Normal adult rat hepatocytes remained viable and functional for at least 43 days when plated on collagen-coated dishes and fed chemically defined medium supplemented with dimethyl sulfoxide (Me2SO). Hepatocytes isolated by collagenase perfusion and cultured in the presence or absence of Me2SO were (i) examined by light and electron microscopy for morphological changes; (it) analyzed for the production of albumin and other plasma proteins; and (iiM) tested by autoradiography for DNA synthesis. Me2SO-treated cells continued to produce specific plasma proteins during the entire culture period; albumin production was consistently high (11)(12)(13)(14)(15)(16)(17)(18)(19) ,.g/ml of culture medium per 24 hr) from day 2 to at least day 43 after plating. Ultrastructural analyses demonstrated that Me2SO-treated hepatocytes resembled those from intact liver in organization of cytoplasmic organelles and cellular junctions. The optimal concentration for observing the morphological and biochemical effects of Me2SO was 2% (vol/vol). We conclude that supplementation of chemically dermed medium with Me2SO enables maintenance ofdifferentiated hepatocytes in culture for extended periods of time.Dimethyl sulfoxide (Me2SO) is a dipolar aprotic solvent that is active in biological systems (1). Addition of 1-2% (vol/vol) Me2SO to the culture medium of Friend virus-induced murine erythroleukemia (MEL) cells for 4-5 days causes 90% of the cells to express characteristics associated with normal erythroid differentiation, including alterations in morphology (2), induction of a-and P-globin synthesis (3, 4), and loss of the capacity for cell division (5). Me2SO-induced differentiation has also been observed in a human leukemia cell line (6) and in cultured fibrosarcoma (7), neuroblastoma (8), human colon carcinoma (9), human lung cancer (10), and murine embryonal carcinoma (11, 12) cell lines.Past efforts to achieve long-term culture of differentiated normal adult hepatocytes have not been successful. Limited proliferation and maintenance of adult hepatocytes can be achieved by supplementing culture medium with serum from partially hepatectomized animals (13) or by plating hepatocytes on liver extracellular matrix and maintaining them in serum-free hormonally defined medium (14). Proliferation also can be achieved by culturing hepatocytes at low cell density in the presence of insulin and epidermal growth factor (EGF), but maintenance of hepatocyte-specific characteristics requires high density or supplementation with hepatic plasma membrane material (15,16).In the present study, we employed a collagen-coated surface and supplemented the culture medium with Me2SO in an attempt to extend the time in vitro that hepatocytes remain biochemically and morphologically differentiated. The addition of Me2SO had a dramatic effect; hepatocytes retaining morphological and biochemical characteristics of normal liver could be maintained in culture for as long as 43 days. Note that Me2SO, used previously to induce differentiation in tumor cells ...

Paratuberculosis is a chronic granulomatous enteritis of domestic and wild ruminants that is caused by Mycobacterium avium subsp. paratuberculosis, a slow-growing intracellular acid-fast bacillus. Animals are usually infected within the first few months of life; however, the chronic wasting and profuse diarrhea that characterize clinical paratuberculosis are usually not observed until 3 or more years after infection (4, 7).M. avium subsp. paratuberculosis enters intestinal tissue through M cells present in the dome epithelium covering the continuous Peyer's patches in the distal ileum of calves and goats (22,32). The discrete Peyer's patches in the intestinal tracts of other mammals and the jejunum of ruminants are secondary lymphoid organs, in which an adaptive immune response can be initiated following exposure to foreign antigens. In contrast, the continuous Peyer's patch in the ruminant ileum is a primary lymphoid organ, wherein B-cell development occurs (36).Fibronectin attachment proteins (FAPs) are a family of fibronectin (FN)-binding proteins present in several species of mycobacteria (25,(28)(29)(30)42). Attachment and internalization of Mycobacterium bovis BCG, M. leprae, and M. avium subsp. paratuberculosis by epithelial cells in vitro have been shown to be dependent on the interaction between FAP and FN (18,29,31). ␤1 integrins have been identified as the host cell receptor for FN-opsonized mycobacteria in vitro (3, 18). M cells are unique among cells of the intestinal epithelium in that they display ␤1 integrins on their luminal faces at high density (6). Thus, the interaction between cell surface integrins and FN bound by organisms may explain why M cells are the portals of entry for M. avium subsp. paratuberculosis. However, adherence and internalization assays with macrophages, monocytes, and epithelial cell lines give only a general picture of the adhesive and invasive potential of mycobacteria. The nature and density of potential host cell receptors vary for each culture system to such an extent that the significance of hostpathogen interactions observed in vitro must be interpreted with caution. Moreover, the distribution of receptors on cells in culture is often markedly different from that on cells in intact tissue, particularly for intestinal epithelial cells (8,15,37). Thus, it is imperative that putative adhesins and invasins be evaluated in in vivo model systems before any degree of significance can accurately be attributed to their expression.The expression of FAP was found to be important for attachment of M. avium subsp. avium to respiratory explants and attachment of M. bovis BCG to murine bladder mucosa in vivo (20,41). However, demonstration of FAP-mediated mycobacterial attachment required exposure of the extracellular matrix. This mechanism is insufficient to explain M-cell targeting by M. avium subsp. paratuberculosis. Thus, the contribution of the FAP of M. avium subsp. paratuberculosis (FAP-P) to the invasive potential of M. avium subsp. paratuberculosis for intestinal

