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Biomedical subjects

T J Goodwin

Publications and source records attributed to T J Goodwin.

At least 37 records · Page 2Linked to original sources
PubMed1997

Induction of carcinoembryonic antigen expression in a three-dimensional culture system.

MIP-101 is a poorly differentiated human colon carcinoma cell line established from ascites that produces minimal amounts of carcinoembryonic antigen (CEA), a 180 kDa glycoprotein tumor marker, and nonspecific cross-reacting antigen (NCA), a related protein that has 50 and 90 kDa isoforms, in monolayer culture. However, MIP-101 produces CEA when implanted into the peritoneum of nude mice but not when implanted into subcutaneous tissue. We tested whether three-dimensional (3D) growth was a sufficient stimulus to produce CEA and NCA 50/90 in MIP-101 cells, because cells grow in 3D in vivo rather than in two-dimensions (2D) as occurs in monolayer cultures. To do this, MIP-101 cells were cultured on microcarrier beads in 3D cultures, either in static cultures as nonadherent aggregates or under dynamic conditions in a NASA-designed low shear stress bioreactor. MIP-101 cells proliferated well under all three conditions and increased CEA and NCA production three- to four-fold when grown in 3D cultures compared to MIP-101 cells growing logarithmically in monolayers. These results suggest that 3D growth in vitro simulates tumor function in vivo and that 3D growth by itself may enhance production of molecules that are associated with the metastatic process.

Animals↗
PubMed1997

Three-dimensional culture of bovine chondrocytes in rotating-wall vessels.

The Rotating-Wall Vessel (RWV) was used to culture chondrocytes for 36 d to observe the influence of low-shear and quiescent culture conditions allowing three-dimensional freedom on growth, differentiation, and extracellular matrix formation. Chondrocytes were freshly isolated from bovine cartilage and placed into the RWV with Cytodex-3 microcarriers. Nonadherent petri dishes were initiated with microcarriers as representative of standard culture conditions. In the RWV, large three-dimensional aggregates (5-7 mm) were formed in suspension. In addition, a large sheet of matrix adhered to the oxygenator core and vessel endcaps. Petri dish culture resulted in the formation of sheets of chondrocytes with no matrix production. Immunocytochemical analyses on histologic sections of tissue obtained from the RWV and the petri dish controls were performed with antibodies against fibronectin, collagen II, chondroitin-4-sulfate, chondroitin-6-sulfate, and vimentin. Results demonstrated increased signal in the RWV material while the petri dishes demonstrated a slight decrease in signal. In addition, differentiated chondrocytes were observed in sections of RWV material through 36 d, while few were observed in the sections of petri dish material. These results indicate that the unique conditions provided by the RWV afford access to cellular processes that signify the initiation of differentiation as well as production of normal matrix material.

Animals↗
PubMed1997

Three-dimensional culture of a mixed mullerian tumor of the ovary: expression of in vivo characteristics.

The Rotating-Wall Vessel (RWV) is a novel in vitro cell culture system used to successfully culture a cell line derived from a heterologous mixed mullerian tumor cell of the ovary. Although the original tumor was comprised of both epithelial and mesodermal components, long-term culture in conventional flasks established a cell line from this tumor with homogeneous epitheliallike growth characteristics (1). Cells from Passage 36 were seeded into a Rotating-Wall Vessel containing Cytodex-3 microcarrier beads. Scanning electron micrographs of tumor cells cultured for 32 d in the RWV showed the presence of heterogeneous cell populations organized into three-dimensional tissuelike architecture. Immunocytochemical analysis confirmed the cellular heterogeneity, as demonstrated by expression of both epithelial and mesenchymal antigens. Reverse transcription polymerase chain reaction amplification demonstrated the presence of mRNA for cellular oncogenes HER-2/neu, H-ras, K-ras, and tumor suppressor p53. Thus, there are two advantages to propagation of tissue in the RWV culture system:(a) tissue diversification representing populations present in the original tumor, and (b) the three-dimensional freedom to organize tissues morphologically akin to those observed in vivo. These data indicate that the RWV culture system is suitable for generating large quantities of ovarian tumor cells in vitro that are amenable to immunocytochemical, oncogenic, morphologic characteristics demonstrated in vivo.

