Putative membrane fatty acid translocase and cytoplasmic fatty acid-binding protein are co-expressed in rat heart and skeletal muscles.

A membrane protein (FAT) homologous to CD36 has recently been implicated in the binding and transport of long-chain fatty acids (FA). Expression of this protein in rat heart, skeletal muscles and in isolated cardiac cells was studied. Changes in expression during development of the heart were also examined. Expression of FAT was compared to that of the cytoplasmic fatty acid-binding protein (H-FABP) to determine whether coexpression, indicative of related biological functions, could be demonstrated. FAT and H-FABP mRNAs showed a similar muscle tissue distribution and similar cellular localization in the heart. During development, heart mRNA levels for both proteins were upregulated in the same way. In conclusion, expression of FAT and H-FABP in muscle tissues and cell-types with high FA metabolism and the upregulation of mRNA levels associated with heart development, when FA utilization increases, support the suggested role of both proteins in FA metabolism.

Fatty acid transfer across the myocardial capillary wall: no evidence of a substantial role for cytoplasmic fatty acid-binding protein.

It has recently been hypothesized that fatty acid (FA) transfer across the myocardial capillary wall is mediated by cytoplasmic fatty acid-binding protein (FABP). Therefore, we studied the type and content of FABP in endothelial cells from rat heart, using molecular biological, immunochemical, and FA-binding assays. Studies were performed on short term cultured endothelial cells, two established endothelial cell lines and ultrathin cryosections from adult rat heart. Northern blotting analysis of endothelial cell RNA failed to detect either heart-type (H-) FABP or liver-type (L-) FABP mRNA, but the reversed transcription-polymerase chain reaction revealed both H- and L-FABP mRNAs, indicating the presence of minor amounts of these mRNAs. Highly sensitive immunochemical assays (sandwich ELISAs) using specific antibodies raised against rat H- or L-FABP showed the contents of these FABP-types in endothelial cells to be 1-5 ng/mg cytosolic protein, which is more than three orders of magnitude lower than the contents of H-FABP in heart or L-FABP in liver. Immuno-electron microscopy also showed that the concentration of H-FABP in endothelial cells is at least two orders of magnitude lower than that in cardiomyocytes. Finally, cytosolic protein samples from endothelial cells revealed no significant FA-binding activity in the 15-kDa region. We conclude that rat heart endothelial cells contain only minor quantities of cytoplasmic FABP and that, therefore, FA transport over the endothelium is mediated by FABP only to a minor extent. It is postulated that aqueous diffusion of FA through the endothelial cytoplasm most likely accounts for the experimentally observed rates of cardiac FA utilization.

Expression of MUC2-mucin in colorectal adenomas and carcinomas of different histological types.

The expression of mucin MUC2 was investigated in normal colonic tissue, in colonic adenomas and in carcinomas of the mucinous and non-mucinous type. The latter were subdivided into carcinomas originating from the adenoma-carcinoma sequence (ACS) and de novo (DN) carcinomas. The expression was assayed by immunohistochemistry with the monoclonal anti-MUC2 antibody CCP58 and by mRNA semiquantitation. MUC2 protein epitope CCP58 was strongly expressed in 21% of normal colonic tissues, in 40% of villous and in 48% of tubular adenomas. Mucinous carcinomas exhibited strong expression in 72%, ACS carcinomas in 21% and DN adenocarcinomas in none of the tumors investigated. Compared with the adjacent non-malignant tissue (transitional mucosa), CCP58 epitope expression in the tumor was higher in 74% of mucinous carcinomas, but equal or lower in 69% of ACS carcinomas and in 100% of de novo carcinomas. The alterations of MUC2 expression detected by immunohistochemistry in adenocarcinomas were confirmed on mRNA level. These data indicate that the MUC2 expression pattern is different in the 3 carcinoma types investigated. MUC2 over-expression occurs in the adenomatous tissue. It is always maintained in mucinous carcinomas, but frequently decreased in non-mucinous ACS carcinomas. DN carcinomas are most frequently associated with decreased expression of MUC2.

Enzyme-linked immunosorbent assay using monoclonal antibodies for identification of mycobacteria from early cultures.

