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Dideoxynucleosides currently in use for anti-HIV therapy have been found to be inefficient in passing through the blood-brain barrier to enter and maintain therapeutic drug levels in brain, a very significant reservoir of HIV. The low bioavailability of these drugs combined with the bone marrow toxicity of AZT (3'-azido, 3'-deoxythymidine, Zidovudine), resulting in anemia and leukopenia, pancreatitis with ddI (2',3'-dideoxyinosine, Didanosine) and painful peripheral neuropathy in case of ddC (2',3-dideoxycytosine, Zalcitabine) are the limiting factors in their use. In addition, the emergence of strains of HIV resistant to AZT, the most commonly used drug, further restricts its use. Thus the control of AIDS and its complications, needs special therapeutic approaches to combat the disease. In order to overcome these limitations, AZT and ddI have been synthesized as ester-linked ceramide- and phosphatidylcholine-linked prodrugs possessing therapeutic attributes lacking in the parent compounds. There is greater uptake and longer retention of these prodrugs in NIH/3T3 cells in vitro. Pretreatment with our prodrugs blocked infection of these cells by Moloney murine leukemia virus (M-MuLV) for an extended period, which the parent drugs failed to do. When human CD4+ HeLa cells were continuously exposed to the AZT prodrug, subsequent infection of these cells by HIV was blocked. Similar results were obtained with NIH/3T3 cells exposed to M-MuLV. AE(6)C, a prodrug of AZT linked to ceramide via a cleavable ester bond and a six carbon linker, was less toxic to both mouse and human bone marrow progenitor cells than free AZT. Most significantly, the prodrugs concentration was greater and the retention longer, in well known sanctuaries for HIV, such as the brain, testes and thymus.

Whole body hyperthermia therapy (WBHT) is the elevation of the core body temperature to 42 degrees C. In vitro studies have confirmed that 42 degrees C is cytocidal for virally infected lymphocytes, and even more effective when heating is repeated 4 days later. The safety and efficacy of two successive sessions of WBHT (4 days apart) was evaluated in 30 patients with AIDS (not on protease inhibitors), randomized to: 1) untreated controls, 2) low temperature WBHT for 1 hour at 40 degrees C and repeated 96 hours later, and 3) high temperature WBHT for 1 hour at 42 degrees C and repeated 96 hours later. The sorbent suspension in the ThermoChem System (HemoCleanse, West Lafayette, IN) system automatically controlled blood phosphate, calcium, and other electrolyte concentrations during WBHT. In 1 year of follow-up after WBHT, there were positive effects of the therapy on frequency of AIDS defining events, Karnofsky score, and weight maintenance. However, effects on plasma HIV RNA and CD4 counts were transient. Two successive WBHT treatments were performed in four patients who were on protease inhibitor/triple drug therapy, but had suboptimal response. In follow-up for 6 months, plasma HIV RNA and CD4 improved after WBHT, and the patients remained clinically well. This WBHT may have specific advantages in patients with suboptimal response to protease inhibitor therapy.

The safety and possible efficacy of extracorporeal whole-body hyperthermia (WBHT) were evaluated in the first FDA-approved feasibility study of WBHT in persons with AIDS. Six gay men, aged 20-50 years, CDC class C-3, underwent 1 h of WBHT at either 40 degrees C or 42 degrees C, employing a system that minimizes the physiological and biochemical changes that occur during WBHT. All subjects had Kaposi's sarcoma (KS), were free of opportunistic infections, and had significant elevations of plasma HIV RNA. During the treatment, there were no adverse side effects and all subjects tolerated WBHT without problems. KS lesions partially regressed immediately following WBHT in all subjects but returned to pretreatment status in five of six patients at 1 week. In subjects treated at 40 degrees C, CD4 counts decreased during the 8-week follow-up period; they remained unchanged, however, following 42 degrees C WBHT. Viral load remained unchanged following WBHT in subjects treated at 40 degrees C. Treatment at 42 degrees C resulted in an immediate reduction in HIV RNA that was not sustained at 1 week post-WBHT. We conclude that WBHT is safe in subjects with advanced HIV disease and that it may have a role in treating HIV infection. A larger controlled trial involving two treatments in less immunocompromised subjects is currently in progress to test this hypothesis.

