Taking advantage of a specially constructed vector, luciferase LuxA and LuxB subunits were connected in frame to different amino acid linkers to reproduce a series of monomeric luciferase enzymes. A comparison of their activities in E. coli cells demonstrated that the length of the linkers positively affected activity. One luciferase fusion gene was expressed in plant cells, and we showed that this gene activity could be monitored directly without destructive sampling.

Amino Acid Sequence↗

PubMedDate not supplied

Human pheochromocytoma cells studied in culture contain large amounts of DSIP-like material.

Delta sleep-inducing peptide (DSIP)-like immunoreactive (LI) material has been detected in nine different human pheochromocytoma tumors by immunocytochemistry. In primary tumors subjected to indirect immunofluorescence a variable number of tumor cells (25-75%) showed positive cytoplasmic labeling after incubation with DSIP antiserum. Tumor cells grown in culture were strongly labeled by the DSIP antiserum with DSIP-LI concentrated to cell bodies. Electron microscopic immunocytochemistry (immunogold labeling) of pheochromocytoma cells demonstrated DSIP-LI over the dense core of secretory granules. The presence of DSIP-LI in several HPLC fractions from conditioned culture media indicates secretion of DSIP-LI from cultured pheochromocytoma cells. The observations suggest that DSIP-LI is synthesized and stored in secretory granules before release. The different HPLC profiles from each of the tumors may reflect differences in processing or turnover of DSIP-LI in pheochromocytoma cells.

Adrenal Gland Neoplasms↗

PubMedDate not supplied

Accelerated acute rejection of the intestinal graft in CD28-deficient mice.

OBJECTIVE: Multiple in vivo studies have shown that the pace and severity of graft rejection is little or not at all changed by deleting CD28 molecules in the recipient. These findings contrast with the effects of monoclonal antibody therapy aimed the same costimulatory target. The objective of the present study was to evaluate how the acute rejection process is affected in CD28-deficient mice using a fully allogeneic, highly immunologically reactive transplant model. METHODS: Heterotopic vascularized small bowel transplants were performed in 24 recipient mice divided into 4 groups: 2 wild-type and 2 knockout groups. Each group consisted of 5 to 7 animals in which BalbC mice were used as intestinal donors to either wild-type C57BL6 or C57BL6 background CD28-deficient recipient mice. Selected endpoints were 3 and 6 postoperative days (POD). Intestinal rejection was evaluated by mucosal laser Doppler flowmetry (expressed in perfusion units) and histology (expressed in rejection grades). RESULTS: Acute rejection occurred in both wild-type and CD28-deficient groups. At POD 3, no significant difference was noted between groups in terms of mucosal perfusion and histology. At POD 6, significant differences in graft mucosal perfusion and histology revealed a more aggressive rejection in the CD28-deficient group compared to the wild-type group. CONCLUSIONS: The present study showed that the severity of intestinal graft rejection responses was amplified by deleting CD28 molecules. Together with data from other studies, these results suggest a different pattern of distribution and/or activation of CD28/B7 receptors in various organs.

Acute Disease↗

PubMedDate not supplied

Laser-Doppler flowmetry in the monitoring of the human intestinal allograft: a preliminary report.

During acute rejection, graft endothelium becomes a prime target for recipient immune cells. Animal studies have shown reduced microvascular perfusion, probably due to increased endothelial-leukocyte interaction and endothelial impairment, leading to graft damage. Using laser-Doppler flowmetry (LDF), we correlated the microvascular blood flow in the intestinal mucosa of five patients receiving multivisceral grafts with clinical events and pathology results. Measurements (n = 75) were performed during the first 4 weeks posttransplantation by inserting the LDF flexible probe through the ileostomy for 25 to 30 cm. Forty-six of the 75 measurements were performed within 24 hours of endoscopy and biopsy. In uncomplicated cases, we recorded a gradual increase in mucosal perfusion during the first week posttransplantation that presumably reflected regeneration after reperfusion injury. Increased mucosal perfusion did not seem to correlate with rejection or other adverse clinical events. Sudden and sustained decreases in mucosal perfusion by 30% or more compared to the previous measurements were associated with septic episodes, rejection, or both. LDF revealed a good sensitivity in monitoring the intestinal microcirculation. It was able to indicate perfusion changes associated with acute rejection. The relatively low specificity of LDF may be compensated by the low invasivity, allowing frequent investigation. LDF may be an additional tool for routine monitoring of intestinal allografts.

Adult↗

PubMedDate not supplied

Ultrastructure and ATPase activity of rat uterine epithelium during blastocyst attachment.

Explore the source record for details and available documents.

Adenosine Triphosphatases↗

PubMedDate not supplied

Gastrointestinal carcinoids--aspects of diagnosis and classification.