A simple enzyme-linked immunosorbent assay (ELISA) for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii has been developed (R. Schöningh, C. P. H. J. Verstijnen, S. Kuijper, and A. H. J. Kolk. J. Clin. Microbiol. 28:708-713, 1990). The test for the routine identification of cultured mycobacteria was introduced in five clinical laboratories located in Tanzania, Thailand, Vietnam, and The Netherlands. The ELISA can be conducted without an ELISA reader since the test can be read visually. The results of identification of 255 strains of the M. tuberculosis complex by microbiological means and by ELISA were compared; the specificity and the sensitivity were 100%. For the M. avium complex, the specificity was 100% and the sensitivity was 64%. All 26 M. kansasii strains tested could be identified as M. kansasii. The ELISA described here proved to be useful in both well- and modestly equipped laboratories and may replace the microbiological method of identification of M. tuberculosis and M. kansasii.

Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation. An easy and low cost alternative to radioactive thymidine incorporation.

The classical in vitro assay for the determination of cell mediated immune responses is the lymphocyte transformation test (LTT) in which cell proliferation is measured by incorporation of radioactive labeled thymidine (3H-TdR). The LTT assay using 3H-TdR is less suited for modestly equipped laboratories as it is costly, laborious and involves the need to handle radioactive isotopes and specialized equipment. Here we describe an improved alternative LTT method which is capable of detecting specific cellular immune reactions (CMI) against (mycobacterial) antigens in vitro. This assay, the bromodeoxyuridine-ELISA LTT test, is simple, less expensive, reproducible and is as sensitive as the 3H-TdR test. The specific advantages of the test are a simple denaturation step and the fact that no radioactive isotopes are needed. The test is specifically suited for research laboratories in tropical countries which study CMI in those human infectious diseases where this arm of the immune response plays a pivotal role in the generation of immunity, e.g., in tuberculosis, leprosy and leishmaniasis.

Enzyme immunoassay for identification of heat-killed mycobacteria belonging to the Mycobacterium tuberculosis and Mycobacterium avium complexes and derived from early cultures.

A simple enzyme-linked immunosorbent assay was developed for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii. Six monoclonal antibodies were used: two (F23-49 and F24-2) were specific for the M. tuberculosis complex, two (F85-2 and F85-10) were specific for the identification of the M. avium complex, one (F126-22) was specific for M. kansasii, and one (F141-3) was broadly reactive and distinguished mycobacteria from other bacteria. In the enzyme-linked immunosorbent assay only 10(6) to 10(7) bacteria derived from early cultures (2 to 3 weeks) were needed for each monoclonal antibody. For the M. tuberculosis complex the sensitivity and specificity were both 100%. For the M. avium complex the specificity was 100% and the sensitivity was 70%. The three M. kansasii strains tested could all be identified as M. kansasii.

Rapid identification of cultured Mycobacterium tuberculosis with a panel of monoclonal antibodies in western blot and immunofluorescence.

This paper describes the identification of cultured mycobacteria with a panel of monoclonal antibodies directed against species-specific epitopes in a Western blot (WB) test and in an immunofluorescence test (IFT). In WB, we identified mycobacteria of the Mycobacterium tuberculosis complex (M. tuberculosis, M. africanum, M. bovis, M. bovis BCG, and M. microti) with 10(8) bacteria. In the IFT, we identified mycobacteria of the M. tuberculosis complex, the M. avium complex (M. avium, M. intracellulare and M. scrofulaceum) and M. kansasii with 10(7) bacteria. Using a panel of 105 mycobacterial patient isolates, we compared identification by WB and IFT with conventional, culture and biochemical identification. Identification of the M. tuberculosis complex in WB had a specificity and sensitivity of 96.3 and 98.3%, respectively. Identification in IFT of the M. tuberculosis complex had a specificity and sensitivity of 94.6% and 89.7%, respectively. Since the identification of mycobacteria with mAb requires only a small number of bacteria, these tests will reduce by several weeks the time necessary for microbiological identification.

Colonic epithelium reactive monoclonal antibodies. Identification and immunohistochemical localization of the target epitopes.

