Search

The effect of trimodality treatment consisting of hyperthermia, cisplatin and radiation was investigated in two cell lines with different sensitivities to cisplatin. Hyperthermia treatment was performed for 1 h at 41 degrees C and 43 degrees C in order to compare the effects of the two temperatures. Clonogenic assays were performed with cisplatin-sensitive SiHa human cervical carcinoma and cisplatin-resistant SW-1573 human lung carcinoma cell lines. Cells were treated with various combinations of hyperthermia, cisplatin and radiation. Radiation was performed after 1 h of simultaneous hyperthermia and cisplatin treatment. Cisplatin exposure was for 1 h or continuous without refreshment of the cisplatin-containing medium. SiHa cells were more sensitive to cisplatin than SW-1573 cells. Hyperthermia at 41 degrees C decreased survival in SW-1573 cells but was not cytotoxic in SiHa cells. Hyperthermia at 43 degrees C decreased survival dramatically in both cell lines with SiHa being the most sensitive. The addition of hyperthermia at 41 degrees C and 43 degrees C to cisplatin treatment led to enhanced cell kill in both cell lines compared with cisplatin alone. Radiosensitization was observed after continuous but not after 1 h of cisplatin treatment. Hyperthermia at 43 degrees C increased radiosensitivity whereas hyperthermia at 41 degrees C did not. A combination of 41 degrees C hyperthermia with continuous cisplatin treatment had an additive effect on SW-1573 cells but enhanced cisplatin radiosensitivity of SiHa cells. In SW-1573 cells trimodality treatment using 43 degrees C hyperthermia enhances cisplatin radiosensitivity. We conclude that hyperthermia at 43 degrees C enhances cisplatin-induced radiosensitization in both cisplatin-sensitive and -resistant cell lines. Hyperthermia at 41 degrees C was also able to increase cisplatin-induced radiosensitivity but only in the cisplatin-sensitive SiHa cell line.

Irradiation of murine subcutaneous stroma before implantation of tumor cells leads to retarded tumor growth. This effect is called Tumor Bed Effect (TBE) and can be used to assess the sensitivity of stromal tissue to radiation. We tested the ability of stromal tissue to recover from X-ray-induced damage as a function of the time interval between X-irradiation and implantation of tumor cells over a period of 195 days. We also assessed the effects of a second test treatment of X-irradiation before implantation to assess residual damage by the first radiation treatment. The tumor bed effect in C57Bl10xDBA2 mice observed after X-ray treatment and implantation of M8013 cells (from a transplantable mouse mammary carcinoma) declines with the time that elapses between X-rays and implantation. Implantation of tumor cells 195 days after initial irradiation of 10 or 20 Gy resulted in a considerably smaller TBE. The half-time of the decay is estimated as about 50 days. The extent of the recovery was then tested in two-fraction experiments, with radiation fractions separated by intervals of 30 or 180 days. In the experiment with re-irradiation at an interval of 30 days after the first radiation dose of 20 Gy hardly any recovery was observed, whereas at an interval of 180 days a considerable recovery was observed. We presume that the recovery in TBE that was observed a long time after the irradiation results from a proliferative stimulus to endothelial cells which takes place during the post-irradiation period. The proliferative response leads to cell death of the X-ray damaged endothelial cells and thereafter these are replaced by healthy cells.

Single high dose rate irradiation of 4 Gy in SW-1573 cells, derived from non-small cell lung cancer, led to increased activities of deoxycytidine kinase (dCK) and thymidine kinase 1 and 2 (TK1 and 2). The activity of dCK increased by approximately 30% between 1 and 5 h after irradiation, after which the activity returned to the level of control cells by 8 h after irradiation. TK1 activity also increased by 30-50% between 1 and 6 h after irradiation. The decline to normal levels of dCK concurred with a further increase in the activity of TK1, 8 h after irradiation. TK2 activity was below control levels during the first 4 h after irradiation but rose 3-fold at 8 and 16 h after treatment. The activities of TK1 and TK2 had returned to approximate control levels 24 h after irradiation. The observation that mitochondrial TK2 activity increased to a very high level after irradiation may indicate that the activity of this enzyme is not only important for the damage to mitochondrial DNA, the increased activity may also be instrumental for repair of damage to nuclear DNA. We presume that the increase in activity of TK1 after irradiation is limited to cells in S-phase. Recruitment of cells into S-phase, to replace cells killed by irradiation, is probably too slow to offer an explanation for the enhanced activity of TK1 8 h after irradiation. The increase in activity of both dCK, and TK1 and 2 might be involved in an adaptive response of the cells to radiation by facilitation of DNA repair. The expression of protein kinase C (PKC) decreased during the first 5 h after irradiation. At 5 h after irradiation the level of expression had decreased by >50%. The decrease in PKC expression is concurrent with the increase in dCK activity. This suggests a role of PKC in the signal transduction from DNA damage to the increase in activity of enzymes instrumental in DNA repair.

