SEARCH · Search PubMed

Results for “ORGANOIDS”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources

PubMedDate not supplied

[Sulfanilamide binding and distribution in the liver cells of rats].

Experiments on white noninbred rats were made to study the binding by isolated organoids and distribution in liver cells of etazole, norsulfazole, sulfamonomethoxine and sulfalene. The latter drug was found to be absorbed by organoids to the greatest degree. Drug complexes with organoids were discovered to be capable of dissociating with a partial release of unbound drug. Intracellular distribution of sulfanilamides is marked by their accumulation primarily by the nuclei and to a lesser degree by the mitochondria and microsomes.

Animals↗

PubMed2000

Adrenal medullary explants as an efficient tool for pain control: adhesive biomolecular components are involved in graft function ex vivo.

Adrenal medullary (AM) tissue transplantation into the central nervous system has been reported as a potential source of opioid peptides and catecholamines, which have analgesic effects useful in the control of chronic pain. Clinical trials, involving allogeneic graft of whole tissue explants into the subarachnoid space of the lumbar spinal cord, have already been reported. The aim of the present study was to determine whether adhesion and function of AM explants were related in some extent and how this relationship could account for improvement of AM tissue in terms of analgesic activity before grafting. Our experiments demonstrated a significant correlation between the adherent state of AM organoids during culture and a sustained secretion of Met-enkephalin and catecholamines by chromaffin cells (CC). These findings suggest that optimal culture condition for AM organoid adhesion can be defined for maintenance of tissue, prior to transplantation. Using immunocytochemistry, flow cytometry, and ELISA assays we showed that different cell adhesion molecules (CAMs) and extracellular matrix ECM proteins were expressed and released by AM cells during culture. Adherent AM organoids expressed increased levels of specific neural CAMs (NCAM and HNK-1 epitope) and integrin chains (beta1, alpha1, alpha2, alpha4, alpha5) and deposited markedly higher levels of fibronectin, but also laminin and collagen IV. Those molecules and probably adhesion processes they control might be involved in the maintenance of the CC-secreting neuroendocrine phenotype through cellular signaling pathways.

Adrenal Medulla↗

PubMed1999

Effects of anastomosis of tissue-engineered neointestine to native small bowel.

BACKGROUND: Our laboratory is investigating the tissue engineering of small intestine using intestinal epithelial organoid units seeded onto highly porous biodegradable polymer matrices. This study investigated the effects of anastomosis of tissue-engineered intestine to native small bowel alone or combined with small bowel resection on neointestinal regeneration. METHODS: Intestinal epithelial organoid units harvested from neonatal Lewis rats were seeded onto biodegradable polymer tubes and implanted into the omentum of adult Lewis rats as follows: (1) implantation alone (n = 9); (2) implantation followed by anastomosis to native small bowel at 3 weeks (n = 11); and (3) implantation after small bowel resection and anastomosis to native small bowel at 3 weeks (n = 8). All constructs were harvested at 10 weeks and examined by histology. Morphometric analysis of the neomucosa was obtained using a computer image analysis program. RESULTS: Cyst development was noted in all animals. All anastomoses were patent at 10 weeks. Histology revealed the development of a vascularized tissue with a neomucosa lining the lumen of the cyst with invaginations resembling crypt-villus structures. Morphometric analysis demonstrated significantly greater villus number, villus height, crypt number, crypt area, and mucosal surface length in groups 2 and 3 compared with group 1, and significantly greater villus number, villus height, crypt area, and mucosal surface length in group 3 compared with group 2 (P < 0.05, ANOVA, Tukey test). CONCLUSION: Intestinal epithelial organoid units transplanted on biodegradable polymer tubes can regenerate into complex tissue resembling small intestine. Anastomosis to native small bowel combined with small bowel resection and anastomosis alone contribute significant regenerative stimuli for the morphogenesis and differentiation of tissue-engineered neointestine.

Anastomosis, Surgical↗

PubMed1989

Distinction between vascular smooth muscle cells and myoepithelial cells in primary monolayer cultures of human breast tissue.

