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Biomedical subjects

L Germain

Publications and source records attributed to L Germain.

At least 91 records · Page 5Linked to original sources
PubMed1989

Acoustic microscopy applied to nonlinear characterization of biological media.

The use of an acoustic microscope as a method for nonlinear characterization of biological media is evaluated. The method consists of measuring the second harmonic signal generated in a sample located at the focus of the acoustic lens. Two experimental configurations were compared: The first is the simple case where the sample is a liquid mixture filling the space between the lens cavity and the receiver; in the second case, the liquid mixture is confined to a thin layer at the focal plane, in order to simulate a real tissue layer. Aqueous solutions of ethylene glycol and methanol were used as liquid samples. Simple theoretical models are presented for each configuration and the predictions show good qualitative agreement with experimental measurements. These results confirm our belief that the acoustic microscope is potentially a good tool for nonlinear B/A imaging of biological systems.

Acoustic Stimulation↗
PubMed1988

Biliary epithelial and hepatocytic cell lineage relationships in embryonic rat liver as determined by the differential expression of cytokeratins, alpha-fetoprotein, albumin, and cell surface-exposed components.

The differentiation patterns of epithelial cells in fetal rat liver were analyzed in situ and in primary culture by indirect immunofluorescence microscopy using polyvalent and monoclonal antibodies directed against cytokeratins with molecular weights of 55,000 (CK55), 52,000 (CK52), and 39,000 (CK39) and against vimentin, albumin, alpha-fetoprotein, and surface-exposed components of bile ductular cells (BDS7) and hepatocytes (HES6). The anti-CK52 antibody, which reacted with biliary ductal cells in the liver of adult rats (Germain et al., Cancer Res., 45:673, 1985; Germain et al., Cancer Res., 48: 368-378, 1988), stained essentially all of the epithelial cells of embryonic day 12 (E12) rat liver. The anti-BDS7 antibody reacted with a few cell foci, which enlarged and became more numerous at later developmental ages. At E12 essentially all of the cells were positive for albumin and alpha-fetoprotein but did not express HES6. In fact HES6 was not detected until E15 in cells with the morphology of immature hepatocytes. By E18 staining with anti-HES6 reached the level of that observed on adult rat hepatocytes. Liver cells isolated from E12 rats were seeded on fibronectin-treated dishes and their response to various combinations of growth- and differentiation-promoting factors was evaluated with respect to their capacity to express either the hepatocytic or the bile ductular phenotype. In medium supplemented with serum, insulin, dexamethasone, and dimethyl sulfoxide, the E12 cells were capable of differentiating in culture to mimic over a 6-day period the sequential phenotypic changes which occur in vivo during normal hepatoontogeny, namely the loss of CK52 and the appearance of HES6. In contrast, the addition of sodium butyrate to the above supplement mixture resulted in the massive expression of BDS7. To further assess the developmental potential of fetal rat liver cells toward the biliary epithelial cell lineage, the in vitro assay was performed using cells isolated from livers of E18 rats and also from 2-day-old (P2) and P14 rats. While a slight expression of BDS7 was induced in cell culture from E18 liver, essentially no expression was observed in cells from postnatal livers. These findings strongly suggest that the emerging hepatic tissue in rat embryo is composed of bipotential progenitor epithelial cells that are capable of differentiating along either the hepatocytic or biliary epithelial cell lineage. These observations constitute a clear demonstration of the plasticity of liver differentiation and also provide a striking example of environmental influences on liver progenitor cell differentiation.

Albumins↗
PubMed1988

Promotion of growth and differentiation of rat ductular oval cells in primary culture.

