E-selectin and very late activation antigen-4 mediate adhesion of hematopoietic progenitor cells to bone marrow endothelium

 Adhesion of CD34⁺ hematopoietic progenitor cells (HPCs) to sinusoidal endothelium probably plays a key role in homing of transplanted CD34⁺ HPCs to the bone marrow (BM). We have investigated the role of various adhesion molecules in the interaction of purified CD34⁺ HPCs derived from BM or peripheral blood (PB) and a human BM-derived endothelial cell line. Adhesion of CD34⁺ HPCs to endothelial cells was measured with the use of a double-color flow microfluorimetric adhesion assay. In this assay, adhesion is measured under stirring conditions, simulating blood flow in sinusoidal marrow vessels. Adhesion of PB CD34⁺ cells to human BM endothelial cells (HBMECs) was observed only after interleukin (IL)-1β prestimulation of the endothelial cells. This adhesion was strongly increased after addition of phorbol-myristate acetate (PMA). Adhesion of PB CD34⁺ cells to IL-1β-prestimulated HBMECs was inhibited by blocking monoclonal antibodies (mAbs) against E-selectin and by neuraminidase treatment of the PB CD34⁺ cells. mAbs against very late activation antigen (VLA)-4 inhibited adhesion only when the E-selectin-mediated interaction was prevented. No clear inhibiting effect was found with blocking mAbs against β₂-integrins. Stimulation with the β₁-integrin-activating mAb, 8A2, induced adhesion of CD34⁺ cells to endothelial cells. In conclusion, stimulation of both endothelial cells and CD34⁺ HPCs is necessary for adhesion of CD34⁺ HPCs to endothelial cells. We furthermore demonstrated that E-selectin and VLA-4 mediated this adhesion. Received: 26 April 1999 / Accepted: 8 February 2000     

Elastic band ligation of hemorrhoids： Flexible gastroscope or rigid proctoscope？

AIM： To compare dgid proctoscope and flexible endoscope for elastic band ligation of internal hemorrhoids.
METHODS： Patients between 18 and 80 years old, with chronic complaints （blood loss, pain, itching or prolapse） of internal hemorrhoids of grade 1-91, were randomized to elastic band ligation by rigid proctoscope or flexible endoscope （preloaded with 7 bands）. Patients were retreated every 6 wk until the cessation of complaints. Evaluation by three-dimensional anal endosonography was performed.
RESULTS： Forty-one patients were included （median age 52.0, range 27-79 years, 20 men）. Nineteen patients were treated with a rigid proctoscope and twenty two with a flexible endoscope. Twenty-nine patients had grade I hemorrhoids, 9 patients had grade 11 hemorrhoids and 3 patients had grade 91 hemorrhoids. All patients needed a minimum of 1 treatment and a maximum of 3 treatments. A median of 4.0 bands was used in the rigid proctoscope group and a median of 6.0 bands was used in the flexible endoscope group （P 〈 0.05）. Pain after ligation tended to be more frequent in patients treated with the flexible endoscope （first treatment： 3 vs 20 patients, P 〈 0.05）. Three- dimensional endosonography showed no sphincter defects or alterations in submucosal thickness. CONCLUSION： Both techniques are easy to perform, well tolerated and have a good and fast effect. It is easier to perform more ligations with the flexible endoscope. Additional advantages of the flexible scope are the maneuverability and photographic documentation. However, treatment with the flexible endoscope might be more painful and is more expensive.     

Direct Versus Competitive Biosensor Immunoassays for the Detection of (Dihydro)Streptomycin Residues in Milk

A monoclonal antibody (MAb) against dihydrostreptomycin (4G8) was developed and its performance compared with a previously developed MAb against streptomycin (4E2) in biosensor immunoassays (BIAs) using a surface plasmon resonance (SPR)-based biosensor (BIACORE 3000). Direct BIAs for the detection of dihydrostreptomycin (DHS; 583 Da) and streptomycin (STREP; 581 Da) were developed by immobilising the MAbs on the sensor chip (CM5). These direct BIAs were compared with competitive inhibition BIAs, using a STREP- protein conjugate immobilized on the chip. The sensitivities of the direct and competitive BIAs for both drugs in buffer were comparable (10-20 ng ml- 1 at 50% binding or inhibition). With milk, interferences, probably due to the nonspecific binding of proteins to the sensor chips, were observed in both BIAs. These interferences could be largely reduced using ultra filtration (UF) as sample pre-treatment. Another option was the use of a reference flow channel to correct for nonspecific binding. Using this option with five times diluted milk, MAb 4G8 was found to be suited for the direct BIA of both drugs with a limit of detection (LOD) of 20 ng ml- 1 and both MAbs could be applied in the competitive BIA format with similar LODs.     

