Combination therapy: lonidamine, hyperthermia, and chemotherapy against the RIF-1 tumor in vivo.

Lonidamine enhances the cytotoxicity in vitro of several conventional antitumor drugs as well as that of hyperthermia (HT). We have investigated the possibility that such enhancement can also be demonstrated in vivo against the RIF-1 tumor system. Two assays were used to examine antitumor activity: tumor growth delay and clonogenicity of cells obtained from tumors from treated animals. We used drug (and HT) doses that by themselves did not achieve significant cell killing. The drugs whose interaction with lonidamine was tested were: cis-diamminedichloroplatinum (CDDP), mitomycin C (MMC), bleomycin, 5-fluorouracil, and nitrosourea. Of these only CDDP and MMC yielded positive data. Both assays gave essentially the same results, showing that antitumor activity reflected direct cell killing. CDDP and MCC activity was also enhanced by HT. When we combined all three modalities, however, the results of the trimodality therapies were no better than that of individual bimodality treatments. These last results suggest that lonidamine and HT have similar mechanisms, most likely inhibition of repair of DNA damage. Our data do suggest that lonidamine may have a role in multidrug therapies that include either CDDP or MMC as a component of the treatments.     

Alteration of pancreatic B-cells in Wistar rats treated with non-diabetogenic doses of cyclosporin A.

We have investigated the effect of non-immunosuppressive cyclosporin A (CS-A) doses on glucose tolerance, pancreatic insulin content, insulin content of isolated islets and insulin secretion in vitro in response to glucose. Within 12 weeks animals treated permanently with a dose of 2.5 mg CS-A/kg b.wt. developed glucose intolerance (but not hyperglycaemia) accompanied by a decrease of pancreatic insulin content due to a decrease of islet insulin content, whereas the relative B-cell volume density was not changed. Isolated islets obtained from rats treated for 4 weeks had a diminished sensitivity for glucose, whereas islets obtained from animals treated for greater than 4 weeks showed a diminished half-maximum and maximum secretion rate. Rats treated for 12 weeks with 1.25 mg CS-A/kg b.wt. developed an impaired glucose tolerance after 8 weeks accompanied by a diminished pancreatic insulin content. Despite continued treatment the pancreatic insulin content was able to increase and the glucose tolerance to normalize, indicating an adaptation of pancreatic B-cells to CS-A. The results support the theory that a potential toxic effect of cyclosporin A can be diagnosed by functional tests (e.g. insulin secretion in response to a stepwise increase of glucose) before the irreversible (e.g. morphological) alterations occur.     

Bone structure changes in hip joint endoprosthesis implantation over the course of many years. A quantitative study

The stability of implanted artificial joints is limited. One main factor for this is the change of the bone tissue near the implant. Therefore we studied femura with stem endoprostheses derived from autopsy. All specimens were cut in horizontal and longitudinal sections. X-rays were made of all sections. Then the specimens were embedded undecalcified and ground to 10 microns. The wellknown phenomenons at the bone cement border were found too. Direct bone cement contact is about 5%. The 4 studied femura show a higher porosity of cortical bone as controls. The loss of cortical bone is up to 40% after 55 months. There is a correlation between new load situation and the localisation of bone loss.     

Effects of hyperthermia on the intracellular pH and membrane potential of Chinese hamster ovary cells

The effects of hyperthermia (exposure to 41-47 degrees C) on the intracellular pH and membrane potential have been studied using Chinese hamster ovary HA-1 cells. Our goal was to determine whether intracellular pH changes or changes in membrane potential correlated with cell killing. The intracellular pH (pHi) was measured using the DMO partitioning technique. A rapid acidification of the intracellular environment was observed at all the elevated temperatures studied. The pHi reached a plateau value of approximately 6.9, and started reversing towards normal values upon prolonged exposure to heat. Similar patterns were seen for delta pH (pHi-pHo). The membrane potential difference (delta psi) was measured using the fluorescence quenching of 3,3-dipropylthio-carbocyanine, and calibrated using a 86Rb+ diffusion potential. We found that delta psi falls to zero only upon prolonged exposure to temperatures above 43 degrees C. When the external pH was changed from normal values the drop in delta psi occurred more readily. Development of thermotolerance resulted in an increase in the time required to make delta psi change by half. The changes in delta psi were shown to be irreversible. When the proton electrochemical gradient (delta mu H+) was calculated using the measured values of delta psi and delta pH, the trends observed were the same as those seen for delta psi. The changes observed for pHi can be accounted for by the changes in the pK values of the components involved in the intracellular buffering. The changes in delta psi and delta mu H+ may reflect the physical breakdown of the transmembrane H+ gradients, which may be the actual mechanical process of cell death. No correlation of cell survival with the measured parameters was observed.     

