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Biomedical subjects

M Toner

Publications and source records attributed to M Toner.

At least 73 records · Page 4Linked to original sources
PubMed1997

Reversible permeabilization of plasma membranes with an engineered switchable pore.

By using an engineered, self-assembling, proteinaceous, 2-nm pore equipped with a metal-actuated switch, a technique to reversibly permeabilize the plasma membrane to small molecules (approximately 1000 Da) has been developed. We have demonstrated the dose-dependent permeabilization of fibroblasts by pores designed to be blocked and unblocked by the addition and removal of microM concentrations of Zn2+. Further, we have shown that the activity of the switch allows permeabilized cells to maintain viability and ultrastructural integrity following the unconstrained flux of small molecules. This ability to control the transmembrane influx and efflux of molecules and thereby vary the intracellular environment yet maintain cell viability will impact an array of biological and medical problems.

3T3 Cells↗
PubMed1997

Autopsy findings in the tongues of 20 patients with AIDS.

An extensive examination of the tongue was performed at autopsy in 20 consecutive patients who had died with AIDS. Abnormalities in the tongue were detected in 18 (90%) of the cases; the commonest lesions were ulceration (11), candidosis (8) and small foci of hyperkeratosis (10). The most extensive lesions were caused by Aspergillus infection (1), non-Hodgkin's lymphoma juxtaposed with Kaposi's sarcoma (1), herpetic infection (1) and candidosis (5). The disease causing death was identified in the tongue in two cases. There was a surprisingly low prevalence of oral hairy leukoplakia, which may be related to anti-viral or retroviral therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Induction of tolerance to hypothermia by previous heat shock using human fibroblasts in culture.

The technique of organ preservation is limited by the amount of time which organs can be hypothermically stored. A potential method to effectively extend reliable storage times involves the conditioning of cells to better withstand hypothermia by previous exposure to a less severe stress. Using human fibroblasts in culture, we have demonstrated that such an approach may be feasible. Subjecting human diploid IMR-90 fibroblasts to 5 h 42.5 degrees C heat shock was found to improve cell survival more than 10-fold to subsequent 4 degrees C hypothermic exposure. The prior heat shock resulted in the increased synthesis of heat shock proteins (HSPs), the absolute concentrations of which were measured by an assay which utilized sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. Both the degree of cold tolerance conferred upon IMR-90 cells and the levels of HSP27 and HSP27 were dependent upon initial heat shock duration. Induced cold tolerance was found to be reversible; longer recovery times at 37 degrees C following heat shock resulted in a loss of this cold-tolerant state as well as a disappearance of HSPs. The fact that the degree of cold tolerance and HSP concentrations showed similar trends with respect to both heat shock time at 42.5 degrees C and subsequent recovery time at 37 degrees C suggests that these proteins may be intimately involved in the induction of cold tolerance.

Cells, Cultured↗
PubMed1996

Extracorporeal plasma perfusion of cultured hepatocytes: effect of intermittent perfusion on hepatocyte function and morphology.

The most promising approaches to developing a temporary bioartificial liver support system involve incorporating cultured primary hepatocytes into an extracorporeal perfusion device. As a result, it is important to characterize both the phenotypic response of these cells during extracorporeal perfusion and the critical factors involved in maintaining differentiated cell function over extended periods of perfusion. In this study, hepatocytes cultured in a collagen sandwich configuration were connected to a rat via a hollow fiber plasma separator and perfused with plasma on line. Perfusions were either continuous for 48 hr or intermittent for up to 174 hr with 6 hr per day of extracorporeal plasma perfusion alternating with 18 hr of culture medium perfusion. During perfusion cell morphology was continuously monitored by time-lapse video microscopy. After the procedure, hepatocytes were returned to static culture and function was evaluated by measuring the rates of urea synthesis daily for 7 days. During plasma perfusion all hepatocytes accumulated cytoplasmic lipid droplets in a time dependent manner. Urea synthesis was maintained at initial levels for up to 20 hr of continuous plasma perfusion. However, urea synthesis rates were reduced by 31 and 52% after 30 and 48 hr of continuous plasma exposure, respectively. With intermittent perfusions, as well as with control cells perfused with culture medium only, urea synthesis rates did not decrease for at least 78 hr of total perfusion. There was no difference between the urea synthesis rates after 48 hr of cumulative plasma exposure time between cells subjected to continuous and intermittent plasma perfusion. These results suggest that cultured hepatocytes may be exposed to plasma for at least 20 hr with no significant reduction in liver-specific function. Furthermore, an intermittent plasma perfusion schedule can be used to divide the useful plasma perfusion time over several days with no adverse effects on cell function.

