The effects of conjugated linoleic acid supplementation on immune function in healthy volunteers.

OBJECTIVE: To assess the effects of dietary supplementation using two isomeric blends of conjugated linoleic acid (CLA) on immune function in healthy human volunteers. DESIGN: Double-blind, randomised, placebo-controlled intervention trial. SUBJECTS AND INTERVENTION: A total of 55 healthy volunteers (n=20 males, n=35 females) were randomised into one of three study groups who received 3 g/day of a fatty acid blend containing a 50:50 cis-9, trans-11: trans-10, cis-12 CLA isomer blend (2 g CLA), and 80:20 cis-9, trans-11: trans-10, cis-12 (80:20) CLA isomer blend (1.76 g CLA) or linoleic acid (control, 2 g linoleic acid) for 8 weeks. RESULTS: Supplementation with the 80:20 CLA isomer blend significantly (P< or =0.05) enhanced PHA-induced lymphocyte proliferation. CLA decreased basal interleukin (IL)-2 secretion (P< or =0.01) and increased PHA-induced IL-2 and tumor necrosis factor alpha (TNF(alpha)) production (P< or =0.01). However, these effects were not solely attributable to CLA as similar results were observed with linoleic acid. CLA supplementation had no significant effect on peripheral blood mononuclear cells IL-4 production, or on serum-soluble intercellular adhesion molecule-1 (sICAM-1) or plasma prostaglandin E2 (PGE2) or leukotreine B4 (LTB4) concentrations. CONCLUSIONS: This study shows that CLA supplementation had a minimal effect on the markers of human immune function. Furthermore, supplementation with CLA had no immunological benefit compared with linoleic acid.

Combined hyperthermia and chlorophyll-based photodynamic therapy: tumour growth and metabolic microenvironment.

The effects of combined and simultaneously applied localised 43 degrees C hyperthermia (HT) and an antivascular bacteriochlorophyll-serine-based photodynamic therapy (Bchl-ser-PDT) on tumour growth and several microenvironmental parameters were examined. Rats bearing DS-sarcomas were allocated to treatment groups: (i) sham-treatment (control), (ii) Bchl-ser-PDT (20 mg kg(-1) i.v.), (iii) localised HT, (iv) Bchl-ser-PDT+HT. The light source used was an infrared-A irradiator, which, by use of appropriate filters, delivered the different ranges of wavelengths required. Following treatment, tumour volume was monitored. The greatest tumour growth inhibition was seen with Bchl-ser-PDT+HT, and subsequent experiments identified the pathophysiological basis for this effect. Red blood cell flux in tumour microvessels declined rapidly upon Bchl-ser-PDT+HT, reaching approximately 10% of initial values by the end of treatment. Similarly, tumour oxygenation worsened, reaching almost anoxic levels by the end of the treatment period. Assessment of metabolic parameters showed a pronounced increase in lactate levels and a decrease in ATP concentrations after combined treatment. The results presented suggest that vascular collapse and flow stasis resulting in a deterioration of tumour oxygenation and a switch from oxidative to glycolytic glucose turnover are key elements in the tumour eradication seen with this novel approach in which an antivascular PDT and HT are combined and simultaneously applied.

Enhanced effects of aminolaevulinic acid-based photodynamic therapy through local hyperthermia in rat tumours.