The life cycle of enveloped viruses is intimately associated with, and influenced by, host cell membrane organization, which is altered by hyperthermia. Hyperthermia-modified Moloney murine leukaemia virus (M-MuLV) release, protein production and intracellular protein processing in a chronically infected cultured murine cell line, C9CL98 (C9). Both 44 degrees C/45 min and 42.8 degrees C/135 min substantially decreased cell-free viral env protein 8-48 h postheating, but virus release and cellular viral protein content increased following 42.8 degrees C/25 min. Proteolytic processing of viral Pr65 gag precursor to p30 gag protein, normally observed within unheated C9 cells, was blocked for at least 8 h after 44 degrees C/45 min. Virus released from heated C9 cells was as infectious to NIH/3T3 cells as was virus from control cells. Cells surviving exposure to 42.8 degrees C/135 min became thermotolerant to decreased virus release from a second heating if delivered 10-48 h after the initial heating. The mechanism by which virus release is blocked after hyperthermia remains to be elucidated.

Hyperthermic treatment reduces protein synthesis and modifies amino acid transport in Escherichia coli. The present study examined the role of nutrient availability on these processes. Cultures of E. coli in log phase were aliquoted into growth medium with or without complete amino acid supplementation and exposed to 37, 44, or 48 degrees C for 10 min. Amino acid supplementation increased radiolabelled arginine uptake at 48 degrees C when compared with unsupplemented cells. Exposure to 48 degrees C also reduced protein synthesis in both groups by at least 50% as reflected by labelled arginine incorporation. Two-dimensional gel electrophoresis indicated that this heat-related decrement in synthesis was most apparent in basic proteins. Total density analysis of the fluorographs demonstrated reductions in basic proteins of 15% at 44 degrees C and 89% at 48 degrees C, while acidic proteins only showed an 80% reduction at 48 degrees C. Amino acid supplementation appears to raise the baseline, but not to modify the final results of hyperthermia-induced inhibition of protein synthesis. The sensitivity of basic protein synthesis seems to be a key event in this process.

We have reviewed the literature on cellular membrane radiobiology over the last ten years and, in particular, report on the development of rapid techniques used to identify damage soon after irradiation. It is clear that damage can now be quantified after low doses, and further refinements can be expected. From the work summarised, it would appear that changes to membranes at low doses may occur soon after damage to other important macromolecules by intercommunicating processes. We believe that there now exists a variety of rapid methods of measuring deposition of damage in vital macromolecules, such as cellular membranes and DNA, which can give a fuller picture of the overall effects of radiation and lead to predictions of eventual cellular mortality.

Considering the lack of effectiveness of current therapies to treat HIV disease, the authors present observations that provide a strong cogent argument for critically designed and meticulously performed clinical trials employing whole body hyperthermia with or without other therapeutic modalities. Only as a result of such clinical trials will it be possible to fairly evaluate the role of hyperthermia as a potential therapy for treatment of chronic HIV infection.

We have re-examined critically the evidence for and against the involvement of membranes in determining the response of cells to acute and chronic heat stress. Although frequently dismissed by many in the past, we believe that the bulk of evidence presented supports the view that physical and compositional alterations of membrane lipid components, both during and subsequent to heat exposure may, at least in part, account for cell adaptation, malfunction and lethality. Our primary goal in this review is to generate renewed interest in testing the validity of this hypothesis.

Hyperthermia-induced cell lethality is thought to be mediated through injury to the cell membrane. Membrane perturbation results in the release of prostaglandins (PG) and leukotrienes (LT). These compounds are potent biological mediators and may modify the tumor microenvironment and therapeutic efficacy. Membrane composition and PG/LT release are influenced by the dietary fatty acids. The relationship between these variables and response to hyperthermia was examined in vitro using murine P388 leukemia cells grown as an ascites in mice provided either saturated fatty acid diet (SFA; 16% beef tallow) or unsaturated fatty acid diet (UFA; 16% safflower oil). Cells were harvested and exposed in vitro to either 37 degrees C or 43.5 degrees C for periods up to 2 hours. Hyperthermic exposure for 2 hours resulted in 40% cell lethality in SFA cells and 55% in UFA cells. The phospholipid and total cholesterol content was higher (33% and 50% respectively) in the UFA versus the SFA cells. Hyperthermia produced a six-fold increase in prostaglandin E2 PGE2 release by SFA cells and a 4.5-fold increase by UFA cells. No LTC4 was detected. Alteration of dietary fat affects cell lethality and PG release following hyperthermic treatment. The increase in phospholipid and cholesterol content of UFA cells may be a response to reduced membrane fluidity.