Animals↗
PubMed1996

Long-term storage of tissues by cryopreservation: critical issues.

The technique of cryopreservation (maintenance of biological samples in a state of 'suspended animation' at cryogenic temperatures), its potential use in tissue engineering applications and current obstacles to the development of effective cryopreservation methods for tissues are reviewed. A didactic overview of the principles of cryobiology and the methodology of cryopreservation is given, with emphasis on the processes of injury to cells during freezing and thawing, and how these are related to the physicochemical and biophysical changes occurring during cryopreservation. Critical issues relevant to the application of cryopreservation methods to tissues are then addressed, including heat and mass transfer limitations in these bulk systems, intrinsic differences between isolated and cultured cells, and mechanisms of freezing injury unique to tissue systems.

Animals↗
PubMed1996

Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function.

Several extracellular matrix (ECM) configurations involving type I collagen and Matrigel were examined for their ability to support differentiated function and polarity of cultured adult rat hepatocytes. Collagen sandwich- and Matrigel-based cultures yielded superior and comparable albumin secretion for at least 2 weeks. In collagen sandwich, hepatocytes were polygonal, and formed multicellular arrays. Collagen sandwich was also found to promote in vivo-like polarization of F-actin, cell adhesion molecules (E-cadherin), and lateral (Na+, K(+)-ATPase, glucose transporter) and apical (dipeptidyl peptidase, aminopeptidase) membrane polarity markers, but not the expression of the gap junction protein connexin 32 and the epidermal growth factor (EGF) receptor. In contrast, hepatocytes cultured in or on Matrigel were more rounded and formed aggregates. Matrigel-based cultures also elicited detectable levels of connexin and EGF receptor and an altered distribution of F-actin, E-cadherin, and apical and lateral membrane proteins. Composite sandwich configurations containing collagen I and Matrigel restored markers lacking in the collagen sandwich, and showed a variable morphology and membrane polarity. Hepatocyte polarity could thus be manipulated by the overall ECM composition. Furthermore, in composite sandwich cultures, these manipulations can be effected largely independent of changes in hepatocyte morphology and albumin secretion.

Actins↗
PubMed1996

Proliferating cell nuclear antigen staining in syndrome and nonsyndrome odontogenic keratocysts.

OBJECTIVES: The relatively aggressive behavior of the odontogenic keratocyst might be expected to correlate with the degree of proliferation of the cyst epithelium. The proliferation rate of odontogenic keratocyst epithelium was studied with the use of an antibody to the proliferating cell nuclear antigen, a nuclear protein expressed in cycling cells. STUDY DESIGN: We used the monoclonal antibody PC10 to examine 41 odontogenic keratocysts (21 associated with the nevoid basal cell carcinoma syndrome, 20 nonsyndrome), and assessed the number of positive nuclei in 1000 cells. Statistical analysis was carried out with analysis of variance and correlation analysis. RESULTS: Positive staining with proliferating cell nuclear antigen was seen in all keratocysts; in addition, there was a significant difference in proliferating cell nuclear antigen positivity between the syndrome and nonsyndrome keratocysts. CONCLUSIONS: The findings support the view that epithelial proliferation plays a significant role in the behavior of odontogenic keratocysts and suggest that the more aggressive behavior in nevoid basal cell carcinoma syndrome may correlate with a higher rate of epithelial proliferation.

Analysis of Variance↗
PubMed1996

Cryobiology of non-human primate oocytes.