Gastrointestinal carcinoids are a heterogeneous group of hormone-producing tumours originating from the diffuse neuroendocrine system of the gastrointestinal mucosa. Recent studies have shown that carcinoid tumours share many of the morphological, biochemical and functional properties of the neuroendocrine cells from which they are derived. Identification of these properties, e.g. specific hormone production, contents of secretory granules or specific proteins of the secretory granules/ vesicles, forms the basis for the diagnosis of carcinoid tumours. Classification of gastrointestinal carcinoids is based on the clinical settings under which the tumours develop, tumour site, as well as the morphological and biochemical characteristics of the tumour. Although generally considered a low grade malignancy, carcinoid tumours show a highly variable clinical course from benign tumours discovered incidentally to highly malignant tumours presenting with disseminated spread and disabling hormonal symptoms. Precise knowledge of tumour location, size and stage, as well as growth pattern and hormonal syndromes, is necessary to predict the behaviour of the tumour and optimise treatment.

Carcinoid Tumor↗

PubMedDate not supplied

Automated immunofluorometric assay for MUC1.

The aim of the present study was to establish a robust, reliable and fully automated immunofluorometric assay for the breast cancer serum marker MUC1. This would further serve as a prototype assay for evaluation of other MUC1 assays based on new antibody combinations. Using time-resolved fluorescence as tracer signal we developed an automated immunofluorometric assay for MUC1 (MUC1 IFMA). This assay was compared with two commercial assays. The CA15-3 EIA (CanAg) which use the same antibodies as the MUC1 IFMA, and the ETI-CA-15-3 K (Sorin) which use the original antibodies defining the CA 15-3 assay. The three assays showed comparable results. The coefficient of variation was below 10% from 9 to 2,400 kU/l for the MUC1 IFMA, from 15 to 250 kU/l for the CA15-3 EIA, and from 25 to 200 kU/l for the ETI-CA-15-3 K assay. At a specificity of 0.94 the overall diagnostic sensitivities for the MUC1-IFMA, CA15-3 EIA and ETI-CA-15-3 K assays were 0.40, 0.37, and 0.38, respectively. When applied to metastatic breast cancer, all assays had sensitivities close to 0.80. There was a close correlation (Spearman rank = 0.99) between results from the new assay and the CA15-3EIA. The new automated assay was not strictly immunometric as we could not achieve conditions where solid phase or tracer antibodies were in apparent excess. However, the assay performed well at a wide range of assay conditions. The automation, which minimizes imprecision in pipetting and handling of samples, and the high capacity of the AutoDELFIA instrument enabling measurement of all samples in a single run, were important aspects for establishing a reliable assay. The principle of the new automated immunofluorometric assay will be used as a rapid and reliable evaluation of a wide range of monoclonal antibody combinations in our search for the optimal MUC1 assay. This new automated immunofluorometric assay will be useful in the rapid and reliable evaluation of a wide range of monoclonal antibody combinations in our search for the optimal MUC1 assay.

Adult↗

PubMedDate not supplied

Epitopes on CA 125 from cervical mucus and ascites fluid and characterization of six new antibodies. Third report from the ISOBM TD-1 workshop.

CA 125 is found in body fluids in a variety of molecular weight forms. The largest species are found in normal abdominal fluid and cervical mucus. The present study therefore incorporated CA 125 derived from these sources as well as ascites fluid to investigate if the source of CA 125 influenced epitope characterization. Ascites-derived CA 125 varied in size from about 190 to about 2,700 kD. Cervical mucus-derived CA 125 treated with ultrasound changed its apparent size from more than 20,000 to 700 kD. Epitope mapping of antibodies was not grossly influenced by the size or source of CA 125 used as target. However, low-molecular-weight CA 125, i.e. ascites fractions CA 17/E, CA 17/F and CA 10/7, did show differences in certain assay combinations and cross-inhibition patterns which probably can be explained by steric effects due to the smaller size compared with the most abundant forms of CA 125 present in serum and other body fluids. The specificity of six new monoclonal antibodies to CA 125 was tested by cross-inhibition and immunometric assay combinations and compared to reference antibodies. One antibody, X306, belonged to the OC125-like antibodies. Four antibodies, X52, X75, X325 and VK8, were M11-like. The sixth antibody, 7C12, reacted with an epitope which was difficult to define. This antibody was inhibited by M11-like antibodies and OV197. However, used as an inhibitor, 7C12 inhibited only itself. We grouped it as an OV197-like antibody, but clearly different from OV197. The topography of epitopes was studied by analyzing all antibody pairs in immunoradiometric assays. These results confirmed the grouping of antibodies described above and are in accordance with previous findings that the highest signal is obtained using an OC125-like antibody or OV197 on the solid phase and an M11-like antibody as tracer. The composition of the sample in terms of high- and low-molecular-weight species of CA 125 was measured, with different responses depending on the antibody pair used. This might be one reason for discrepancies between assay results for CA 125 using different assays.

Antibodies, Monoclonal↗