Background: Radiation is an effective anti-cancer therapy but leads to severe late radiation toxicity in 5%-10% of patients. Assuming that genetic susceptibility impacts this risk, we hypothesized that the cellular response of normal tissue to X-rays could discriminate patients with and without late radiation toxicity., Methods and Findings: Prostate carcinoma patients without evidence of cancer 2 y after curative radiotherapy were recruited in the study. Blood samples of 21 patients with severe late complications from radiation and 17 patients without symptoms were collected. Stimulated peripheral lymphocytes were mock-irradiated or irradiated with 2-Gy X-rays. The 24-h radiation response was analyzed by gene expression profiling and used for classification. Classification was performed either on the expression of separate genes or, to augment the classification power, on gene sets consisting of genes grouped together based on function or cellular colocalization.X-ray irradiation altered the expression of radio-responsive genes in both groups. This response was variable across individuals, and the expression of the most significant radio-responsive genes was unlinked to radiation toxicity. The classifier based on the radiation response of separate genes correctly classified 63% of the patients. The classifier based on affected gene sets improved correct classification to 86%, although on the individual level only 21/38 (55%) patients were classified with high certainty. The majority of the discriminative genes and gene sets belonged to the ubiquitin, apoptosis, and stress signaling networks. The apoptotic response appeared more pronounced in patients that did not develop toxicity. In an independent set of 12 patients, the toxicity status of eight was predicted correctly by the gene set classifier., Conclusions: Gene expression profiling succeeded to some extent in discriminating groups of patients with and without severe late radiotherapy toxicity. Moreover, the discriminative power was enhanced by assessment of functionally or structurally related gene sets. While prediction of individual response requires improvement, this study is a step forward in predicting susceptibility to late radiation toxicity.

Radiation to the head-neck region may damage the thyroid gland, leading to hypothyroidism or thyroid carcinoma. Outcomes of radiation protection by lowering plasma thyroid-stimulating hormone (TSH) have thus far been ambiguous. Our aim was to evaluate the radioprotective effect of inhibiting the thyroid gland's activity during x-radiation. For this purpose, of 80 5-week old Wistar rats, 64 received cervical irradiation with 15 Gy (single dose). During irradiation, endocrine intervention was done, using thyroxine (T(4)), T(4) plus iodine, or iodine alone compared to placebo. During the endocrine interventions and follow-up, TSH and T(4) concentrations were measured periodically. Histologic examination of thyroid, pituitary gland, or the hypothalamus and any suspect lymph nodes, lungs, and liver was performed after 6 and 54 weeks. It was found that during the endocrine intervention, plasma levels of TSH were lower in rats given T(4) and higher in rats given iodine. After 6 and 54 weeks, no significant reduction in hypothyroidism or thyroid carcinoma was found between the different groups of rats given any endocrine intervention or no intervention. In conclusion, the administration of T(4), iodine or the combination during x-irradiation does not protect against radiation-induced thyroid damage.

Purpose: Cisplatin was found to radiosensitize SW-1573 cells by inhibition of PLDR. Therefore, it was investigated whether cisplatin combined with gamma-radiation leads to an increase in the number of chromosomal aberrations or apoptotic cells compared with radiation alone., Methods: Confluent cultures of the human lung carcinoma cell line SW-1573 were treated with 1 microM cisplatin for 1 h, 4 Gy gamma-radiation, or a combination of both. Cell survival was studied by the clonogenic assay. Aberrations were analysed by FISH in prematurely condensed chromosomes (PCC) and the induction of apoptosis by counting fragmented nuclei., Results: A radiosensitizing effect of cisplatin on cell survival was observed if time for PLDR was allowed. An increased number of chromosomal fragments were observed immediately after irradiation compared with 24 h after irradiation whereas color junctions are only formed 24 h after irradiation. No increase in chromosomal aberrations was found after combined treatment, but a significantly enhanced number of fragmented nuclei were observed when confluent cultures were replated after allowing PLDR., Conclusion: The inhibition of PLDR by cisplatin in delayed plated SW-1573 cells did not increase chromosomal aberrations, but increased the induction of apoptosis.

Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo "unlimited" division. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retains the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 1-3 weeks. Colonies are fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope. A method for the analysis of radiation dose-survival curves is included.

Purpose: Mismatch repair (MMR) proficiency has been reported to either increase or decrease radioenhancement by 24-h incubations with gemcitabine. This study aimed to establish the importance of MMR for radioenhancement by gemcitabine after short-exposure, high-dose treatment and long-exposure, low-dose treatment., Methods and Materials: Survival of MMR-deficient HCT116 and MMR-proficient HCT116 + 3 cells was analyzed by clonogenic assays. Mild, equitoxic gemcitabine treatments (4 h, 0.1 microM vs. 24 h, 6 nM) were combined with gamma-irradiation to determine the radioenhancement with or without recovery. Gemcitabine metabolism and cell-cycle effects were evaluated by high-performance liquid chromatography analysis and bivariate flow cytometry., Results: Radioenhancement after 4 h of 0.1 microM of gemcitabine was similar in both cell lines, but the radioenhancement after 24 h of 6 nM of gemcitabine was reduced in MMR-proficient cells. No significant differences between both cell lines were observed in the gemcitabine metabolism or cell-cycle effects after these treatments. Gemcitabine radioenhancement after recovery was also lower in MMR-proficient cells than in MMR-deficient cells., Conclusion: Mismatch repair proficiency decreases radioenhancement by long incubations of gemcitabine but does not affect radioenhancement by short exposures to a clinically relevant gemcitabine dose. Our data suggest that MMR contributes to the recovery from gemcitabine treatment.