Oval cells emerging in rat liver at the early period of 3-methyl-4-dimethylaminoazobenzene treatment constitute a mixed epithelial cell compartment with respect to alpha-fetoprotein (AFP) and cytokeratin differential expression, and include a subpopulation which exhibits a phenotype intermediate between ductular cells and hepatocytes (Germain et al., Cancer Res., 45:673-681, 1985). In the present study we have examined the developmental potential of ductular oval cells in primary culture and after in vivo transfer. The use of monoclonal and polyclonal antibodies directed against cytokeratins of Mr 39,000 (CK39), 52,000 (CK52), and 55,000 (CK55) and vimentin, and also monoclonal antibodies against exposed surface components of oval cells (BDS7) and normal hepatocytes (HES6) allowed us to establish the ductular phenotype of the oval cells. A highly enriched preparation of oval cells was obtained by perfusion/digestion of the liver with collagenase, treatment of the cell suspension with trypsin and DNase, selective removal of hepatocytes by panning using the anti-HES6 antibody, and cell separation by isopyknic centrifugation in a Percoll gradient. The procedure yielded about 8 x 10(7) cells, of which 95% expressed CK39, CK52, and BDS7, 84% gamma-glutamyl transpeptidase, and 5% albumin and AFP. The primary response of cultured oval cells to various combinations of growth and differentiation promoting factors was evaluated with respect to their capacity to initiate DNA synthesis as measured by [3H]thymidine labeling from day 1 to 3, and/or to produce albumin and AFP and express tyrosine aminotransferase. Culture in the presence of either serum or clot blood extract resulted in a low proliferative activity with less than 5% of the nuclei being labeled. Over a 5-day period, fusion of a large portion of the oval cells led to multinucleated cells. When the cells were cultured in the presence of an elaborate combination of supplements [minimum essential medium containing 1 mM pyruvate, 0.2 mM aspartate, 0.2 mM serine, 1 mM tyrosine, 1 mM proline, 1 mM phenylalanine and supplemented with 20% clot blood extract, 10 ng/ml oxidized bile acids, 17 microM bilirubin, 10 ng/ml cholera toxin, 1 microM dexamethasone, 2.5 micrograms/ml insulin, 50 mM beta-mercaptoethanol, and 5 micrograms/ml transferrin (medium MX)], the labeling index increased to around 30% and the level of cell fusion greatly decreased. The addition of dimethyl sulfoxide further enhanced the initiation of DNA synthesis, while sodium butyrate acted as an inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Experimental allergic encephalomyelitis: a neurourological study.

To assess the neurourological disturbances produced by the perivascular inflammatory process in the acute phase of experimental allergic encephalomyelitis, we carried out a controlled study. Forty-six New Zealand male rabbits were inoculated with an encephalitogenic preparation. Twenty-seven of them developed neurological deficits in an average of 9.3 days postinoculation. A sample of 19 subjects was used as a control. The subjects were studied within 2 days after the manifestation of the first sign of encephalomyelitis. An average of eight successive cystosphincterograms were recorded. Their entire central nervous system was removed intact and suspended in 10% neutral buffered Formalin solution for 2 weeks, then cut into segments, embedded in paraffin, and finally stained with hematoxylin-eosin and Luxol Fast Blue. The results demonstrated a significant reduction of mean vesical capacity, maximum urethral pressure, and mean urethral pressure in the group with encephalomyelitis. Furthermore, we found a significant negative correlation between the average number of cellular perivascular infiltrates in the spinal sacral segment and urethral pressures. The results showed a significant negative correlation between supreamesencephalic inflammation and mean vesical capacity.

Animals↗
PubMed1988

Increased blood concentration of des-Arg9-bradykinin in experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is a delayed cell-mediated autoimmune reaction to myelin basic protein. The neuropathology of this acute inflammatory process is characterized by cellular infiltration of the perivascular nervous parenchyma which increases the blood-brain barrier permeability. There is good evidence for an activation of the kallikrein-kinin system during inflammation. In order to assess blood concentrations of kinins in a group of 21 New Zealand rabbits in the acute phase of EAE and a group of 12 normal controls, we performed a sensitive and specific radioimmunoassay. All subjects in the encephalomyelitic group were inoculated with 0.05 ml of an antigenic preparation. They all developed neurological deficits on average 30 days later and within 4 days 10 ml of arterial blood were withdrawn for radioimmunoassay of kinins. On the same days their central nervous system was dissected and prepared for staining and histological study. We demonstrated a significant difference in bradykinin blood concentration between the encephalomyelitic (170.6 pg/ml) and the control (245.8 pg/ml) groups. There is also a significant difference in des-Arg9-bradykinin blood concentration between the encephalomyelitic (168.0 pg/ml) and the control (96.1 pg/ml) groups. These results suggest an activation of circulating carboxypeptidases involved in the transformation of bradykinin into des-Arg9-bradykinin during EAE.

Animals↗
PubMed1986

Blood levels of kinins in experimental allergic encephalomyelitis.