The responses to various stresses involved with cryopreservation protocols were investigated using non-human primate oocytes. Fluorescence microscopy was used to assess the status of the F-actin microfilament system of rhesus monkey oocytes after exposure to different concentrations of glycerol. The F-actin organization around the cortex and in the transzonal processes was modified by exposure to 1.0 ot 2.0 M glycerol at ambient temperature. These effects were reduced significantly when exposure to glycerol was combined with cooling to O degrees C. Cynomolgus monkey oocytes were also subjected to hyperosmotic stress and observed for morphological changes. An irregular shrinkage phenomenon was observed with germinal vesicle or metaphase I but not metaphase II (MII) oocytes. The irregular shrinkage became uniform and spherical when the oocytes were pretreated with ethyleneglycol-bis-(beta-aminoethyl ether)N,N,N'N' tetraacetic acid (EGTA) before exposure to hypertonic solution. Also, in-vitro-matured MII oocytes from cynomolgus monkeys were used to determine crucial biophysical parameters for freezing primate oocytes. The permeability of oocyte plasma membrane to water, Lpg, and its activation energy, ELp, were determined between 0 and -12 degrees C in the absence of cryoprotective additives. The Lpg was found to be 3.8x10(-14) m3N/s and the ELp was 141.5 kJ/mol. the pre-exponential kinetic and exponential thermodynamic parameters of intracellular ice formation were determined to be 8x108 m2/S and 2. 2x10(9) K5 respectively. By combining models of water transport and intracellular ice formation, the cumulative fraction of oocytes with intracellular ice as a function of the cooling rate was also predicted, and it was shown to correlate reasonably with experimental observations.

Actins↗
PubMed1996

Fertilization and development of mouse oocytes cryopreserved using a theoretically optimized protocol.

Rational design of a cryopreservation protocol was demonstrated by using theoretical models of the cryopreservation process to develop an optimal freezing protocol for mouse oocytes. A coupled mechanistic model of the processes of freeze-induced cell dehydration and intracellular ice formation was developed, and cryomicroscopical measurements of intracellular ice formation kinetics were used to determine biophysical parameters required by the model, and to test model predictions of the freezing behaviour of mouse oocytes. A simple phenomenological model for oocyte damage resulting from exposure to concentrated electrolyte and cryoprotectant solutions during cryopreservation was obtained by defining a cost function equal to the duration of the freezing protocol. A two-step freezing protocol was theoretically optimized by using a sequential simplex algorithm to minimize the cost function, subject to the constraint that the predicted probability of intracellular ice formation remain below 5%, yielding a putative optimum at the cooling rate B = 0.59 degrees C/min, and plunge temperature Tp = -67 degrees C. By systematically varying B and Tp about these values in experiments with mouse oocytes cryopreserved in 1.5 M dimethyl sulphoxide, the maximal recovery of intact oocytes with a normal morphology (82%) was obtained for B = 0.5 degrees C/min and Tp = -80 degrees C. Further evaluation of the fertilizability and developmental capacity of oocytes cryopreserved using the optimized protocol yielded cleavage to the 2-cell stage in 65% of oocytes inseminated, and blastocyst formation in 50% of these 2-cell embryos.

Animals↗
PubMed1996

Changes in membrane integrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol.

A systematic approach was taken to assess and optimize a protocol for intracellular vitrification by introducing high concentrations of the cryoprotectant agent (CPA) ethylene glycol (EG) into unfertilized murine oocytes. The effects of EG on membrane integrity, microfilament organization, and developmental potential were evaluated. During exposure to 0.5-2 M EG, oocytes showed maximum shrinkage to 55.5% of the isotonic volume within the first minute and reexpanded to their initial volume within 15 min. Transferral of oocytes to higher concentrations of EG (4-8 M EG) for 1-5 min after 15 min of equilibration at 2 M EG was tolerated well. Microfilament organization appeared normal after this equilibration period. During prolonged exposure (> 5 min) to high concentrations of EG (> 4 M), membrane blebs were noticed on the surface of the cells, and microfilament distribution was disturbed. After treatment with 6 M EG and vitrification with 6 M EG + f2p40.5 M sucrose, there were no significant differences in development to the two-cell and blastocyst stages between CPA-treated, vitrified, and control oocytes. These results indicate that EG is an effective CPA for mouse oocyte vitrification protocols without any observed compromise in morphology and developmental functions.

Actin Cytoskeleton↗
PubMed1996

Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration.