The cytotoxicity of cisplatin, applied alone or in combination with hyperthermia, to mouse mammary adenocarcinoma cells (M8013S) was studied with the cells either treated in medium [Eagle's minimum essential medium (MEM), supplemented with 10% foetal bovine serum, 100 IU/ml penicillin, 200 mM glutamine and 0.35 g/l NaHCO(3)] or in Hank's balanced salt solution (HBSS) without serum. To study the role of platinum uptake by the M8013 cells in cytotoxicity, uptake was determined under conditions similar to those used in the survival experiments. Our results show that hyperthermia (30 min at 43 degrees C) enhances the toxicity of cisplatin. Enhanced toxicity by heat treatment is not observed with the cells in HBSS. The thermal enhancement of effects of cisplatin to cells in MEM with serum is clearly related to an enhanced uptake of cisplatin. A novel observation is that in order to obtain a considerable thermal enhancement of the cytotoxic effect of cisplatin, the exposure of the cells to the drug is required not only during the hyperthermic treatment but the exposure has to be maintained for at least 2 h after hyperthermia. These same conditions are also required for enhanced uptake of cisplatin. The present results may indicate that cisplatin has to be bound to some serum component in order to facilitate an 'active' uptake. Hyperthermia leads to a considerable intracellular accumulation of cisplatin, relative to the extracellular concentration. This accumulation takes place during exposure to cisplatin but after heat treatment.

The role of EGR-1 in potentially lethal damage repair (PLDR) was studied. Induction of the early response protein EGR-1 and survival after ionizing radiation of two human tumour cell lines after culturing for 48 h in serum-deprived medium was investigated. The glioblastoma cell line (Gli-6) and a lung carcinoma cell line (SW-1573) were selected as these cell lines differ considerably in the degree of PLD repair after radiation. In both cell lines induction of EGR-1 protein was observed between 30-120 min after treatment with 10 Gy in serum-deprived cultures. In cells growing in medium with normal serum no induction of EGR-1 was observed. No difference in EGR-1 expression levels between the two cell lines was detected. Linear-Quadratic analysis of the survival curves showed a much larger difference between the values of alpha after immediate and delayed plated cells of the cultures in normal serum as compared to cells cultured in serum-deprived medium. The cells cultured in serum-deprived medium showed much larger difference between the values of beta. This indicates that induction of EGR-1 is correlated with a reduction of repair of lethal lesions (PLDRalpha) and with an increase of repair of sublethal lesions (PLDRbeta).

The radiosensitizing potential of gemcitabine (2',2'-difluoro-2'-deoxycytidine) was studied in combination with pulsed low dose-rate irradiation. The experiments were carried out with a human lung carcinoma cell line SW1573. These were irradiated at pulsed low dose rate (p-LDR); the average dose rate was 1 Gy/h. In the experiments with gemcitabine, this drug was applied for 24 h at a concentration of 10 nM prior to irradiation. The response of the cells to treatment was tested by using the standard clonogenic assay. Next to the cell-killing effects, damage to chromosomes was also assayed by using by whole chromosome Fluorescent In Situ Hybridization (FISH). Damage in chromosomes 2 and 18 was visualized by whole chromosome FISH and scored according to the PAINT method. A clear enhancement of the effects of radiation on cell survival was observed by preincubation of the cells with gemcitabine. The enhancement factor obtained from the p-LDR data was 1.7, which is much lower than the enhancement factor of 2.9 at high-dose rate. We did not observe a consistent increase in color junctions concomitant with radiosensitization. In chromosome 2, a small increase, and in chromosome 18, a decrease, in the number of color junctions was observed after radiation combined with gemcitabine compared to irradiation alone. These differences were not statistically significant. However, for the (unstable) acentric chromosome fragments from both chromosomes, significant changes were observed: In the case of chromosome 2, an increase, and in the case of chromosome 18, a decrease. So these results indicate that gemcitabine has no large and consistent effect on the repair of genomic lesions that induce secondary chromosome breaks. Although it is clear that gemcitabine-induced radiosensitization can be expected when it is combined with brachytherapy, as with radiation at a high-dose rate, the mechanism of radiosensitization is so far not evident, and further experiments will be needed to elucidate this.

Background and Purpose: To determine the cytotoxicity of, and radioenhancement by, gemcitabine on a glioma cell line grown as a monolayer and as spheroid cultures., Material and Methods: We used a human glioma cell line, Gli-6, which originated from a biopsy specimen of a patient with a glioblastoma multiforme. Spheroids of Gli-6 were prepared by seeding a single cell suspension on agarose-coated Petri dishes. Clonogenic and growth delay assays were used to determine radio-chemosensitivity of monolayer cultures. The growth delay assay was used to determine that of Gli-6 spheroid cultures., Results: Spheroid cultures were found to be more resistant to irradiation with/or without gemcitabine than monolayer cultures. Whereas gemcitabine significantly enhances the radiation effect of exponentially growing Gli-6 monolayer cultures at minimal cytotoxic concentrations (10 nM, 24 h), no enhancement was seen in confluent monolayer cultures and in large spheroids at the same concentration. In small spheroids no enhancement was observed at a low-dose gemcitabine (10 nM for 24 h), but an enhancement was observed at higher concentrations (100 nM for 24 h)., Conclusion: Gemcitabine can lead to enhancement of the effects of X-irradiation in both monolayer as spheroid glioblastoma cultures. The lack of enhancement in confluent monolayer cultures supports the view that cell cycle distribution of cells is important in radiosensitisation by gemcitabine