Geraniol, an acyclic end product of a plant isoprene pathway and a pyrophosphorylated intermediate in plant and animal pathways, caused a concentration-dependent increase in the population doubling time of murine P388 leukemia cells in suspension culture and of B16 melanoma cells in monolayer culture. The suppression of the growth of P388 cells by geraniol (0-0.9 mM) and by mevinolin (0-0.25 microM), a competitive inhibitor of mevalonate biosynthesis, was reversed by the addition of 0.5 mM mevalonolactone to the growth medium. Flow cytometry of asynchronous B16 cells grown with geraniol (0-0.15 mM) revealed a population characterized by larger cells with altered nuclear characteristics. Over the course of four studies, dietary geraniol increased the 50% survival time of mice by 10, 29, 33, and 50% following the i.p. transfer of P388 cells. The results of the latter study showed that, following the i.p. transfer of 1 x 10(5) P388 cells, the control group of female C57BL x DBA/2 F1 mice had a 50% survival time of 24 days and a maximum survival of 27 days. Mice fed a diet containing 0.1% geraniol for 14 days prior to and following the P388 cell transfer had a 50% survival time of 36 days, and 20% of the mice remained free of tumors during the 50-day trial. These studies support the possibility that monoterpenes and other isoprenoid products of plant metabolism are in part responsible for the anticarcinogenic actions of diverse fruits, vegetables, and cereal products.

In Escherichia coli K1060 grown at 37 degrees C we observed that the uptake of both L-[3H]leucine and L-[35S]methionine was inhibited by exposure of the cells to 48 degrees C. The calcium channel blockers diltiazem and verapamil, and the anti-arrhythmic agent quinidine, inhibited the uptake of L-[3H]leucine at both 37 degrees C and 48 degrees C. Verapamil also inhibited the uptake of L-[35S]methionine at 37 degrees C, but at 48 degrees C protected against some of the heat-induced decrease in the uptake of this amino acid. The local anaesthetic procaine markedly inhibited the uptake of both labelled amino acids at temperatures between 37 degrees C and 48 degrees C. Amino acid uptake and cell killing were not correlated.

Total membranes from Escherichia coli cells grown in different fatty acid-supplemented media have been examined by 31P NMR at different pH values. The isolated inner and outer membranes were also studied and compared to the liposomes formed with the corresponding extracted lipids. While the liposomes show structures that are correlated with lipid composition, degree of fatty acid unsaturation, and pH, the membrane structure is mainly bilayer. The presence of two bilayer phases characterized by different chemical shift anisotropy values (delta nu csa) is detectable at neutral pH; a perturbation of the bilayer phase characterized by the smallest delta nu csa is produced by low pH. Moreover, an isotropic peak is always present in the membrane NMR spectra: its attribution to cardiolipin molecules is discussed on the basis of digestion experiments with phospholipase C.

Altering the biophysical characteristics of cell membranes by diet and membrane perturbing agents markedly influences thermosensitivity of cells. Likewise, manipulation of viral envelopes either by altering their lipid composition by diet or by the use of agents that perturb the lipid envelope influence infectivity of enveloped viruses and the progression of viral disease. The use of hyperthermia and envelope modification as a combined approach to treat AIDS has until now neither been suggested nor attempted. On the basis of my previous work and a review of the literature, I theorize that the combination of hyperthermia with procedures designed to alter the viral envelope will likely result in an increased viral sensitivity and be useful clinically for treatment of patients with enveloped viral diseases such as AIDS.

Concern about radiation induced nephropathy results in a dose limiting constraint in some applications of radiation therapy. An understanding of the etiology of radiation therapy. An understanding of the etiology of radiation nephropathy is essential if attempts to alter the time course or extent of the pathology are to be successful. In an attempt to gain a better understanding of this disease process, and to see if it could be altered by dietary manipulation, young adult male Sprague-Dawley rats were unilaterally nephrectomized, after which the remaining kidney was exteriorized and exposed to 14 Gy of X rays. Non-irradiated control animals had their remaining kidney exteriorized for a comparable length of time. Five days after irradiation, the animals were switched from standard lab rations to isocaloric diets of differing protein content. Diets used included 4%, 20% and 50% protein and the 4% and 20% diets given in combination with 0.9% NaCl drinking water. For all the diet groups, irradiated animals had median survival times shorter than their corresponding non-irradiated controls. Within the irradiated groups, the ranking of the median survival times was: 4% + 0.9% NaCl greater than 4% greater than 20% + 0.9% NaCl = 20% greater than 50%. The differences in survival among the irradiated groups were significant at the 0.01 level. These data indicate that kidney response to irradiation can be altered by manipulation of dietary protein levels. Such information may have clinical application.

