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Biomedical subjects

L M Reid

Publications and source records attributed to L M Reid.

At least 19 recordsLinked to original sources
PubMed2004

Analysis of Fusarium graminearum mycotoxins in different biological matrices by LC/MS.

The purpose of this study was to develop an LC/MS assay to accurately detect three mycotoxins produced by Fusarium graminearum in various matrices. Using different LC conditions, deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), and zearalenone (ZEN) were detected in four different matrices (fungal liquid cultures, maize grain, insect larvae and pig serum). The sensitivity of MS detection allowed us to detect concentrations as low as 8 ppb of DON and 12 ppb of ZEN. A very small quantity of matrix was therefore necessary for successful analysis of these toxins and a variety of experimental situations were successfully investigated using this technique. Production of 15-ADON and butenolide was monitored in a liquid culture of F. graminearum under controlled conditions. Using simple extraction procedures, the differential accumulation of DON and 15-ADON was followed in inoculated maize genotypes varying in susceptibility to F. graminearum. Toxicokinetic studies were carried out with maize insect pests reared continually on artificial diets containing ZEN and suggested that larvae may possess the ability to degrade ZEN. Finally, persistence of DON was assessed in pigs fed diet supplemented with DON, results indicated that DON accumulates quickly in pig blood and then levels decline progressively for 12 hours thereafter. The LC/MS study reported here is very useful and flexible for the detection of these mycotoxins in different media and at very low concentrations.

Animals↗
PubMed2001

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on fetal mouse urinary tract epithelium in vitro.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces hydronephrosis by altering the differentiation and proliferation of ureteric epithelial cells in the fetal C57BL/6N mouse urinary tract. This study tests the hypothesis that the late fetal urinary tract epithelial cells respond to TCDD with increased proliferation and that the responses do not require contributions from other maternal or fetal tissues. This was achieved by exposing late gestation fetal urinary tract cells to TCDD in an in vitro model. Isolated ureteric cells from gestation day (GD) 18 fetal ureters were plated in medium supplemented with trace elements, a complex mixture of lipids, a defined mixture of purified hormones and growth factors. Both epithelial and mesenchymal cells remain viable under these conditions. The cultures were exposed to 0.1% dimethylsulfoxide (DMSO), 1x10(-8), 1x10(-9) or 1x10(-10) M TCDD. Exposure to 1x10(-10) M TCDD did not affect the cultures, while 1x10(-8) and 1x10(-9) M TCDD supported epithelial, but not mesenchymal, cell survival and stimulated epithelial cell proliferation and differentiation. The TCDD-exposed cells expressed high levels of keratin and little or no vimentin, confirming that the cells, which survive and differentiate are epithelial. However, after continuous exposure to epidermal growth factor (EGF), the TCDD-induced stimulation of ureteric epithelial growth could not be detected. In conclusion, this study demonstrates that late gestational ureteric cells respond to TCDD in vitro with the stimulation of epithelial cell growth and differentiation.

Abnormalities, Drug-Induced↗
PubMed2001

Isolation, maintenance, and characterization of human pancreatic islet tumor cells expressing vasoactive intestinal peptide.

Tissue from a vasoactive intestinal peptide (VIP)-secreting human tumor has been used to establish and characterize human neuroendocrine primary cell cultures from which permanent, clone-derived cell lines have been established. Viable cells were obtained by enzymatic and mechanical dissociation of freshly resected pancreatic islet tumor and hepatic metastatic tumor tissues. Aliquots of tumor cells were established ex vivo under culture conditions including porous substrata coated with type IV collagen and laminin and a low serum, hormonally defined culture medium. The small (<10 microm) rounded, grape-like cells had a very slow growth rate of doubling times estimated at several weeks or more. After several passages, morphologically uniform cells were derived that strongly expressed neuroendocrine markers of synaptophysin and synaptobrevin. Although chromogranin A and VIP had somewhat weaker expression, both demonstrated phorbol ester-stimulated secretion. The morphologic and secretory properties were maintained by the cells for nearly 2 years in culture. The establishment of this novel VIP-secreting human neuroendocrine cell line (HuNET) makes available a culture model with which to study a transformed version of this pancreatic islet cell type and offers approaches by which to establish islet tumor cell lines.