Purpose: To determine whether measurement of colour junctions by fluorescent in situ hybridisation (FISH) can predict radiosensitisation of gemcitabine (2'-2'-difluorodeoxycytidine)., Methods: Human lung carcinoma cells (SW-1573) were irradiated with X-rays with or without incubation of 10 n M gemcitabine for 24 h. Cell survival was determined with clonogenic assay. Colour junctions were measured by whole chromosome FISH of chromosomes 2 and 18 and were scored according to the PAINT method., Results: A clear radiosensitisation by gemcitabine was observed on cell survival. A significant decrease in the number of colour junctions was observed after gemcitabine treatment compared with radiation treatment alone. The correlation between colour junction induction and cell survival was high for both with and without gemcitabine, but the gemcitabine-sensitised curve did not coincide with the non-sensitised curve., Conclusions: Gemcitabine-induced radiosensitisation is not predicted by induction of colour junctions in cultured SW-1573 cells. This reduces the clinical applicability of this predictive assay for radiotherapy in combination with gemcitabine.

A 24-year-old patient was admitted to the intensive care unit because he had swallowed about 20 cocaine packets 48 hours before admission; he also complained of abdominal cramps, perspiration and dizziness. The patient reported that he had not defecated since swallowing the packets. Abdominal X-ray revealed only coprotasis. On conservative therapy with bowel irrigation, two packets were eliminated, after which a second abdominal X-ray revealed several cocaine packets in the colon. Four days afterwards, the cocaine packets in the colon had not progressed despite adequate bowel irrigation. The patient now showed signs of mild cocaine intoxication (hallucinations and tachycardia). It was therefore decided to perform a laparotomy. Via a sigmoidotomy, 7 intact packets were removed; another 3 had already ruptured and were empty. The rupture of 3 cocaine packets in this patient was probably not fatal because of the sedation with midazolam and because the patient had diarrhoea as a result of the extensive irrigation, so that a large proportion of the cocaine was probably not absorbed. This case also shows that the presence of foreign bodies cannot be established adequately by an abdominal X-ray if there is coprostasis.

Purpose: To determine cross-resistance to anti-tumor treatments in 2',2'difluorodeoxycytidine (dFdC, gemcitabine)-resistant human tumor cells., Methods and Materials: Human lung carcinoma cells SW-1573 (SWp) were made resistant to dFdC (SWg). Sensitivity to cisplatin (cDDP), paclitaxel, 5-fluorouracil (5-FU), methotrexate (MTX), cytarabine (ara-C), and dFdC was measured by a proliferation assay. Radiosensitivity and radioenhancement by dFdC of this cell panel and the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000 were determined by clonogenic assay. Bivariate flowcytometry was performed to study cell cycle changes., Results: In the SWg, a complete deoxycytidine kinase (dCK) deficiency was found on mRNA and protein level. This was accompanied by a 10-fold decrease in dCK activity which resulted in the >1000-fold resistance to dFdC. Sensitivity to other anti-tumor drugs was not altered, except for ara-C (>100-fold resistance). Radiosensitivity was not altered in the dFdC-resistant cell lines SWg and AG6000. High concentrations (50-100 microM dFdC) induced radioenhancement in the dFdC-resistant cell lines similar to the radioenhancement obtained at lower concentrations (10 nM dFdC) in the parental lines. An early S-phase arrest was found in all cell lines after dFdC treatment where radioenhancement was achieved., Conclusions: In the dFdC-resistant lung tumor cell line SWg, the deficiency in dCK is related to the resistance to dFdC and ara-C. No cross-resistance was observed to other anti-tumor drugs used for the treatment in lung cancer. Sensitivity to ionizing radiation was not altered in two different dFdC-resistant cell lines. Resistance to dFdC does not eliminate the ability of dFdC to sensitize cells to radiation.

After pulsed low dose rate irradiation the activities of deoxycytidine kinase and thymidine kinase 1 and 2 were increased 1.5-2-fold 6 h after treatment. Twenty-four hours after treatment the activities of these enzymes had returned to control levels. We presume that the increase of enzyme activities is part of an adaptive response to irradiation and that this increase could be an explanation for the increased survival in the initial part of the SW-1573 cell survival curve. The observation that not only S-phase specific thymidine kinase 1 but also mitochondrial thymidine kinase 2 increases, implies that both these enzymes play a role in an adaptive response of cells to irradiation.