Bioreactors↗
PubMed1997

Establishment of a three-dimensional human prostate organoid coculture under microgravity-simulated conditions: evaluation of androgen-induced growth and PSA expression.

A novel in vitro human prostate cancer model was established by using a coculture technique in which isolated human prostate fibroblasts were observed to grow as a mixed culture with isolated human prostate cancer cells (LNCaP) on microcarrier beads under microgravity-simulated conditions. This model appears to be promising and deserves further exploration because: (a) cocultured human prostate fibroblasts and cancer epithelial cells appear to undergo patterns of histogenesis similar to those observed in human prostate tumors and (b) unlike the conventional cell culture on plastic dishes, cocultured human prostate fibroblasts and LNCaP cells in microgravity-simulated conditions responded to the inductive signals of growth and differentiation from dihydrotestosterone in a manner similar to that observed in the in vivo condition. These results offer an opportunity to examine molecular mechanisms of cellular signaling in response to androgen stimulation during normal and aberrant human prostate development. The microgravity-simulated three-dimensional prostate epithelial cell culture with prostate fibroblasts can be further explored as an ideal in vitro model for the study of normal and neoplastic prostate development. This model could also be adopted as a drug screening program for the discovery of novel therapeutic agents in the treatment of human prostate cancer and benign hyperplastic growth.

Bioreactors↗
PubMed1997

Changes in gravity inhibit lymphocyte locomotion through type I collagen.

Immunity relies on the circulation of lymphocytes through many different tissues including blood vessels, lymphatic channels, and lymphoid organs. The ability of lymphocytes to traverse the interstitium in both nonlymphoid and lymphoid tissues can be determined in vitro by assaying their capacity to locomote through Type I collagen. In an attempt to characterize potential causes of microgravity-induced immunosuppression, we investigated the effects of simulated microgravity on human lymphocyte function in vitro using a specialized rotating-wall vessel culture system developed at the Johnson Space Center. This very low shear culture system randomizes gravitational vectors and provides an in vitro approximation of microgravity. In the randomized gravity of the rotating-wall vessel culture system, peripheral blood lymphocytes did not locomote through Type I collagen, whereas static cultures supported normal movement. Although cells remained viable during the entire culture period, peripheral blood lymphocytes transferred to unit gravity (static culture) after 6 h in the rotating-wall vessel culture system were slow to recover and locomote into collagen matrix. After 72 h in the rotating-wall vessel culture system and an additional 72 h in static culture, peripheral blood lymphocytes did not recover their ability to locomote. Loss of locomotory activity in rotating-wall vessel cultures appears to be related to changes in the activation state of the lymphocytes and the expression of adhesion molecules. Culture in the rotating-wall vessel system blunted the ability of peripheral blood lymphocytes to respond to polyclonal activation with phytohemagglutinin. Locomotory response remained intact when peripheral blood lymphocytes were activated by anti-CD3 antibody and interleukin-2 prior to introduction into the rotating-wall vessel culture system. Thus, in addition to the systemic stress factors that may affect immunity, isolated lymphocytes respond to gravitational changes by ceasing locomotion through model interstitium. These in vitro investigations suggest that microgravity induces non-stress-related changes in cell function that may be critical to immunity. Preliminary analysis of locomotion in true microgravity revealed a substantial inhibition of cellular movement in Type I collagen. Thus, the rotating-wall vessel culture system provides a model for analyzing the microgravity-induced inhibition of lymphocyte locomotion and the investigation of the mechanisms related to lymphocyte movement.

Animals↗
PubMed1997

Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor.

