Biomedical subjects

J G Baust

Publications and source records attributed to J G Baust.

At least 19 recordsLinked to original sources

PubMed2004

Progress toward optimization of cryosurgery.

Cryosurgery for diverse neoplastic and non-neoplastic diseases has expanded in applicability in recent years, especially since intraoperative ultrasound became available as a method of monitoring the process of tissue freezing. However, persistence of disease after presumably adequate cryosurgical treatment has disclosed deficiencies in the technique, perhaps due to faulty application of the freeze-thaw cycles or due to shortcomings in the imaging method. Clearly cryosurgical technique is less than optimal. The optimal dosimetry for tissue freezing, the recent improvements in imaging techniques, and the need for adjunctive therapy are defined in this review, which assesses the progress toward improving the efficacy of cryosurgery.

Cryosurgery↗

PubMed2001

Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins.

The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.

Amino Acid Sequence↗

PubMed2001

Chemo-cryo combination therapy: an adjunctive model for the treatment of prostate cancer.

Despite continuing research and the development of alternate therapeutic options, prostate cancer remains problematic. Chemotherapy has played a minor role as a treatment option due to its lack of efficacy. Whereas cryotherapy has received renewed attention as a treatment modality, it too fails to offer an absolute curative option. Previously, we reported on the utilization of a therapeutic model, which, in combination, increases cell death in a canine renal cell model. Based upon that study, we investigated a combination therapy model as an alternative for the treatment modality for prostate cancer. We hypothesized that the combination of chemotherapy and cryosurgery would result in enhanced cell death, thereby presenting a more effective treatment of prostate cancer. A human prostate cancer cell (PC-3) model was exposed to 5-fluorouracil (5-FU) for 2 and 4 days (prefreeze), freezing (-5 to -100 degrees C), or a combination of the two treatments, and each was assessed for effectiveness over a 2-week posttreatment period. Additionally, investigation into the mechanisms of cell death initiated by the respective therapies was performed through DNA cleavage analysis. For chemotherapy, cultures exposed to 5-FU (2-4 days) yielded a 15-25% loss in cell survival. For cryotherapy, cultures exposed to a temperature window of -5 to -20 degrees C yielded an initial 5-70% loss of viability but cells propagated over time. Cultures exposed to temperatures of -25 to -80 degrees C yielded a 90-99% (+/-4.5%) initial loss in viability with repopulation observed by 12 days postthaw. Cells frozen to -100 degrees C yielded 100% (+/-0.3%) loss of viability and exhibited no signs of propagation. For chemo-cryo therapy, combination treatment at milder temperatures (-5 to -25 degrees C) resulted in an enhanced loss of cell viability compared to that for either treatment alone. Combination treatment at lower temperatures (-40 to -80 degrees C) resulted in a complete loss of cell viability. DNA fragmentation analysis at 48 h posttreatment revealed that dead (detached) cells treated with 5-FU died primarily through apoptosis, whereas dead cells from freezing (-15 degrees C) alone died primarily through freeze-rupture and necrosis. Detached cell analysis from combination treatment at -15 degrees C revealed the presence of apoptotic, necrotic, and freeze-rupture cell death. Scanning electron micrographs of cells exposed to freezing contributing to cell death. These data demonstrate that the combination of 5-FU at sublethal doses and freezing temperatures improves human prostate cancer cell death efficacy. Further, we suggest that chemo-cryo therapy offers a potential alternative treatment for the control and eradication of prostate cancer.

Animals↗

PubMed2001

A molecular basis of cryopreservation failure and its modulation to improve cell survival.