The possibility of enhancing aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) by simultaneous application of localised hyperthermia (HT) was evaluated. Treatments of rat DS-sarcomas included: (i) control, (ii) ALA administration (375 mg kg(-1), i.p.), no illumination, (iii) 'nonthermal' illumination, (iv) ALA-PDT: that is, ALA administration, 'nonthermal' illumination, (v) localised HT, 43 degrees C, 60 min (vi) ALA-PDT+HT: ALA administration with full spectrum irradiation resulting in ALA-PDT and HT. Tumour volume was monitored for 90 days or until a target volume (3.5 ml) was reached. No differences were seen between the first three groups, with all tumours reaching the target volume by 8-11 days. A total of 13 and 15% of tumours did not reach the target volume by day 90 following HT or ALA-PDT treatment, respectively. ALA-PDT+HT showed the greatest antitumour effect (P=0.0001), with 61% of the tumours not reaching the target volume. Viability and in vitro growth were also assessed in cells from tumours excised after treatment. ALA-PDT+HT reduced the fraction of viable tumour cells by 85%, and in vitro culture showed pronounced growth delay compared to control cells. These results demonstrate an enhanced antitumour effect upon ALA+HT, which appears to involve direct cell toxicity rather than solely vascular damage.

Tumour tissue monitoring during photodynamic and hyperthermic treatment using bioimpedance spectroscopy.

Electrical bioimpedance spectroscopy is a fast and relatively easily applicable method for tissue characterization. In the frequency range up to 10 MHz, current conduction through tissue is mainly determined by tissue structure, i.e. the extra- and intra-cellular compartments and the insulating cell membranes. Therefore, changes in the extra- and intra-cellular fluid volumes are reflected in the impedance spectra. Investigations of tumours (DS sarcoma, implanted on the hind foot dorsum of rats) during treatment with localized hyperthermia (HT), photodynamic therapy (PDT) and the combination of these two components were carried out using impedance spectroscopy in the frequency range of 37 Hz to 3.7 MHz. Data collected reveal totally different, but characteristic, behaviour patterns for the three treatments. HT caused a slow increase in conductance at high frequencies (G(HF)) and in the extracellular space index (ECSI), indicating an increase in extracellular fluid volume and in total fluid content. With PDT, G(HF) increased immediately upon commencement of irradiation and was accompanied by a distinct decrease in ECSI, indicating the development of a pronounced intracellular oedema.

2-Methoxyestradiol enhances reactive oxygen species formation and increases the efficacy of oxygen radical generating tumor treatment.

In an investigation of the antitumor effects of 2-methoxyestradiol (2-ME) in combination with other reactive oxygen generating treatments, 2-ME (0.5 microM) was found to completely inhibit cell proliferation of rat DS-sarcoma cells in vitro, with 71% of cells dying after exposure to 5 microM 2-ME. Concentration-dependent increases in ROS-formation, lipid peroxidation and mitochondrial changes were also observed, and an elevation in caspase-3 activity resulted in DNA fragmentation and apoptosis. Combination of 2-ME with hypoxanthine and xanthine oxidase enhanced in vitro cytotoxicity. In vivo, 2-ME caused a slight inhibition of tumor growth, with no tumors cured. Combination of 2-ME treatment with localized 44 degrees C hyperthermia, respiratory hyperoxia and xanthine oxidase caused a tumor growth delay with 51% of tumors cured. These results suggest that amplifying the levels of reactive oxygen species within tumor tissue with substances such as 2-ME may prove to be a promising strategy for adjuvant treatment of solid tumors.

Water-filtered infrared-A radiation: a novel technique for localized hyperthermia in combination with bacteriochlorophyll-based photodynamic therapy.

A novel application of an infrared-A (IR-A) radiation source equipped with a water-filter in the radiation path is described, which allows for tumour treatment with a simultaneous combination of localized hyperthermia (HT) and bacteriochlorophyll-serine (Bchl-ser) based photodynamic therapy (PDT). Using this system, the IR-A radiation was used to heat tumours to 43 degrees C for 60 min, while at the same time activating the Bchl-ser which was injected i.v. at a dose of 20 mg/kg, 10 min following commencement of HT. The growth of tumours undergoing this combined therapy was compared to that of tumours undergoing HT alone or sham-treated controls. Within the 90 day observation period, 100% of tumours in sham-treated animals, 80% in HT-treated animals and only 17% in HT + Bchlser-treated animals reached the end point target volume of 3.5 ml. Thus, the tumour growth inhibition effect of HT can be substantially enhanced by combination with Bchl-ser-PDT. This novel technique has proved to be well-tolerated, easy to apply and should be suitable for treatment of superficial malignancies, especially where hypoxic tumour areas are present.