Disruption of the integrity of tumor cellular membranes has been proposed as an initiating event in hyperthermic cell death. Thermosensitivity measured by the shift in the harmonic mean of tumor regrowth delay of CA755 mammary adenocarcinomas grown in the hind legs of male BDF1, mice increased 22% when the hosts were fed a diet enriched in polyunsaturated fatty acids. Although the diet elicited the anticipated increase in tumor membrane phospholipid polyunsaturated fatty acids, the proportion of total unsaturated fatty acids decreased and the proportion of membrane-rigidifying saturated fatty acids increased. Concomitantly, the concentrations of cholesterol and phospholipid phosphorus increased and the ratio of phosphatidylethanolamine to phosphatidylcholine decreased, presumably to counter the effect of the change in the fatty acid pattern. In host liver membranes, the diet-mediated increase in proportion of polyunsaturated fatty acids was not accompanied by an increase in the proportion of rigidifying saturated fatty acids. Instead, the homeoviscous adaptation consisted of decreases in monounsaturated fatty acids and cholesterol concentration and an increase in the phosphatidylethanolamine-phosphatidylcholine ratio. Addition of a natural inhibitor of cholesterol biosynthesis to the polyunsaturated fatty acid enriched-diet reversed the diet-mediated increase in the phosphatidylethanolamine-phosphatidylcholine ratio of host liver membranes. Tumor membrane lipids from hosts fed the combined dietary factors were characterized by the formentioned rigidifying increase in saturated fatty acids and compensatory decrease in the phosphatidylethanolamine-phosphatidylcholine ratio. The inhibitor reversed the compensatory increases in cholesterol and phospholipid phosphorus concentrations. As a consequence the thermosensitivity of tumors bearing this perturbed membrane was increased.

When Escherichia coli are exposed to heat stress, the majority of proteins in the process of synthesis at the time of heat stress are rapidly translocated to the outer membrane of the bacterium. The synthesis of most of these proteins appears to take place on membrane-bound polyribosomes. With the temperature shift, overall protein synthesis is inhibited while the synthesis of a small group of proteins is initiated. These proteins are not translocated, but remain in the cytosolic compartment, and they are identifiable as heat-stress proteins. Both the translocation phenomenon and the retention of heat-stress proteins in the cytosolic compartment in proximity to the nucleoid could counteract the effects of heat stress. The translocated proteins may operate by stabilizing the outer membrane prior to the induction of heat-stress proteins and the latter, which are confined to the cytoplasmic compartment, may serve to protect the integrity of the nucleoid structures.

The effect of ferrous ion-ascorbate and X-radiation on multilamellar liposomes, composed of either completely saturated, unsaturated or a mixture of saturated and unsaturated fatty acids, is reported. Lipid composition is shown to be of critical importance in determining the extent to which peroxidation occurs. Liposomes composed of the mixture of saturated and unsaturated fatty acids are peroxidized to a lesser extent by ferrous ion-ascorbate. The reduced peroxidation is apparently the result of an inhibition mechanism shown by the saturated lipid component. In contrast, liposomes composed of mixed lipids do not reduce the level of peroxidation induced by ionizing radiation. These results show that the composition of liposomes plays a role in determining the extent to which peroxidation occurs when a chemical oxidant is employed, but composition is a negligible factor when ionizing radiation is the oxidant.

Drugs can be released from lipid vesicles by pH-change in calf, horse or human serum when pH-sensitive trigger molecules are incorporated in the vesicle lipid bilayer. The lipid composition is so chosen that the drug release is best performed at 37 degrees C. Specific drug targeting is envisaged to loci of the body with lower than physiological pH, such as primary or metastatic tumors.

Sonicated unilamellar liposomes containing phosphatidylethanolamine and palmitoylhomocysteine fuse rapidly when the medium pH is lowered from 7 to 5. Liposome fusion was demonstrated by (i) mixing of the liposomal lipids as shown by resonance energy transfer, (ii) gel filtration, and (iii) electron microscopy. The pH-sensitive fusion of liposomes was observed only when palmitoylhomocysteine (greater than or equal to 20 mol%) was present in the liposomes. The presence of phosphatidyl-ethanolamine in the liposomes greatly enhanced fusion whereas the presence of phosphatidylcholine inhibited fusion. During fusion of liposomes containing phosphatidylethanolamine and palmitoylhomocysteine (8:2, mol/mol), almost all of the encapsulated calcein was released. Inclusion of cholesterol (40 mol%) in the liposomes substantially decreased leakage without impairing fusion.