The requirement for more effective cryopreservation (CP) methodologies in support of the emerging fields of cell bioprocessing and cell therapy is now critical. Current CP strategies appropriately focus on minimizing the damaging actions of physicochemical stressors and membrane disruption associated with extra- and intracellular ice formation that occurs during the freeze-thaw process. CP protocols derived from this conceptual paradigm, however, yield suboptimal survival rates. We now provide the first report on the identification of delayed-onset cell death following CP and the significance of modulating molecular biological aspects of the cellular responses (apoptosis) to low temperature as an essential component to improve postthaw outcome. In this study we quantitatively examined the molecular basis of cell death associated with CP failure in a canine renal cell model. In addition, we report on the significant improvement in CP outcome through the modulation of these molecular mechanisms by the utilization of an organ preservation solution. HypoThermosol. Further, the utilization of HypoThermosol as the preservation medium and the modulation of molecular-based cell death have led to a paradigm shift in biologic preservation methodologies. The recognition of molecular mechanisms associated with CP-induced cell death offers the promise of improved CP of more complex and/or fragile biological systems such as stem cells, engineered tissues, and human organs.

Animals↗

PubMed2000

Cell viability improves following inhibition of cryopreservation-induced apoptosis.

A new concept in cryopreservation solution design was developed that focuses on the use of an intracellular-type, hypothermic maintenance medium coupled with additives that inhibit cryopreservation-induced apoptosis. HypoThermosol' (HTS), a hypothermic (4 degrees C) maintenance medium utilized in the long-term storage of cell, tissue, and organ systems, was tested for cryoprotective capability on a renal cell line (Madin-Darby Canine Kidney cells). HTS and HTS derivatives were tested against conventional cell culture medium (Dulbecco's Minimal Essential medium, DME) as the cryoprotectant carrier solution because (1) cells are exposed to an extended state of hypothermia during the freeze-thaw process, and (2) HTS is designed to protect cells exposed to a hypothermic state. Cells separately cryopreserved in either HTS or DME + 5% dimethyl sulfoxide (DMSO) yielded equivalent 24-h postthaw survival (approximately 30%) and 5-d recovery (approximately 90%). Cells cryopreserved in CryoStor CS 5, a HTS derivative containing 5% DMSO, yielded approximately 75% 24-h postthaw survival and recovery to 100% within 3 d. DNA gel electrophoresis was performed to determine the mechanisms of cell death contributing to cryopreservation failure. Cells preserved in DME (DMSO-free) died primarily through necrosis, whereas cells preserved in either DME + 5% DMSO, HTS, or CryoStor CS 5 died through a combination of apoptosis and necrosis. This observation led to the inclusion of an apoptotic inhibitor designed to improve cryopreservation outcome. MDCK cells cryopreserved in CryoStor CS 5 supplemented with an apoptotic inhibitor (Caspase I Inhibitor V), hereafter termed CryoStor CS 5N, resulted in a 24-h postthaw survival and recovery rate exceeding that of any other cryoprotective solution tested (85%). We conclude that: (1) the use of HTS (a dextran-based, intracellular-type solution) without DMSO can yield postthaw viability equivalent to that of standard DMSO-based cryopreservation methods, (2) postthaw viability can be significantly increased through the use of an intracellular-type solution in conjunction with DMSO, (3) the use of HTS allows for cryopreservation to be accomplished with reduced levels of cryoprotectants, and (4) the regulation of apoptosis is essential for the improvement of cryopreservation outcome.

Animals↗

PubMed1999

Endoscopic cryotherapy: experimental results and first clinical use.

BACKGROUND: Cryotherapy or the application of extreme cold has many potential applications in gastroenterology including tissue destruction and hemostasis but until now its development has been prevented by the lack of a delivery device suitable for use through the endoscope. We report here our experience with prototype devices using both liquid nitrogen driven by a cryosurgical system and cryogenic refrigerants (nitrous oxide and carbon dioxide) at or near ambient temperature. METHODS: Cryotherapy was applied to the distal esophageal mucosa of dogs via a flexible catheter passed through an upper endoscope. In other dogs, cryotherapy was used for hemostasis in a bleeding ulcer model. The procedure was also used for palliation in a 58-year-old man with unresectable adenocarcinoma of the stomach with pyloric channel obstruction. RESULTS: Freezing of the superficial mucosa was nearly instantaneous. All dogs survived the procedure and appeared to thrive. Histologic evaluation revealed significant necrosis of the superficial epithelial layer accompanied by a fibrinocellular infiltrate on the surface. These markers of acute injury subside by the fourth to sixth day and are replaced by regenerating epithelium, a process that is virtually complete by day 10. In the hemostasis experiments, bleeding ceased immediately after cryospraying of the lesions but resumed on thawing in most cases. Application of cryotherapy in the patient resulted in reduction of the pyloric mass with no immediately apparent adverse effects. CONCLUSIONS: These data, although preliminary, demonstrate the feasibility of endoscopic cryotherapy using a simple hand-held device. This device has broad potential for use in gastroenterology including ablation of superficial epithelium, debulking of large tumors and hemostasis.