Nerve biopsy findings in Niemann-Pick type II (NPC)

The severe infantile form of Niemann-Pick disease type II was diagnosed in a 4-year-old girl and confirmed by demonstrating in cultured skin fibroblasts a deficiency of low-density lipoprotein-stimulated cholesterol ester synthesis of < 5% of normal. Electrodiagnostic studies revealed changes of a predominantly demyelinating motor and sensory polyneuropathy. Light microscope and ultrastructural examination of a peroneal nerve biopsy showed unique changes. Compacted myelin sheaths were disproportionately thin with marked globular irregularities in single teased nerve fibres and evidence of chronic demyelination. The majority of axons were preserved but axonal spheroids and cytoskeletal abnormalities akin to neuroaxonal dystrophy were noted. Membrane-bound multilobulated lysosomal inclusions of floccular and electron-dense material were present in Schwann cells (SC), endoneurial fibroblasts, macrophages, pericytes and endothelial cells. SC of myelinated fibres were stuffed with whorls of concentric osmiophilic membranous profiles and electron-lucent material. The findings are diagnostic and differ from those of classical Niemann-Pick disease.     

Membrane lipids of B16 melanoma cells and heat-resistant variants

Membrane lipid composition and fluidity of a series of B16 melanoma cell variants with increased resistance to heat were analysed for changes within the lipid component that may contribute to the acquisition of heat resistance. Within one series of heat-resistant lines the cholesterol content of the cells decreased as their heat resistance increased. The most heat-resistant line, WH75, had 40 per cent less cholesterol than the parent line. No change in the composition of phospholipid fatty acids was found. An increased level of membrane fluidity in WH75 was demonstrated by electron paramagnetic resonance using 5- or 12-doxyl stearic acid. When challenged by heat the increase in membrane fluidity was similar for WH75 and for the parent line. Thus the increased heat resistance of the variants is probably not due to their ability to adapt to heat challenge by increasing membrane thermostability. The inverse relationship between heat resistance and cholesterol content was not demonstrated in two other series of heat-resistant variants. The cholesterol decrease, therefore, is not a universal response of cells as they acquire heat resistance.     

Anti-phospholipid and anti-DNA antibodies are not associated with the elevated release of circulatory fetal DNA in pregnancies affected by preeclampsia.

OBJECTIVES: We have previously shown that the levels of circulatory fetal DNA are elevated in preeclampsia and that these increases correspond to disease severity. Several reports have indicated that increased levels of antiphospholipid (anti-PL) and anti-DNA antibodies may be associated with preeclampsia, in particular with the severe forms of the disorder. Since the release of cell-free DNA by the placenta is attributed to some form of cell death or damage and as anti-PL and anti-double-stranded DNA (dsDNA) antibodies have been proposed to lead to placental damage, we have studied the relationship between these parameters in preeclampsia. METHODS: Circulating fetal DNA levels in samples taken from pregnant women with mild (n = 12) or severe (n = 12) preeclampsia and from normal pregnant controls (n = 35) were quantified using a Taqman real-time Polymerase Chain Reaction (PCR) assay. The Anti-PL antibodies (IgG and IgM) were assayed by anticardiolipin ELISA and by commercial anti-beta2-Glycoprotein I (GPI) ELISA kits. Anti-dsDNA antibodies (IgG and IgM) were analyzed by a commercially available anti-dsDNA ELISA kit. RESULTS: No correlation could be drawn with the quantity of circulatory fetal DNA in the samples analyzed and corresponding anti-PL or anti-dsDNA antibody levels. Furthermore, no significant difference existed between the levels of these antibodies in the two study groups and the control cohort. CONCLUSION: Our data suggest that the mechanism leading to the increased release of cell-free circulatory DNA from the placenta does not involve trophoblast damage mediated by these agents. Our analysis also questions the reported involvement of anti-PL and anti-DNA antibodies in preeclampsia.