Exposure of Escherichia coli to heat resulted in 1) selective inhibition of protein synthesis, 2) synthesis of heat shock proteins, and 3) altered subcellular distribution of newly synthesized proteins. Either 5 min or 1 h at 48 degrees C increases outer membrane proteins of Coomassie Blue-stained gels. After 1 h, there was a loss of stained proteins from the soluble fraction. Much greater changes in the distribution of radiolabeled (newly synthesized) proteins were observed, with marked increases in the number of outer membrane protein species and a corresponding loss of soluble fraction proteins. Three major species of radiolabeled proteins from heat-treated cells remain in the soluble fraction; these proteins have apparent Mr 56,000, 69,200, and 79,400. Cells were labeled with L-[35S] methionine at either 37 or 48 degrees C and chased with non-radiolabeled methionine before a temperature shift to either 48 or 37 degrees C, respectively. Only proteins synthesized at elevated temperature participated in translocation. It is suggested that heat disordering of membrane lipids promotes interlipidic connections between the inner and outer membrane providing pathways for protein movement to the outer membrane and may be the mechanism whereby a cell quickly responds to environmental temperature stress. The response does not require but may trigger synthesis of mRNA.

The response of E. coli K1060 D-lactate dehydrogenase (D-LDH), an enzyme located in the cytoplasmic membrane, was studied following 42.5 degrees C hyperthermia and/or gamma-irradiation. The inactivation of D-LDH following the above treatment was used as a tool to probe the role of membrane proteins in the radiation and/or heat sensitivity of cells. No correlation between loss of enzyme activity and cell killing was found, suggesting that D-LDH does not play an important role in hyperthermic cell survival. The results obtained in combined hyperthermia and gamma-irradiation treatments on loss of D-LDH activity and E. coli cell killing suggest that an interaction between heat and radiation occurs at the membrane structure level. Moreover, when cells were heated at 42.5 degrees C in the presence of 10 mM procaine-HCl, both cell killing and loss of D-LDH activity were enhanced. The involvement of membrane structure in the heat sensitivity of cells is strongly indicated by the latter observations. The opposite effect was observed when procaine was present during irradiation in oxic conditions, suggesting that procaine itself can also act as a scavenger towards OH-induced radicals.

The enhancement of killing by gamma-irradiation, which is seen when E. coli K1060 are cooled below the transition temperature of their membrane lipids, is blocked by procaine-HCl. These data are consistent with the hypothesis that increased killing associated with irradiation at 0 degree C is the result of membrane microviscosity increases, since procaine is known to fluidize membranes. A cooling enhancement ratio (c.e.r.) is defined as the ratio of radiation D0 at 22 degrees C to its value at 0 degree C. The c.e.r. for oxygen-bubbled cells is 1.5 and for nitrogen-bubbled cells is 2.1. In the presence of 25 mM procaine the respective c.e.r. values are 1.08 and 1.29. The oxygen enhancement ratio (o.e.r.) at 22 degree C is 3.43 and at 0 degree C is 2.45. The addition of procaine does not change the o.e.r. Thus, the temperature effect on o.e.r. does not appear to be related to membrane fluidity.

Hyperthermic sensitivities of Escherichia coli B/r and Bs-1 were determined for lag-, midlog-, and stationary-phase cells at 47, 48, and 49 degrees C. In both strains midlog-phase cells were strikingly more heat sensitive (100-fold greater killing after 4 h at 48 degrees C) than stationary-phase cells, with intermediate sensitivity for lag-phase cells. In contrast to the reported difference in the radiation sensitivity between these two strains, very little difference in heat sensitivity was seen. Patterns of fatty acid composition of both strains were very similar at each phase of growth. From midlog to stationary phase, 16:1 and 18:1 unsaturated fatty acids decrease from 16 and 30% to 0.5 and 3%, respectively, while the C17 and C19 cyclopropane fatty acids increase from 7 and 3% to 22 and 25%, respectively. Concomitant with these changes in fatty acid composition, substantially higher membrane microviscosity values were recorded for stationary-phase cells. Total membrane microviscosity was positively associated with the C17 and C19 cyclopropane fatty acid composition and with cell survival following hyperthermia. In contrast to hyperthermic sensitivity, radiation survival differences between B/r and Bs-1 are little affected by growth stage. We propose that these results are consistent with a critical influence of membrane lipids on cellular hyperthermic sensitivity and further that the target sites for radiation and hyperthermia are different in these cells.