Growth patterns of a number of human tumor cell lines that from three-dimensional structures of various architectures when cultured without carrier beads in a NASA rotary cell culture system are described and illustrated. The culture system, which was designed to mimic microgravity, maintained cells in suspension under very low-shear stress throughout culture. Spheroid (particulate) production occurred within a few hours after culture was started, and spheroids increased in size by cell division and fusion of small spheroids, usually stabilizing at a spheroid diameter of about 0.5 mm. Architecture of spheroids varied with cell type. Cellular interactions that occurred in spheroids resulted in conformation and shape changes of cells, and some cell lines produced complex, epithelial-like architectures. Expression of the cell adhesion molecules, CD44 and E cadherin, was upregulated in the three-dimensional constructs. Coculture of fibroblast spheroids with PC3 prostate cancer cells induced tenascin expression by the fibroblasts underlying the adherent prostate epithelial cells. Invasion of the fibroblast spheroids by the malignant epithelium was also demonstrated.

Bioreactors↗
PubMed1997

pCal, a highly unusual Ty1/copia retrotransposon from the pathogenic yeast Candida albicans.

Retrotransposons are mobile genetic elements. They can transpose via the reverse transcription of mRNA into double-stranded DNA (dsDNA) followed by the insertion of this dsDNA into new sites within the host genome. The unintegrated, linear, dsDNA form of retrotransposons is usually very rare. We report here the isolation of a retrotransposon from Candida albicans which is unusual in this respect. This element, which we have named pCal, was first identified as a distinct band when uncut C. albicans DNA was examined on an agarose gel. Sequence analysis of the cloned element revealed that it is a retrotransposon belonging to the Ty1/copia group. It is estimated that pCal produces 50 to 100 free, linear, dsDNA copies of itself per cell. This is a much higher level of expression than even that of the system in which Ty1 is expressed behind the highly active GAL1 promoter on a high-copy-number plasmid (about 10 copies per cell). Another unusual feature of pCal is that its Pol enzymes are likely to be expressed via the pseudoknot-assisted suppression of an upstream, in-phase stop codon, as has been shown for Moloney murine leukemia virus.

Amino Acid Sequence↗
PubMed1994

Simulated microgravity does not alter epithelial cell adhesion to matrix and other molecules.

Microgravity has advantages for the cultivation of tissues with high fidelity; however, tissue formation requires cellular recognition and adhesion. We tested the hypothesis that simulated microgravity does not affect cell adhesion. Human colorectal carcinoma cells were cultured in the NASA Rotating Wall Vessel (RWV) under low shear stress with randomization of the gravity vector that simulates microgravity. After 6-7 days, cells were assayed for binding to various substrates and compared to cells grown in standard tissue culture flasks and static suspension cultures. The RWV cultures bound as well to basement membrane proteins and to CEA, an intercellular adhesion molecule, as control cultures did. Thus, microgravity does not alter epithelial cell adhesion and may be useful for tissue engineering.

Basement Membrane↗
PubMed1994

Audit on myocardial infarction in a district general hospital: is there room for improvement in diagnostic accuracy?

The proportion of patients with an acute myocardial infarction (MI) whose diagnosis was missed on admission was accessed. The admitting consultants were then tested to see if they could diagnose the patients correctly when they were shown the admission histories and electrocardiograms (ECGs). Twenty-six per cent of patients with a final diagnosis of MI were not correctly diagnosed on admission. Fifty-one per cent of all patients did not receive thrombolytic therapy (TT) mainly because the diagnosis was not made on admission. A smaller proportion of these patients were admitted to the coronary care unit (CCU). The consultants only correctly diagnosed an average of 7.3 of the 20 patients who were mis-diagnosed on admission and would have prescribed TT to an average of 4.3 of these 20 patients. A significant proportion of patients had a diagnosis of MI missed on admission and therefore did not receive TT. Therefore, another aid to diagnosis such as serum creatinine phosphokinase (CPK) measurement should be available as an emergency test.

