P. Sminia

Expression of Transforming Growth Factor (TGF)-β1, -β2, and -β3 Isoforms and TGF-β Type I and Type II Receptors in Multiple Sclerosis Lesions and Human Adult Astrocyte Cultures

It is known that the pleiotropic cytokine transforming growth factor beta (TGF-beta) has a regulatory role in the process of tissue repair and remodelling following injury. As reports on these molecules in multiple sclerosis (MS) lesion with different lesional activity are rare, we studied the cellular localization of TGF-beta1, -beta2, and -beta3 isoforms, and TGF-beta receptor type I (TGF-betaR-I) and TGF-betaR-II expression by immunohistochemistry on postmortem brain tissue from MS and normal control cases. To validate the TGF-beta staining results we demonstrated that cultured human adult astrocytes that produce biological active TGF-beta2, and to a lesser extent TGF-beta1, were immunoreactive for all 3 TGF-beta isoforms. Moreover, at mRNA level TGF-beta1 was detected in MS and normal control brain tissue. In normal control brain tissue, TGF-beta isoforms were expressed in ramified microglia and TGF-beta2, and -beta3 on neuronal cells in the gray matter TGF-betaR-I and TGF-betaR-II expression was found on endothelial cells, astrocytes, microglia, and neurons. In active demyelinating MS lesions a strong to intense immunoreactivity was detected for all 3 TGF-beta isoforms in perivascular and parenchymal (foamy) macrophages and in hypertrophic astrocytes. Strong immunoreactivity for TGF-betaR-I and TGF-betaR-II was found on macrophages in both parenchymal and perivascular areas and on hypertrophic astrocytes and endothelial cells in active demyelinating MS lesions. In chronic active and inactive MS lesions, all 3 TGF-beta isoforms and their receptors were strongly expressed in hypertrophic astrocytes. Our findings strongly suggest that the expression of the various TGF-beta isoforms and their receptor types found in MS lesions with different cellular activity participate in reactive processes leading to the formation of chronic MS lesions.

Effect of hyperthermia on the central nervous system: A review

Experimental data show that nervous tissue is sensitive to heat. Animal data indicate that the maximum tolerated heat dose after local hyperthermia of the central nervous system (CNS) lies in the range of 40-60 min at 42-42 x 5 degrees C or 10-30 min at 43 degrees C. No conclusions concerning the heat sensitivity of nervous tissue can be derived from clinical studies using localized hyperthermia. The choice whether or not to exceed the critical heat dose, as derived from laboratory studies, in clinical practice is very much dependent on the clinical situation such as the anatomical site and volume of the tissue involved, and prior therapy. Data on clinical application of whole body hyperthermia (WBH) show that nervous tissue can withstand a slightly higher heat dose than after localized heating, which might be the result of developing thermal resistance during treatment. Expression of thermotolerance was observed in the spinal cord of laboratory animals. After WBH in man at a maximum between 40 and 43 degrees C for 6 h-30 min CNS complications were reported, but other complications seemed to be more life-threatening. Most studies indicate that impairment of the CNS after WBH was not due to direct heat injury to the brain or spinal cord, but was secondary as a result of physiological changes. Heat, at least if applied shortly after X-rays, enhances the response of nervous tissue to radiation. Neurotoxicity of chemotherapeutic drugs does not seem to be a limiting complication in hyperthermia if combined with chemotherapy, but only few data are available. The limited clinical experience shows that safe hyperthermic treatment of CNS malignancies or tumours located close to the CNS seems feasible under appropriate technical conditions with adequate thermometry and taking the sensitivity of the surrounding normal nervous tissue into account.

Optimisation of intraperitoneal cisplatin therapy with regional hyperthermia in rats