The sensitivity of the solid mammary adenocarcinomas CA755 and MtGB, grown in the medial aspect of the hind legs of host mice, to local hyperthermia (43.5 +/- 0.1 degrees C for 1 hr) was increased by feeding the host mice a diet enriched in linoleic acid. The enhanced sensitivity was expressed only when the diet was fed for 15 days prior to the tumor transfer. Infusion of lidocaine into the tumor immediately before the hyperthermic exposure enhanced the thermal sensitivity of the controls but not of the linoleic acid-enriched tumors. Sensitivity was analyzed by tumor growth rates and growth delay following exposure. The fatty acid patterns revealed that the proportion of polyunsaturated fatty acids (20:4 and 22:6) decreased reciprocally with increased linoleic acid in the liver phospholipids, whereas in the tumor all polyunsaturated fatty acids increased at the expense of monounsaturated fatty acids. These studies suggest that dietary lipids affected tumor cell sensitivity to hyperthermia.

In studies using Escherichia coli we have shown that new protein species appear in the outer membrane fraction with concomitant losses of nascent proteins from the soluble and inner membrane fractions following heat exposure. Of the various explanations for this phenomenon, temperature-induced membrane disorganization appeared the most likely. It is suggested that heat mimics the action of the signal sequence of a protein on the lipid bilayer allowing non-signal-sequence-containing proteins to be translocated. To test this hypothesis we grew E. coli K1060 cells, an unsaturated fatty acid requiring auxotroph, supplemented during growth with fatty acids of varying chain length in an attempt to determine whether biological membranes of varying ability to maintain their bilayer configuration could be constructed. The rationale being that such membranes would allow us to determine whether differences in translocation would occur in cells grown at the same temperature supplemented with either 16:1 or 20:1 unsaturated fatty acids when the cells were subjected to a series of thermal insults. Protein translocation occurred to a greater extent and at lower temperatures in cells supplemented with the longer chain fatty acid. Treatment of outer membranes with either 1 M salt, 6 M urea or high pH and studies determining fluorescent polarization values by scanning up and down through a series of temperatures ranging from 15 to 49 degrees C indicate that the proteins translocated by heat to the outer membrane are integral. Protein translocation may represent an adaptive response to an altered environment enabling the cell to respond to stress by stabilizing its outer membrane.

Survival of E. coli K1060 is enhanced when they are heated at 47 degrees C in pH 6 medium as compared to pH 7.4. At pH 6 nitrogen bubbling and 10 mM procaine did not increase hyperthermic killing. The membrane content of phosphatidylethanolamine is about 80 per cent of the total of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin phospholipids. The polar headgroup of this lipid is highly sensitive to pH changes in this pH range. The changes in electrostatic charge with its secondary effects on membrane-protein relationships may explain resistance to hyperthermic killing. Thus, the difference in response to lowered pH of bacterial, compared to mammalian cells may be revealing membrane-related phenomena critical to hyperthermic killing. Also increased levels of cardiolipid were observed in linolenic acid grown cells which could be an 'attempt' to stabilize their membranes.

Ease of transfusion and high long-term survival rate were obtained when whole blood was administered via the corpus cavernosum of the penis and removed from the orbital sinus of male C57Bl mice. When 2 volumes of blood are replaced approximately 14% of pre-transfusion red cells remain after hematocrit corrections are made. The post-transfusion hematocrit levels dropped 19%, probably the result of leakage, which is difficult to avoid.

Previous work has utilized spleen colony formation to evaluate the fractional survival of AKR leukemia and normal bone marrow cells after in vitro heat exposure. An inherently greater sensitivity of neoplastic cells to thermal killing, as compared to normal syngeneic stem cells, has been established both at 41.8 degrees C and 42.5 degrees C. Normal bone marrow colony-forming units were assayed in lethally irradiated (750 cGy) mice. Leukemic colony-forming units were assayed in nonirradiated mice. Using this methodology, the authors demonstrated that the differential effect of hyperthermia on AKR murine leukemia and AKR bone marrow cells can be further enhanced by the addition of lidocaine or thiopental to incubation mixtures. These findings may have application to autologous bone marrow transplantation in humans.