Adult↗
PubMed2001

Soft, porous poly(D,L-lactide-co-glycotide) microcarriers designed for ex vivo studies and for transplantation of adherent cell types including progenitors.

Our laboratory is undertaking tissue engineering of liver using enriched liver progenitor cells. We report here our ongoing study to design biodegradable and biocompatible three-dimensional substratum supports of both natural and synthetic polymeric materials suitable to the adhesion, growth, and differentiation of adult and progenitor liver cells for their transplantation, and for the development of a bioartificial liver assist device. Porous biocompatible and biodegradable microcarriers of diameter 20-40 microm and 100-300 microm were prepared from (alpha-hydroxy) acid family of polymers. Human hepatoma cell line HepG2 and adult rodent liver cells were found to attach to collagen-coated surface of poly(D,L-lactide-co-glycotide) microcarriers. HepG2 cells attached to the degradable microcarriers remained viable and underwent growth expansion, forming three-dimensional cell-degradable microcarrier colonies in culture. These cell-degradable microcarrier colonies may undergo further growth expansion, thus providing a viable approach for three-dimensional organogenesis of tissue.

Animals↗
PubMed2001

Effect of flow configuration and membrane characteristics on membrane fouling in a novel multicoaxial hollow-fiber bioartificial liver.

A novel "multicoaxial hollow fiber bioreactor" has been developed consisting of four concentric tubes, the two innermost tubes are called hollow fibers. Bioartificial livers are created by culturing liver progenitors in the space between the two innermost hollow fibers and with culture media contained in the two compartments (intracapillary and extracapillary) sandwiching the cell compartment. The outermost compartment is used for gas exchange. A hydrodynamic model has recently been established to predict the optimum hydraulic permeability and bioreactor operational parameters to create the physicochemical environment found in the liver acinus. However, perfusion with serum-free hormonally-defined media and inoculation of cells introduces membrane fouling into the equation, and this parameter must be incorporated into the model. Using commercially available semipermeable hollow fibers (1 mm [0.65 microm pores] and 3 mm [0.1 microm pores] outer diameters [o.d]), the primary cause of resistance is the middle hollow fiber. Preliminary studies using bioreactors inoculated with isolated rat hepatocytes and perfused with serum-containing culture media demonstrated that the middle hollow fiber is the primary site of fouling, and this fouling ultimately causes cell mortality by blocking the transfer of nutrients. Experiments were performed to determine the best commercially available middle hollow fiber for construction of bioreactors and two 3-mm outer-diameter middle hollow fibers were compared: polypropylene and polysulfone, with 0.2 microm and 0.1 microm pore sizes, respectively. Dead-ended and cross flow configurations were compared for their effectiveness at reducing membrane fouling in the middle hollow fiber by determining the change in resistance with time. The results demonstrate that the 0.2-microm pore size polypropylene hollow fiber is the best choice for construction of the multicoaxial hollow-fiber bioreactor, and that cross flow results in two orders of magnitude lower resistance than dead-ended flow after 36 h.

Animals↗
PubMed2001

Hepatic progenitors and strategies for liver cell therapies.

Liver cell therapies, including liver cell transplantation and bioartificial livers, are being developed as alternatives to whole liver transplantation for some patients with severe liver dysfunction. Hepatic progenitors are proposed as ideal cells for use in these liver cell therapies given their ability to expand extensively, differentiate into all mature liver cells, have minimal immunogenicity, be cryopreservable, and reconstitute liver tissue when transplanted. We summarize our ongoing efforts to develop clinical programs of hepatic progenitor cell therapies with a focus on hepatic stem cell biology and strategies that have emerged in analyzing that biology.

