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Biomedical subjects

M Toner

Publications and source records attributed to M Toner.

At least 109 records · Page 6Linked to original sources
PubMed1991

Cryopreservation of isolated hepatocytes: intracellular ice formation under various chemical and physical conditions.

Kinetics of intracellular ice formation (IIF) for isolated rat hepatocytes was studied using a cryomicroscopy system. The effect of the cooling rate on IIF was investigated between 20 and 400 degrees C/min in isotonic solution. At 50 degrees C/min and below, none of the hepatocytes underwent IIF; whereas at 150 degrees C/min and above, IIF was observed throughout the entire hepatocyte population. The temperature at which 50% of hepatocytes showed IIF (50TIIF) was almost constant with an average value of -7.7 degrees C. Different behavior was seen in isothermal subzero holding temperatures in the presence of extracellular ice. 50TIIF from isothermal temperature experiments was approximately -5 degrees C as opposed to -7.7 degrees C for constant cooling rate experiments. These experiments clearly demonstrated both the time and temperature dependence of IIF. On the other hand, in cooling experiments in the absence of extracellular ice, IIF was not observed until approximately -20 degrees C (at which temperature the whole suspension was frozen spontaneously) suggesting the involvement of the external ice in the initiation of IIF. The effect of dimethyl sulfoxide (Me2SO) on IIF was also quantified. 50TIIF decreased from -7.7 degrees C in the absence of Me2SO to -16.8 degrees C in 2.0 M Me2SO for a cooling rate of 400 degrees C/min. However, the cooling rate (between 75 and 400 degrees C/min) did not significantly affect 50TIIF (-8.7 degrees C) in 0.5 M Me2SO. These results suggest that multistep protocols will be required for the cryopreservation of hepatocytes.

Animals↗
PubMed1990

Water transport and estimated transmembrane potential during freezing of mouse oocytes.

The kinetics of water transport and the changes in transmembrane potential during freezing of mouse oocytes in isotonic phosphate buffered saline (PBS) were simulated using thermodynamic models. The permeability to water at 0 degree C, Lpg, and the activation energy, ELp, of metaphase II mouse oocytes from B6D2F1 mice were determined to be 0.044 +/- 0.008 micron/min-atm and 13.3 +/- 2.5 kcal/mol during freezing at 2 degrees C/min. The inactive cell volume was determined to be 0.214 with a correlation coefficient of 0.995, indicating that the oocytes closely follow the ideal Boyle-van't Hoff relation. The mean value of the oocyte diameter was 79.41 +/- 4.62 microns. These results were used to predict the behavior of mouse oocytes under various freezing conditions. The effect of the cooling rate on the cell volume and cytoplasm undercooling was investigated. The changes in transmembrane potential were also investigated during freezing of mouse oocytes. The computer simulations showed that at the beginning of the freezing process (-1 degrees C), the fast growth of ice in the extracellular solution causes a sharp increase of the membrane potential. It is predicted that the change in membrane potential is substantial for almost all cooling rates. Estimations show that values as high as -90 mV may be reached during freezing. The hyperpolarization of the membrane may cause orientation of the dipoles within the membrane. For membrane proteins with 300 debye dipole moment, the theoretical prediction suggests that the percentage of dipoles aligned with the membrane potential increases from 16% at 0 degrees C prior to freezing to 58% at -8 degrees C after seeding of the external ice followed with a cooling at 120 degrees C/min.

Animals↗
PubMed1990

A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration.

Current methods of cryopreservation of hepatocytes in single cell suspensions result in low overall yields of hepatocytes, demonstrating long-term preservation of hepatocellular functions. A novel culture method has recently been developed to culture liver cells in a sandwich configuration of collagen layers in order to stabilize the phenotypic expression of these cells in vitro (J. C. Y. Dunn, M. L. Yarmush, H. G. Koebe, and R. G. Tompkins, FASEB J. 3, 174, 1989). Using this culture system, rat hepatocytes were frozen with 15% (v/v) Me2SO to -70 degrees C, and stored at approximately -100 degrees C. Following rapid thawing, long-term function was assessed by measuring albumin secretion in culture for 7-14 days postfreezing. Comparison was made with cryopreservation of liver cells in single cell suspensions. Cryopreservation of liver cells in suspension resulted in only a 2% yield of cells which could be successfully cultured; albumin secretion rates in these cultured cells over 48 hr were 26-30% of secretion rates for nonfrozen hepatocytes. Freezing cultured liver cells in the sandwich configuration after 3, 7, and 11 days in culture maintained 0, 26, and 19% of the secretion rates of nonfrozen hepatocytes, respectively. Morphology of the cryopreserved cells appeared grossly similar to cells without freezing; however, this morphological result was patchy and represented approximately 30% of the cells in culture. These results represent the first demonstration of any quantitative long-term preservation of hepatocellular function by cryopreservation, suggesting that cultured hepatocytes can survive freezing and maintain function.