The mechanism of hyperthermic killing, a component of some cancer therapy, is not known. Cell-survival curves during hyperthermic exposure can be used to elucidate the effects of membrane modifying procedures on cell death. Experiments were designed to test whether procedures that were reported to increase membrane fluidity also resulted in increased killing on hyperthermic exposure. An E. coli K12 mutant, K1060, is used to predictably alter the degree and amount of unsaturated fatty acids incorporated into membranes. Changing from an 18:1 to an 18:3 unsaturated fatty acid increases killing. Decreasing the amount of unsaturated fatty acid cells incorporated by increasing growth temperature decreases killing. Procaine, a drug known to decrease membrane viscosity, increases heat killing. These data are most simply explained by the hypothesis that membrane disorganization occurs as a result of temperature increasing to a point where a lipid transition causes a membrane structural change, which results in cell-death.

When pH-sensitive molecules are incorporated into liposomes, drugs can be specifically released from these vesicles by a change of pH in the ambient serum. Liposomes containing the pH-sensitive lipid palmitoyl homocysteine (PHC) were constructed so that the greatest pH differential (6.0 to 7.4) of drug release was obtained near physiological temperature. Such liposomes could be useful clinically if they enable drugs to be targeted to areas of the body in which pH is less than physiological, such as primary tumors and metastases or sites of inflammation and infection.

Immediately following unilateral nephrectomy the remaining kidney of juvenile male Sprague-Dawley rats was sham irradiated or irradiated to doses of 14-30 Gy. Following irradiation the animals were placed on isocaloric diets of either 20 or 4% protein. Median life spans for the animals on the low protein diet were significantly increased compared to the median life spans on the 20% protein diet. Serum urea nitrogen (SUN) levels were periodically measured in rats from each of the experimental groups. SUN levels in the irradiated rats fed the 20% protein diet increased significantly over unirradiated controls as a function of time. In contrast animals fed the 4% protein diet showed no significant changes in SUN levels irrespective of the size of radiation dose and time post irradiation. Renal protective factors calculated as the ratio of 80% survival times for animals fed the 20% protein diet compared to animals fed the 4% protein diet can be calculated to be 2.3 at 18 Gy and 2.8 at 22 Gy. Likewise, a SUN protective factor calculated as the ratio of percentage of nonirradiated control SUN values for the two diets (SUN 20% irradiated) (SUN 20% nonirradiated) (SUN 4% irradiated) (SUN 4% nonirradiated) is 2.4 for 18 Gy and 3.9 for 22 Gy.

In an attempt to optimize the chemotherapeutic treatment of mouse tumor Sarcoma 180, liposomes containing cis-dichlorodiammineplatinum(II) (PDD), having transition temperatures of few degrees higher than the rectal temperature of mice, were used in combination with local hyperthermia. The uptake of radioactive PDD by tumors heated for 1 hr at 42 degrees was almost four-fold greater when the drug was associated in liposomes than if administered as free drug. Uptake of liposome-administered radioactive platinum by liver was twice that obtained with free PDD, whereas its incorporation by the kidney was the same by either method of drug administration. The effect of various combinations of hyperthermia, drug-containing liposomes, and free PDD on tumor growth was also studied. Treatment with liposome-associated PDD plus local heating resulted in a dose-modifying factor of 7 when compared with free drug and no hyperthermia. The dose-modifying factor was 2.5 when PDD liposomes and heat were compared within free drug and heat. Thus, PDD could be specifically released from liposomes by heat and resulted in both a greater drug uptake and a delayed tumor growth following treatment. Potential normal tissue toxicity problems, however, still need to be resolved before clinical application of this combined modality will be possible.

Metastasizing and non-metastasizing transplantable mammary tumors were implanted into female W/Fu rats. A pair of tumors were employed, the SMT-2A and MT-W9B. When these tumors were exposed to water bath heating at 43.5 degrees C for 60 minutes, a significantly longer tumor-free growth delay was obtained in the metastasizing tumor compared to its non-metastasizing counterpart. The protein to phospholipid ratio and the content of arachidonic acid was lower in the metastasizing tumor than in the non-metastasizing one. By way of apparent compensation, the metastasizing tumor contained more linoleic and stearic acid. These observations suggest a relation between metastasizing capacity, thermal sensitivity, and membrane composition.