Atrophy and Defects Detection of the External Anal Sphincter: Comparison Between Three-Dimensional Anal Endosonography and Endoanal Magnetic Resonance Imaging

Purpose Using endoanal magnetic resonance imaging, atrophy of the external anal sphincter can be established. This aspect has not been thoroughly investigated using three-dimensional anal endosonography. The purpose of this study was to compare prospectively three-dimensional anal endosonography to magnetic resonance imaging in the detection of atrophy and defects of the external anal sphincter in patients with fecal incontinence. In addition, we compared both techniques for anal sphincter thickness and length measurements. Materials and Methods Patients with fecal incontinence underwent three-dimensional anal endosonography and magnetic resonance imaging. Images of both endoluminal techniques were evaluated for atrophy and defects of the external anal sphincter. External anal sphincter atrophy scoring with three-dimensional anal endosonography depended on the distinction of the external anal sphincter and its reflectivity. External anal sphincter atrophy scoring with magnetic resonance imaging depended on the amount of muscle and the presence of fat replacement. Atrophy score was defined as none, moderate, and severe. A defect was defined at anal endosonography by a hypoechogenic zone and at magnetic resonance imaging as a discontinuity of the sphincteric ring and/or scar tissue. Differences between three-dimensional anal endosonography and magnetic resonance imaging for the detection of external anal sphincter atrophy and defects were calculated. In addition, we compared external anal sphincter thickness and length measurements in three-dimensional anal endosonography and magnetic resonance imaging. Results Eighteen patients were included (median age, 58 years; range, 27–80; 15 women). Three-dimensional anal endosonography and magnetic resonance imaging did not significantly differ for the detection of external anal sphincter atrophy (P = 0.25) and defects (P = 0.38). Three-dimensional anal endosonography demonstrated atrophy in 16 patients, magnetic resonance imaging detected atrophy in 13 patients. Three-dimensional anal endosonography agreed with magnetic resonance imaging in 15 of 18 patients for the detection of external anal sphincter atrophy. Using the grading system, 8 of the 18 patients scored the same grade. Three-dimensional anal endosonography detected seven external anal sphincter defects and magnetic resonance imaging detected ten. Three-dimensional anal endosonography and magnetic resonance imaging agreed on the detection of external anal sphincter defects in 13 of 18 patients. Comparison between three-dimensional anal endosonography and magnetic resonance imaging for sphincter thickness and length measurements showed no statistically significant concordance and had no correlation with external anal sphincter atrophy. Conclusion This is the first study that shows that three-dimensional anal endosonography can be used for detecting external anal sphincter atrophy. Both endoanal techniques are comparable in detecting atrophy and defects of the external anal sphincter, although there is a substantial difference in grading of external anal sphincter atrophy. Correlation between three-dimensional anal endosonography and magnetic resonance imaging for thickness and length measurements is poor. Inconsistency between the two methods needs to be evaluated further. Supported in part by grant No. 945-01-013 from the Netherlands Organization for Health Research and Development. Presented in part at the United European Gastroenterology Week, Prague, Czech Republic, September 25 to 29, 2004. Reprints are not available.     

Endosonography in anorectal disease: an overview

Anorectal endosonography (AE), which was introduced 20 years ago, derives from the study of urology. It was first used to evaluate rectal tumours and later also to investigate benign disorders of the anal sphincters and pelvic floor. The technique is easy to perform, it has a short learning curve and causes no more discomfort than a routine digital examination. A rotating probe with a 360 degrees radius and a frequency between 5 and 16 MHz is introduced to the rectum and then slowly withdrawn so that the pelvic floor and subsequently the sphincter complex are seen. Recently, it has become possible to reconstruct three-dimensional images. AE has been used for almost every possible disorder in the anal region and has increased our insight into anal pathology. The clinical indications for AE are: 1. Faecal incontinence in patients when surgery is an option. AE can show sphincter defects with excellent precision. There is a perfect correlation with surgical findings. Studies comparing AE with endoanal magnetic resonance imaging (MRI) have shown that both methods are equally good for demonstrating defects in the external anal sphincter; the internal anal sphincter is better visualized with AE. After sphincter repair, the effect is directly related to the decrease in the sphincter defect. 2. Perianal fistulae. AE has been shown to be accurate in staging perianal cryptoglandular fistulae and fistulae in Crohn's disease. When there is an external fistula opening, H2O2 can be introduced with a plastic infusion catheter. The tract then becomes visible as a hyperechoic lesion ("white"). It has been shown that this corresponds well with surgical findings. It is equally sensitive as endoanal MRI. Since recurrent cryptoglandular fistulae are complex in 50% and Crohn's fistula in 75%, it is mandatory to perform AE preoperatively in these patients to avoid missed tracts during surgery and subsequent recurrences. 3. Rectal tumors. In low tubulovillous adenomas or malignant polyps considered removable locally, confirming the local resectability (T0 or T1) is mandatory. Although larger rectal and more advanced tumours can be evaluated with AE, MRI is more sensitive in staging nodal involvement. 4. Anal carcinoma for staging. AE has been shown to stage better than the classical TNM classification for both local extension and prognosis. In conclusion, AE images the internal and external anal sphincter with high accuracy. It is easy to perform and is of particular value in the diagnosis of anal incontinence and perianal fistulae. It is excellent in staging anal carcinoma and can also be used in staging rectal carcinoma, especially very low large malignant polyps.     

