John G. Baust

Ten-year biochemical disease control for patients with prostate cancer treated with cryosurgery as primary therapy.

OBJECTIVES Prostate cryosurgery has been increasingly used for the management of localized prostate cancer since its introduction in a minimally invasive form in the early 1990s. We performed a retrospective study of the largest and most mature patient group treated with cryosurgery reported thus far. METHODS We retrospectively analyzed the data from 370 patients treated consecutively from 1991 to 1996 with a focus on the determination of biochemical disease-free survival for a group of patients with T1 to T3 prostate cancer who had undergone prostate cryosurgery as primary monotherapy. Only patients with no previous radiotherapy, hormonal therapy, or surgery were included. RESULTS The median follow-up was 12.55 years. Using a nadir plus 2 ng/dL definition, Kaplan-Meier analysis demonstrated a biochemical disease-free survival rate at 10 years of 80.56%, 74.16%, and 45.54% for low, moderate, and high-risk groups, respectively. The 10-year negative biopsy rate was 76.96%. CONCLUSIONS The results for this pilot group of patients who underwent percutaneous prostate cryosurgery monotherapy demonstrated biochemical disease-free survival rates that overlap with those of similar groups of patients treated under similar circumstances using other types of nonextirpative monotherapy.

Mechanisms of cryoprotection in freezing tolerant animal systems

Abstract The theoretical mechanisms of freezing protection afforded by the natural occurrence of glycerol in an adult, freezing-tolerant insect have been considered in light of recent findings. While unequivocal identification of the specific site or sites of action of glycerol is yet obscured in a maze of interactions, it is apparent that a multicellular system (organism) that naturally possesses glycerol in high, nonlethal concentrations, that can maintain activity in the presence of tissue ice, that can survive frequent and prolonged freeze-thaw encounters, and finally, that can regulate cryoprotectant levels in the presence of changing environmental conditions should be a major focus of future studies. The carabid beetle, Pterostichus brevicornis has been found to regulate glycerol levels in response to fluctuating ambient temperatures even while frozen. Hemolymph freezing points and whole-body supercooling points correlated well with changes in glycerol. Freezing and supercooling points decreased 0.9 ° C per 4 g/100 ml increase in glycerol. An interpretation of the data accumulated on insect studies and integrated with data from other multicellular systems supports the theory that a single site of freezing concept as applied to considerations of cryoprotection and cryoinjury may not be realistic. Each level of events occurring during the freezing process in glycerolated and nonglycerolated animal systems has been discussed. There appears little room for speculative separation and isolation of the site of action for the events of freezing (and thawing) represent a continuum with changes in each parameter directly dependent upon the entire sequence.

Influence of low temperature acclimation on cold hardiness in the beetle, Pterostichus brevicornis

Abstract Temperature acclimation studies on adult P. brevicornis have demonstrated time-temperature rates of change in haemolymph glycerol, glucose, and trehalose content. Fluctuations in glycerol have been correlated with changes in haemolymph freezing points and whole body supercooling points. During spring acclimation at 0°C, glycerol was lost at a rate of 0·3 g% per hr with the rate of loss increasing to 0·7 g% per hr at 23°C. Supercooling and freezing point changes were directly related and correlated with adjustments in lower lethal temperatures. Specimens acclimated to low temperatures accumulated glycerol at rates dependent upon temperature and demonstrated an anticipatory response (synthesis overshoot). Concomitant changes in freezing and supercooling points were observed. Attempts to substantiate the probable carbohydrate source of glycerol during overwintering were not substantiative.

Supercooling phenomenon and water content independence in the overwintering beetle, Coleomegilla maculata

Abstract Insect supercooling phenomena observed during overwintering have been coupled with the seasonality of water and nucleator content and feeding behaviour. However, the strength of the conclusion has in most cases rested on presumption and model systems since nucleator content is not readily quantifiable. The ladybird, Coleomegilla maculata is an overwintering adult insect that fasts for weeks before hibernation, does not accumulate identifiable cryoprotectants during cold exposures, and is freezing intolerant. This species would be expected to overwinter with a constancy of ice nucleators subject to relative increases due to winter dehydration and decreases in supercooling ranges. However, acclimatization and laboratory acclimation experiments have demonstrated both temporal and temperature independence of water content while supercooling levels varied substantially. Supercooling ranges were narrow during warm exposures with a unimodal peak at −6·3°C (±0·2 S.E.). Progressive temperature reductions yielded phasic shifts to multimodal ranges until a low temperature peak of −18·4°C (±0·5 S.E.) was attained. Induced feedings of controlled nucleator substances resulted in predictable supercooling variations while ‘nucleator free’ diets yielded results consistent with natural populations.

Cold-Hardiness in the Antarctic Tick, Ixodes uriae

Ixodes uriae White (Ixodidae, Acarina) is the predominant tick on the Antarctic peninsula. This species has a circumpolar distribution in both hemispheres and is associated with or known to parasitize 48 species of seabirds. Large colonies of 1,000 or more individuals of all life stages were found beneath rocks on the periphery of Adélie penguin rookeries near Palmer Station, Anvers Island. All life stages (egg, larva, nymph, and adult) were intolerant of freezing. Engorged nymphs and larvae had supercooling points between -18 and -20 C. Eggs had the lowest supercooling points (-28.7 C), while adults had the highest values (from -7 to -13 C). Acclimation to temperatures between -12 and +25 C for 2 wk had no effect on the supercooling point of engorged immobile nymphs. Desiccation of engorged nymphs to 80% of their initial weight resulted in no change in supercooling points or glycerol levels. In January, engorged nymphs enter a state of apolysis and lose mobility. Correlated with this change is an increase in cold tolerance as evidenced by a decrease in supercooling points from -11.5 to -19.5 C. This species exhibits the greatest range of thermal tolerance, from -30 to 40 C, reported for any Antarctic terrestrial arthropod. Except for a short period associated with feeding, I. uriae remains in a permanent state of cold-hardiness throughout the year.