Extracellular matrix (ECM) geometry is an important modulator of cell polarity and function. For example, 3-dimensional matrices often more effectively induce differentiated cell function than traditional 2-dimensional substrates. The effect of ECM topology can be investigated in a controlled fashion using a technique whereby cells cultured on a single surface are overlaid with a second layer of ECM, thereby creating a "sandwich" configuration. Confluent monolayers of epithelial or endothelial cells overlaid in this fashion often reorganize into structures that are reminiscent of their native tissue. In the case of hepatocytes, the overlay causes a dramatic reorganization of the cytoskeleton, adoption of in vivo-like morphology and polarity, and expression of a wide array of liver-specific functions. In this short review, we use the sandwiched hepatocyte culture system to illustrate the effect of ECM geometry on cellular function. Pertinent studies are summarized in the context of defining the parallels, strengths, and limitations of this culture system as an in vitro model to study the physiology and morphogenesis of liver tissue. We also explore some of its potential uses as a model to study liver pharmacology and toxicology, and for the development of liver preservation techniques and liver-assist devices.

Animals↗
PubMed1996

Mandibular invasion diagnosed by SPECT.

To date no investigation has proven accurate and reliable for assessing mandibular invasion by carcinoma prior to surgery. This prospective study compared a new imaging modality, single photon emission computed tomography (SPECT), with clinical examination and high resolution CT scanning to evaluate the sensitivity and specificity of each method in detecting mandibular invasion by squamous cell carcinoma. Twenty-nine patients (21 men, 8 women) with an oral cavity/oropharyngeal carcinoma were studied. All underwent primary surgery and/or radiotherapy where indicated. Resected mandibles were decalcified and examined for tumour infiltration. Imaging studies were read independently by two experienced observers. Clinical examination predicted bone invasion with a sensitivity of 90% and a specificity of only 25%. CT had a sensitivity of 89% and a specificity of 57% while SPECT imaging had a sensitivity of 100% and a specificity of 29%. Using Fisher's exact test and the Kappa statistic for pairwise comparisons between each method, SPECT imaging was complementary to CT in the pre-operative assessment of mandibular invasion.

Aged↗
PubMed1996

Exfoliated malignant cells in glove and instrument washings following head and neck surgery.

Loco-regional recurrence following resection of squamous cell carcinoma of the upper aero-digestive tract is a major problem. Cells exfoliated from malignant tumours have been considered capable of implanting and giving rise to local recurrence or metastatic deposits. A prospective study was performed examining washings from the gloves and instruments used in major head and neck operations to determine if exfoliated cells were indeed present. Following removal of the main tumour specimen all surgeons washed their hands in a solution containing a cell fixative and all instruments were soaked in the solution. Samples were then centrifuged and stained using a modified PAP technique. On occasions nuclear fragments consistent with squamous cell carcinoma were present in both glove and instrument washings. We recommend that all surgeons change gloves and instruments following removal of the main tumour specimen and prior to irrigation of the operative field with a tumoricidal agent.

Carcinoma, Squamous Cell↗
PubMed1996

A rapid microwave-in situ hybridization method for the definitive diagnosis of oral hairy leukoplakia: comparison with immunohistochemistry.

As a diagnostic technique, in situ hybridization requires a long processing time, a degree of expertise and may be difficult to handle routinely in some laboratories. To simplify the in situ hybridization method, we have modified a microwave in situ hybridization technique and applied it to oral hairy leukoplakia (OHL) biopsies obtained from 10 HIV-seropositive patients (definitively diagnosed by a conventional in situ hybridization technique) with appropriate controls. It was necessary to design a novel chamber to avoid drying of sections during the hybridization step. This modified microwave in situ hybridization technique was equispecific and equisensitive to the conventional technique and it shortens the hybridization time from overnight incubation to 14 minutes. To determine the sensitivity of our microwave in situ hybridization method we applied it to previously documented tongue tissue obtained from an AIDS autopsy without clinical evidence of OHL, but found to contain Epstein-Barr virus (EBV) by conventional in situ hybridization. This tissue specimen acted as a low EBV copy number, positive control. The sensitivity of immunohistochemistry using three different commercial detection kits was compared to that of in situ hybridization on the same tissues, following optimisation steps. This included the use of 2 cycles of primary and biotinylated secondary antibodies (antibody double cycling). Clearly positive signals for EBV were detected in all OHL biopsies with the Vectastain Elite ABC and the Histostain-SP kits. The sensitivity of the three commercial detection kits was evaluated at immunohistochemistry level by their application to the low-EBV copy number positive control specimen. Signals for EBV antigen in the low copy number positive control specimen were obtained only with the Vectastain Elite ABC kit. This indicates that, in this application, use of the Vectastain Elite ABC kit gives comparable sensitivity for immunohistochemistry to that found by in situ hybridiation.