Animals↗
PubMed1990

Obstructive colitis. Ulceroinflammatory lesions occurring proximal to colonic obstruction.

The term "obstructive colitis" refers to ulceroinflammatory lesions occurring in the colon proximal to an obstructing or potentially obstructing lesion. We studied nine cases identified over a 9-month period. The patients were predominantly female (only one was male), elderly (mean age, 73), and usually had hypertension, diabetes, or other prior chronic illness. The colonic obstruction was due to adenocarcinoma in seven cases and to diverticular disease in two cases. Areas of colitis occurred either as circumscribed ulcers 0.5-2 cm in diameter (three cases) or as confluent circumferential lesions 8-25 cm in length; they were always separated from the more distal obstructing lesions by a segment of normal colon measuring 2.5-35 cm (mean, 14.6 cm). The involved area of colon was usually only mildly dilated; it exhibited moderate thickening of the wall and had a granular luminal surface accentuated in areas by deeper longitudinal or transverse ulcers. Often there were scattered pseudopolyps, and the margin separating the lesion from normal mucosa was well demarcated and irregular. In one case, two distinct separate areas of colitis were present; in another, the appendix was acutely inflamed. Microscopically, the lesions were composed of granulation tissue with a mixed acute and chronic inflammatory infiltrate that replaced the mucosa and often the submucosa; sometimes it extended into the muscularis propria, with associated peritonitis and perforation. Many of the features of obstructive colitis suggest an ischemic origin, probably mainly due to hypoperfusion following raised intramural pressure, but altered fecal flora may have a synergistic effect. The features of the disease are usually sufficiently characteristic to distinguish it from Crohn's disease and other forms of colitis. Complications include peritonitis, perforation and breakdown of anastomoses made through involved segments of colon that may appear externally normal at surgery.

Aged↗
PubMed1988

Adsorption of cations to phosphatidylinositol 4,5-bisphosphate.

We investigated the binding of physiologically and pharmacologically relevant ions to the phosphoinositides by making 31P NMR, electrophoretic mobility, surface potential, and calcium activity measurements. We studied the binding of protons to phosphatidylinositol 4,5-bisphosphate (PIP2) by measuring the effect of pH on the chemical shifts of the 31P NMR signals from the two monoester phosphate groups of PIP2. We studied the binding of potassium, calcium, magnesium, spermine, and gentamicin ions to the phosphoinositides by measuring the effect of these cations on the electrophoretic mobility of multilamellar vesicles formed from mixtures of phosphatidylcholine (PC) and either phosphatidylinositol, phosphatidylinositol 4-phosphate, or PIP2; the adsorption of these cations depends on the surface potential of the membrane and can be described qualitatively by combining the Gouy-Chapman theory with Langmuir adsorption isotherms. Monovalent anionic phospholipids, such as phosphatidylserine and phosphatidylinositol, produce a negative electrostatic potential at the cytoplasmic surface of plasma membranes of erythrocytes, platelets, and other cells. When the electrostatic potential at the surface of a PC/PIP2 bilayer membrane is -30 mV and the aqueous phase contains 0.1 M KCl at pH 7.0, PIP2 binds about one hydrogen and one potassium ion and has a net charge of about -3. Our mobility, surface potential, and electrode measurements suggest that a negligible fraction of the PIP2 molecules in a cell bind calcium ions, but a significant fraction may bind magnesium and spermine ions.

Adsorption↗
PubMed1986

Osteomyelitis with methicillin-resistant Staphylococcus aureus.

We describe 10 patients with hospital-acquired osteomyelitis due to methicillin-resistant Staphylococcus aureus. The patients were posttraumatic and eight had a foreign body in situ at the site of infection. Vancomycin therapy in association with radical debridement was followed by clinical and radiological cure in eight patients at 2-3.5 years follow-up, in two of whom a foreign body was left in situ. Only minor adverse effects of vancomycin therapy (one rash, two thrombophlebitis) were seen.

Adult↗
PubMed1986

Myocardial hypertrophy, fibrosis and infarction following exposure of the heart to radiation for Hodgkin's disease.