Animals↗
PubMed2000

Clonogenic hepatoblasts, common precursors for hepatocytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen.

An in vitro colony-forming assay and flow cytometry were used to identify rat hepatoblasts as being classical MHC class I, RT1A(l-), OX18(low) intercellular adhesion molecule 1 (ICAM-1)(+). Inducible differentiation toward biliary lineage was observed in most colonies derived from single RT1A(l-) progenitors, proving their bipotentiality. These findings demonstrate the antigenic profile of clonogenic hepatoblasts and proof of their bipotency. Furthermore, whereas colony formation of adult hepatocytes required epidermal growth factor, clonal growth of hepatoblasts was potentiated without epidermal growth factor. The adult hepatic colonies consisted of RT1A(l+)OX18(+)ICAM-1(++) cells. These results indicate that hepatoblasts possess unique characteristics as compared with adult hepatocytes harboring significant proliferative activity. The phenotypic identity of hepatoblasts and the clonal culture system have relevance for identifying hepatic stem cells from adults, for studying liver development, and for cell therapy based on hepatic progenitors.

Animals↗
PubMed2000

Pre-harvest accumulation of deoxynivalenol in sweet corn ears inoculated with Fusarium graminearum.

Three types of commercial sweet corn hybrids [surgary (su1), shrunken or 'supersweet' (sh2) and surgary enhancer (se1)] were silk channel inoculated in 1996 and 1997 with a macroconidial suspension of Fusarium graminearum to determine how early the mycotoxin deoxynivalenol accumulates in kernels. Disease symptoms rapidly developed on all hybrids and were apparent 4 days after inoculation. Symptoms stabilized by 28 days after inoculation. Toxin levels were greater than 1 microgram/g in kernels as early as 2 weeks after silk emergence and rapidly increased to extremely high levels. Susceptibility in all hybrids decreased as the silk dried out. Deoxynivalenol concentrations were correlated to disease severity. There was some indication that the sh2 genotype was more susceptible than the su1 or se1 genotypes. These results suggest that improvement needs to be made in sweet corn with respect to resistance to gibberella ear rot.

Enzyme-Linked Immunosorbent Assay↗
PubMed1999

Expansion conditions for early hepatic progenitor cells from embryonal and neonatal rat livers.

Long-term primary cultures were established from fetal or neonatal livers by using cell suspensions depleted of red blood cells and by culturing the cells in hormonally defined medium containing dimethyl sulfoxide. Two distinct populations of hepatic progenitor cells were evident in the cultures, based on morphology, proliferative ability, and liver-specific gene expression. Most colonies consisted of immature hepatic progenitors: small, blastlike cells, weakly expressing alpha-fetoprotein, albumin, and gamma-glutamyltranspeptidase, and showing evidence of proliferation as measured by bromodeoxyuridine incorporation. At the perimeter of these colonies of immature cells and forming some colonies by themselves were more mature hepatic progenitor cells: larger cells, with increased cytoplasmic to nuclear ratios, little proliferation, and strongly expressing albumin, alpha-fetoprotein, and gamma-glutamyltranspeptidase. The latter two proteins were localized to the bile canalicular membranes of these cells. Glycogen deposits were present in the mature cells from day 14 embryos after eight days of culture. Thus, DMSO treatment of hepatic parenchymal progenitors provides a novel system for studies of liver development.

Albumins↗
PubMed1999

Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events.

