in non-human primates. These primates do not share the human predilection for colitis and subsequent development of colorectal cancer. CEA expression has not been fully evaluated in a lower-order primate, the cotton-top tamarin (Saguinus oedipus), an animal model for colitis and colorectal cancer. We found increased levels of CEA in both colonic washings and tissues of these animals using a commercially available kit, CEA AIA-PACK (Tosoh Medics, Foster City, CA). In contrast, we observed that other CEA kits failed to detect CEA in tamarins. To elucidate the nature of the CEA-like protein detected, we used the two component monoclonal antibodies used in the CEA AIA-PACK kit, and identified the reactive molecules by Western blotting. A band of approximately M(r) 50,000 was found to be common to samples from both humans and the tamarins. Minimal binding was observed with NCA antibody. We conclude that a CEA-like molecule shared by humans and tamarins may play a role in the pathogenesis of colitis and cancer in both species.

Carcinoembryonic antigen family of adhesion molecules in the cotton top tamarin (Saguinus oedipus).

Humans and the cotton top tamarin, a model for colitis and colorectal cancer, share carcinoembryonic antigen (CEA) moieties. We quantified CEA in colonic washings and extracts in both, and CEA bands were confirmed by Western blot. We compared CEA-family expression in tissues and serum in the tamarin with that of the common marmoset, which develops colitis but not cancer. CEA levels are higher in tamarin washings compared with humans, and higher than in marmosets extracts (P<0.005). CEA molecular species appear to be specific, and human CEA-family member epitopes are also found in these primates. The higher CEA levels in the tamarin may reflect the overall higher cancer prevalence.

Detection of carcinoembryonic antigen in colonic effluent by specific anti-CEA monoclonal antibodies.

To characterize the CEA in colonic effluent, anti-CEA monoclonal antibody COL-4 was used in a qualitative radioimmunoassay in both fractionated and unfractionated colonic effluent. Both effluent samples and tissue extracts, were subjected to Western blotting and tissue sections to immunohistochemistry. Quantitative levels of colonic effluent CEA were determined by a kit (Abbott-EIA). Higher mean values of COL-4 binding activity were seen only in patients with a past history of polyps (P < 0.01). Quantitated CEA correlated with the presence of colorectal cancer (CRC) as compared to normal subjects, (1133 +/- 875 vs. 459 ng/ml +/- 602, P < 0.05) but not when standardized for protein content. COL-4 reacted with an 180,000 M(r) CEA in the effluent and activity was associated with membrane fraction of the effluent, but bore no relation to the immunohistological staining. We conclude that CEA is detectable in colonic effluent and is membrane associated, but the overlapping values in effluent samples do not make this a useful test in the diagnosis of CRC.

Adenoma-derived antibody, Adnab-9 recognizes a membrane-bound glycoprotein in colonic tissue and effluent material from patients with colorectal neoplasia.

The binding in pre-colonoscopic effluent of Adnab-9, a monoclonal antibody raised against colonic adenomas, was evaluated for specificity in the diagnosis of colorectal cancer. A heterogeneous group of 58 patients was evaluated by ELISA. Effluent samples and tissue extracts were subjected to Western blotting or ELISA to confirm specificity. Immunohistochemistry was performed on the cancer tissue sections. The proportion of positive effluent binding was higher in the cancer when compared to the normal group (P = 0.036). A dominant 87 M(r) band was found in adenoma extracts and some effluent samples. Adnab-9 binding in effluent samples predominated in membrane-bound fractions. Immunohistochemistry showed no specific staining in the cancer cells. The antigen recognised is a glycoprotein shown by effects of N-glycanase digestion and not cross-reactive with carcinoembryonic antigen. Non-gastro-intestinal tissue extracts did not bind Adnab-9. The major 87 M(r) adenoma-derived antigen may be found in effluent material, particularly in the membrane-bound fraction.

Increased expression of a putative adenoma-associated antigen in pre-colonoscopic effluent of patients with colorectal cancer.

A monoclonal antibody Adnab-9, was raised against antigens derived from benign polyps of the colon. Adnab-9 was tested against pre-colonoscopic effluent material obtained from groups of patients with a macroscopically normal colonscopic examination, histologically confirmed adenomatous polyps and patients with colorectal cancer (CRC). The resultant binding levels displayed little overlap between the CRC group and the normal, and the difference was statistically significant. Since this putative early neoplasia associated antigen is essentially not expressed in CRC extracts, it may originate from a region of the colon predisposed to neoplasia, increasing in expression as the tendency to malignancy progresses, useful in the diagnosis of early stage malignancy.

