Constitutive expression of inducible nitric oxide synthase in the normal human colonic epithelium.

BACKGROUND: Inducible nitric oxide synthase (iNOS) in the human colon is considered expressed only in inflammatory states such as ulcerative or collagenous colitis. As subtle iNOS labelling was previously observed in some colonic mucosal biopsies from a heterogeneous group of controls with non-inflamed bowel, we studied whether bowel preparation with bisacodyl or polyethylene glycol prior to sigmoidoscopy might induce iNOS expression. METHODS: Ten healthy, non-smoking male subjects were investigated. Mucosal biopsies were taken from the sigmoid colon prior to bowel preparation and again 12 h after rectal administration of bisacodyl or polyethylene glycol in randomized order. Expression of iNOS protein was quantified by Western blot analysis and localized by immunohistochemistry. RESULTS: iNOS was expressed in the colonic mucosal biopsies from all subjects and localized in the epithelial cells, particularly at the luminal border of the epithelial cells and more pronounced in the crypt epithelium. The expression of iNOS was unaffected by bowel preparation with bisacodyl or polyethylene glycol. CONCLUSIONS: iNOS is constitutively expressed in the normal colonic epithelium. The results suggest that synthesis of iNOS protein is unaffected by bowel preparation with the secretagogue laxative, bisacodyl, or polyethylene glycol.

Cytokeratin-positive cells in preoperative peripheral blood and bone marrow aspirates of patients with colorectal cancer.

UNLABELLED: Detection of cytokeratin-positive cells in bone marrow and peripheral blood may have prognostic significance in cancer patients. Furthermore, a correlation between uPAR expression on micrometastases and patient prognosis has been suggested. However, in patients with colorectal cancer, preoperative detection and characterization of tumour cells in bone marrow and peripheral blood, using an immunocytochemical approach, have not yet been substantiated as a prognostic tool. METHODS: Forty-one bone marrow aspirates and 38 peripheral blood aspirates, obtained preoperatively from patients with colorectal cancer, were immunocytochemically screened for cytokeratin-positive cells. Where cytokeratin-positive cells were observed, an additional microslide was double immunostained for simultaneous detection of cytokeratin and uPAR/CD87. RESULTS: Cytokeratin-positive cells were observed in 4 out of 41 bone marrow aspirates (10%). None of the isolated cytokeratin-positive cells expressed uPAR. In peripheral blood, no cytokeratin-positive cells were observed. CONCLUSION: The present study does not support the introduction of a routine immunocytochemical identification of cytokeratin-positive cells in preoperatively obtained bone marrow aspirates or peripheral blood from patients with colorectal cancer.

Expression of nitric oxide synthases and effects of L-arginine and L-NMMA on nitric oxide production and fluid transport in collagenous colitis.

BACKGROUND AND AIMS: Luminal nitric oxide (NO) is greatly increased in the colon of patients with collagenous and ulcerative colitis. To define the source and consequence of enhanced NO production we have studied expression of NO synthase (NOS) isoforms and nitrotyrosine in mucosal biopsies from these patients. In addition, effects on colonic fluid transfer caused by manipulating the substrate of NOS were studied in patients with collagenous colitis. PATIENTS: Eight patients with collagenous colitis, nine with active ulcerative colitis, and 10 with uninflamed bowel were included. METHODS: Expression of NOS isoforms was quantified by western blotting. Inducible NOS (iNOS) and nitrotyrosine were localised by immunohistochemistry. Modulation of NOS activity by topical N(G)-monomethyl-L-arginine (L-NMMA) or L-arginine was assessed during perfusion of whole colon. Plasma and perfusate nitrite/nitrate (NOx) was measured by Griess' reaction. RESULTS: Both in collagenous and ulcerative colitis, expression of iNOS was 10(2)-10(3) higher (p<0.001) than in uninflamed bowel and localised primarily to the epithelium. Endothelial NOS was evenly expressed in all groups while neuronal NOS was undetectable. Nitrotyrosine was markedly expressed in active ulcerative colitis but rarely detected in collagenous colitis and never in uninflamed bowel. In collagenous colitis, the output of NOx was markedly increased compared with uninflamed bowel (283 (58) v <37 nmol/min; p<0.01) and fluid was net secreted. L-NMMA reduced the output of NOx by 13-66% (95% confidence intervals) and secretion of fluid by 25-109% whereas L-arginine increased the output of NOx by 3-39% and secretion of fluid by 15-93%. CONCLUSIONS: In collagenous colitis, as opposed to ulcerative colitis, upregulation of iNOS occurs in the absence of nitrotyrosine formation and mucosal damage. Excess generation of NO may be the primary cause of diarrhoea in this condition.

The use of the CELLection kit in the isolation of carcinoma cells from mononuclear cell suspensions.