We report on the discrimination of vascular smooth muscle cells and myoepithelial cells in primary cultures of human breast tissue. Breast tissue was disaggregated enzymatically and the resulting organoids seeded in monolayer culture on collagen-coated plastic in serum-free medium CDM3a. Two main types of organoids were present after enzymatic digestion. One resembled small blood vessels and the other interlobular ducts or acini of the breast gland epithelium. Within 3 to 8 d after plating the organoids migrated into typical monolayer islets. These monolayer islets were evaluated using phase contrast microscopy and further tagged with monoclonal antibodies for immunocytochemical demonstration of Factor VIII-related antigen, muscle iso-forms of actin, type IV collagen, vimentin, desmin, and keratins. It is concluded that vascular smooth muscle cells resembled myoepithelial cells by expressing vimentin filaments, depositing type IV collagen, and showing immunoreactivity to muscle iso-forms of actin. However, whereas vascular smooth muscle cells were associated with endothelial cells and sometimes expressed desmin, myoepithelial cells appeared together with luminal epithelial cells and expressed cytokeratins.

Actins↗

PubMed1989

cAMP levels in proliferating rat mammary epithelial cells in vitro and in vivo.

Agents that elevate intracellular cAMP levels are required for growth of many cell types in culture including normal rat mammary epithelial (RME) cells. To determine if the intracellular levels of cAMP that result from stimulation by agents such as cholera toxin (CT) or prostaglandin E-1 (PGE-1) are within the physiological range, cAMP levels were determined in RME cells growing in primary culture and compared to levels measured in freshly isolated mammary epithelium. The results indicate that the cAMP levels of mammary epithelial organoids obtained from 45-day-old virgin rats are 4 to 6 pmol/10(6) cells. Growth of RME cells in primary culture in the presence of CT results in cAMP levels of approximately 15 to 20 pmol/10(6) cells early in culture when cells are proliferating rapidly. As cells approach confluence, cAMP concentrations decrease to levels observed in fresh organoids. CT-stimulated cAMP levels appear to be within the range of those found in pregnant mammary epithelium in vivo. Growth of RME cells in medium supplemented with PGE-1 instead of CT results in cAMP levels equivalent to those found in fresh mammary epithelial organoids and under these conditions the growth rate is approximately half that found in CT-stimulated cells. These results indicate cAMP to be a positive regulator of cell growth in vivo at levels that are within the physiological range.

Alprostadil↗

PubMed1992

Phenotypic responses of mouse mammary tumours and normal mammary epithelium cultured in collagen gels: correlation with tumour type and progression.

Mammary tumours in female BR6/Icrf mice and the corresponding contralateral normal mammary glands were disaggregated with collagenase and the epithelial structures released ('organoids') separated from other cellular components by filtration. The organoids were established in primary culture in a collagen matrix and the outgrowths obtained were studied by light microscopy and time-lapse cinemicroscopy. The pattern of three-dimensional outgrowths produced was found to be specific to the original tissue. Organoids from normal tissue formed a characteristic outgrowth designated Pattern A. Normal tissue from pregnant mice formed an additional characteristic outgrowth (Pattern A') which has not been described previously. Pregnancy-dependent tumours produced a distinctive phenotypic outgrowth designated Pattern D, whereas pregnancy-independent tumours gave a different distinctive Pattern B as well as a unique specific outgrowth designated Pattern C. Outgrowths of Pattern D from a pregnancy-dependent tumour were removed from culture and implanted into a syngeneic female mouse. Tumours arising in the host were found to be pregnancy-independent and showed phenotypic outgrowths in subsequent culture of pregnancy-independent Patterns B and C. The results show that the type of outgrowths in these cultures correlates with the biology of the tissue in vivo and that changes in tumour progression in vivo are accompanied by alterations in phenotypic outgrowths in culture.

Animals↗

PubMed2005

Engraftment of mucosal stem cells into murine jejunum is dependent on optimal dose of cells.

