Expression of interleukin-6, leukemia inhibitory factor and their receptors by colonic epithelium and pericryptal fibroblasts.

BACKGROUND AND AIM: The cellular configuration of the human colon suggests a predetermined organization that creates specific microenvironments. The role of pericryptal fibroblasts in this microenvironment has been the subject of considerable speculation. This study examined the expression of growth factors and their receptors by colonic crypt epithelium and pericryptal fibroblasts. METHODS AND RESULTS: Pericryptal fibroblast cells were isolated and cultured from decrypted human colonic mucosa. The pericryptal fibroblast cells expressed messenger RNA (mRNA) for interleukin-6 (IL-6), leukemia inhibitory factor (LIF), LIF receptor alpha, and the common coreceptor glycoprotein 130 (GP130), but not the IL-6 receptor alpha. Interleukin-6 protein expression was confirmed by the analysis of conditioned medium and immunohistochemistry. In comparison, normal colonic epithelial cells express mRNA for LIF but not IL-6 as well as the receptors for GP-130, IL-6 receptor alpha but not LIF receptor alpha. As cultures of normal human colonic epithelial cells were not available, the conditioned medium was assayed from established colon carcinoma cell lines and demonstrated a secretion of LIF but not IL-6 protein. CONCLUSION: The expression of reciprocal cytokine and receptor expression suggest that there is a paracrine relationship between pericryptal fibroblasts and colonic epithelium.

Conditionally immortalized mouse hepatocytes for use in liver gene therapy.

BACKGROUND AND AIMS: The use of hepatocytes for gene therapy is limited by the difficulty of maintaining and altering primary liver cells in culture. A conditionally immortalized mouse hepatocyte cell line has been developed which can be passaged indefinitely at the permissive temperature (33 degrees C), but fails to proliferate and dies at the non-permissive temperature (39 degrees C) in vitro. METHODS: Hepatocytes were harvested from a 6 week-old male transgenic mouse ('immortomouse') carrying a thermolabile SV40 Large T gene, using a modified two-step collagenase perfusion method, and serially passaged at 33 degrees C for more than 1 year. To assess the ability of immortohepatocytes to engraft and populate mouse liver, cells were infused into partially hepatectomized congenic mice via the portal vein (n = 10) or the spleen with (n = 2) and without (n = 2) partial hepatectomy. The ability to transfect immortohepatocytes was assessed using the reporter gene enhanced green fluorescent protein (EGFP). RESULTS: All immortohepatocytes in culture stained positive by immunohistochemistry for the hepatocyte markers albumin, AFP, CK8 and CK18. In early cultures a proportion of cells also stained strongly for the biliary epithelial markers CK7 and CK19. Late cell cultures were negative for M2PK and CK7 and stained variably with anti-CK19 antibodies. Cells transferred to the non-permissive temperature of 39 degrees C ceased proliferation and died within 1 week in vitro. Large T DNA was detected in the liver of all postoperative mice up to 2 weeks post-hepatocellular transplantation via PCR and Southern blot analysis. The immortohepatocytes were easily transfected with a reporter gene. CONCLUSIONS: Immortohepatocytes can survive in vivo after transfer to liver, and will be useful as a model for hepatic gene therapy.

Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans.

BACKGROUND & AIMS: The factors controlling the proliferation and differentiation of the colonic mucosa are unknown and have proved difficult to identify mainly because of a lack of in vitro methods for studying the proliferative cells of the mucosa. METHODS: We have developed a novel method of preparing a viable single-cell suspension from isolated crypts and cloning these single cells. RESULTS: We have obtained clonogenic growth from this single-cell suspension with an average of 1 colony per 10(5) cells in control cultures. Addition of conditioned medium from the LIM1863 colon carcinoma cell line increased the mean colony number to 11 +/- 3 per 10(5) cells. The cells forming the colonies are still viable after 4 weeks in culture. The epithelial nature of the cells was confirmed by ultrastructural and immunohistochemical methods with staining for keratin 8 and 18 and anti-human epithelial membrane-specific antigen and a positive result on polymerase chain reaction for keratin 19. CONCLUSIONS: We have successfully cloned single cells from disaggregated colonic crypts from both human and murine colonic mucosa. We have also demonstrated the presence of an active clonogenic factor in the conditioned medium of a colon carcinoma cell line. Assays show that the clonogenic activity in the conditioned medium is not caused by the presence of any of the epidermal growth factor family of growth factors. This is the first report of a clonogenic assay for epithelial cells of normal colonic mucosa.