Adenocarcinoma↗

PubMed1999

Cryosurgical effects on growing vessels.

Cryosurgical treatment of unresectable hepatic malignancies has proven beneficial in adults. Concerns regarding its use in children include the effect on growth and the risk of injury to adjacent structures. To test the effect of cryoablation on adjacent vascular structures in a growing animal, liquid nitrogen cryoablation was performed on a juvenile murine model. Sprague Dawley rats underwent double freeze-thaw cryoablation of the abdominal aorta with interposed liver tissue. Serial sacrifices were performed over 120 days. Comparisons were made with sham-operated controls. Overall, animal growth paralleled that of sham controls through all time points. Gross examination of aortic diameter also showed similar growth in vessel size between the groups. Histologic analysis demonstrated injury after cryoablation with smooth muscle cell vacuolization, followed by cell death. Aortic media layer collapse resulted from cellular loss, however, elastin fiber composition was maintained. Aortic patency was preserved despite evidence of cellular injury and aortic wall remodeling. An associated thermal sink effect on the opposing wall was identified. After cryoablation adjacent to the abdominal aorta in adolescent rats, vascular patency is maintained and animal growth and structural function is preserved, despite cellular injury and wall compression. These observations suggest that cryoablation may be a useful treatment adjunct in young subjects.

Animals↗

PubMed1995

A new solution for life without blood. Asanguineous low-flow perfusion of a whole-body perfusate during 3 hours of cardiac arrest and profound hypothermia.

BACKGROUND: The benefits of hypothermia for preventing ischemic injury are well known, but its application in surgery to protect the whole body during procedures requiring circulatory arrest is currently limited to < 1 hour at 15 degrees C using 50% hemodilution. In a significant departure from previous methods, we have developed a technique of asanguineous blood substitution with low-flow perfusion and cardiac arrest at < 10 degrees C in a canine model. Our approach has been to design a hypothermic blood substitute that would protect the brain and visceral organs during several hours of bloodless perfusion. Two different solutions have been designed to fulfill separate requirements in the procedure. METHODS AND RESULTS: With the use of extracorporeal cardiac bypass, 14 adult dogs were exsanguinated during cooling; 11 dogs were blood substituted using in combination the "purge" and "maintenance" solutions (group 1), and 3 dogs were perfused throughout with the "purge" solution alone as controls (group 2). After cardiac arrest, the solutions were continuously circulated for 3 1/2 hours by the extracorporeal pump (flow rate, 40 to 85 mL.kg-1.min-1; mean arterial blood pressure, 25 to 40 mm Hg). The temperature was maintained at < 10 degrees C (nadir, 6.6 +/- 0.1 degrees C) for 3 hours, and the hematocrit was kept at < 1% before controlled rewarming and autotransfusion. In the experimental group, the heart always started spontaneously in the temperature range of 11 degrees C to 27 degrees C, and 8 animals have survived long-term (current range, 14 to 110 weeks) without any detectable neurological deficit. In contrast, two control animals survived after extensive and aggressive cardiac resuscitation efforts; after surgery they exhibited transient motor and sensory deficits for approximately 1 week. Evaluation of biochemical and hematological parameters showed only a transient and inconsequential elevation in enzymes (eg, brain, liver, cardiac) in group 1 compared with the markedly greater elevations in group 2. For example, immediate postoperative values (mean +/- SEM) for lactate dehydrogenase were 114 +/- 10 for group 1 versus 490 +/- 210 for group 2 (P < .03); for SGOT, values were 93 +/- 18 for group 1 versus 734 +/- 540 for group 2 (P < .05). On day 1 for creatine kinase (CK), the group 1 value was 7841 +/- 2307 versus 71,550 +/- 2658 for group 2 (P = .03), and for CK-BB, the group 1 value was 108 +/- 22 versus 617 +/- 154 for group 2 (P = .03). Neurological evaluation using deficit scores (NDS) was based on a modification of the Glasgow Coma Scale score: 0, normal; 1, minimal abnormality; 2, weakness; 3, paralysis; 4, coma; and 5, death. At days 1 and 2 after surgery, NDS (mean +/- SEM) were 0 +/- 0 for the experimental group versus 1.5 +/- 0.5 for the control group. At days 3 and 7 after surgery, NDS were 0 +/- 0 for group 1 versus 1.0 +/- 1.0 for group 2. CONCLUSIONS: The faster neurological recovery of dogs treated with the "intracellular-type" maintenance solution supports the biochemical data showing the benefits of this type of blood substitute for extending the safe limits of hypothermic cardiac arrest procedures to > 3 hours.