The purpose of this study was to optimise intraperitoneal chemotherapy by combining this modality with regional hyperthermia. In vitro data demonstrated that both the uptake of cisplatin into CC531 tumour cells and cytotoxicity were increased at temperatures of 40 degrees C (factor 4) and 43 degrees C (factor 6) compared to 37 degrees C. The increase of intracellular platinum concentration correlated well with the decrease in survival of these cells. In vivo, rats were treated intraperitoneally with cisplatin (5 mg/kg) in combination with regional hyperthermia of the abdomen (41.5 degrees C, 1 h). The mean (S.D.) temperature in the peritoneal cavity was 41.5 (0.3) degrees C and outside the peritoneal cavity 40.5 (0.3) degrees C. Enhanced platinum concentrations were found in peritoneal tumours (factor 4.1) and kidney, liver, spleen and lung (all around a factor 2.0), after combined cisplatin-hyperthermia treatment. The platinum distribution in peritoneal tumours was more homogeneous after the combined treatment than after cisplatin alone, possibly due to increased penetration of cisplatin into peritoneal tumours. Pharmacokinetic data demonstrated an increased tumour exposure for unfiltered platinum in the peritoneal cavity (area under the curve [AUC] increased from 339 mumol/l/min to 486 mumol/l/min at 37 degrees C and 41.5 degrees C, respectively), and for total and ultrafiltered platinum in the blood. The AUC for total platinum increased from 97.9 to 325.8 mumol/min and for ultrafiltered platinum from 22.2 to 107 mumol/l/min at 37 degrees C and 41.5 degrees C respectively. The latter might be due to a slower elimination of platinum from the blood. The combined treatment, intraperitoneal cisplatin and regional hyperthermia, also increased toxicity. The thermal enhancement ratio (TER) using lethality as endpoint was 1.8.

Hyperbaric Oxygen Therapy for Cognitive Disorders after Irradiation of the Brain

Purpose: Analysis of the feasibility and effect of hyperbaric oxygen treatment (HBO) on cognitive functioning in patients with cognitive disorders after irradiation of the brain. Patients and Method: Seven patients with cognitive impairment after brain irradiation, with an interval of at least 1.5 years after treatment, were treated with 30 sessions of HBO in a phase I–II study. A comprehensive neuropsychological test battery was performed before treatment, at 3 and 6 months thereafter. Patients were randomized into an immediate treatment group and a delayed treatment group. The delayed group had a second neuropsychological test at 3 months without treatment in that period and started HBO thereafter. Results: All eligible patients completed the HBO treatment and the extensive neuropsychological testing. One out of seven patients had a meaningful improvement in neuropsychological functioning. At 3 months there was a small, but not significant benefit in neuropsychological performance for the group with HBO compared to the group without HBO treatment. Six out of seven patients eventually showed improvement after HBO in one to nine (median 2.5) of the 31 tests, although without statistical significance. Conclusion: HBO treatment was feasible and resulted in a meaningful improvement of congitive functioning in one out of seven patients. Overall there was a small but not significant improvement.Hintergrund: Es wurden die Durchführbarkeit und Wirksamkeit der hyperbaren Oxygenierung (HBO) auf die Gehirnfunktion bei Patienten mit kognitiven Störungen nach Hirnbestrahlung untersucht. Patientengut und Methode: In einer Phase-I/II-Studie wurden sieben Patienten mit kognitiven Störungen nach einem minimalen Intervall von 1,5 Jahren 30 HBO-Behandlungen unterzogen. Neuropsychologische Tests wurden vor HBO sowie 3 und 6 Monate nach Abschluss der Behandlung durchgeführt. Patienten wurden randomisiert in eine sofortige und eine späte Behandlungsgruppe. Die späte Behandlungsgruppe wurde nach 3 Monaten zum zweiten Mal neuropsychologisch getestet ohne zwischenzeitliche HBO-Behandlung. Nach diesem zweiten Test begann in dieser Gruppe die HBO. Ergebnisse: Alle Patienten konnten die vorgeschriebene HBO-Therapie und das umfangreiche neuropsychologische Testprogramm abschließen. Einer von sieben Patienten zeigte eine bedeutsame Verbesserung der neuropsychologischen Funktion. Die HBO-Gruppe zeigte im Vergleich zu einer nicht mit HBO behandelten Kontrollgruppe nach 3 Monaten eine leichte, statistisch nicht signifikante Verbesserung. Bei sechs der insgesamt sieben HBO-Patienten waren Verbesserungen nachweisbar in einem bis neun (median 2,5) der 31 Tests, die statistisch jedoch nicht signifikant waren. Schlussfolgerung: HBO ist bei Patienten nach Hirnbestrahlung durchführbar und erzielte bei einem von sieben Patienten eine bedeutsame Verbesserung der kognitiven Funktion. Insgesamt zeigte sich eine leichte, statistisch nicht signifikante Verbesserung der getesteten Hirnfunktionen.