Aged↗
PubMed1993

Three-dimensional growth and differentiation of ovarian tumor cell line in high aspect rotating-wall vessel: morphologic and embryologic considerations.

Cancer of the ovary is the leading cause of death from gynecologic malignancy. To understand better these aggressive tumors, the development of in vitro models to study human ovarian cancer is critical. However, the establishment of long-term cell lines has been difficult, due to the generalized poor survival of patient tumor cells grown in primary culture. Satisfactory culture systems for ovarian tumor cells have therefore been limited. To study cellular interactions involved in the growth and differentiation of these tumors, a cell line was established from a mixed müllerian tumor of the ovary. This cell line, designated LN1, was cultured on microcarrier beads in the high aspect rotating-wall vessel. The tumor cells grown in this vessel readily proliferated without a requirement for cocultivation with a supportive cell layer. Evaluation of cellular morphology by phase contrast light microscopy and scanning electron microscopy revealed the presence of three-dimensional multicellular aggregates consisting of multiple cell-coated beads bridged together, as well as scattered aggregates of LN1 cells proliferating as spheroids free in suspension. In contrast to conventional culture systems, culture in the high aspect rotating-wall vessel facilitated the generation of multiple cell types that could be recovered. These results illustrate the ability of this culture system to provide the biological conditions necessary for pluripotent cell growth.

Biotechnology↗
PubMed1993

Prospects for use of microgravity-based bioreactors to study three-dimensional host-tumor interactions in human neoplasia.

Microgravity offers unique advantages for the cultivation of mammalian tissues because the lack of gravity-induced sedimentation supports three-dimensional growth in batch culture in aqueous medium. Bioreactors that simulate microgravity but operate in unit gravity provide conditions that permit human epithelial cells to grow to densities approaching 10(7) cells/ml on microcarriers in suspension, in masses up to 1 cm in diameter, and under conditions of low shear stress. While useful for many different applications in tissue culture, this culture system is especially useful for the analysis of the microenvironment in which host matrix and cells interact with infiltrating tumor cells. Growth in the microgravity-based bioreactor has supported morphological differentiation of human colon carcinoma cells when cultured with normal human stromal cells. Furthermore, these co-cultures produced factors that stimulated goblet cell production in normal colon cells in an in vivo bioassay. Early experiments also suggest that the microgravity environment will not alter the ability of epithelial cells to recognize and associate with each other and with constituents of basement membrane and extracellular matrix. These findings suggest that cells grown in bioreactors that simulate aspects of microgravity or under actual microgravity conditions will produce tissues and substances in sufficient quantity and at high enough concentration to promote characterization of molecules that control differentiation and neoplastic transformation.

Biotechnology↗
PubMed1993

Reduced shear stress: a major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity.

BHK-21 cells were cultured under various shear stress conditions in an Integrated Rotating-Wall Vessel (IRWV). Shear ranged from 0.5 dyn/cm2 (simulated microgravity) to 0.92 dyn/cm2. Under simulated microgravity conditions, BHK-21 cells complexed into three-dimensional cellular aggregates attaining 6 x 10(6) cells/ml as compared to growth under 0.92 dyn/cm2 conditions. Glucose utilization in simulated microgravity was reduced significantly, and cellular damage at the microcarrier surface was kept to a minimum. Thus, the integrated rotating wall vessel provides a quiescent environment for the culture of mammalian cells.

Animals↗
PubMed1993

Rotating-wall vessel coculture of small intestine as a prelude to tissue modeling: aspects of simulated microgravity.