Animals↗

PubMed1995

A comparative assessment of cryosurgical devices: application to prostatic disease.

OBJECTIVES: To determine the comparative freezing ability of the Cryotech (CT) and AccuProbe (CMS) cryosurgical systems. METHODS: Four conditions designed to model clinical situations were produced: (1) Single-probe performance in water at 17 degrees C; (2) five-probe performance in water at 17 degrees C; (3) single-probe performance in gel at 22 degrees C; and (4) single-probe performance in bovine liver. Parameters evaluated included temperatures at various time points (rates to and final low temperature), configuration of a freeze zone, and shaft freezing characteristics. In addition, isotherms were measured at predetermined distances from the center of the freeze zone. RESULTS: Both systems provided freezing of various media under operational conditions. In water, the CMS 3-mm probe delivered more rapid freezing temperature rates than the 3-mm CT probe, with a 110 degrees C difference in probe surface temperature. In gel, the CMS probe increased freeze volume fourfold versus a twofold increase for the CT probe. In bovine liver, there was nearly equivalent performance with respect to geometry of the freeze ball. Extrapolation of the CT cooling curve indicated temperature equivalence at 30 minutes. A larger shaft diameter 4.9-mm CT probe produced results similar to the CMS probe in all the tested media. In addition, the freeze configuration of the CMS probe was spherical; the CT configuration was more cylindrical. CMS probe (equivalent diameter) tip temperatures were on average 100 degrees C lower. CONCLUSIONS: Our tests demonstrated differences between the CMS and CT probe. The major differences are in the configuration of the freeze zone and shaft freezing. In equivalent conditions, the CMS 3-mm probe delivered more rapid cooling rates, a more spherical freeze ball, and lower absolute temperatures than the CT 3-mm probe. The larger CT probe produces equivalent freezing temperatures to the CMS probe, albeit with a more spherical shape. However, these in vitro systems may not adequately reflect varied prostate morphology. Further research is under way to determine if these differences affect relative efficacy of cryotherapy of the prostate.

Animals↗

PubMed1992

Biochemical modification of plasma ice nucleating activity in a freeze-tolerant frog.