BACKGROUND: Transplantation of intestinal mucosal stem cells is an important step in the development of intestinal gene therapy and treatment of intestinal mucosal diseases. We hypothesized that engraftment rates increase proportionally with increasing doses of seeded stem cells and increasing jejunal débridment. MATERIALS AND METHODS: Intestinal mucosal organoids were harvested from neonatal mice carrying a green-fluorescent protein (GFP) transgene and transplanted into adult GFP(-) mice (n = 66). In recipients, two jejunal segments (1.5 cm) were isolated with their blood circulation left intact with anastomosis of the distal and proximal segments to restore continuity. Debridement of native enterocytes was performed by perfusing luminally with ethylene diamine tetraacetc acid solutions for 20 min. A total of 5,000, 10,000, or 25,000 organoids were then seeded. Three weeks later, cross sections (n = 398) of the segments were evaluated for the presence of GFP(+) neomucosa using fluorescence microscopy. Additional segments were débrided for 30 and 40 min (n = 83). Other conditions were not tested because of the rate of high mortality in these experiments. RESULTS: The group seeded with 10,000 organoid units at 20 min showed the highest engraftment of GFP(+) epithelium. Engraftment was improved by increasing débridment times at this seeding density. Overall mortality was 70%. CONCLUSIONS: These findings suggest that there is an optimal seeding density of stem cell clusters for enhanced engraftment in this model. Mortality prohibited complete testing of all combinations of seeding density and debridement times.

Animals↗

PubMed2006

Prolonged maintenance of neointestine using subcutaneously implanted tubular scaffolds in a rat model.

Tissue-engineered small intestine offers a possible alternative to long-term parenteral nutrition or intestinal transplantation in patients with short bowel syndrome. The aim of this study was to investigate the prolonged development of neointestine grown on subcutaneously implanted scaffolds. Tubular polylactide-coglycolide (PLGA) scaffolds were implanted into adult Lewis rats. Four weeks after scaffold implantation, a suspension of organoid units was delivered to the lumen of each scaffold. Organoid units were manufactured from small intestine harvested from neonatal Lewis rats by partial digestion using collagenase and dispase. Scaffolds were removed at 4, 8, and 12 weeks after organoid unit implantation, processed to paraffin, and sectioned. Hematoxylin and eosin staining demonstrated well-developed and well-differentiated intestinal mucosa and a vascularised submucosa within the scaffolds at 4, 8, and 12 weeks. Appearances were similar to native small intestine. Immunohistochemistry performed using primary antibody against proliferating cell nuclear antigen, a marker for cellular proliferation, demonstrated positively staining cells within the mucosa and submucosa at all time points. In the mucosal layer these positively staining cells were found primarily in the crypts. These findings show that neointestinal mucosa can be maintained for at least 12 weeks on a subcutaneous PLGA scaffold, and the presence of actively proliferating cells at 12 weeks suggests potential for further development beyond this.

Animals↗

PubMed1997

Formation of spheroid structures in a human colon carcinoma cell line involves a complex series of intercellular rearrangements.

The structural remodelling of tissues that occurs in vivo during animal morphogenesis can often prove difficult to study. Here we investigate the organizational processes of the LIM 1863 colon carcinoma cell line as it transforms from a single-cell stage into multicellular spherical structures called 'organoids'. The organoids can be dissociated into a viable single-cell suspension when cultured in calcium-depleted medium, and then induced to reform the organoid structure by the readdition of calcium. Previous studies have shown that initial cell attachment under these conditions is characterized by a novel mechanism of cell engulfment termed 'clutching'. This investigation reveals the subsequent appearance of junctional complexes between groups of 'clutched' cells prior to lumen formation, and the ultimate 'declutching' of entrapped cells as a means of cell rearrangement. Intact actin filaments but not microtubules were required for the initial clutching events, while inhibition of microtubule polymerization resulted in aberrant apical protein polarization, but did not affect the development of a luminal space within the spheroids. Single cells exhibited pools of intracellular microvilli contained in vacuolar apical compartments, which were resistant to the effects of cytoskeleton-disrupting drugs. However, these structures did not seem to be responsible for the swift development of the luminal surface observed in these cells. Two other cell lines, MDCK and DU 4475, were found to exhibit similar clutching conformations when induced to form three-dimensional structures, suggesting that this may be a widespread mechanism of cell rearrangement that reflects the process of organ morphogenesis in vivo.

Carcinoma↗

PubMed1989

Expression of c-myc, c-Ha-ras1, and c-erbB-2 proto-oncogenes in normal and malignant human breast epithelial cells.