We have produced a small library of colonic mucosa and colorectal carcinoma reactive monoclonal antibodies (MoAbs) by immunizations with extracts of human colon cancer tissue and a human colon cancer cell line. Hybridoma supernatants were tested on (normal and neoplastic) human tissues by immunoperoxidase methods to evaluate organ or tissue specificity. Initial biochemical characterization of the target antigens was performed by gelpermeation chromatography, Western blotting and competition assays. Based upon the immunoreactivity patterns and the characteristics of the antigen four groups of MoAbs could be distinguished. The first group concerns the antibodies PARLAM 3, 9 and 10. These antibodies react with an 87 kDa protein moiety in high molecular weight (2-5 x 10(6) Da) glycoproteins. In intestinal and colon mucosa these antibodies showed diffuse binding with goblet cells. In colon carcinoma decreased reactivity with these MoAbs was found. The second group consists of antibodies PARLAM 8, 12 and 13. These antibodies react with large (greater than 5 x 10(6) Da) glycoproteins, most likely with carbohydrate epitopes. By immunohistochemistry in normal colon mucosa the antibodies all show granular supranuclear reactivity with goblet cells. These antibodies show increased reactivity with colon adenomas and adenocarcinomas. A third group is formed by PARLAM 2, which also reacts with a large (greater than 5 x 10(6) Da) glycoprotein, showing a granular distribution in goblet cells. In colon carcinomas more extensive expression is found than in normal colonic mucosa. Finally, the fourth group consists of PARLAM 11, which also reacts with a large (greater than 5 x 10(6) Da) glycoprotein, located in the brush border of colonic columnar cells. These antibodies might be useful tools for the analysis of the expression of mucin related glycoproteins in normal, preneoplastic and neoplastic colon mucosa.

Culturing and xenografting of primary colorectal carcinoma cells: comparison of in vitro, and in vivo model and primary tumor.

In a series of 61 primary colorectal carcinomas, we attempted to determine which primary tumor characteristics correlated with the possibility to continuously maintain tumor cells in vitro or in vivo, and to what extent the characteristics of a primary tumor were maintained in vitro or as xenograft. Four continuous cell lines and 10 serially transplantable tumors were obtained. Only one cell line could be maintained both in vitro and in vivo. Tumors that had metastized and tumors in the proximal colon showed a higher success for in vivo and in vitro growth. DNA analysis showed that in most xenografts the DNA index was identical to that the primary tumor. However, in some cases tumor cell subpopulations were lost or genetically variant new subpopulations were generated. In general, the degree of differentiation in the xenografts corresponded with the least differentiated areas in the primary tumor. Xenografts appeared to display comparable of antigen expression.

Xenografting of normal colonic mucosa in athymic mice.

Colonic mucosa was implanted subcutaneously into nude mice in order to investigate the potential use of xenografts as an in vivo model system for the study of colonic epithelial proliferation and differentiation. The xenografts were followed for 5 weeks. Proliferation was studied by bromodeoxyuridine (BrdU) incorporation followed by visualization of the DNA-synthesizing cells with an anti BrdU monoclonal antibody. Differentiated cells were visualized by histochemical staining of goblet- and enterochromaffin cells and of columnar cells by immunoperoxidase staining for carcinoembryonic antigen (CEA), secretory component, and serotonin. In different strains of mice different observations were made. Less immunocompetent strains (Balb c nu/nu, NMRI nu/nu) developed abscesses at the site of the xenograft as well as wasting disease. These phenomena almost never occurred in CD-1 nu/nu mice. The success rate was highest in CD-1 nu/nu mice. After about 1 week, only crypt base cells remained vital and started to repopulate crypts with epithelial cells showing normal colonic differentiation features such as CEA, secretory component, and serotonin immunoreactivity and mainly sulphomucin production. After longer periods of time, crypts started to form cyst-like structures due to accumulation of secretion products and dead cells. DNA-synthesizing cells were seen in the basal areas of recognizable crypts and in the cyst-like structures in a random distribution. These results indicate that normal colon mucosa xenografted into nude mice maintains its proliferative and differentiating capacity for at least 5 weeks.

Influence of implantation site on growth, antigen expression and metastatic potential of human colonic cancer HT29 and 5583 xenografts in nude mice.