Feasibility of curative radiotherapy with a concomitant boost technique in 33 patients with non-small cell lung cancer (NSCLC)

Thirty-three patients with an inoperable NSCLC were treated with a dose of 60 Gy/20 fractions/25 days, using a concomitant boost technique. A dose of 40 Gy/2 Gy/25 days was given to the tumor area and a part (15 patients) or the whole (18 patients) mediastinum. During each session a simultaneous boost to the tumor of 1 Gy was administered. Moderate acute oesophageal toxicity was observed in 7/33 patients (22%). One out of 33 patients developed serious late oesophageal toxicity. A correlation between the oesophageal toxicity, absorbed oesophageal dose of irradiation and length of the elective field was observed. Five out of 33 patients developed subacute radiation pneumonitis grade 2 or 3. In selected patients with inoperable NSCLC radiotherapy, with a dose of 60 Gy/20 fractions/25 days, using a concomitant technique is feasible.

Pulse Frequency in Pulsed Brachytherapy Based on Tissue Repair Kinetics

PURPOSE Investigation of normal tissue sparing in pulsed brachytherapy (PB) relative to continuous low-dose rate irradiation (CLDR) by adjusting pulse frequency based on tissue repair characteristics. METHOD Using the linear quadratic model, the relative effectiveness (RE) of a 20 Gy boost was calculated for tissue with an alpha/beta ratio ranging from 2 to 10 Gy and a half-time of sublethal damage repair between 0.1 and 3 h. The boost dose was considered to be delivered either in a number of pulses varying from 2 to 25, or continuously at a dose rate of 0.50, 0.80, or 1.20 Gy/h. RESULTS The RE of 20 Gy was found to be identical for PB in 25 pulses of 0.80 Gy each h and CLDR delivered at 0.80 Gy/h for any alpha/beta value and for a repair half-time > 0.75 h. When normal tissue repair half-times are assumed to be longer than tumor repair half-times, normal tissue sparing can be obtained, within the restriction of a fixed overall treatment time, with higher dose per pulse and longer period time (time elapsed between start of pulse n and start of pulse n + 1). An optimum relative normal tissue sparing larger than 10% was found with 4 pulses of 5 Gy every 8 h. Hence, a therapeutic gain might be obtained when changing from CLDR to PB by adjusting the physical dose in such a way that the biological dose on the tumor is maintained. The normal tissue-sparing phenomenon can be explained by an increase in RE with longer period time for tissue with high alpha/beta ratio and fast or intermediate repair half-time, and the RE for tissue with low alpha/beta ratio and long repair half-time remains almost constant. CONCLUSION Within the benchmark of the LQ model, advantage in normal tissue-sparing is expected when matching the pulse frequency to the repair kinetics of the normal tissue exposed. A period time longer than 1 h may lead to a reduction of late normal tissue complications. This theoretical advantage emphasizes the need for better knowledge of human tissue-repair kinetics.

Hypofractionated radiation induces a decrease in cell proliferation but no histological damage to organotypic multicellular spheroids of human glioblastomas

The aim of this study was to examine the effect of radiation on glioblastoma, using an organotypic multicellular spheroid (OMS) model. Most glioblastoma cell lines are, in contrast to glioblastomas in vivo, relatively radiosensitive. This limits the value of using cell lines for studying the radiation effect of glioblastomas. The advantage of OMS is maintenance of the characteristics of the original tumour, which is lost in conventional cell cultures. OMS prepared from four glioblastomas were treated with hypofractionated radiation with a radiobiologically equivalent dose to standard radiation treatment for glioblastoma patients. After treatment, the histology as well as the cell proliferation of the OMS was examined. After radiation, a significant decrease in cell proliferation was found, although no histological damage to the OMS was observed. The modest effects of radiation on the OMS are in agreement with the limited therapeutic value of radiotherapy for glioblastoma patients. Therefore, OMS seems to be a good alternative for cell lines to study the radiobiological effect on glioblastomas.