The pathology of acute experimental allergic encephalomyelitis is characterized by perivascular cellular infiltrates in the central nervous system. Our hypothesis is that this immuno-pharmacological process can activate the circulating and tissular kallikrein-kinin systems. We studied 21 New Zealand rabbits inoculated with a homogenate of guinea-pig spinal cord in complete Freund's adjuvant. Each animal underwent radioimmunoassay for arterial blood bradykinin, des-Arg9-bradykinin and des-Phe8-des-Arg9-bradykinin during the acute or subacute clinical phase of encephalomyelitis. Immediately after blood sampling, all animals had their complete central nervous system dissected out and prepared for staining and histological study. The number of hematoxylin-eosin-stained perivascular cellular infiltrates was counted at eight levels of the central nervous system, from the hypothalamic to sacral spinal segments. We demonstrated a positive significant (P less than or equal to 0.02) correlation (r = 0.55) between the number of perivascular cellular infiltrates and blood level of the biologically active kinin bradykinin.

Animals↗
PubMed1986

Cell of origin of distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective expression.

The cell of origin of the nonparenchymal epithelioid cells that emerge in liver cell cultures is unknown. Cultures of rat hepatocytes and several types of nonparenchymal cells obtained by selective tissue dispersion procedures were typed with monoclonal antibodies to rat liver cytokeratin and vimentin, polyvalent antibodies to cow hoof cytokeratins and porcine lens vimentin, and monoclonal antibodies to surface membrane components of ductular oval cells and hepatocytes. Immunoblot analysis revealed that, in cultured rat liver nonparenchymal epithelial cells, the anti-rat hepatocyte cytokeratin antibody recognized a cytokeratin of relative mass (Mr) 55,000 and the anti-cow hoof cytokeratin antibody reacted with a cytokeratin of Mr 52,000, while the anti-vimentin antibodies detected vimentin in both cultured rat fibroblasts and nonparenchymal epithelial cells. Analyses on the specificity of anti-cytokeratin and anti-vimentin antibodies toward the various cellular structures of liver by double immunofluorescence staining of frozen tissue sections revealed unique reactivity patterns. For example, hepatocytes were only stained with anti-Mr 55,000 cytokeratin antibody, while the sinusoidal cells reacted only with the anti-vimentin antibodies. In contrast, epithelial cells of the bile ductular structures and mesothelial cells of the Glisson capsula reacted with all the anti-cytokeratin and anti-vimentin antibodies. It should be stressed, however, that the reaction of the anti-vimentin antibodies on bile ductular cells was weak. The same analysis on tissue sections using the anti-ductular oval cell antibody revealed that it reacted with bile duct structures but not with the Glisson capsula. The anti-hepatocyte antibody reacted only with the parenchymal cells. The differential reactivity of the anti-cytokeratin and anti-vimentin antibodies with the various liver cell compartments was confirmed in primary cultures of hepatocytes, sinusoidal cells, and bile ductular cells, indicating that the present panel of antibodies to intermediate filament constituants allowed a clear-cut distinction between cultured nonparenchymal epithelial cells, hepatocytes, and sinusoidal cells. Indirect immunofluorescence microscopy on nonfixed and paraformaldehyde-fixed cultured hepatocytes and bile ductular cells further confirmed that both anti-hepatocyte and anti-ductular oval cell antibodies recognized surface-exposed components on the respective cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Differential cytokeratin and alpha-fetoprotein expression in morphologically distinct epithelial cells emerging at the early stage of rat hepatocarcinogenesis.