In order to study the interaction between tumors and host environmental factors, we xenografted cells from the human colonic carcinoma cell lines HT29 and 5583-S in the subcutis, cecum, spleen and liver of nude mice and compared growth characteristics, metastatic potential and some phenotypic features of the xenografts in these sites. No remarkable differences were observed between the tumors at different inoculation sites in regard of their expression of carcinoembryonic antigen and secretory component or type of mucin produced. Also the proportion of DNA synthesizing cells as determined by bromodeoxyuridine incorporation appeared to be comparable in the studied implantation sites. Local invasive growth characteristics and metastatic potential, however, showed marked differences. Subcutaneous and cecal xenografts frequently showed tumor cell invasion into the surrounding tissue, vasoinvasive growth and discontinuous basement membrane deposition, whereas splenic and hepatic implants demonstrated more encapsulation, no invasion of blood vessels and more continuous basement membrane deposition. Subcutaneous xenografts produced no metastasis. With HT29 cells liver and lymph node metastases occurred frequently from the splenic as well as the cecal xenografts. 5583 cells regularly produced liver metastases from the splenic xenografts, whereas no metastasis from cecal xenografts were observed. We conclude that although the patterns of invasive growth and the metastatic potential differ for various implantation sites, antigen expression and cell kinetic features of tumor implants are hardly influenced by the site of inoculation.

Comparison of phenotypic and genotypic features in human primary large bowel carcinomas and lymph node metastases.

In order to test the contention that metastasis is a selective process and that therefore metastases might show a more restricted pattern of phenotypic and genotypic characteristics than primary tumors, we compared the expression of carcinoembryonic antigen, Ca 19-9, secretory component, serotonin, and mucin production as well as flow cytometric data on DNA content and percentage of S-phase cells in 87 primary large bowel carcinomas and their lymph node metastases. In a majority of the cases primary tumors and their metastases were largely identical with regard to the examined phenotypic features. In discrepant cases, however, metastases did not invariably show a more restricted pattern than primary tumors, indicating high differentiational plasticity of primary and metastatic colorectal cancer cells. In contrast, in a number of cases genotypic discrepancies were observed. We conclude that phenotypic characteristics of colorectal cancer cells cannot be used to study the pathogenesis of lymph node metastasis. Genotypic studies, however, suggest that lymphogenic metastasis may be a selective event.

The establishment and characterization of two new cell lines derived from a single human colonic adenocarcinoma.

Two cell lines with different in vitro growth characteristics were established from a single mucinous colonic adenocarcinoma. Epithelial cells of the line 5583-E demonstrated anchorage-dependent growth while those of line 5583-S were anchorage-independent and grew as multicellular floating spheroids. Both cell lines shared common characteristics with respect to the expression of differentiation markers (secretory component, carcinoembryonic antigen), mucins and karyotype (trisomy 12 and 14, marker chromosome) but also showed consistent differences. In nude mice 5583-S cells formed moderately differentiated mucinous adenocarcinomas with high carcinoembryonic antigen and mucin production, whereas 5583-E xenografts were poorly differentiated and almost entirely failed to produce carcinoembryonic antigen and mucins. The plating efficiency of 5583-E cells appeared to be greater and doubling time shorter than those of 5583-S cells. Furthermore, 5583-E cells showed an extra isochromosome, 1q. The cell lines were genotypically and phenotypically stable over a period of 2 years. Our results reemphasize that multiple cell lines with heterogeneous phenotypic and genotypic characteristics can be obtained from a single primary tumor.

Vitamin K-dependent carboxylase in skin.

Vitamin K-dependent carboxylase is demonstrated in skin microsomes from humans, rats, rabbits, and mice. This enzyme converts a number of distinct protein-bound glutamic acid residues into gamma-carboxyglutamic acid residues, which strongly interact with Ca++ ions. The enzymatic activity (expressed per mg protein) in skin is about 20% of that in liver. Vitamin K-dependent carboxylase is present in both epidermal and dermal tissue. It is demonstrated that warfarin treatment in mice results in an accumulation of noncarboxylated precursor proteins in both dermal and epidermal microsomes. Most probably this effect of warfarin is not restricted to mice, but occurs also in the skin of patients under oral anticoagulant therapy. A possible relation between vitamin K-dependent skin carboxylase and the gamma-carboxyglutamic acid-containing protein in calcified nodules from patients with scleroderma and dermatomyositis is discussed.