A 35 year old man was treated for stage IIA Hodgkin's disease by radiation to the upper thorax, axillae and neck. Three years later he presented with intractable and ultimately fatal congestive heart failure. Autopsy revealed massive biventricular hypertrophy with widespread subendocardial fibrosis and myocardial infarction, but with little coronary artery disease. Such a complex of features has not previously been described after radiation therapy and cannot be adequately explained by other known causes of heart muscle disease. Ventricular hypertrophy with extensive subendocardial fibrosis may be part of the spectrum of radiation heart disease.

Adult↗
PubMed1978

Metabolic and cardiovascular adjustment to arm training.

Maximal and submaximal metabolic and cardiovascular measures and work capacity were studied in control (n = 7) and experimental (n = 9) subjects (S's) during arm work prior to and following 10 wk of interval arm training. These measures were oxygen uptake (VO2), minute ventilation (VE), heart rate (HR), respiratory exchange ratio (R), cardiac output (Q), stroke volume (SV), and arteriovenous oxygen difference ((a--v)O2 diff). In addition, maximal oxygen uptake (VO2max) was measured in both groups during treadmill running. Experimental S's showed significant increases (P less than 0.01) in peak VO2 (438 ml.min-1), max VE (17.7 l.min-1), max (a--v)O2 diff (20.8 ml.l-1), and work time (9.2 min) during arm ergometry, while maximum values of Q, SV, HR, and R remained unchanged. In addition, submaximal heart rates were significantly lower during arm ergometry after training. VO2max during treadmill running remained essentially unchanged. No changes in metabolic and physiological measures were noted for the controls after the 10-wk training period. The results support the concept of training specificity for VO2max, and indicate that the improvement in peak VO2 in arm ergometry reflects enhanced oxygen utilization due to an expanded (a--v)O2 diff.

Adult↗
PubMed1978

Specificity of run training on VO2 max and heart rate cganges during running and swimming.

To further evaluate the specificity of aerobic training, maximum physiologic measures (VO2, VE, HR, and R) and submaximal exercise heart rate were determined in control (N = 8) and experimental (N = 11) subjects prior to and following 10-week interval run training. Experimental subjects significantly increased (P less than 0.01) treadmill VO2 max by 252 ml O2 or 6.3%. This was siginificantly larger (P less than 0.01) than the 87 ml O2 or 2.6% increase (P less than 0.05) observed during swimming. Max HR decreased significantly in both forms of exercise. In addition, heart rate at two submaximal work levels during running and swimming was significantly lower after training. No changes in metabolic and physiologic measures were demonstrated for the controls after the 10-wk period. These results further support the concept of the specificity of the metabolic adaptation to aerobic training and strongly suggest that local adaptations in skeletal muscle significantly contribute to improvement in VO2 max. However, running may produce a general training adaptation in maximal and submaximal heart rate.

Adolescent↗
PubMedDate not supplied

Intracellular ice formation during the freezing of hepatocytes cultured in a double collagen gel.

During freezing, intracellular ice formation (IIF) has been correlated with loss in viability for a wide variety of biological systems. Hence, determination of IIF characteristics is essential in the development of an efficient methodology for cryopreservation. In this study, IIF characteristics of hepatocytes cultured in a collagen matrix were determined using cryomicroscopy. Four factors influenced the IIF behavior of the hepatocytes in the matrix: cooling rate, final cooling temperature, concentration of Me2SO, and time in culture prior to freezing. The maximum cumulative fraction of cells with IIF increased with increasing cooling rate. For cultured cells frozen in Dulbecco's modified Eagle's medium (DMEM), the cooling rate for which 50% of the cells formed ice (B50) was 70 degrees C/min for cells frozen after 1 day in culture and decreased to 15 degrees C/min for cells frozen after 7 days in culture. When cells were frozen in a 0.5 M Me2SO + DMEM solution, the value of B50 decreased from 70 to 50 degrees C/min for cells in culture for 1 day and from 15 to 10 degrees C/min for cells in culture for 7 days. The value of the average temperature for IIF (TIIF) for cultured cells was only slightly depressed by the addition of Me2SO when compared to the IIF behavior of other cell types. The results of this study indicate that the presence of the collagen matrix alters significantly the IIF characteristics of hepatocytes. Thus freezing studies using hepatocytes in suspension are not useful in predicting the freezing behavior of hepatocytes cultured in a collagen matrix. Furthermore, the weak effect of Me2SO on IIF characteristics implies that lower concentrations of Me2SO (0.5 M) may be just as effective in preserving viability. Finally, the value of B50 measured in this study indicates that cooling rates nearly an order of magnitude faster than those previously investigated could be used for cryopreservation of the hepatocytes in a collagen gel.