In understanding mechanisms of liver repopulation with transplanted hepatocytes, we studied the consequences of hepatic polyploidization in the two-thirds partial hepatectomy model of liver regeneration. Liver repopulation studies using genetically marked rodent hepatocytes showed that the number of previously transplanted hepatocytes did not increase in the liver with subsequential partial hepatectomy. In contrast, recipients undergoing partial hepatectomy before cells were transplanted showed proliferation in transplanted hepatocytes, with kinetics of DNA synthesis differing in transplanted and host hepatocytes. Also, partial hepatectomy caused multiple changes in the rat liver, including accumulation of polyploid hepatocytes along with prolonged depletion of diploid hepatocytes, as well as increased senescence-associated beta-galactosidase and p21 expression. Remnant hepatocytes in the partially hepatectomized liver showed increased autofluorescence and cytoplasmic complexity on flow cytometry, which are associated with lipofuscin accumulation during cell aging, and underwent apoptosis more frequently. Moreover, hepatocytes from the partially hepatectomized liver showed attenuated proliferative capacity in cell culture. These findings were compatible with decreased proliferative potential of hepatocytes experiencing partial hepatectomy compared with hepatocytes from the unperturbed liver. Attenuation of proliferative capacity and other changes in hepatocytes experiencing partial hepatectomy offer novel perspectives concerning liver regeneration in the context of cell ploidy.

Animals↗
PubMed1998

Maturation-dependent gene expression in a conditionally transformed liver progenitor cell line.

We have isolated a conditionally transformed liver progenitor cell line with phenotypic similarities to both hepatoblasts (bipotent embryonic liver cells that give rise to hepatocytes and intrahepatic biliary epithelial cells) and liver epithelial cells (primitive hepatic cells isolated from adult livers capable of generating both hepatocytic and biliary lineages). Cell line L2039 was derived from E14 fetal mouse liver after transformation with temperature-sensitive SV-40 large T antigen. At 33 degrees C, these cells have an epithelial morphology with a high nucleocytoplasmic ratio and express both hepatocytic and biliary genes, including albumin, alpha-fetoprotein, glutamine synthetase, insulinlike growth factor II receptor, fibronectin and laminin, and cytokeratins 8 and 19, a set of markers characteristic for hepatoblasts. The presence of cytokeratin 14, vimentin, and several oval-cell antigens link cell line L2039 to nonparenchymal liver epithelial cell populations thought to contain progenitor cells. Serum-free, hormonally defined media conditions and extracellular matrix requirements were determined for growth and differentiation of this cell line. During culture on type IV collagen at 39 degrees C, L2039 cells cease dividing and demonstrate hepatocytic differentiation with the assumption of a hepatocytelike morphology and glucocorticoid-dependent regulation of liver-specific genes, including albumin, alpha-fetoprotein, phosphoenolpyruvate carboxykinase, and liver-enriched transcription factors. The number of albumin-positive cells increases during culture at 39 degrees C, indicating that L2039 cells convert from a prehepatocytic to a hepatocytic phenotype. Under conditions specific for hepatocytic differentiation, C/EBPs were expressed and differentially regulated, with C/EBPbeta and C/EBPdelta upregulated early and C/EBPalpha only slightly expressed after 7 d, indicating that C/EBPalpha may not be a crucial factor in commitment to the hepatocytic phenotype.

Animals↗
PubMed1998

Analysis of Fusarium toxins in maize and wheat using thin layer chromatography.

Thin layer chromatography (TLC) methods for identifying and quantifying deoxynivalenol (DON), fumonisin B1 (FB1) and zearalenone in grain samples were compared to immunoassay (ELISA) and high performance liquid chromatography (HPLC) methods to determine the reliability of the less expensive TLC. There was a very good agreement between levels of DON measured by TLC and competitive-direct ELISA, and between levels of fumonisin B1 measured by TLC and HPLC, over a wide range of concentrations. Correlation coefficients (Pearson's) were 0.978, 0.914 and 0.953 for DON in maize, DON in wheat and FB1 in maize respectively. A lower correlation coefficient (r = 0.672) was obtained when zearalenone was quantified by TLC and HPLC. Possible reasons for this are discussed. A cost comparison of the various methods revealed that TLC was the least expensive for sample analysis. It is recommended that researchers choose which analytical method to use based upon individual considerations of cost and precision.