Modulation of tumor oxygenation.

There is a large body of evidence suggesting that deficiencies in the O2 supply of tumors exist due to restrictions (i) in the O2 delivery by perfusion and/or diffusion, and (ii) in the O2 transport capacity. Whereas the former are mostly based on inadequate and heterogeneous microcirculatory functions, the latter are predominantly due to tumor-associated anemia. Possible uses and limitations of measures are discussed which can increase the microvascular O2 content and thus may preferentially serve to enhance diffusion-limited O2 availability. In addition, means are described for improving and increasing the uniformity of microcirculation thus possibly enhancing perfusion-limited O2 delivery. Reducing cellular respiration rate should be of benefit in both pathophysiological conditions. Because both types of O2 limitation coexist in solid tumors, appropriate combinations should be aimed at eradicating tumor hypoxia which is present in at least one third of cancers in the clinical setting.

Enhancement of oxidative cell injury and antitumor effects of localized 44 degrees C hyperthermia upon combination with respiratory hyperoxia and xanthine oxidase.

The effects of respiratory hyperoxia (RH) and xanthine oxidase (XO) during localized hyperthermia (HT) were investigated by determining markers of oxidative damage to lipids and proteins and tumor growth. Anesthetized rats with s.c. DS-sarcomas underwent one of the following treatments: (a) localized saline-bath HT (60 min, 44 degrees C); (b) HT + RH (100% O2); and (c) HT + RH + XO (15 units/kg i.v.). Sham-treated animals served as controls. Tumors were investigated for: (a) thiobarbituric acid-reactive substance formation and protein-bound 4-hydroxynonenal, as indicators of lipid peroxidation; (b) reactive oxygen-mediated protein modifications; (c) apoptosis; and (d) tumor volume growth. Upon treatment, increases in thiobarbituric acid-reactive substances, protein-bound 4-hydroxynonenal, protein-associated carbonyl functions, and number of cells undergoing apoptosis were found in tumor tissue, together with an inhibition of tumor growth. When treatment groups were compared, effects in the group HT + RH + XO were generally most pronounced. These findings indicate that the antitumor effect of HT is at least partially mediated through the selective induction of lipid peroxidation and oxidative injury in tumor cells, leading to apoptosis. This effect was enhanced by adding RH or RH + XO, presumably due to enhanced tissue damage following an increased formation of reactive oxygen species, with higher levels of lipid peroxidation and protein oxidation.

Localized hypothermia: impact on oxygenation, microregional perfusion, metabolic and bioenergetic status of subcutaneous rat tumours.

The effect of localized hypothermia on microcirculatory and metabolic parameters in s.c. DS sarcomas on the hind foot dorsum of Sprague-Dawley rats was investigated. Tumours were cooled by superfusion of the tumour surface with cooled saline solution to 25 degrees C or 15 degrees C. Control tumours remained at 35 degrees C. These temperatures were maintained for 30 min. In tumour oxygenation measurements, hypothermia at 25 degrees C and 15 degrees C caused progressive decreases in the size of the fraction of pO2 measurements between 0 and 2.5 mmHg together with a reduction in pO2 variability. No significant changes in median or mean pO2 or in the fraction of pO2 measurements between 0 and 5 mmHg, and 0 and 10 mmHg were observed. Using laser Doppler flowmetry, red blood cell flux was found to decrease significantly upon 25 degrees C or 15 degrees C hypothermia treatment to 67% and 37% of starting values respectively, whereas no significant changes were seen in control tumours over the whole observation period. Viscosity was measured in blood and plasma samples over a range of temperatures and was found to increase with decreasing temperature. Assessment of tumour glucose levels showed an increased concentration of glucose following 15 degrees C hypothermia, an observation consistent with a 'slowing down' of glycolysis. No changes in lactate or adenylate phosphate levels were observed. As a way of improving tumour oxygenation, localized hypothermia may therefore be a useful means of radiosensitization.