[Osteopenia, marrow atrophy and capillary circulation. Comparative studies of the human iliac crest and 1st lumbar vertebra]. / Osteopenie, Markatrophie und Kapillarversorgung. Vergleichende Untersuchungen am menschlichen Beckenkamm und 1. Lendenwirbelkörper.

Histological and histomorphometrical measurements were made on normal and on osteoporotic bone from the first lumbar vertebra and the iliac crest of 15 cases. A decrease in cancellous bone was found in both. This was accompanied by a reduction in haematopoietic marrow and a corresponding increase in fat cells. In addition, the numbers of arterial capillaries and sinuses per unit area were also reduced. There is a greater overall vascularization in the first lumbar vertebra than in the iliac crest, even though the amount of cancellous bone is normally less in the former. However, the arborization of the terminal vessels in the iliac crest is more extensive than that in the first lumbar vertebra. These observations provide support for the participation of a vascular component in the pathogenesis of osteoporosis.

[Twelve theses on the pathogenesis of primary and secondary osteoporosis based on histological observations]. / Zwölf histobioptische thesen zur pathogenese der primären und sekundären osteoporose.

In spite of a well defined clinical syndrome and a wealth of biochemical information, the pathogenesis of primary osteoporosis is still uncertain. Microscopic evaluation of semithin sections of 1727 bone biopsies from patients suffering from "idiopathic" osteoporosis and 288 from patients with secondary osteoporosis has lead to the recognition of a pathogenetic relationship between changes of the bone marrow capillaries and atrophy of the trabecular bone in these groups. A new hypothesis is proposed for the structural and functional role of the bone marrow capillaries in normal and abnormal osseous remodelling; this is based on comparative morphometric analysis of normal cases and of hypo- and hyperplastic changes of the myelogenous and osseous tissues in various haematological and bone disorders. The hormonal and nervous regulation of the microcirculation of bone marrow may offer a new approach to the understanding and cure of so-called idiopathic osteoporosis.

[Familial occurrence of chronic lymphocytic leukemia: report of three siblings (author's transl)]. / Familiär-gehäuftes auftreten der chronischen lymphatischen Leukämie. Bericht über 3 erkrankte Brüder

Chronic lymphocytic leukemia was diagnosed in three siblings. Familiar occurrence speaks in favor of a predisposition in this disorder of unknown etiology. According to the present status of knowledge endogenous - genetically determined - factors combined with exogenous factors (viruses, radiation exposure, toxic substances) participate in the etiopathogenesis of the different forms of leukemia.

[Haematological and immunological findings in bone marrow aplasia (author's transl)]. / Hämatologische und immunologische Befunde bei Knochenmarkaplasie

The cellularity of the bone marrow, the number of blood cells and the immunoglobulins were investigated in 51 patients suffering from bone marrow aplasia. In this disease not only is a haematopoietic organ disturbed, but disorders of the lymphocyte and of the monocyte-systems can be detected too: the majority of patients show lymphocytopenia and monocytopenia. Immunologic reactions seem to be involved in the pathogenesis of bone marrow aplasia. The plasma cells of the bone marrow are elevated in nearly all of the patients, lymphocytes in every second case. One or several fractions of immunoglobulins are increased in about 50% and diminished in 25% of the patients.