Short-term cultures of normal human mammary epithelial cells were used to determine the extent to which c-myc, c-Ha-ras1, and c-erbB-2 proto-oncogenes were expressed in proliferating normal cells. This level of expression was compared with that of primary tumor cells, malignant effusion cells, or permanently established breast cancer cell lines. Pure preparations of epithelial organoids from seven different reduction mammoplasty tissue samples yielded proliferating normal epithelial cells upon short-term tissue culture. In every sample, proto-oncogene transcript levels increased upon short-term culture of the epithelial cells. These levels often exceeded by 10-fold the levels measured in uncultured organoids from the same tissue. In four of the seven cultured normal breast samples, at least one of the proto-oncogenes increased its expression to a level equaling or exceeding that found in a proliferating breast cancer cell line, MCF7. One effusion metastasis sample and two primary ductal adenocarcinomas were also examined for proto-oncogene expression. The effusion metastasis sample expressed high levels of c-erbB-2 messenger RNA, in accord with its amplified gene copy number; otherwise, the levels of proto-oncogene transcripts were low in unprocessed tumor and uncultured organoids, but they increased with proliferation of the tumor cells in culture. These results indicate that the variable expression of these proto-oncogenes observed in breast biopsy specimens needs to be controlled for cellular growth rate or proliferation index. Furthermore, these findings suggest that dysregulated proto-oncogene expression, rather than overexpression per se, needs to be evaluated as a possible mechanism contributing to the development of human breast cancer.

Blotting, Northern↗

PubMed1995

Stromal tumors of the duodenum. A histologic and immunohistochemical study of 20 cases.

Using cell size, cell density, and microscopic growth pattern, 20 duodenal stromal tumors were initially separated into benign and malignant categories. The 10 histologic benign tumors had uniform spindle cells, low cellularity, and an organoid pattern. All had round eosinophilic collagen blobs scattered among the spindle cells, were 4.5 cm or less in maximum diameter, and had two or fewer mitoses per 50 high-power fields (HPF). None metastasized or recurred during a median follow-up of 7 years. In contrast, the 10 histologically malignant tumors were highly cellular, all had two or more mitoses per 50 HPF, and all but one had diameters of 4.5 cm or greater, the exception being 4 cm. Eight cases also had benign-appearing areas, usually submucosal. Eight patients died with disease a median of 31 months after resection, almost all with liver metastases. One patient is alive with metastasis at 13 years. The patient with the 4-cm malignant tumor is disease free at 49 months. All 15 cases were strongly vimentin positive, 11 had S-100 protein, and seven had the CD34 marker. None were desmin or actin positive. No immunophenotype separated benign from malignant. The proliferation marker, proliferating cell nuclear antigen, correlated with histologic diagnosis and clinical outcome, but Ki-67 did not. Based on light microscopic features alone, benign and malignant duodenal stromal tumors can be separated from each other. Tumors with large cells and an organoid pattern are predictably benign; in this study, these tumors measured 4.5 cm or less in diameter and had fewer than 2 mitoses per 50 HPF. Highly cellular tumors with small cells and little or no organoid pattern are malignant. They usually have a diameter greater than 4.5 cm and more than two mitoses per 50 HPF, and they are usually fatal. Immunostaining for cytoplasmic proteins and proliferation markers offers no additional prognostic information to the light microscopic appearances. These conclusions apply only to duodenal tumors; whether they also apply to stromal tumors of the jejunum and ileum is not known.

Adult↗

PubMed1997

Renal oncocytoma: a reappraisal of morphologic features with clinicopathologic findings in 80 cases.