Bromodeoxyuridine (BrdUrd)-induced radiosensitization of two different tumour cell lines was compared at equal levels of thymidine replacement. Human lung carcinoma cells (SW-1573) and human colorectal carcinoma cells (RKO) were grown for 48 h in the presence of respectively 1 microM BrdUrd and 4 microM of BrdUrd in order to obtain equal levels of BrdUrd into the DNA. In SW cells the level of thymidine replacement by BrdUrd was 6.7+/-0.5% and in RKO cells this was 7.1+/-0.8. Cell survival after irradiation with single doses up to 8 Gy, was determined with clonogenic assay. The magnitude of BrdUrd-induced radiosensitization was determined by analyzing radiation-dose survival curves with the linear-quadratic formula [S(D)/S(0)=exp-(alphaD+betaD2)]. In the SW cells BrdUrd radiosensitization led to a significant increase of the linear parameter, alpha, determining the initial slope of the survival curves, by a factor of about 2. In the RKO cells BrdUrd increased the value of alpha by a factor 1.4. This suggests that repair of potentially lethal damage (PLD) is inhibited. In both cell lines the quadratic term, beta, strongly influencing the high dose region of the survival curves, was not altered by sensitization by BrdUrd. The increase of alpha is of interest for clinical applications as BrdUrd sensitizes tumour cells after low doses of radiation.

Purpose: To investigate the schedule-dependency of 2',2'difluorodeoxycytidine (dFdC, Gemcitabine) combined with hyperthermia (HT), in vitro as well as in vivo., Materials and Methods: Rat R-1 rhabdomyosarcoma cells were treated with various concentrations of dFdC for 70 min, 4 h and 24 h. After various time intervals HT (60 min at 43 degrees C) was applied. Cell survival was determined by clonogenic assays. Female Wag/Rij rats bearing R-1 tumours on the hind limbs were treated with dFdC (20 mg/kg), with locally applied HT (60 min at 43 degrees C) or with a combined treatment using different time intervals (0, 24 and 48 h). Tumour growth delay (TGD) and normal tissue toxicity were assessed., Results: With dFdC alone, significant cytotoxicity was observed after a 24 h-exposure. Except for the 24 h-exposure, HT reduced the cytotoxicity of dFdC in simultaneous applications. An enhanced cytotoxic effect was found when HT was applied 20 h after a 4 h-incubation with dFdC. In vivo, HT applied 48 h after dFdC-administration resulted in potentiation of the effect of dFdC with respect to TGD without an increase in toxicity., Conclusions: The efficacy of dFdC combined with HT is schedule-dependent both in vitro and in vivo. The addition of HT enhances the effectiveness of dFdC in the R-1 tumour model.

Cell-cycle checkpoints are thought to govern the cellular response to external stimuli. The involvement of the p53 tumour-suppressor protein and the retinoblastoma protein (pRb) in the cell-cycle checkpoint in G1 phase is well established. However, little is known about the importance of these G1 checkpoint regulators in hyperthermia-induced cytotoxicity. Such information is relevant because of the clinical application of hyperthermia in combination with chemotherapy or with radiotherapy. The effects of p53 or pRb inactivation were studied in a well-established isogenic system using the human colorectal carcinoma cell line (RKO). The cells were treated with clinically relevant heat doses (60 min at 40-43 degrees C). Cell survival, cell-cycle redistribution and induction of apoptosis were investigated. Survival of the p53-inactivated transfectants was higher than that of the wild-type p53 cells. The pRb-inactivated transfectants showed an intermediate sensitivity to hyperthermia. All transfectants showed G2 arrest after hyperthermia and the appearance of a sub-G1 population. The induction of apoptosis was inhibited in p53-inactivated and pRb-inactivated transfectants. These results suggest that p53 and/or pRb status may be an important determinant of the clinical response to hyperthermia.

Bacterial sepsis remains a frequent complication after liver transplantation. We previously reported the results of a pilot study that suggested that low expression of HLA-DR on monocytes is a predictive marker for the occurrence of sepsis. We have studied the value of this marker in an additional cohort of patients, and have analyzed the relation of HLA-DR expression with the use of immunosuppressive agents. 20 adult liver transplantation patients were prospectively monitored during the first 4 weeks after transplantation. All were treated according to standard protocols. The percentage of monocytes expressing HLA-DR was measured by flow cytometry. In addition, the effects of incubation of monocytes with prednisolone in vitro on the expression of HLA-DR was determined in 7 healthy volunteers. Seven patients developed bacterial sepsis after a median 15 (range 10-20) days after transplantation. HLA-DR expression was significantly lower in these patients on days 7, 14, 21, and 28 after transplantation compared with non-septic patients. The percentage of HLA-DR positive monocytes was 30% or less, 3 (1-8) days before onset of sepsis. On day 7 after transplantation, HLA-DR expression on 50% or less of monocytes had a positive predictive value for sepsis of 71%, whereas the negative predictive value was 85%. Patients who developed sepsis received significantly more prednisolone. Incubation with prednisolone in vitro lowered the expression of HLA-DR in a dose-dependent manner. We conclude that low HLA-DR expression on monocytes is a marker for a high risk of subsequent sepsis in liver transplantation patients. This high risk may be (at least partly) related to the dose of prednisolone.

The clinical application of hyperthermia in the treatment of oncological patients is based on a strong biological rationale: hyperthermia is an effective cell-killing agent, the effects of which can be induced selectively in tumour tissue. The mechanism of action of hyperthermia is complementary to that of radiotherapy (and chemotherapy). Furthermore, hyperthermia enhances the effects of radiotherapy (and of chemotherapy). In the approximately 20 years that hyperthermia was clinically investigated, much progress was made in the techniques of application. The clinical results achieved so far show that the therapeutic gain by adding hyperthermia can indeed be substantial.