Previously, we reported that local lidocaine infusion of a CA 755 mammary adenocarcinoma growing in C57BL X DBA/2 F1 mice, when combined with local heating for 1 hr in a 43.5 degrees water bath, significantly increased survival and inhibited tumor growth more than heating alone. Because of its clinical implications, systemic lidocaine was tested in the above model system and in a murine fibrosarcoma tumor model. An equivalent supraadditive, tumor-inhibitory effect of heat and lidocaine was obtained with both systemically and intratumor-administered lidocaine. The serum levels of lidocaine necessary to achieve tumor regression were within the therapeutic range for the control of arrhythmia in humans. Several treatment schedules, varying the mode of drug delivery, were evaluated. The effects of treatment on tumor growth characteristics were analyzed using an extension of the Cox survival model.

The quantitative distribution of cell surface alterations of heated P388 ascites tumour cells was determined by scanning electron microscopy. Cells harvested from host animals maintained on a standard rodent chow diet or one high in saturated fatty acids responded differently, to identical hyperthermic treatment in vitro, to cells obtained from animals on a highly unsaturated diet. The morphological response of cells from chow fed animals was modified by addition to the incubation medium, of procaine, a membrane-active drug. The pattern of response observed after these cells were heated in the presence of procaine resembled that seen following heat treatment of ascites cells obtained from animals fed diet high in unsaturated fatty acids. These data are consistent with the hypothesis that a cell's response to hyperthermic insult is related to its membrane fluidity at the time of treatment.

In our view, the initial effect of hyperthermia on cells is the disorganization of the membrane lipid. Such disorganization alters the membrane's biophysical properties leading to passive changes in transmembrane permeability, shifts in surface charge, and altered stereoorganization of macromolecules associated with the membrane. For example, the passive permeability changes could account for the observed increase in the association of non-histone proteins to chromatin. Surface charge changes resulting from relative changes in the phospholipids could shift the concentration and types of membrane-bound proteins. Such events could initiate hyperthermic cell death. Membranes of tumour cells are characterized by elevated cholesterol. Such differences in cholesterol concentration with their attendant shift of biophysical characteristics could explain the variation in heat sensitivity between cell lines and within the cell cycle. Further support for lipids being the initial target comes from our studies demonstrating enhanced thermosensitivity when anaesthetics are present. Thermosensitivity of solid tumours is not further influenced by lidocaine in host animals fed diets enriched in linoleic acid, a diet which markedly modifies fatty acid, phospholipid patterns and cholesterol concentration of cellular membranes. One should recognize that global measures of change, such as the ratio of unsaturated to saturated fatty acids, in unsaturated fatty acid index, or percentage of unsaturated fatty acids, may not accurately reflect changes in fatty acid patterns which are related to changes in thermosensitivity. For example, we now recognize that double bond location with respect to the headgroup must be considered as well as the relative content of unsaturated fatty acids in the membrane. Further studies bearing on the role of diet and anaesthetics on cell killing and on metastatic spread are needed. An increased understanding of the relationship of membrane biophysics and biochemistry correlated with how cells respond to heat could aid in elucidating the mechanisms of cell death. Such knowledge could provide a more rational basis for cancer therapy. The association between pyrexia and tumour regression was noted as early as 1866 by Busch who observed neoplasm remission in patients afflicted with severe erysipelas (Busch, 1866). Over the past 80 years clinicians have on occasion treated tumours by heat alone or, more recently, in combination with radiotherapy (Dickson, 1979; Jensen, 1903).(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of membrane fluidity on hyperthermic cell killing has been investigated in ascites tumor cells. Membrane lipid composition of P388 ascites tumor cells were modified by feeding host animals with diets containing either unsaturated fatty acids (UFA) or saturated fatty acids (SFA). Both kinds of ascites were heat treated in vitro at 37, 42 or 43.5 degrees C for 30 or 60 min. The cell killing effect of hyperthermia was tested by transplantation of cells into recipient mice and survival examined. While at 37 and 42 degrees C for 1 h, there was no difference in cell killing of the two types of ascites, elevating the temperature to 43.5 degrees C the survival was significantly longer on transplantation of ascites of UFA diet. This effect was potentiated by membrane-fluidizing drugs. On the addition of 1 mM procaine during 1 h treatment at 43.5 degrees C, the ascites of SFA diet killed 80% of mice, while the ascites of UFA diet left all the mice alive at least for 3 months. Scanning electron-microscopic observations of the treated cells was performed in parallel and showed close correspondence with the results of survival studies. In conclusion, the increase of membrane fluidity by incorporating more of UFA or by the addition of membrane-fluidizing drugs or especially by the combination of both, the sensitivity of cells to heat enhanced. These experiments support the hypothesis that membranes are a target for hyperthermia.