Animals↗
PubMedDate not supplied

A stable long-term hepatocyte culture system for studies of physiologic processes: cytokine stimulation of the acute phase response in rat and human hepatocytes.

Prior studies on the in vitro hepatic acute phase response have involved either hepatoma cell lines or conventional short-term cultures of primary hepatocytes. No data are available on the response of primary hepatocytes in stable long-term culture systems. In this study, the acute phase response of rat and human hepatocytes in a new long-term culture system was examined in response to interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF-alpha). The cultured cells were sandwiched between two layers of collagen in a (double-gel) configuration which has been shown to preserve both hepatocyte function and morphology over prolonged periods of time. The stability of this culture configuration enabled us to investigate, for the first time, the temporal aspects of the response in addition to the effects of the mediators on protein secretion. Exposure of rat hepatocytes to IL-6 after culture for 16 days resulted in a 2-fold reduction of albumin secretion and a 15-fold increase in the secretion rates of fibrinogen and alpha 2-macroglobulin. In all instances, the peak response occurred at 48 h after IL-6 exposure, and all protein secretion rates returned to pretreatment values within 5 days posttreatment. Changes in the mRNA levels of these proteins in response to IL-6 corresponded with those changes seen with the secreted products, indicating pretranslational regulation. Administration of IL-1 beta to rat hepatocyte produced a similar decline of albumin secretion and a 5-fold increase of fibrinogen secretion, whereas alpha 2-macroglobulin secretion remained undisturbed.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute-Phase Reaction↗
PubMedDate not supplied

Microfabrication of hepatocyte/fibroblast co-cultures: role of homotypic cell interactions.

Cell-cell interactions are important in embryogenesis, in adult physiology and pathophysiology of many disease processes. Co-cultivation of parenchymal and mesenchymal cells has been widely utilized as a paradigm for the study of cell-cell interactions in vitro. In addition, co-cultures of two cell types provide highly functional tissue constructs for use in therapeutic or investigational applications. The inherent complexity of such co-cultures creates difficulty in characterization of cell-cell interactions and their effects on function. In the present study, we utilize conventional "randomly distributed" co-cultures of primary rat hepatocytes and murine 3T3-J2 fibroblasts to investigate the role of increasing fibroblast density on hepatic function. In addition, we utilize microfabrication techniques to localize both cell populations in patterned configurations on rigid substrates. This technique allowed the isolation of fibroblast number as an independent variable in hepatic function. Notably, homotypic hepatocyte interactions were held constant by utilization of similar hepatocyte patterns in all conditions, and the heterotypic interface (region of contact between cell populations) was also held constant. Co-cultures were probed for synthetic and metabolic markers of liver-specific function. The data suggest that fibroblast number plays a role in modulation of hepatocellular response through homotypic fibroblast interactions. The response to changes in fibroblast number are distinct from those attributed to increased contact between hepatocytes and fibroblasts. This approach will allow further elucidation of the complex interplay between two cell types as they form a functional model tissue in vitro or as they interact in vivo to form a functional organ.

3T3 Cells↗
PubMedDate not supplied

Cellular micropatterns on biocompatible materials.

We present a method to produce micropatterns of cells on tissue culture substrates. A network of deep elastomeric microchannels defining the desired pattern is sealed onto the surface of interest, and a protein template is created by injecting sub-milliliter quantities of protein solution into the microchannels. Protein adsorbs only on the areas that were exposed to the microflow. After the channels are flushed and the elastomer is removed, cells attach only on the protein template. Micropatterns of collagen or fibronectin were used to selectively adhere cells on various biomedical polymers and on heterogeneous or microtextured substrates. Since the bare substrate areas remain apt for seeding other, more adhesive cell types such as fibroblasts, we were able to create micropatterned co-cultures. Our method allows for inexpensive patterning of a rich assortment of biomolecules, cells, and surfaces under physiological conditions.

Animals↗
PubMedDate not supplied

Stabilization of active recombinant retroviruses in an amorphous dry state with trehalose.

The disaccharide trehalose is found to be effective for stabilization of active recombinant retroviruses in an amorphous dry state achieved through ambient-temperature vacuum dehydration of retroviral supernatants. Studies revealed that trehalose is a significantly better desiccation protectant than sucrose, glucose, and dextran: dextran has essentially no protective effect on retroviral survival after drying and rehydration. X-ray diffractometry of the retroviral supernatant dried with trehalose demonstrated its amorphous nature. The ability to dehydrate retroviral stocks at ambient temperatures into a stable glassy state will have a profound effect for researchers and commercial biotechnology companies which supply retroviral vectors for human gene therapy and basic research.

3T3 Cells↗