Histopathological changes in the spinal cord after 434 MHz microwave hyperthermia in the cervical region of the rat

The time-course of thermal damage to the spinal cord was studied after hyperthermia of the cervical region in the rat. Local 434 MHz microwave heating of the spinal cord (cervical 5-thoracic 2) was obtained by using a ring-shaped applicator. Heat treatment at 42.9 degrees C (+/- 0.4 degrees C) for 38 min resulted in neurological symptoms, ranging from uncoordinated use of the forelegs to paralysis and death in 90 per cent (28/31) of the animals. Histological sectioning of the treated part of the spinal cord was performed immediately, 4, 24 h and 3, 7, 14 and 28 days after hyperthermia. Immediately and 4 h after treatment, neurons in the grey matter of the cord were affected and vacuolization was observed in the white matter. In animals with paralysis 1 day after treatment, we noticed neuronal degeneration, myelin pallor and sometimes haemorrhagic foci in white and grey matter. As a reaction to the thermal injury, gliosis was observed and invasion of macrophages and lymphocytes (day 3-14). Animals which had severe neurological symptoms at day 1 after hyperthermia, but had recovered completely 28 days after treatment, showed focal scar formation and demyelination in the spinal cord. The observed neurological complications correlated well with the localization of the observed histopathological changes in the cervical spinal cord.

Additive cytotoxic effect of cisplatin and X-irradiation on human glioma cell cultures derived from biopsy-tissue

Purpose: Investigation of the in vitro cytotoxic effect of X-rays, either alone or combined with cisplatin on early passage cell cultures derived from human glioblastoma multiforme biopsy tissue. Materials and methods: Fresh tumour specimens from four patients were processed to cell cultures. The U373 glioma cell line was used as a reference. Early passage cell cultures were X-irradiated (0–8 Gy) either alone or in combination with cisplatin (0.5–1 μg/ml). Cell survival was determined by either clonogenic assay or the colorimetric MTT assay. Survival curves were generated and mathematically analysed using the linear quadratic model, to obtain the radiosensitivity parameters α, β, and SF2, i.e., the Surviving Fraction after 2 Gy. Results: Two patient-derived glioma cell cultures and the U373 cell line showed rather high SF2 values of 0.61–0.72 in the clonogenic assay, indicating relative high radiation resistance. Cisplatin alone (1 μg/ml) reduced cell survival by 10–30% (n=4). When combined with irradiation, a clear additive cytotoxic effect of cisplatin was demonstrated by the unaltered value of the α-parameter for reproductive cell death. Conclusion: Cisplatin exerted an additive rather than radiosensitising cytotoxic effect in uncharacterised patient derived glioma cell cultures.

Neurological observations after local irradiation and hyperthermia of rat lumbosacral spinal cord

PURPOSE Investigation of the effects of hyperthermia on the radiation response of rat lumbosacral spinal cord with respect to: (a) incidence of paralysis, (b) latency, (c) histopathology, and (d) tumor induction. METHODS AND MATERIALS Rat lumbosacral spinal cord with the cauda equina was single-dose irradiated with 15 to 32 Gy of x-rays. Hyperthermia for 30 min at a spinal cord temperature of 41.1, 42.3, and 42.6 +/- 0.4 degrees C was applied 5 to 10 min after irradiation by means of a 434 MHz microwave applicator. Animals were observed for 21 months while recording myelopathy and development of tumors. RESULTS The latent period for hind leg paralysis decreased with increasing radiation dose from 359 +/- 31 days (n = 9) after 20 Gy to 200 +/- 4 days (n = 5) after 32 Gy. Hyperthermia enhanced the radiation response of the lumbosacral spinal cord as evidenced by shortening of the latent period for paralysis and a decrease in the biological effective dose. After 20 Gy followed by 30 min 41.1 degrees C, latency was diminished to 214 +/- 16 days (n = 7, p < 0.001 vs. 20 Gy alone). The ED50 was 21.1 Gy, which was diminished to values between 16 and 17 Gy if radiation was followed by hyperthermia, giving a thermal enhancement ratio between 1.24 and 1.32. Histopathological examination of the spinal cord after combined treatment of x-rays and hyperthermia showed necrosis of nerve roots. Irradiation with 16, 20, 24, and 28 Gy (n = 77) alone led to tumor induction in 17 +/- 8% of the animals (pooled data). If followed by hyperthermia (n = 96), it was increased to 33 +/- 12% (p < 0.01). Most tumors induced by radiation and hyperthermia were sarcomas. CONCLUSION First, the radiation response of rat lumbosacral spinal cord was enhanced by heat. Second, latency for paralysis was shortened in the lower dose range. Third, no difference in pathology between x-rays alone or in combination with hyperthermia. Fourth, hyperthermia did increase radiation carcinogenesis.