The levels of TNF, IL-1 and IL-6 in circulating blood female WAG/Ry rats were assessed in relation to treatment with localized hyperthermia of the right hind leg or with whole-body hyperthermia (WBH). After a localized treatment for 30 min at 43 or 44 degrees C no detectable increase in levels of IL-6 or TNF was obtained. Hyperthermia for 30 min at 45 degrees C led to an elevated level of IL-6 of 19.4 +/- 5.2 U/ml above the control level of 24 h after treatment. Levels of IL-1 were never higher than those in control animals that received only anaesthesia. Anaesthesia induced a peak level of approximately 131 U/ml IL-1 6 h after treatment. Serum levels of IL-1 and IL-6 are enhanced after WBH. IL-1 reaches a peak level already during WBH about 15 after reaching 41.5 degrees C. IL-6 levels were not enhanced during WBH but 1 h after WBH a clear peak was observed. Anaesthesia with sham WBH did not lead to enhanced IL-6 levels but enhanced IL-1 levels were clearly detected. We did not detect TNF in any sample after WBH. It is concluded from the present results that IL-6 is not induced by a 'standard' treatment of localized hyperthermia as used in oncotherapy (i.e. 60 min at 43 degrees C) to such a high level locally that this is reflected in increased levels in circulating blood. WBH at clinically relevant temperatures leads to enhanced levels of IL-1 and IL-6. The difference in IL-6 response after WBH or localized hyperthermia probably is related to the fact that in WBH also the bone marrow is treated. This may lead to stimulation of this important stem cell compartment of the peripheral blood. The sequence of appearance of IL-1 and IL-6 after hyperthermia is akin to the sequence in an inflammatory response. However, the experiments with sham treatment show that IL-1 may appear in the circulating blood not followed by IL-6. These results indicate that enhanced IL-1 levels may reflect a stress reaction of the animal related to the (sham) treatment. Enhanced levels of IL-1 after WBH correlate with enhanced levels of ACTH in the circulating blood.

The support of the Michigan State Medical Society is appreciated as we implement the Michigan Children's Immunization Registry. I look forward to collaborating on the registry and other initiatives to quickly raise Michigan's immunization rate for two-year-olds from 64%, the lowest in the nation, to 100%. This is a responsibility that we all bear, in both the public and private sectors. To say that our national status is a professional embarrassment is to understate the seriousness of this circumstance. We must fulfill our obligation to fully immunize all children by age two.

The right part of the median lobe of the liver of female Wistar rats was irradiated, 12.5 or 25 Gy, at a field size of 15 x 20 mm. The central part of the irradiated liver lobe was fixed and used for the estimation of the collagen protein ratio by means of the Sirius Red-Fast Green extraction method, immediately, 8, 16 or 32 weeks after irradiation. No significant increase in collagen content could be demonstrated in this time range, both after irradiation at 12.5 Gy and at 25 Gy. Partial hepatectomy according to Higgins led to rapid regrowth of the remaining liver lobes. The right lobe grew out rapidly to replace the median lobe. Two days after partial hepatectomy the right lobe was irradiated at the same field size. Measurement of the collagen protein ratio in this experiment did not show a significant increase 8, 16 or 32 weeks after irradiation. However, the 25 Gy group did not survive long enough to obtain data at 16 or 32 weeks. The animals in this latter experiment suffered from ascites before dying. Experimentally induced cholestasis was obtained by ligation and partial resection of the common bile duct. After two weeks of cholestasis the bile flow was restored by Roux-en-Y choledochojejunostomy. The effect of irradiation 2 days after repair surgery was studied. Without irradiation the collagen protein ratio is increased. Irradiation of the right part of the median lobe led to a relatively enhanced collagen content in this lobe. Our results indicate that radiation itself does not lead to a significantly enhanced degree of fibrosis in the liver. However when an increase in collagen content was induced by cholestasis, the partial "dilution" of enhanced fibrosis as a result of proliferation of liver parenchyma cells following repair surgery was inhibited by irradiation.

Difluorodeoxycytidine (dFdCyd, gemcitabine) was tested for cytotoxicity in cultured human lung-cancer cells SW1573 in combination with 1 hr hyperthermia at 43 degrees C. The results show that the timing is extremely important. Simultaneous application led to decreased cytotoxicity, whereas an interval of 20 or 24 hr between exposure to dFdCyd and hyperthermia led to enhanced cell killing. The decrease in cytotoxicity after simultaneous hyperthermia and dFdCyd probably results from inhibition of activation of dFdCyd to the triphosphate metabolite. The enhanced cytotoxicity in sequential application of dFdCyd and hyperthermia is not caused by accumulation of cells in a sensitive cell-cycle phase. Our results show that the G1 phase becomes relatively abundant 20 hr after exposure to 0.1 microM dFdCyd, approximately 48% versus 31% in control cultures. Presumably, inhibition by hyperthermia of repair of DNA damage plays a role. Our results confirm earlier data with regard to reutilization of activated dFdCyd at high cell density. dFdCyd was clearly more toxic to SW1573 cells at 4 x 10(5) cells per dish than at 400 cells per dish. This reutilization of activated drug is evidently not a restricted property of a particular cell line and may add to the value of the drug in cancer treatment.