Recently, we reported the presence of ice nucleating activity, apparently proteinaceous, in the plasma of a freeze-tolerant frog, Rana sylvatica, collected in autumn and spring. Although this protein has not been purified, its ice nucleating behavior can act as an internal reference for tests that attempt to modify its ability to nucleate ice formation. If the addition of a chemical reagent alters the temperature of ice crystallization compared with the control, it can be assumed that protein modification may have occurred. The ice nucleating protein in R. sylvatica showed resistance to proteolysis with four different proteases although there was a significant reduction in the temperatures of nucleation with these treatments (ANOVA P less than 0.001). However, ice nucleating activity was lost when plasma was treated with the addition of urea or N-bromosuccinimide. Modification of protein sulphydryl groups with iodoacetamide did not affect the crystallization temperature (Tc) but treatment with iodoacetic acid resulted in a significant increase in Tc of plasma. An abrupt loss of ice nucleating ability was observed in plasma samples after heating above 87 degrees C. Anomalous potentiation of ice nucleating activity occurred when the plasma was heated to and held at temperatures between 67-75 degrees C.

Acclimatization↗

PubMed1991

Calorimetric analysis of antifreeze glycoproteins of the polar fish, Dissostichus mawsoni.

Solutions of antifreeze glycoproteins 1 through 5 and 8 were analyzed for activity by differential scanning calorimetry. With a scan rate of 1 degree C min-1, antifreeze glycoproteins 1-5 (20 mg/ml) revealed antifreeze activity with a delay in the freeze exotherm during cooling in the presence of ice. Antifreeze glycoprotein 8 (60 mg/ml), however, did not reveal antifreeze activity. When a 0.1 degree C min-1 scan rate was used, glycoproteins 1-5 again yielded a delay in the freeze onset, but the exotherm consisted of multiple events. At the slower scan glycoprotein 8 revealed an initial freeze followed by multiple exothermic events resembling those of glycoproteins 1-5. Thermograms exhibiting antifreeze activity had an initial shoulder in the exotherm direction upon cooling followed by a delay before the exotherm. The shoulders were correlated with c-axis ice growth observed in visual methods. The glycoprotein antifreezes had a linear increase in activity with decreased ice content.

Animals↗

PubMed1991

Vitreous domains in an aqueous ribose solution.

Aqueous solutions containing d-ribose demonstrate the ability to form more than one vitreous domain when exposed to low temperatures. Differential scanning calorimetry revealed two glass transitions (at Tgs of -63 and -43 degrees C) upon cooling and warming at a constant rate of 5 degrees C.min-1. The bulk water of the solution crystallizes at -18 degrees C (Tc). Heat capacity and enthalpy changes, and the derivatives for each thermal event, are calculated. Relaxation studies on the observed Tgs produced anticipated overshoots characteristic of the presence of glassy states.

Calorimetry, Differential Scanning↗

PubMed1991

Physical aging of glassy state: DSC study of vitrified glycerol systems.

After annealing during the glass transition temperature (Tg) range or at sub-Tg temperatures, a "Tg overshoot" has been observed by differential scanning calorimetry in vitrified 55 and 80% (W/W) glycerol solutions. The temperature dependence of the overshoot is most pronounced immediately below the Tg and always increases in amplitude with lengthened annealing periods, whether annealed during Tg range or below. Of particular note is the anomalous shift in devitrification temperature (Td) to lower temperatures following increased time of annealing at sub-Tg temperatures. The Tg obtained on warming also occurs at higher temperatures with increased annealing times. The results allow us to suggest the existence of the time and temperature dependency of the glassy state. Accordingly, it may be desirable to maintain vitrified biological systems at temperatures sufficiently below Tg so that the extent of relaxation in the glass system is avoided or minimized during cryopreservation. The relaxation effects must be taken into account prior to warming a biological system after a long-term cryopreservation. These effects should also be considered in studies of vitrification solutions.

Calorimetry, Differential Scanning↗

PubMed1991

Ultra-rapid freezing by spraying/plunging: pre-cooling in the cold gaseous layer.