The various liver cell populations emerging during the transitory reappearance of alpha-fetoprotein (AFP) in serum of 3'-methyl-4-dimethylaminoazobenzene-ingesting rats were analyzed in situ and in vitro on isolated cell preparations in terms of their cytokeratin and AFP expression using single and double indirect immunofluorescence microscopy. A polyclonal guinea pig antibody raised against cow hoof prekeratin, which recognized a Mr 52,000 cytokeratin, was found to react with bile ductular epithelial cells and oval cells but not with hepatocytes. A monoclonal antibody against a Mr 55,000 cytokeratin reacted not only with bile ductular and oval cells but also with hepatocytes. In contrast, a polyclonal antibody against porcine eye lens vimentin reacted with sinusoidal cells and stroma cells. To assess further the heterogeneity of the emerging cell populations, liver cells were isolated after 4 weeks of treatment and fractionated according to cell size and ploidy level into 4 fractions (I to IV) by velocity sedimentation at 1 X g. A cell-type analysis using AFP and albumin as functional markers revealed the presence of AFP-producing cells in Fraction IV at a mean velocity equivalent to that of newborn diploid rat hepatocytes, whereas most of the albumin-producing cells were distributed in Fractions I to III at velocities similar to those of adult tetraploid rat hepatocytes. A similar analysis based on the differential expression of Mr 52,000 and Mr 55,000 cytokeratins and vimentin in bile ductular and other diploid epithelial cells, hepatocytes, and mesenchymal cells showed that large cells in Fractions I to III were tetraploid hepatocytes, whereas viable cells present in Fraction IV were diploid epithelial cells and mesenchymal cells in proportion of 62 and 38%, respectively. These cell populations could be resolved further by changing the sedimentation time. A subsequent examination of the Mr 55,000 cytokeratin-containing diploid epithelial cells in Fraction IV using double immunofluorescence microscopy resolved three cell populations with respect to Mr 52,000 cytokeratin and AFP expression, namely, two cell populations expressing either protein marker and a third one containing both markers. These results suggest a ductular origin of oval cells and a possible relation to immature hepatocytes.

Animals↗
PubMed1984

Hormonal regulation of glucose transport in contracting skeletal muscle from normal and diabetic rats.

Using the hindlimb perfusion system, we have studied glycogenolysis and glucose transport in resting and contracting skeletal muscle from normal and diabetic rats. Glucose transport was measured using the glucose analogue 2-deoxyglucose. The muscles were treated for 15 minutes with either saline solution, insulin (1 mU/mL) or epinephrine (10(-7) mol/L) at rest and during electrical stimulation. In the resting muscle, basal glycogen was lower in diabetic rats (25 v 40 mumol/g), was not affected by insulin in either group, and was decreased by epinephrine in normal rats (to 26 mumol/g) but not in diabetic rats. Basal glucose transport was identical in the two groups (7.5 mumol/100 g/minute), was stimulated by insulin to a greater extent in diabetics (fivefold) than in normal rats (threefold), but was unaffected by epinephrine in either group. In contracting muscle, glycogen was decreased by 10 mumol/g in normal and diabetic rats independently of hormonal treatment. Basal glucose transport was not affected by muscle contraction with or without epinephrine, whereas the stimulatory effect of insulin on this process was blunted by such contraction (result, 10 mumoles/100 g/minute). We conclude that in normal and diabetic rats, muscle contraction stimulates glycogen breakdown independently of hormonal treatment; muscle contraction per se does not increase glucose transport; and finally, contracting muscle uses glycogen rather than circulating glucose as energy substrate.

Animals↗
PubMedDate not supplied

Characterization of a 150 kDa accessory receptor for TGF-beta 1 on keratinocytes: direct evidence for a GPI anchor and ligand binding of the released form.

Transforming growth factor-beta (TGF-beta) is a key modulator of epidermal development and homeostasis, and has been shown to potently regulate keratinocyte migration and function during wound repair. There are three cloned TGF-beta receptors termed type I, type II, and type III that are found on most cell types. The types I and II are the signaling receptors, while the type III is believed to facilitate TGF-beta binding to the types I and II receptors. Recently, we reported that in addition to these receptors, human keratinocytes express a 150 kDa TGF-beta 1 binding protein (r150) which forms a heteromeric complex with the TGF-beta signaling receptors. This accessory receptor was described as glycosyl phosphatidylinositol-specific anchored based on its sensitivity to phosphatidylinositol phospholipase C (PIPLC). In the present study, we demonstrate that the GPI-anchor is contained in r150 itself and not on a tightly associated protein and that it binds TGF-beta 1 with an affinity similar to those of the types I and II TGF-beta signaling receptors. Furthermore, the PIPLC released (soluble) form of this protein is capable of binding TGF-beta 1 independently from the signaling receptors. In addition, we provide evidence that r150 is released from the cell surface by an endogenous phospholipase C. Our observation that r150 interacts with the TGF-beta signaling receptors, together with the finding that the soluble r150 binds TGF-beta 1 suggest that r150 in either its membrane anchored or soluble form may potentiate or antagonize TGF-beta signaling. Elucidating the mechanism by which r150 functions as an accessory molecule in TGF-beta signaling may be critical to understanding the molecular mechanisms underlying the regulation of TGF-beta action in keratinocytes.

Activin Receptors, Type I↗