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.

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.

The influence of cisplatin on the sensitivity of the rat sciatic nerve to local hyperthermia was investigated. Rats received 1.7 mg/kg cisplatin i.p., twice a week for 6 weeks, up to a cumulative dose of 20.4 mg/kg. After termination of cisplatin treatment, a 5 mm segment of the nerve was locally heated at a temperature of 45 degrees C (5-30 min). Loss of motor function was assessed by means of the toe-spreading test, 24 h post heating. The calculated ED50 for control nerves was significantly (p < 0.01) larger than the ED50 for cisplatin treated rats; 16.3 +/- 1.1 min vs. 10.9 +/- 1.1 min. This indicates that nerves from cisplatin treated rats were more sensitive to heat than nerves from control rats (dose modifying factor = 1.5 +/- 0.2). Histopathological investigation of nerves after heat alone or after heat preceded by cisplatin confirmed these differences and showed that edema, vascular damage and axonal degenerative changes of axons and myelin sheaths occurred at lower heat doses when compared to control nerves. Recovery studies showed that cisplatin treatment before hyperthermia caused a delay in recovery from motor function loss of about 6 days. Cisplatin treatment after hyperthermia had no influence on recovery from motor function loss.

The influence of local hyperthermia on the uptake of cisplatin in the rat cervical spinal cord was investigated. After single intraperitoneal or intravenous injection of cisplatin (5 mg/kg body weight), the spinal cord region cervical 5-thoracic 2 was heated for 60 min at mean (S.D.) 41.2 (0.4) degrees C or 40 min 42.4 (0.3) degrees C using a 434 MHz microwave heating device. One day after treatment with either hyperthermia alone, cisplatin alone or the combination, none of the animals expressed neurological symptoms. The spinal cord was dissected and platinum levels were measured by flameless atomic absorption spectroscopy. No difference was found in uptake of platinum in the spinal cord between control- and heat treated animals. In a second series of experiments, the spinal cord was heated for 30-60 min. during a 2 h infusion of cisplatin. One day after treatment at 42.3 degrees C for 60 min, neither motor nor sensory functions were affected and platinum levels did not differ significantly between control and treated animals. Also, platinum levels measured in the spinal cord immediately after cisplatin infusion were not influenced by heat treatment at 42.1 or 43.0 degrees C for 30 min. However, after a heat dose of 60 min 43 degrees C, cisplatin uptake was significantly increased (P less than 0.001) by a factor of 2.8 (1.3). The data demonstrate that mild hyperthermia has no effect on the uptake of cisplatin in the spinal cord, while an injurious heat dose leads to a significant increase in cisplatin uptake. The present findings indicate that, in case of treatment of tumours of the central nervous system with hyperthermia and cisplatin, a treatment which might be toxic for the tumour is well tolerated by the normal nervous tissue.

The right hind legs of mice wee heated in a waterbath at 44 degrees C. The animals were killed at various time intervals after exposure. Tissue damage was studied histologically. After 15 min exposure light and reversible changes were seen including oedema and some neutrophilic inflammatory infiltration immediately after treatment. After 30 min exposure an extensive inflammatory infiltrate and strong oedema were seen during the first days after treatment. Adjacent to areas in the skin with strong oedema extensive muscular necrosis was observed. The muscular tissue regenerated almost completely in three weeks. After 60 min heating the histological picture was dominated by massive necrosis of muscle, subcutaneous fat tissue and skin during the first week after treatment followed by local ulceration. From about the 7th day after treatment regeneration of the epithelium started and granulation tissue could be observed in the margin of the ulceration. Healing of the skin was completed at about day 21 after treatment. Our results indicate that heat induced tissue damage in some tissues is due to a direct effect on the cells composing the tissue (e.g., fat cells in subcutaneous fat) but that, in most other tissues (e.g., muscle and skin) it is a consequence of damage to the vasculature.

After a 500 mus laser flash a 120 mus phase in the decay of delayed fluorescence is visible under a variety of circumstances in spinach chloroplasts and subchloroplast particles enriched in Photosystem II prepared by means of digitonin. The level of this phase is high in the case of inhibition of oxygen evolution at the donor side of Photosystem II. Comparison with the results of Babcock and Sauer (1975) Biochim. Bio-phys. Acta 376, 329-344, indicates that their EPR signal IIf which they suppose to be due to Z+, the oxidized first secondary donor of Photosystem II, is well correlated with a large amplitude of our 120 mus phase. We explain our 120 mus phase by the intrinsic back reaction of the excited reaction center in the presence of Z+, as predicted by Van Gorkom and Donze (1973) Photochem. Photobiol. 17, 333-342. The redox state of Z+ is dependent on the internal pH of the thylakoids. The results on the effect of pH in the mus region are compared with those obtained in the ms region.

N-Acetylcysteine is currently being considered as a possible selective protector against pulmonary toxicity resulting from X-rays or chemotherapeutic treatment, but its clinical application awaits evidence that it does not interfere with the efficient killing of tumor cells. The capacity of N-acetylcysteine to protect against the antitumor activity of X-rays and of bleomycin was evaluated in a clonogenic cell-survival assay using SW-1573 human squamous lung carcinoma cells as a tumor model. Using the highest non-toxic dose of N-acetylcysteine (incubation for 2 days in the continuous presence of 10 mM) no effect on clonogenic cell killing by X-rays or bleomycin treatment could be detected, even though a twofold enhancement of endogenous glutathione was effectuated. Our data thus indicate that clinically relevant concentrations of N-acetylcysteine are incapable of protecting tumor cells against clonogenic killing by X-rays and by bleomycin.