A thermophysical model is established to analyse the influence of pre-cooling of a biological specimen in the cold gas layer associated with spray-freezing techniques. The basic principles governing the process of pre-cooling are provided. It is concluded that pre-cooling is one of the major limiting steps in attaining an overall ultra-rapid cooling rate. Pre-cooling has a substantial influence on the nature of the final frozen specimens. In order completely to avoid crystallization before entry into the liquid cryogen and maximize the overall cooling rate of the specimen, precautions should be taken to control the height of the gaseous layer and the size of the specimen. The probability of the specimen being frozen in the cold gaseous layer is reduced by increasing the entry speed. The effectiveness, however, becomes less marked at speeds greater than 10 m s. In order to minimize the risk of misinterpreting the measured cooling rate, it is necessary to specify the pre-cooling conditions. The pre-cooling effect is much more evident in liquid helium than in cryogens such as propane, ethane, Freon 12, 13 and 22.

Freezing↗

PubMed1990

Ice nucleating activity in the blood of the freeze-tolerant frog, Rana sylvatica.

Although the presence of antifreeze and ice nucleating agents in the hemolymph of insects has been well documented, there have been no reports of either of these types of agent in vertebrates. The technique of differential scanning calorimetry was used to examine the blood, serum, and plasma of a freeze-tolerant frog, Rana sylvatica, for the presence of antifreeze protein activity. Results demonstrate the absence of antifreeze protein but the presence of an ice nucleating agent that may serve as a functional component of the overwintering strategy of this species. Ice nucleating activity was detected in samples of cell-free blood, serum, and plasma, suggesting that the agent is a soluble component and possibly plasma protein. To our knowledge, the identification of ice nucleating activity in this freeze-tolerant vertebrate is novel.

Adaptation, Physiological↗

PubMed1989

Effect of cooling rate on the survival of larvae, pupariation, and adult emergence of the gallfly Eurosta solidaginis.

Freeze-tolerant third instar larvae of the gallfly Eurosta solidaginis were cooled at 10, 5, 1, and 0.1 degrees C min-1 to -40 degrees C and then warmed to +5 degrees C at 1 degree C min-1. After cooling and warming the larvae were transferred to 21 degrees C and the survival of larvae, success of pupariation, and adult emergence were monitored at daily intervals in comparison to an uncooled control sample. The percentage emergences of flies from larvae cooled at 10, 5, 1, and 0.1 degree C min-1 and in the control were 7, 13, 37, 77, and 67%, respectively. A number of flies in each group emerged with malformed (unextended) wings and an unretracted ptilinum on the head capsule. The percentage emergences of normal flies at the four cooling rates and from the control were 3, 0, 17, 47, and 57%. At 48 hr after exposure all larvae in each treatment were alive. First mortality was observed between 48 and 72 hr after cooling and increased with time at each cooling rate. Mortality was apportioned into four phases of development: larva, pupariation, and early and late pupae. Mortality commenced earlier at the faster cooling rates; at 10 degrees C min-1, 37% of the sample died as larvae and a further 20% failed to complete pupariation, whereas at 0.1 degree C min-1, only 3% died as larvae and 97% formed a puparium.

Animals↗

PubMed1988

Differential scanning calorimetric analysis of antifreeze protein activity in the common mealworm, Tenebrio molitor.

Antifreeze proteins (AFP) are able to inhibit the growth of ice-crystals at temperatures below the equilibrium freezing point (Tf) of hemolymph. The analysis of AFP activity has commonly involved the use of direct microscopic observation of a sample following inoculation with ice. The resulting activity, defined as the amount of thermal hysteresis observed between Tf and the subsequent rapid growth of ice, has been reported to range up to 7 degrees C. However, most studies report high level of variation, possibly due to ice-crystal size variability and the presence of non-visible ice nuclei. We describe a new method of analysis of AFP activity using differential scanning calorimetry (DSC). DSC analysis reveals much higher activity, up to 10 degrees C, with less variation observed within a sample, and is not subject to the difficulty of accurate assessment of ice-crystal volume.

Animals↗

PubMed1985

Fecundity and longevity of houseflies after space flight.

After 7 days of space flight house flies, Musca domestica, exhibited similar longevity, but a reduction in reproductive output as compared to earthbound controls. This reduction was not observed in later generations. These data suggest that space flight directly alters the rate of ovarian development, but that this effect is not genetically transmitted.

Animals↗