Blood flow, oxygenation, and bioenergetic status of tumors after erythropoietin treatment in normal and anemic rats.

Growth, blood flow, oxygenation, and bioenergetic status of experimental tumors were investigated in normal (control) and anemic animals after administration of recombinant human erythropoietin (rhEPO). DS sarcomas were implanted s.c. onto the hind foot dorsum of Sprague-Dawley rats. Tumor-associated anemia was induced by the development of an i.p. hemorrhagic ascites. rhEPO (1000 IU/kg) was administered s.c. three times per week over 14 days, after which it was found to have significantly increased hematocrit values in both normal and anemic animals. Tumor growth in anemic animals was slower than in normal animals, and rhEPO administration did not influence tumor growth in either group. Tumor blood flow in anemic animals was lower than in control animals and was only increased in larger tumors in animals in which anemia was prevented by prophylactic rhEPO application. Tumor oxygenation, determined using polarographic needle electrodes and oxygen partial pressure histography, was poorer in anemic animals than in normal animals. This reduction could be reversed partially, but not compensated fully by rhEPO treatment in smaller tumors (< or = 1.4 ml). These changes suggest that rhEPO, by improving tumor oxygenation, may increase the efficacy of standard radiotherapy in anemic animals and may be of use in anemic tumor patients in whom the success of radiotherapy or O2-dependent chemotherapy might be limited by tumor hypoxia.

Tumour-growth inhibition by induced hyperglycaemia/hyperlactacidaemia and localized hyperthermia.

The present study was undertaken to exploit pathophysiological properties of solid tumours for a tumour-specific therapy. Experiments were carried out on DS-sarcomas implanted s.c. in the hind foot dorsum of Sprague Dawley rats. Treatment strategies included tumour acidification, lactate accumulation and disturbance of the microcirculation by induced systemic hyperglycaemia/hyperlact-acidaemia (15-25/10 mmol/L; for 60 min) as well as localized hyperthermia (water-bath; 43 degrees C, 30 min.). A special infusion solution was developed for the systemic treatment containing glucose, lactic acid and organic buffer without inorganic ions. Growth kinetics of tumour volume and animal survival were taken as endpoints in order to quantify therapeutic efficiency. After a single treatment with combined modalities, i.e., with hyperglycaemia/hyperlactacidaemia and hyperthermia, approximately 50% of the tumours showed complete remission in three independent series of experiments; around 40% of the animals survived more than two months. In the untreated control group, all animals died from the disease within 10-15 days after tumour implantation. The overall effect on tumour volume changes of the combined therapy was supra-additive compared to that of treatment with hyperthermia or hyperglycaemia/hyperlactacidaemia alone. However, treated animals either showed a dramatic response to the combination of treatments with complete tumour remission or hardly responded at all, justifying a subdivision into responders and non-responders. Pathophysiological mechanisms responsible for this behaviour have to be elucidated in future studies. Nevertheless, the present study represents an approach to an efficient tumour therapy with a potential application in clinical oncology in the not too distant future.

Changes in microregional perfusion, oxygenation, ATP and lactate distribution in subcutaneous rat tumours upon water-filtered IR-A hyperthermia.