Renal oncocytoma has several features that overlap with other renal neoplasms with a preponderance of granular cytoplasm, such as chromophobe, granular, and papillary renal cell carcinomas. Lack of knowledge of this entire spectrum of eosinophilic renal cell neoplasms has led to several misconceptions in the literature regarding renal oncocytoma. These include the "grading of oncocytomas," "metastatic oncocytomas," and the impression that renal oncocytoma is usually low grade and lacks prominent nucleoli. In order to further characterize the histologic features and embelLish diagnostic criteria, we evaluated 93 tumors from 80 patients. Four tumors were bilateral and two were multifocal. The mean age was 67.2 years (32-89 years), men were more commonly affected (3.1:1), and 82.7% tumors were incidental findings. Grossly, the tumors were mahogany brown, lacked necrosis, and averaged 4.4 cm in size (range 0.6-15 cm). Histologically, renal oncocytoma was composed of an exclusive or predominant component of acidophilic cells with three architectural patterns of disposition: (a) The "classic" pattern (57.5%), composed of a characteristic nested or organoid arrangement of cells, each surrounded by a distinct reticulin framework; (b) a "tubulocystic pattern" (6.3%) with numerous closely packed cystically dilated tubular structures; and (c) "mixed pattern" (36.2%), which had both the organoid and tubulocystic patterns. A gross or microscopic scar was noted in 53.8% cases, and histologically a distinctive myxoid and/or hyalinized stroma separated nests of cells. Generally, the nuclei of renal oncocytoma were round with uniform nuclear contours. Nearly half of the tumors had prominent nucleoli (42.5% had prominent nucleoli equivalent to Fuhrman's grade III or IV). Pleomorphism was absent in 50% of cases but was conspicuous in 12.5% of cases including foci of bizarre cells. Other atypical features included perinephric fat involvement (11.3%), renal parenchymal invasion not associated with desmoplasia (10%), and hemorrhage (31.3%). Renal oncocytoma by definition lacks areas of clear cell carcinoma, significant lesional necrosis, or conspicuous papillary formations. Ancillary features noted included normal-appearing renal tubules within the lesion (15%), intranuclear holes (20%), psammoma bodies (7.5%), and foam cells (7.5%). 15% of tumors were locally excised, and 85% resulted in radical nephrectomy. Mean follow-up of 7.6 years (range 15-200 months) showed no evidence of recurrence, metastasis, or death due to tumor. In conclusion, renal oncocytoma, herein described, is a benign neoplasm and therefore does not merit a nuclear grading scheme. It has unique histologic features including an organoid and tubulocystic architecture, myxoid or hyalinized stroma, and occasionally some atypical findings including nuclear pleomorphism, prominent nucleoli, and adjacent renal parenchymal and perinephric fat involvement.

Adenoma, Oxyphilic↗

PubMed1990

Growth factors specifically alter hair follicle cell proliferation and collagenolytic activity alone or in combination.

A three-dimensional culture model for isolated murine pelage hair follicles in a type I collagen gel has been utilized to study the effects of selected growth factors on follicle cell proliferation and release of collagenolytic factors. Cultured follicle organoids differentially express cytokeratins 6 and 14 in a pattern suggesting they contain cells of the outer root sheath, inner root sheath and follicle matrix. Using incorporation of [3H]thymidine as a measure of proliferation, follicle organoids show a peak of DNA synthesis between day 1 and 5 of culture, depending on plating density, and then have a low rate of DNA synthesis. Thymidine incorporation is stimulated by transforming growth factor-alpha (TGF-alpha) in a dose-dependent response. Only peripheral cells presumably of the outer root sheath, incorporate thymidine in basal or stimulated conditions. TGF-beta 1 and TGF-beta 2 inhibit constitutive cell proliferation and oppose growth stimulation by TGF-alpha. Hair follicles lyse the collagen gel matrix when exposed to certain cytokines. Epidermal growth factor (EGF) and TGF-alpha stimulate gel lysis, but TGF-beta 1, TGF-beta 2 and cholera toxin do not. Other skin-derived cells, such as interfollicular epidermal cells, dermal fibroblasts, or combinations thereof, do not lyse gels in this culture model even when exposed to growth factors. Combinations of EGF or TGF-alpha with TGF-beta 1 or TGF-beta 2 are synergistic for collagenase release. These cytokines stimulate release of multiple species of matrix metalloproteinases, but the 92-kDa and 72 kDa type IV procollagenases and their activated derivatives predominate on zymograms. In cytokine-stimulated follicles, both peripheral and centrally located cells in the organoids express the 72-kDa type IV collagenase and a similar immunostaining pattern is present in developing follicles in vivo. Thus growth factors appear to work in concert for certain hair follicle responses and in opposition for others. These combined actions may play a role in different phases of hair follicle development that require cell replication and invasion into the deeper dermis.

Animals↗

PubMed1985

A subclass of luminal epithelial cells in the human mammary gland, defined by antibodies to cytokeratins.