Attachment and ingestion ofJohne's disease poses a significant economic threat to the dairy cattle industry (13, 21). Johne's disease is a granulomatous enteritis of ruminants that is caused by Mycobacterium avium subsp. paratuberculosis. M. avium subsp. paratuberculosis invades macrophages that reside in ileal Peyer's patches, subsequently resulting in granuloma formation. Infiltration of intestinal lamina propria by inflammatory cells and an increase in the size and number of granulomas disrupt intestinal absorption, resulting in wasting and death. Animals are usually infected before 6 months of age; however, the cachexia and profuse diarrhea that are the hallmarks of Johne's disease are not observed until several years postinfection.The ability of a microorganism to bind fibronectin (FN) may potentially facilitate its colonization of the host through attachment to the extracellular matrix in areas of epithelial damage. Furthermore, because several host cell integrins have binding sites for FN, the ability of a microorganism to bind soluble FN establishes a bridge between the organism and the host cell cytoskeleton, a condition necessary for the internalization of the microbe by the cell (3, 6). Fibronectin attachment proteins (FAPs) comprise a family of FN-binding glycoproteins that are expressed by several species of mycobacteria (16)(17)(18)(19)24). Expression of FAP is critical for the attachment of Mycobacterium bovis BCG and M. avium subsp. avium to tissue in vivo and ex vivo (11,24). Moreover, the internalization of M. bovis BCG and Mycobacterium leprae by cultured epithelial cells was shown to be a FAP-dependent process (10, 18).M. avium subsp. paratuberculosis also expresses a FAP (designated FAP-P) which mediates soluble FN binding (19). However, unlike the FAPs of other mycobacteria, FAP-P is not present on the surface of the organism (19). Thus, the manner in which FAP-P engages FN may sequester the cell binding domain from host cell receptors. As a result, binding and ingestion of M. avium subsp. paratuberculosis by host cells would be FN independent.To examine the effect of the interaction of FAP-P and FN on the ability of M. avium subsp. paratuberculosis to attach to and invade epithelial cells, FN-opsonized and nonopsonized organisms were used to infect T-24 human bladder carcinoma cells (a gift from J. S. Schorey, University of Notre Dame, South Bend, Ind.) and Caco-2 human intestinal adenocarcinoma cells (supplied by M. Popielarczyk, Purdue University, West Lafayette, Ind.). T-24 cells were propagated as described previously (18 M. avium subsp. paratuberculosis cultures were centrifuged for 15 min at 1,600 ϫ g. The bacterial pellet was mixed thoroughly by vigorous pipetting and vortexing in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (Sigma) to generate a single-cell suspension and resuspended in a volume of PBS containing 0.05% Tween 20 sufficient to yield 5 ϫ 10 6 CFU/ml. Five milliliters of bacterial suspension was centrifuged as described above, and the pellet was resuspende...

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Abstract. The feasibility of coupling an agar culture enrichment step with gene amplification (ACE-PCR) as a means to improve turnaround time and detect Mycobacterium paratuberculosis (Mpt) in the presence of contaminants was investigated. Fecal samples from 463 Pennsylvania dairy cows were cultured in duplicate sets. One replicate from each set was processed and interpreted according to standard culture (SC) protocol, whereas cultures from the second replicate were harvested at 6 weeks postinoculation; DNA extracts from the harvested material were evaluated by a polymerase chain reaction (PCR) test for the Mpt-specific IS900 gene. One hundred seventy-six of 463 culture sets were positive by either method. One hundred sixty-five of these (94%) were ACE-PCR positive, and 151 (86%) were positive by SC. Eleven SC-positive samples were ACE-PCR negative, and 9 ACE-PCR-positive samples were negative by SC; these discrepancies could be a consequence of a low organism burden (Յ5 organisms/g) or slow growth rate of Mpt in cultures of these samples. One hundred thirty-nine of 463 culture sets (30%) were reported as inconclusive because of culture contamination according to SC protocol; 16 of these (11.5%) were ACE-PCR positive. Seventy-four ACE-PCR-positive sets (42% of all positives) were negative or inconclusive by SC at 6 weeks postinoculation. Agar culture enrichment prior to IS900 PCR testing significantly improves Mpt culture turnaround time and sensitivity.