Carboxylic Acids↗
PubMed1998

Phenotypic characterization of rat hepatoma cell lines and lineage-specific regulation of gene expression by differentiation agents.

Hepatoma cell lines can be characterized by their expression of hepatocyte- and biliary-specific genes and by their response to differentiating agents in a lineage-dependent manner. These characteristics can be used to map the maturational lineage position of the cell lines. Tissue-specific gene expression and regulation by heparin, dimethylsulfoxide (DMSO), and sodium butyrate (SB) were examined in three rat hepatoma cell lines and two rat liver epithelial cell lines. Based on antigenic profiles and gene expression in serum-supplemented medium, the hepatoma cell lines could be organized in distinct categories of hepatic differentiation. All three hepatomas expressed the following five genes: gamma-glutamyl transpeptidase (GGT), glutathione-S-transferase pi (Yp), glutamine synthetase, and alpha 5 and beta 1 integrin. Cell line H4AzC2 also expressed alpha-fetoprotein (AFP), albumin. IGF II receptor, and the biliary/oval cell antigens OC.2 and OC.3, a phenotype characteristic of fetal hepatocytes. FTO-2B cells lacked AFP, OC.2, and OC.3 but expressed albumin and IGF II receptor in addition to the five commonly expressed genes, consistent with a more hepatocyte-like phenotype. Cell line H5D-7 expressed neither albumin nor the IGF II receptor, but did express OC.2, OC.3, and alpha 3 integrin in addition to the five commonly expressed genes, characteristic of biliary epithelial cells. Regulation of gene expression by heparin, DMSO, and SB was examined in cells cultured in hormonally defined medium. The patterns of regulation of AFP, albumin, GGT, and Yp were dependent upon the state of differentiation of the cell. FTO-2B cells regulated genes in a manner similar to that of E16 fetal hepatocytes, H4AzC2 regulated genes characteristic of both hepatocytic and biliary lineages, and H5D.7 regulated only biliary genes. Suppression of GGT by DMSO was uniformly observed. The three cell lines expressed equal amounts of HNF-4, but FTO-2B cells expressed more HNF-3 beta and less HNF-3 alpha, while the reverse was true of H4AzC2 and H5D.7 cells.

Animals↗
PubMed1997

Putative liver progenitor cells: conditions for long-term survival in culture.

Oval cells proliferate extensively in the livers of animals exposed to oncogenic insults, are bipotent and are believed to be related to the so far unidentified liver stem cell. In normal liver, cells antigenically related to oval cells and expressing liver and epithelial markers are considered to be liver progenitor cells. We isolated, by fluorescence-activated cell sorting or magnetic bead sorting, cells expressing the oval cell antigens OC.2 or OC.3 from the liver of normal newborn or day 12 embryonal age rats. Magnetic bead sorting of positive cells was as efficient as fluorescence-activated cell sorting. A two-chamber culture system was devised in which cells were plated onto transwell filters coated with type IV collagen and cultured in a serum-free Ham's F12 medium supplemented with free fatty acids and bovine serum albumin. Under these conditions, cells remained viable for up to 6 weeks and their antigenic phenotype was unchanged throughout. Approximately 30% of sorted cells expressed epithelial and/or liver-specific markers. Growth factors mitogenic for epithelial cells and hepatocytes did not elicit cell proliferation. These results provide an important background for further studies designed to determine the biological significance of OC.2+ and OC.3+ cells in normal liver, to test the liver stem cell hypothesis and to develop protocols for the expansion in vitro of normal liver progenitors.

Animals↗
PubMed1997

Early fetal development of lung vasculature.

