Extracorporeal whole body hyperthermia treatments for HIV infection and AIDS.

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.

Effect of whole-body hyperthermia on AIDS patients with Kaposi's sarcoma: a pilot study.

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.

Effect of heat on viral protein production and budding in cultured mammalian cells.

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.

Effects of heat and amino acid supplementation on the uptake of arginine and its incorporation into proteins in Escherichia coli.

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.

Recent developments in the radiobiology of cellular membranes.

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.

Shedding light on the use of heat to treat HIV infections.

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.

Role of cellular membranes in hyperthermia: some observations and theories reviewed.

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.

Effect of diet on hyperthermia-induced cell lethality and prostaglandin release.

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.

Concentration-dependent increase of murine P388 and B16 population doubling time by the acyclic monoterpene geraniol.

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.

Effects of heat and other agents on amino acid uptake in Escherichia coli.

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.

Effects of decreased pH on membrane structural organization of Escherichia coli grown in different fatty acid-supplemented media: a 31P NMR study.

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.

An approach to AIDS therapy using hyperthermia and membrane modification.

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.

Major E. coli heat-stress protein do not translocate: implications for cell survival.

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.

Influence of membrane-lipid composition on translocation of nascent proteins in heated Escherichia coli.

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.

Influence of protein nutrition on dose-survival relationship following rat kidney irradiation.

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.

A comparison of X-irradiation and ferrous ion-ascorbate on oxidation of phosphatidylglycerols in multilamellar liposomes.

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.

Heat sensitivity and membrane properties of metastasizing and non-metastasizing rat mammary tumors.

Paired lines of metastasizing and non-metastasizing transplantable rat mammary tumors were studied for their sensitivity to hyperthermia. The metastasizing TMT-081 and SMT-2A tumors were markedly more sensitive to heat injury as measured by tumor growth delay than were their non-metastatic counterparts MT-100 and MT-W9B. The metastasizing MT-081 tumor was also significantly more sensitive than the SMT-2A tumor. The differences in heat sensitivity were not the result of differences in intra-tumor temperatures attained during water bath heating. A more likely explanation for the variable response obtained with these tumors after heat treatment may be the inherent differences in stability and composition of their cellular membranes, particularly the plasma membrane.

Sensitivity of tumour cells to heat and ways of modifying the response.

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)