Two monoclonal antibodies, BA16 and BA17, have been developed using a detergent-insoluble extract of human mammary epithelial organoids as immunogen. Indirect immunofluorescent staining of cultured cells showed that the component reacting with the antibodies was filamentous and the intensity of staining was stronger in mitotic cells. Immunoblotting of cell extracts showed that both antibodies react with only one band of 40 X 10(3) molecular weight, which was present in keratin-enriched extracts of cells or organoids. Furthermore, the tissue distribution of the component reacting with the antibodies was that predicted for human keratin 19. The antibodies showed differences in the intensity of staining of cells or tissue sections fixed and prepared in different ways indicating that they reacted with different epitopes. The pattern of expression of the 40 X 10(3) Mr keratin by normal mammary epithelial cells was investigated by immunoperoxidase staining of tissue sections, cultured milk cells, and organoids of different sizes cultured in collagen gels. It was found that basal or myoepithelial cells did not express this keratin. Some heterogeneity of expression of this component was seen in luminal epithelial cells, found almost exclusively in the smaller structures. These cells did, however, express other keratins characteristic of luminal cells. The distribution in the mammary tree of the luminal cells that did not express the 40 X 10(3) Mr keratin appears to be similar to that expected for cells with the proliferative potential to produce new terminal ductal lobular units or an increase in branching of existing terminal ductal lobular units. It is shown that these cells have considerable proliferative potential by the fact that they form large colonies in milk cell cultures.

Animals↗

PubMed2001

Permanent phenotypic and genotypic changes of prostate cancer cells cultured in a three-dimensional rotating-wall vessel.

A three-dimensional (3D) integrated rotating-wall vessel cell-culture system was used to evaluate the interaction between a human prostate cancer cell line, LNCaP, and microcarrier beads alone, or microcarrier beads previously seeded with either prostate or bone stromal cells. Upon coculture of LNCaP cells with microcarrier beads either in the presence or in the absence of prostate or bone stromal cells, 3D prostate organoids were formed with the expected hormonal responsiveness to androgen, increased cell growth, and prostate-specific antigen production. In this communication, we define permanent phenotypic and genotypic changes of LNCaP cells upon coculture with microcarrier beads alone, or with microcarrier beads previously seeded with either prostate or bone stromal cells. Most notably, we observed selective genetic changes, i.e., chromosomal losses or gains, as evaluated by both conventional cytogenetic and comparative genomic hybridization, in LNCaP sublines derived from the prostate organoids. Moreover, the derivative LNCaP cells appear to have altered growth profiles, and exhibit permanent and stable changes in response to androgen, estrogen, and growth factors. The derivative LNCaP sublines showed increased anchorage-independent growth rate, and enhanced tumorigenicity and metastatic potential when inoculated orthotopically in castrated athymic mice. Our results support the hypothesis that further nonrandom genetic and phenotypic changes in prostate cancer epithelial cells can occur through an event that resembles "adaptive mutation" such as has been described in bacteria subjected to nutritional starvation. The occurrence of such permanent changes may be highly contact dependent, and appears to be driven by specific microenvironmental factors surrounding the tumor cell epithelium grown as 3D prostate organoids.

Animals↗

PubMed1992

Effects of cholera toxin on human colon carcinoma cell lines.

This study reports on changes in morphology and membrane transport in 5 human colon carcinoma cell lines treated with cholera toxin (CT). Three of the cell lines that grew as monolayers (LIM 1215, LIM 1899, LIM 2099) and 1 that grew as floating clumps (LIM 2408) did not show morphological changes after CT treatment. However, cell line LIM 1863 that grows as floating "crypt-like" organoids showed rapid and distinctive changes in morphology and membrane transport after CT treatment. At 1 and 6 hrs after CT treatment, light and transmission electron microscopy revealed rapid dilatation of the central lumen of organoids and the appearance of 2 populations of apical vesicular inclusions. The first population was unusual in being non-membrane bound and limited by fuzzy filamentous material. The second population was membrane bound. Scanning electron microscopy at 1-6 hr after CT treatment showed swelling and loss of surface microvilli on some, but not all, cells. At 24 hr after CT treatment the majority of organoids showed evidence of fluid accumulation and small apical vesicles coalesced to form large single vacuoles that obliterated normal cell morphology. By 48 hr, continued swelling produced extreme attenuation of the plasma membrane with cells taking on an "endothelial cell-like" appearance. The response to CT was dose-dependent. Uptake studies using 86Rubidium and blocking studies using ouabain and amiloride indicated that CT is acting on the Na+/K+ ATPase membrane pump to cause the increased fluid uptake by LIM 1863 cells. This study is the first to report specific morphological changes in intestine-derived cells in response to CT.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma↗

PubMed1999

T-cell receptors and autoimmune thyroid disease--signposts for T-cell-antigen driven diseases.