Despite its relevance to a variety of congenital anomalies, the earliest stages of lung vascular development are poorly understood. In other organs, two processes have been identified: vasculogenesis, the development of blood lakes in mesenchyme, and angiogenesis, the branching of new vessels from preexisting ones. In the present study we established the events in the development of the lung's vasculature in Swiss-Weber mouse fetuses between 9 and 20 days gestation, using light microscopy (LM), transmission electron microscopy (TEM), barium-gelatin angiograms, and scanning electron microscopy (SEM) of Mercox (methyl methacrylate) vascular casts. Three features were identified: (1) central sprouting or angiogenesis for up to approximately seven generations (counting the artery to each lung as first generation); (2) the formation of peripheral lakes by vasculogenesis; and (3) the development of communications between the central and peripheral systems. At 9 days gestation, intercellular spaces were apparent in the lung mesenchyme; these were formed by discharge of vesicles from mesenchymal cells, which then regrouped to provide "endothelial" cells lining the spaces. The isolated lakes coalesced to form sinusoidal spaces of irregular profile. At 12 days gestation, the earliest time at which were able to make a cast, sprouting of arteries and veins from the central pulmonary vascular trunks was apparent. Between 13 and 14 days gestation the earliest connection between the peripheral and central spaces was identified. Such connections became more numerous and dense by term. Similar images seen on examination of human fetal lung sections by LM indicated that similar processes occur in the vascular development of the human lung.

Animals↗
PubMed1997

[Stem cell-fed maturational lineages and epithelial organogenesis].

Stem cell-fed maturational lineages have long known to exist in rapidly proliferating tissues such as bone marrow, gut and epidermis. Recent studies support the hypothesis that stem cell-fed maturational lineages occur also in quiescent tissues. In this review is presented evidence for this hypothesis using liver as one of the model systems representative of quiescent tissues. In addition, studies are summarized indicating that control of growth and tissue-specific gene expression is dependent on maturational lineage mechanisms operating dynamically and sometimes synergistically in combination with gradients of regulatory signals that include hormones, growth factors and extracellular matrix components. Tissue engineering for optimal maintenance of cells and tissues ex vivo and for the development of bioartificial organs will depend on use of cells at specific maturational lineage stages, seeding them onto substrata of specific mixtures of extracellular matrix components, and culturing them in hormonally and nutritionally defined media.

Cell Differentiation↗
PubMed1996

Isolated peripheral pulmonary artery stenoses in the adult.

BACKGROUND: Isolated peripheral pulmonary artery stenosis (PPS) in the adult is rare and frequently unsuspected. We review in this article our experience with 12 adult patients with isolated PPS, half of whom had been previously diagnosed with chronic pulmonary thromboembolic disease. METHODS AND RESULTS: The presentation, evolution, and management of 12 adults with isolated PPS, 17 to 51 years of age (mean, 36.2 +/- 9.7 years), were evaluated. Presenting symptoms were dyspnea and fatigue. Three patients had New York Heart Association (NYHA) functional class III or greater. Lung perfusion scans revealed multiple segmental abnormalities in flow distribution in all patients. Oxygen desaturation at rest was present in 4 patients. At catheterization, right ventricular (RV) pressure was suprasystemic in 2 patients, systemic in 1, and more than half-systemic in 7. All had multiple bilateral non-uniform stenoses in segmental and subsegmental arteries. Balloon pulmonary angioplasty (BPA) to decrease RV hypertension and improve pulmonary flow distribution was performed in 11 patients. After BPA, vessel diameter increased > 50% in 10 patients, distal pulmonary artery pressure increased > or = 30% in 6, and RV pressure decreased > 30% in 5. One patient died shortly after BPA as a result of pulmonary hemorrhage. Immediate procedural success was achieved in 9 of 11 patients. At a mean follow-up period of 52 +/- 32 months, 7 patients had sustained symptomatic improvement (NYHA class I-II). CONCLUSIONS: We describe a severe syndrome of isolated PPS in the adult that mimics chronic pulmonary thromboembolic disease. Pulmonary hemodynamics and angiography are required for definitive diagnosis. BPA may offer these patients successful short-term reduction in RV hypertension and alleviation of symptomatology.

Adolescent↗