Monoclonal antibody Adnab-9 defines a preneoplastic marker in epithelium at risk for adenocarcinoma of the small intestine.

Unlike colorectal cancer, risk markers for adenocarcinoma of the small intestine (ASI) have not been identified. Because the demographic and pathological features of both of these diseases are similar, immunohistochemistry was performed using monoclonal antibodies for three colonic premalignant markers, Adnab-9 (recognizes a colonic adenoma epitope), CaCo3/61, and FBB2/29 (small intestine proteoglycans expressed ectopically in colonic neoplasms), in normal and neoplastic small intestinal epithelium, and the results were compared with normal controls. Adnab-9 was also examined in 20 familial adenomatous polyposis (FAP) patients, a population known to be at an increased risk for ASI. Immunohistochemistry in normal and neoplastic tissue (adenoma, adenocarcinoma) from 18 patients with primary adenocarcinoma of the small intestine was compared with normal small intestine from 10 nonneoplastic controls. Four of 10 (40%) cases of normal small intestinal epithelium from controls were mildly positive in less than 10% of crypts, versus strong staining (>50% of crypts) in 16 of 18 (89%) patients with adenocarcinoma, and in 17 of 20 (85%) patients with FAP (P<.05). Adnab-9 predominantly stained Paneth cells as well as rare crypt and basal villous goblet cells. Adenomatous epithelium from the adenocarcinoma cases and adenomas from the FAP patients showed staining of Adnab-9 in 63% and 78% of cases, respectively. Only 17% of adenocarcinomas were positive for Adnab-9. In contrast, neither CaCo3/61 nor FBB2/29 showed any significant differences in the degree of staining in normal small intestinal epithelium in patients with adenocarcinoma compared with controls. Enhanced Adnab-9 staining in normal small intestinal epithelium from patients who harbor adenocarcinoma, and in FAP patients, supports its role as a risk marker of small intestinal neoplasia.

Cellular signals regulating antibiotic sensitivities of bacteria.

The effects of bacterial masses upon the drug resistance of neighboring bacteria were investigated. The experiments were performed with plastic Petri dishes divided into two identical compartments. A growing mass of Bacillus subtilis (signal emitter cell) in one compartment exerted enhancing effects upon the erythromycin and streptomycin resistance of Bacillus carboniphilus (signal recipient) cells, sparsely seeded in the other compartment, through the plastic wall and the air. These effects of the growing mass of cells are attributed to the emission of "sonic" signals.

Exfoliative colonic cytology. A simplified method of collection and initial results.

Exfoliative colonic cytology for the diagnosis of colorectal cancer has been largely abandoned due to (1) the widespread use of colonoscopy, (2) the cumbersome methods of cell collection and (3) the occasional difficulty of interpreting the cytologic findings in the presence of inflammatory bowel disease or adenomas. This paper describes a newly formulated bowel preparation for routine colonoscopy, based on imbibing 2 L to 4 L of a balanced electrolyte solution, in which the recovered precolonoscopic effluent (using a convenient disposable collecting kit) yielded cells for cytologic evaluation from 70% of a group of 80 patients at high risk for large bowel neoplasia. Cytology demonstrated neoplastic cells in most cases of endoscopically proven cancer. These results suggest that colonic exfoliative cytology may be useful as a supplemental test to routine colonoscopy. This could be enhanced by further methodologic modifications to the collecting and cytologic methods; large long-term studies are needed to evaluate the potential usefulness of colonic exfoliative cytology.

Evidence supporting an independent association between childhood physical abuse and lifetime suicidal ideation.

A regionally representative Canadian sample was used to investigate the gender-specific relationship between childhood physical abuse and lifetime suicidal ideation. The prevalence of suicidal ideation was about five times higher in abused men and women compared with their nonabused counterparts. After controlling for five clusters of potentially confounding factors (adverse childhood conditions, socioeconomic factors, health behaviors, psychosocial stressors/chronic illnesses, and mental health), childhood physical abuse was significantly associated with suicidal ideation (OR(adjusted) women = 4.48, 95% CI = 3.32-6.04; men = 3.57, 95% CI = 2.08-6.14). These findings suggest childhood physical abuse is independently associated with suicidal ideation and highlight the importance of providing preventative treatment to childhood abuse survivors.