The human autoimmune thyroid diseases (AITDs) are characterized by profuse infiltrates of both CD4+ and CD8+ T cells. The intrathyroidal T-cell-receptor repertoire in Graves' disease, more than in Hashimoto's disease, has been shown to be biased as evidenced by phenotypic analysis and by the use of a restricted T-cell-receptor variable (V) gene repertoire seen in both TCR alpha and beta chains. Evidence for a bias in the T-cell repertoire has also been observed in animal models of induced and spontaneous autoimmune thyroiditis. We found a similar phenomenon of autoimmune thyroid-related T-cell bias in thyroid-humanized scid mice. In these studies we transplanted lymphocyte-depleted thyrocytes and autologous peripheral lymphocytes from AITD patients with a basement membrane preparation which allowed the formation of an artificial thyroid which we have called an "organoid". T-cell clonal expansion was present in these artificial mixed-cell organoids which appeared to mimic the in vivo process. Such clonal expansion was suggestive of an antigen-driven immune response and could also be identified in thyroid tissue from patients with Graves' disease. Our data on scid mice grafted with human mixed-cell thyroid organoids, therefore, suggested that the major antigens driving T-cell selection in patients with AITD were most likely to be thyroid specific. These antigens include thyroglobulin, thyroid peroxidase, and the receptor for thyroid stimulating hormone (TSHR) on the surface of thyroid epithelial cells and we found significant T-cell proliferation to synthetic TSHR peptides in patients with AITD as compared with normals. Our search for a TCR recognition motif for the autoantigen TPO did not reveal any specific sequence motifs. Instead, analysis of the physico-chemical characteristics i.e. hydrophobicity of the amino acids in the CDR3 (N) region of the TCR alpha chain, revealed a strong negative linear correlation between strength of stimulation and the average hydrophobicity of N-region amino acids. This led us to hypothesize that lower affinity T-cell clones were commonly more hydrophobic in their CDR3 alpha region amino acids in keeping with potential crossreactivity of such T cells as a consequence of promiscuous, hydrophobic CDR3 regions. This phenomenon would be analogous to polyreactive, natural autoantibodies which tend to be crossreactive and 'sticky'. Thus, the physico-chemical characteristics of the TCR alpha CDR3 region supported the interaction with antigen/MHC by potentially cross-reactive T cells of low affinity. It would seem likely that such low-affinity autoreactive T-cell populations serve as a pool of potentially pathogenetic cells. These cells would be able to respond to an insult which, via a number of possible mechanisms such as molecular mimicry, would initiate a thyroid lymphocytic infiltration in an antigen-driven fashion with intrathyroidal T-cell expansion and a marked bias in the utilization of T-cell-receptor V genes.

Animals↗

PubMed1992

[Primary culture in chemically defined medium of human fetal mammary epithelial cells within reconstituted matrix].

A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal human fetal mammary epithelial cells within an extracellular matrix preparation. Human fetal mammary epithelial cells were isolated from the mammary glands of human female fetuses, 17 to 39 weeks-old. The "organoids" were embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm (EHS) sarcoma according to the method of Hahm and Ip. "Organoids" were grown in either serum-free medium or in medium with fetal calf serum (FCS). The "organoid" proliferated over a 2 to 3 weeks culture period and remained viable for 1 or 2 months within the basement membrane matrix in serum free medium. Several types of colonies were observed; including alveolar-like budding clusters obtained from cultures of mammary gland from fetuses of over 20 weeks age, units with ductule-like projections and stellate-type colonies. Cell proliferation was dependent on the culture medium (with FCS no proliferation was obtained) and on the substratum (without matrix, significantly less growth and development occurred). These types of colonies are obtained when a glandular differentiation of cells budding from the malpighian epithelium is observed. Light microscopic and transmission electron microscopic studies were undertaken at the time of culture. This unique system using normal fetal mammary epithelial cells thus provides a model in which the regulation of human mammary development can be investigated.

Breast↗