CEA-specificity of CEA-reactive monoclonal antibodies. Immunochemical and immunocytochemical studies.

Four carcinoembryonic antigen (CEA) reactive monoclonal antibodies Parlam 1, 4, 5 and 6 were studied in respect to reactivity with CEA and its cross-reacting antigens (NCA-1, NCA-2, BGP). In immunochemical studies (ELISA and SDS-PAGE followed by immunoblotting) Parlam 4 did not react with these crossreacting antigens, whereas Parlam 1, 5, and 6 demonstrated variable reactivity with these antigens. As expected, by immunocytochemistry Parlam 1, 5, and 6 stained bile canaliculi in the liver (due to crossreactivity with BGP), pneumocytes and splenic tissue (NCA-1) to an extent comparable with the results in biochemical tests. In contrast with the immunochemical observations, however, Parlam 4 showed slight but distinct reactivity with splenic granulocytes (NCA-1) and hepatic bile canaliculi (BGP). Relative epitope specificity of the monoclonal antibodies was tested in blocking experiments. These showed that Parlam 1 and 5 detect the same epitope on CEA as they block each others binding completely. In other combinations monoclonal antibodies block each others binding only partially, indicating that they detect different or partially overlapping epitopes. These results suggest that CEA specific epitopes may partly overlap with epitopes on crossreacting antigens. In this context, we propose that on crossreacting antigens epitopes exist that are structurally similar to epitopes on CEA or that some epitopes on CEA may consist of a spatial configuration which involves CEA specific as well as non-CEA specific structures. The antibody will show the highest affinity towards CEA, as this molecule contains the uniquely matching or complete epitope and lower affinity towards the crossreacting antigen with an imperfectly matching or only partially available epitope.

The response of plasma prolactin to suckling during normal and prolonged lactation in the rat.

In dams which had been kept isolated from pups for 8-10 h, the magnitude of the suckling-induced prolactin rise in the plasma was studied in relation to intensity of suckling stimulus and lactational age of the mother. At midlactation the response of prolactin evoked by suckling was enhanced as litter size increased. Suckling of 2 pups induced a greater prolactin rise in dams adjusted to 2 pups than in dams adjusted to 8 pups. Suckling of 8 pups caused a greater prolactin rise in dams which had been adjusted to an 8-pup litter, than in rats with a 2-pup litter. At late and prolonged lactation the rise of prolactin in the plasma induced by the suckling stimulus of 8 pups was significantly lower than at midlactation. Injection of perphenazine after a period of suckling induced a moderate increase of plasma prolactin in dams at midlactation, and a similar increase in dams at late lactation and at day 42 of lactation. It is concluded that in the first half of lactation the number of pups, i.e. the intensity of the suckling stimulus, is an important factor in determining the magnitude of the prolactin response to suckling. The lower response of plasma prolactin to suckling in late lactation is neither caused by a decrease in suckling stimulus from the pups nor by an increase in prolactin clearance; it is probably due to a gradual reduction in prolactin synthesizing and releasing capacity of the pituitary, brought on by a desensitization of the neural or neuroendocrine system to suckling stimuli as lactation proceeds.

Intraperitoneal infusion of EDTA in the rat blocks completely the prolactin rise in the plasma during suckling.

Lactating rats supplied with chronically indwelling intravenous (i.v.) and intraperitoneal (i.p.) catheters were allowed to suckle after a night of isolation from their offspring. The effect of infusions of saline and EDTA, given i.v. or i.p. on the suckling-induced rise of plasma prolactin was evaluated by radioimmunoassay. i.v. saline and EDTA did not affect the normal prolactin rise induced by suckling. However, i.p. EDTA completely blocked this rise, while the weight gain of the pups during the suckling period was only slightly depressed. i.p. EDTA had no suppressive effect on the perphenazine-induced rise of plasma prolactin and caused only a moderate inhibition of the prolactin rise in the plasma evoked by thyrotropin-releasing hormone and by brief exposure to ether. The experiments indicate, that because i.p. EDTA probably stimulates dopamine only slightly, it must inhibit prolactin secretion via another mechanism. This study, therefore, strongly supports the idea that more than one hypothalamic factors affect prolactin release during suckling.