The effect of hyperthermia on microcirculatory and metabolic parameters in s.c. DS-sarcomas of different sizes on the hind foot dorsum of SD-rats was investigated. Hyperthermia was carried out using a novel water-filtered, infrared-A radiation technique. Heating was performed at a rate of 0.5 degrees C/min until 44 degrees C was achieved in the tumour centre, which was maintained for 60 min. Using a multichannel laser Doppler flowmeter, red blood cell flux could be assessed continuously and at several sites within the tumour tissue simultaneously. Substantial inter-site variations in laser Doppler flux (LDF) were observed during hyperthermia which were independent of tumour size, site of measurement, and temperature at the site of measurement, indicating that single site measurements of tumour LDF are poor predictors of the mean response of a tumour to hyperthermia. When mean LDF was considered, decreases in red blood cell fluxes occurred that were more pronounced the greater the tumour volume. In no case was vascular stasis observed. Hyperthermia did not affect tumour oxygenation substantially. Microregional and global assessment of lactate and ATP concentrations demonstrated increased lactate and decreased ATP levels following hyperthermia. Tumour glucose levels were increased following hyperthermia, possibly due to an enlarged distribution space resulting from development of interstitial oedema. Changes in lactate and ATP levels and the lack of changes in tumour oxygenation suggest a modification of energy metabolism following hyperthermia in the form of increased ATP hydrolysis, intensified glycolysis and impaired oxidative phosphorylation.

Water-filtered infrared-A radiation: a novel technique to heat superficial tumors.

A novel device consisting of an infrared-A (= ultrared-A) radiation source equipped with a water filter in the radiation path is described which allows for the therapeutic heating of superficial experimental and human tumors. Preliminary studies with agar phantoms showed that heating in the presence of the water-cuvette avoids intolerable overheating in the very superficial layers. This effect can be further enhanced by surface cooling with room air such that a stratification of the temperature distribution can be achieved. In subsequent experiments, temperature distributions were recorded in the x-, y- and z-axis of superficial rodent tumors. The results obtained confirm those from the phantom experiments, showing that therapeutically relevant temperatures (T > or = 42 degrees C) could be achieved through the tumor mass to a depth of approximately 1.2 cm. Temperature homogeneity is comparable to that seen in superficial tumors undergoing water-bath hyperthermia. This novel technique has proved to be reliable, is well-tolerated, is easy to apply, and is easily accessible to a larger number of potential users for the local heating of superficial malignancies.

Changes in hepatic blood flow during regional hyperthermia.

The influence of liver hyperthermia on hepatic arterial and portal venous blood flow to tumour and normal hepatic tissue was examined in a rabbit VX2 tumour model. Hyperthermia was delivered by 2450 MHz microwave generator to exteriorized livers in 18 rabbits. Blood flow was measured in both portal vein and hepatic artery using radioactive tracer microspheres before, during and 5 min after intense (greater than 43 degrees C) hyperthermia. During hyperthermia a decrease in total liver blood flow was composed primarily of a decrease in hepatic arterial blood flow to tumour tissue. Tumours were supplied almost exclusively by the hepatic artery and thus total tumour blood flow was significantly depressed during heating. The decreased tumour blood flow persisted after the cessation of hyperthermia and was indicative of vascular collapse in the tumour tissue. Temperature differentials in tumour compared to normal tissue ranged from 5 degrees C to 8 degrees C during hyperthermia because of the lower tumour blood flow. The portal vein exerted minimal influence on temperatures attained in the tumour tissue during hyperthermia but would have mediated normal liver tissue heat loss.

Low-dose systemic doxorubicin in combination with regional hepatic hyperthermia.

The influence of regional liver hyperthermia in conjunction with systemic doxorubicin administration was examined in a rabbit VX2 tumour model. Hyperthermia was delivered by 2450MHz microwave generator to the exteriorised livers of the rabbits to provide a thermal dose equivalent of 43 degrees C for 30 minutes. Animals receiving doxorubicin infusion were treated with a total of 1.2 mg/kg over a 3 day protocol through an ear vein. Rabbits were divided into 4 groups; a no treatment control, hyperthermia alone, doxorubicin alone and hyperthermia immediately preceded by doxorubicin. The tumour mass, 10 days post treatment was significantly (P less than 0.0001) reduced in all treatment groups. However, the mean tumour mass in the combination treatment group was also significantly lower than both treatments alone (P less than 0.001). This increased response was not accompanied by any signs of increased systemic or local toxicity associated with any treatment.