Retrospective analysis of old-age colitis in the Dutch inflammatory bowel disease population

AIM： To describe the characteristics of Dutch patients with chronic.inflammatory bowel disease （IBD） first diagnosed above 60 years of age-a disease also known as old-age colitis （OAC） and to highlight a condition that has a similar appearance to IBD, namely segmen- tal colitis associated with diverticular disease （SCAD）. METHODS： A retrospective longitudinal survey of patient demographic and clinical characteristics, disease characteristics, diagnostic methods, management and course of disease was performed. The median follow-up period was 10 years. RESULTS： Of a total of 1100 IBD patients attending the Department of Gastroenterology, 59 （50） [median age 82 years （range 64-101）; 25 male （42%）] were identified. These patients were diagnosed with ulcerative colitis （n = 37, 61%）, Crohn＇s disease （n = 14, 24%）, and indeterminate colitis （n = 8, 15%）. Remission was induced in 40 （68%） patients within a median interval of 6 mo （range 1-21） and immunosuppressive therapy was well tolerated. Histological evaluation based on many biopsy samples and the course of the disease led to other diagnosis, namely SCAD instead of IBD in five （8%） patients. CONCLUSION： OAC is not an infrequent problem for the gastroenterologist, and should be considered in the evaluation of older patients with clinical features suggestive of IBD. Extra awareness and extensive biopsy sampling are required in order to avoid an erroneous diagnosis purely based on histological mimicry of changes seen in SCAD, when diagnosing IBD in the presence of diverticulosis coli.     

Sulphonamide Antibodies: From Specific Polyclonals to Generic Monoclonals

Polyclonal antibodies (PAbs) against eight different sulphonamides were raised in rabbits. The aromatic amino group, common to all sulphonamides, was used for linking the different sulphonamides to the carrier proteins (bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH)) and enzyme (horseradish peroxidase (HRP)), using different coupling procedures. The competitive direct ELISAs (cdELISAs) developed with these antisera and HRP-conjugates showed high sensitivity (0.2- 8.0 ng ml^-1 at 50% inhibition) and high specificity. The performances of these antibodies were compared with PAbs raised in mice against two sulphonamide derivatives (N¹ -[4-(carboxymethyl)-2-thiazolyl]sulphanilamide (TS) and N¹-[4-methyl-5-\[2-(4-carboxyethyl-1-hydroxyphenyl)]-azo-2-pyridyl]sulphanilamide (PS)) linked to proteins (BSA and KLH) in such a way that the common aromatic amino group was distal to the protein. In competitive indirect ELISAs (ciELISAs), these PAbs recognized several structurally different sulphonamides. The PAbs from mice immunized with TS-BSA reacted with sulphonamides containing thiazolyl, thiadiazolyl, pyridazinyl and isoxazolyl groups. The PAbs from mice immunized with PS-KLH reacted with sulphonamides containing pyrimidinyl, pyridazinyl, quinoxalinyl and pyridinyl groups. The spleen cells of the mice were fused with myeloma cells to obtain monoclonal antibodies (MAbs) producing hybridomas. So far, with only one of the mice (immunized with TS-BSA), this resulted in four different MAbs which recognized several sulphonamides. By use of the best MAbs (27G3A9B10 and 4E10B12B6E12) and an optimized ciELISA protocol, eight structurally different sulphonamides showed 50% inhibition at concentrations less than 100 ng ml^-1 or 5 ng/well. However, other relevant sulphonamides (such as sulphadimidine, sulphatroxazole and sulphachloropyrazine) were detected at a high level only.