A study was performed to evaluate in vitro the sensitivity, specificity and variability of a new immunomagnetic microbead isolation technique which provides subsequent immunological staining of captured carcinoma cells. In a mixture of peripheral blood mononuclear cells (PBMCs) and human carcinoma cells the epithelial cancer cells were isolated with the Dynal((R)) RAM IgG1 CELLection Kit using Dynabeads M-280 coated with a rat monoclonal antibody (Mab) against mouse IgG1. The rat Mab was biotinylated and attached to Dynabeads via streptavidin and a DNA linker. The anti-epithelial monoclonal mouse antibody Ber-EP4 was used as the primary capture antibody. In order to permit phenotyping of the isolated carcinoma cells the magnetic beads were removed from the carcinoma cells by DN'ase digestion of the DNA linker between the magnetic bead and the secondary antibody. In an ex vivo model system an average recovery of approximately 60% of a human colon carcinoma cell line HCC-2998 seeded in 5.10(6) PBMCs was obtained, and the recovered cells could subsequently be immunologically stained for the surface antigen CD87 (urokinase plasminogen activator receptor). No positive stained cells were found in control experiments with PBMCs without carcinoma cells. Despite a relatively low recovery, the described method will be valuable for the detection of carcinoma cells in cytospin preparations with subsequent phenotyping of the cells for expression of surface antigens. Depending on the chosen antibodies, the method may be useful for the isolation and characterisation of other cell types in various cell suspensions.

Immunoglobulin and enzyme-conjugated dextran polymers enhance u-PAR staining intensity of carcinoma cells in peripheral blood smears.

The presence of disseminated carcinoma cells in bone marrow and peripheral blood has prognostic importance in patients with carcinomas. Much evidence indicates that dissemination of tumor cells may depend on activation of a variety of degradative enzymes. A strong positive correlation has been shown between the expression of tumor cell proteases and tumor invasion. Therefore, phenotypic characterization of disseminated carcinoma cells for expression of protease activators might define the invasive potential of the cells. We present an immunocytochemically enhanced staining method that allows phenotyping of disseminated carcinoma cells in bone marrow and peripheral blood smears. In the first step, the cells were incubated with antibodies against urokinase plasminogen activator receptor (u-PAR) and subsequently with secondary antibodies conjugated to peroxidase-labeled dextran polymers. A brown color reaction was developed with diaminobenzidine as chromogen. In the second step, the cells were incubated with alkaline phosphatase-conjugated murine monoclonal antibodies against a common cytokeratin epitope and a red color reaction was developed with new fuchsin as substrate. This method allows simultaneous and unambiguous immunolabeling of intracellular cytokeratin and of u-PAR intracellularly and on the surface of carcinoma cells. This novel approach can be used for detection and phenotyping of carcinoma cells in blood smears for u-PAR or, presumably, for any other heterogeneously expressed antigen on the surface of the detected cells.

Immunodeficiency in HIV-2 infection: a community study from Guinea-Bissau.

In a community study in Guinea-Bissau, West Africa, 47 HIV-2-seropositive cases and 87 matched controls were evaluated immunologically using immuno-alkaline phosphatase linked to avidin-biotin complex for the assessment of CD4 and CD8 status. HIV-2-seropositive individuals had significantly lower total numbers of CD4 cells and CD4/CD8 ratios, 38% having a total number of CD4 cells less than or equal to 0.5 x 10(9)/l and 36% having a CD4/CD8 ratio less than or equal to 0.8. Total numbers of CD4 cells less than or equal to 0.5 x 10(9)/l or CD4/CD8 ratio less than or equal to 0.8 were found in 53% of the HIV-2 seropositives compared with 11% among controls [odds ratio (OR) = 7.3; 95% confidence interval (CI): 3.1-17.1]. Lymphadenopathy was significantly more frequent among HIV-2 seropositives than among controls (OR = 3.4; 95% Cl: 1.5-7.6). HIV-2 seropositives with lymphadenopathy had significantly fewer lymphocytes (P = 0.008) and lower total CD4 (P = 0.029) and total CD8 number (P = 0.011) than HIV-2 seropositives without lymphadenopathy. This study indicates that HIV-2 has a significant immunosuppressive effect.

Labelling of T cell subsets under field conditions in tropical countries. Adaptation of the immuno-alkaline phosphatase staining method for blood smears.

Immuno-alkaline phosphatase (AP) staining for T cell subsets (CD4 and CD8) of smears from fingerprick blood functioned well under tropical climatic conditions when smears were stored frozen with silica gel before being labelled. Unlabelled smears were stored for up to 12 months and could be transferred abroad without antigenic damage. Identical total CD4 and CD8 counts were obtained on venous and capillary blood, when compared using a FACS analyser. Although the AP method gave somewhat higher total CD4 and CD8 counts, the ratio remained the same. The major advantages of the method are: (i) no expensive equipment is required, (ii) only minute amounts of blood are needed, and (iii) slides can be stored for long periods before labelling and can be preserved for later reading. The method is suitable for community studies where there is a need for assessing the immune status of the population.

Nutritional assessment of psychogeriatric patients.

The nutritional status of 91 patients on four psychogeriatric wards was assessed by anthropometric measurements and determination of circulating proteins. The patients had low mean values for weight, arm muscle circumference, plasma albumin and serum transferrin. Indicators of malnutrition were combined to define the nutritional status in each individual. Energy and/or protein undernutrition was found in 30% and obesity in 4%. Energy undernutrition was more common than protein deficiency. Undernutrition was quite common during the first year of hospitalization and did not correlate with the duration of hospital stay. Subjects with their own teeth had a lower prevalence of undernutrition than edentulous patients. Food intake was similar in patients with and without undernutrition. The possible interactions between malnutrition and chronic psychiatric disorders in the elderly are discussed.