Effects of temperature cycling on cryoprotectant profiles in the goldenrod gall fly, Eurosta solidaginis (Fitch)

Variations in the levels of the two primary cryoprotectants (glycerol and sorbitol) found in Eurosta solidaginis were examined following successive cryophase: thermophase alterations (4 days cold: 3 days warm) through the overwintering period (September-January). Glycerol synthesis was shown to be insensitive to temperature cycling in early winter larvae, while later winter larvae responded with increased synthesis during exposures to cold (0°C). Sorbitol concentrations were directly affected by acclimation temperature in each month with mean levels rising at 0°C and decreasing at +20°C. Rates of synthesis (cryophase) and catabolism (thermophase) of sorbitol generally increased from September to January. Such changes in the capacity for cryoprotectant synthesis and degradation over the course of a winter season have not previously been reported for any species. Data are presented that demonstrate both the ability and adaptative flexibility of this species to cycle cryoprotectant content in a manner conducive to overwintering survival in a thermal-variable environment.

Temperature-dependent activation of differential apoptotic pathways during cryoablation in a human prostate cancer model

Background:Critical to the continual improvement of cryoablation efficacy is deciphering the biochemical responses of cells to low-temperature exposure. The identification of delayed-onset cell death has allowed for the manipulation of cellular responses through the regulation of apoptosis. We hypothesized that in addition to delayed apoptotic events associated with mild subfreezing temperatures (10 to −25 °C), cells exposed to ultra-low temperatures (<−30 °C) may undergo rapid, early-onset apoptosis.Methods:Human prostate cancer model and cells (PC-3) were exposed to temperatures of −60, −30 and −15 °C to simulate a cryoablative procedure. Using a combination of flow-cytometry, fluorescent microscopy and western blot analyses, samples were assessed at various times post thaw to identify the presence, levels and the pathways involved in cell death.Results:Exposure to temperatures <−30 °C yielded a significant apoptotic population within 30 min of thawing, peaking at 90 min (∼40%), and by 6 h, only necrosis was observed. In samples only reaching temperatures >−30 °C, apoptosis was not noted until 6–24 h post thaw, with the levels of apoptosis reaching ∼10% (−15 °C) and ∼25% (−30 °C) at 6 h post thaw. Further, it was found that early-onset apoptosis progressed through a membrane-mediated mechanism, whereas delayed apoptosis progressed through a mitochondrial path.Conclusions:These data demonstrate the impact of apoptotic continuum, whereby the more severe cryogenic stress activated the extrinsic, membrane-regulated pathway, whereas less severe freezing activated the intrinsic, mitochondrial-mediated path. The rapid induction and progression of apoptosis at ultra-low temperatures provides an explanation as to why such results have not previously been identified following freezing. Ultimately, an understanding of the events and signaling pathways involved in triggering apoptosis following freezing may provide a path for selective induction of the rapid-onset and delayed programmed cell death pathways in an effort to improve the overall cryoablation efficacy.

Scorpion Cold Hardiness

The scorpion Centruroides vittatus is periodically exposed to freezing temperatures in its natural habitat. It survives such stress by employing physiological mechanisms characteristic of the freeze-tolerant survival strategy used by some insects. Studies over a 12-mo period revealed that scorpions collected in the field and exposed to freezing temperatures in the laboratory froze at high subzero temperatures. High percentages of these animals recovered after freezing. Neither polyols nor thermal hysteresis proteins, which may serve as antifreezes, were found in sufficiently high levels in the hemolymph to substantially lower body supercooling points. Instead, potent ice-nucleating activity compartmentalized in the gut induced ice formation. Ice-nucleating activity peaks in winter months and appears to be proteinaceous. This is the first reported observation of freeze tolerance and ice-nucleating agents in scorpions.

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.

Vitrification Capability of Metal Mirror Ultra-Rapid Cooling Apparatus: A Theoretical Evaluation

A theoretical heat sink model was developed to assess the limitations and potential of the “bounce-free” metal mirror cooling methodology now used to vitrify biological materials. Calculations indicated that the current copper and liquid nitrogen (LN2)-based slamming apparatus can vitrify a water sample to approximately 11 microns in depth. Liquid helium (LHe) can enhance the vitrification depth another 4 microns. Approximate 18 microns can be vitrified by virtue of liquid helium and silver. The vitrification efficiency of the apparatus is directly related to the initial temperature and the penetration coefficient of the heat sink. The best combinations able to effect maximum heat transfer for the purpose of vitrification appears to be in the following ordering: Tin (LHe) > Lead (LHe) > Silver (LHe) > Aluminium (LHe) > Copper (LN2). A maximum vitrification depth of approximately 23 microns can be achieved when pure tin is used as the heat sink material, and the sink’s initial temperature is maintained at 4 K or below. Gold and sapphire are less efficient than pure copper as a heat sink at any bath temperature. Diamond is less efficient than copper if its initial temperature is lower than 120 K.