A new low shear stress, low turbulence microcarrier culture system has been developed at NASA's Johnson Space Center that permits large-scale three-dimensional tissue culture. Tissue culture bioreactors called rotating-wall vessels were used in conjunction with multicellular cocultivation to develop a unique in vitro tissue-modeling system. Normal small intestine epithelium and mesenchymal cells were cocultivated on Cytodex-3 microcarriers and were initiated in two phases. Normal small intestine mesenchymal cells were inoculated into the rotating-wall vessel at 2 x 10(5) cells/ml and allowed to attach and proliferate for 2 to 3 days. Normal small intestine epithelium was then added at an innoculum of 2 x 10(5) cells/ml and cultivation continued for 30 to 40 days. These cocultures attained cell numbers of 4-6 x 10(6) cells/ml and differentiated to form tissue-like masses of 0.4-0.5 cm with minimal necrosis. The masses displayed apical brush borders, differentiated epithelial cells, cellular polarity, extracellular matrix, and basal lamina. Verification of mesenchymal and epithelial cell expression was determined by immunocytochemistry and scanning electron microscopy. These data suggest that the rotating-wall vessel affords a new tissue culture model for investigation of growth, regulatory, and differentiation processes within normal tissues.

Biomarkers↗
PubMed1992

Cell culture for three-dimensional modeling in rotating-wall vessels: an application of simulated microgravity.

High-density, three-dimensional cell cultures are difficult to grow in vitro. The rotating-wall vessel (RWV) described here has cultured BHK-21 cells to a density of 1.1 X 10(7) cells/ml. Cells on microcarriers were observed to grow with enhanced bridging in this batch culture system. The RWV is a horizontally rotated tissue culture vessel with silicon membrane oxygenation. This design results in a low-turbulence, low-shear cell culture environment with abundant oxygenation. The RWV has the potential to culture a wide variety of normal and neoplastic cells.

Animals↗
PubMed1992

Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels.

A new low shear stress microcarrier culture system has been developed at NASA's Johnson Space Center that permits three-dimensional tissue culture. Two established human colon adenocarcinoma cell lines, HT-29, an undifferentiated, and HT-29KM, a stable, moderately differentiated subline of HT-29, were grown in new tissue culture bioreactors called Rotating-Wall Vessels (RWVs). RWVs are used in conjunction with multicellular cocultivation to develop a unique in vitro tissue modeling system. Cells were cultivated on Cytodex-3 microcarrier beads, with and without mixed normal human colonic fibroblasts, which served as the mesenchymal layer. Culture of the tumor lines in the absence of fibroblasts produced spheroidlike growth and minimal differentiation. In contrast, when tumor lines were co-cultivated with normal colonic fibroblasts, initial growth was confined to the fibroblast population until the microcarriers were covered. The tumor cells then commenced proliferation at an accelerated rate, organizing themselves into three-dimensional tissue masses that achieved 1.0- to 1.5-cm diameters. The masses displayed glandular structures, apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development, internalized mucin, and structural organization akin to normal colon crypt development. Differentiated samples were subjected to transmission and scanning electron microscopy and histologic analysis, revealing embryoniclike mesenchymal cells lining the areas around the growth matrices. Necrosis was minimal throughout the tissue masses. These data suggest that the RWV affords a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissue.

Adenocarcinoma↗
PubMed1992

Mania in association with hydrocortisone replacement for Addison's disease.

A 32 year old woman with untreated primary adenocortical insufficiency presented with prominent depressive symptoms. Upon treatment with physiological doses of glucocorticoids she developed a self-limiting acute manic illness. We suggest that up-regulation of hippocampal glucocorticoid receptors as a consequence of prolonged hypocortisolaemia resulted in increased cerebral sensitivity to exogenous corticosteroids, which are known to precipitate manic symptoms in some normal individuals when given in supraphysiological doses.

Acute Disease↗
PubMed1992

Naproxen-associated vasculitis.

Three cases of digital vasculitis attributable to naproxen ingestion are reported. The vasculitic changes were reversed by withdrawal of the drug. These three cases emphasize the need to consider drug sensitivity as a cause of unexplained digital vascular lesions.

Acute Disease↗