AIDS-Related Opportunistic Infections↗
PubMed1995

Effects of hypothermia on the function, membrane integrity, and cytoskeletal structure of hepatocytes.

To increase the storage time of livers for transplantation, a better understanding of hypothermia-induced hepatocyte damage is necessary. To this end, we have characterized the effects of hypothermia on long-term function and cytoskeletal organization of hepatocytes cultured in the collagen sandwich configuration, which maintains the expression of liver-specific functions for several weeks. In these studies, cultured hepatocytes (maintained at 37 degrees C for 7 days) were exposed to 4 degrees C in Leibovitz-15 (L15), University of Wisconsin (UW) solution, or L15 supplemented with 2.5 g% polyethylene glycol (PEG) for various time periods followed by a return to normothermia. When L15 medium was used, the long-term albumin secretion rate of cultured hepatocytes was decreased by 50% after 4 h, and by 95% after 24 h of exposure to 4 degrees C. Amorphous precipitates of F-actin and fragmented short microtubules were also observed after 4 and 12 h of hypothermia, respectively. Similar results were obtained when hepatocytes were stored in UW solution. However, in L15 supplemented with PEG, no significant reduction in long-term albumin secretion rates and intact actin and microtubule morphology was observed even after 24 h of exposure to 4 degrees C. The membrane integrity and long-term albumin secretion of hepatocytes stored in the presence of PEG were decreased to approximately 50% only after 48 h of exposure to 4 degrees C. Thus, PEG may be a useful additive in preservation solutions for hepatocytes in hepatocyte-based liver support systems and for intact tissue as well.

Actins↗
PubMed1995

Dynamics of photoinduced cell plasma membrane injury.

We have developed a video microscopy system designed for real-time measurement of single cell damage during photolysis under well defined physicochemical and photophysical conditions. Melanoma cells cultured in vitro were treated with the photosensitizer (PS), tin chlorin e6 (SnCe6) or immunoconjugate (SnCe6 conjugated to a anti-ICAM monoclonal antibody), and illuminated with a 10 mW He/Ne laser at a 630 nm wavelength. Cell membrane integrity was assessed using the vital dye calcein-AM. In experiments in which the laser power density and PS concentration were varied, it was determined that the time lag before cell rupture was inversely proportional to the estimated singlet oxygen flux to the cell surface. Microscopic examination of the lytic event indicated that photo-induced lysis was caused by a point rupture of the plasma membrane. The on-line nature of this microscopy system offers an opportunity to monitor the dynamics of the cell damage process and to gain insights into the mechanism governing photolytic cell injury processes.

Cell Line↗
PubMed1995

Dynamics of cell membrane permeability changes at supraphysiological temperatures.

A quantitative fluorescent microscopy system was developed to characterize, in real time, the effects of supraphysiological temperatures between 37 degrees and 70 degrees C on the plasma membrane of mouse 3T3 fibroblasts and isolated rat skeletal muscle cells. Membrane permeability was assessed by monitoring the leakage as a function of time of the fluorescent membrane integrity probe calcein. The kinetics of dye leakage increased with increasing temperature in both the 3T3 fibroblasts and the skeletal muscle cells. Analytical solutions derived from a two-compartment transport model showed that, for both cell types, a time-dependent permeability assumption provided a statistically better fit of the model predictions to the data than a constant permeability assumption. This finding suggests that the plasma membrane integrity is continuously being compromised while cells are subjected to supraphysiological temperatures.

3T3 Cells↗