Effects of different fractionation schedules of irradiation were studied in an experimental mammary adenocarcinoma transplanted in the back of mice. The number of fractions per day varied from one to three, keeping the daily dose constant. It was found that an increase of the number of fractions per day did not necessarily lead to a decrease in the tumour response, as might be expected for the same total dose. At a twelve-day overall time, three fractions per day did not give such a good result as one fraction per day, but at a nineteen-day overall time three fractions per day were about as good as one fraction per day; and below 45 Gy total dose, the result with three fractions per day even seemed to be better. The effects of actinomycin D administered during the irradiation treatment show that schedules with three fractions per day possibly take better advantage of repair of sublethal and potentially lethal damage than schedules with one fraction per day. Administration of misonidazole during the irradiation treatment led to a dose-modifying effect of 1.2 in one fraction per day schedules but had no significant effect in three fractions per day schedules. The present results may provide guidelines for clinical application of irradiation schedules with more than one fraction per day.

Prompt and delayed chlorophyll fluorescence have been studied in broken spinach chloroplasts at pH values down to 2.6. No direct effect of low pH on the primary charge separation in Photosystem II was observed. The irreversible inactivation of a secondary electron donor in a narrow pH range around pH 4.5 was demonstrated. At lower pH values the photooxidized form of a more primary electron donor, revealed by its efficient fluorescence quenching, was reduced with a half time of about 200 mus, 25% by another electron donor and 75% by back reaction with the reduced acceptor. The electron donation had a half time of 800 mus and was practically irreversible. The back reaction had a pH dependent half time: about 270 mus at pH 4 and increasing towards lower pH. The competition of both reactions resulted in a net efficiency of the charge separation at pH 4 of 25%, increasing towards lower pH.

The heat sensitivity and the time-temperature relationship of non-tolerant and thermotolerant M8013 cells treated at different pHs in either culture medium (including serum) or Hanks' salts solution (HBSS) were compared. The cells were growing asynchronously. Arrhenius plots for non-tolerant cells heated in culture medium pH 7.35 showed two linear parts below and above the transition temperature (Ttrans). The inactivation energies below and above Ttrans were respectively 2980 and 490 kJ/mole. With thermotolerant cells under the same conditions the inactivation energy was approximately constant over the range 42-46 degrees C at 890 kJ/mole. The cells were more sensitive to heat treatment at low pH or in HBSS. Moreover, it appeared that the expression of thermotolerance was strongly dependent on medium conditions: the thermotolerance ratio (TTR, ratio between slopes of survival curves of thermotolerant and normal cells) was much lower at low pH or in cells heated in HBSS. Generally a high TTR observed in experiment with fractioned hyperthermia at temperatures above Ttrans correlated fairly well with a high inactivation energy below Ttrans from the Arrhenius plot derived from data from experiments with the same cells that were not made thermotolerant before treatment.

The response of mouse feet to irradiation and heat was studied 90 days after a first treatment with X-rays, hyperthermia or both. Residual damage after a single dose of 20-30 Gy enhanced both the acute reaction and "late" deformity following a second treatment with radiation or hyperthermia. There was often a larger "memory" of the first radiation treatment for late deformity compared with the acute skin response, especially in the case of retreatment by hyperthermia. Prior treatment of the foot with a moderate heat dose (60 min at 44 degrees C), which by itself did not lead to deformity, had only a small effect on the response to retreatment with irradiation or heat, both with respect to the acute and "late" response. Residual damage after more severe hyperthermia (90 min at 44 degrees C) obscured the evaluation of deformity after a second treatment with radiation or hyperthermia. Feet treated with irradiation followed immediately or after 3 days by heat, show a larger "memory" when retreated with hyperthermia than with irradiation, both with regard to the acute and "late" response. Experiments using misonidazole indicated that the oxygenation status of previously treated skin (pretreatment not leading to deformity) had not changed significantly.

Lysosomes of cultured mammalian cells, derived from a transplantable murine mammary adenocarcinoma, irreversibly lose their capacity to accumulate the fluorescent dye acridine orange after hyperthermia. As acridine orange may be regaraded as a fluorescent probe of the internal pH of the lysosomes, we may conclude that the ability of lysosomes to maintain a low internal pH is destroyed by hyperthermia. The effects of hyperthermia on lysosome fluorescence and on cell survival show several similarities: in both cases hyperthermia is more effective at low pH, below pH 7.0, and CCP (carbonylcyanide-m-chlorophenylhydrazone) enhances effects at low pH, but has no clear effect at pH 8.0. This leads to the conclusion that effects on lysosomes are an important and early event in cellular injury caused by hyperthermia. The activation energy, however, obtained for the effects of hyperthermia on lysosome fluorecence is about a factor of two lower athan the activation energy reported for cell survival after hyperthermia. This suggests that the effect on lysosomes is not directly caused by hyperthermia but is triggered by some other hyperthermia-induced cellular damage.