Regional cerebral metabolic rate (positron emission tomography) during inhalation of nitrous oxide 50% in humans.

BACKGROUND: Recent studies in man have shown that cerebral blood flow increases during inhalation of nitrous oxide (N2O), a finding which is believed to be a result of an increased cerebral metabolic rate (CMR). However, this has not previously been evaluated in man. METHODS: Regional CMR(glu) (rCMR(glu)) was measured three dimensionally with positron emission tomography (PET) after injection of 2-(18F)fluoro-2-deoxy-D-glucose in 10 spontaneously breathing men (mean age 31 yr) inhaling either N2O 50% in O2 30% or O2 30% in N2. RESULTS: Global CMR(glu) in young men was 27 (3) micromol 100 g(-1) min(-1) [mean (SD)]. Inhalation of N2O 50% did not change global CMR(glu) [30 (5) micromol 100 g(-1) min(-1)] significantly, but it changed the distribution of the metabolism in the brain (P<0.0001 analysis of variance). Compared with inhalation of O2 30% in N2, N2O 50% inhalation increased the metabolism in the basal ganglia [14 (17)%, P<0.05] and thalamus [22 (23) %, P<0.05]. There was a prolonged metabolic effect of N2O inhalation seen on a succeeding PET scan with oxygen-enriched air (P<0.0001) performed 1 h after the N2O administration. CONCLUSIONS: Inhalation of N2O 50% did not change global CMR(glu), but the metabolism increased in central brain structures, an effect that was still present 1 h after discontinuation of N2O.

Genetic intratumour heterogeneity in high-grade brain tumours is associated with telomere-dependent mitotic instability.

Glioblastoma multiforme (GBM) and other high-grade brain tumours are typically characterized by complex chromosome abnormalities and extensive intratumour cytogenetic heterogeneity. The mechanisms behind this diversity have been little explored. In this study, we analysed the pattern of chromosome segregation at mitosis in 20 brain tumours. We found an abnormal segregation of chromatids at mitosis through anaphase bridging (10-25% of anaphase cells) in all 10 GBMs. Anaphase bridging was also found in two medulloblastomas (7-15%), one anaplastic astrocytoma (17%) and one oligodendroglioma (6%). These tumours showed a relatively high degree of cytogenetic complexity and heterogeneity. In contrast, cell division abnormalities were not found in low-grade brain tumours with less complex karyotypes, including two pilocytic astrocytomas and two ependymomas. Further analysis of two GBMs by fluorescence in situ hybridization with telomeric repeat probes revealed excessive shortening of TTAGGG repeats, indicating dysfunctional protection of chromosome ends. In xenografts established from these GBMs, there was a gradual reduction in cytogenetic heterogeneity through successive passages as the proportion of abnormally short telomeres was reduced and the frequency of anaphase bridges decreased from >25% to 0. However, bridging could be reintroduced in late-passage xenograft cells by pharmacological induction of telomere shortening, using a small-molecule telomerase inhibitor. Telomere-dependent abnormal segregation of chromosomes at mitosis is thus a common phenomenon in high-grade brain tumours and may be one important factor behind cytogenetic intratumour diversity in GBM.

Inhibition of inducible nitric oxide synthase enhances anti-tumour immune responses in rats immunized with IFN-gamma-secreting glioma cells.

Interferon gamma (IFN-gamma) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN-gamma has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN-gamma (N32-IFN-gamma), could enhance the anti-tumour immune response. Initially, both a selective iNOS, l-N(6)-(1-Iminoethyl)-l-lysine (l-NIL), and non-selective, N-nitro-l-arginine methyl ester (l-NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both l-NIL and l-NAME enhanced proliferation and production of IFN-gamma from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, l-NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32-IFN-gamma, and administered NOS inhibitors by intraperitoneal (i.p.) mini-osmotic pumps, only splenocytes of rats treated with l-NIL, but not l-NAME, displayed an enhanced proliferation and production of IFN-gamma when re-stimulated with N32 tumour cells. Based on these findings, l-NIL was administered concurrently with N32-IFN-gamma cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN-gamma-based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects.

Dynamic susceptibility contrast-enhanced perfusion magnetic resonance (MR) imaging combined with contrast-enhanced MR imaging in the follow-up of immunogene-treated glioblastoma multiforme.

PURPOSE: To assess the value of the combined use of dynamic susceptibility contrast-enhanced perfusion magnetic resonance imaging (MRI) and conventional contrast-enhanced MRI for the follow-up of treatment of glioblastoma multiforme (GBM). MATERIAL AND METHODS: 79 examinations were performed in six surgically and immunogene-treated patients and two surgically treated patients. Ratios of the relative cerebral blood volume (rCBV) in lesions and in the contralateral normal-appearing white matter were calculated. The regions with elevated rCBV were compared with those with contrast enhancement. Tissue specimens from surgical biopsies and autopsies were studied histopathologically. RESULTS: The lesion-to-normal rCBV ratios were high in the tumors prior to operation (7.3 to 18.2) as well as in the recurrent tumors (1.6 to 13.2). The volumes of the regions with elevated rCBV were similar to those with contrast enhancement in 63 of the 79 examinations. However, in 11 of 79 examinations, the regions with high rCBV were smaller than the regions with contrast enhancement ("mismatch"). In two samples from the immunogene-treated patients this was correlated with the histopathological finding of malignant tumor with numerous proliferating GBM vessels with multiple minimal lumina, sometimes thrombotized or ruptured. These vessels may have increased permeability with contrast enhancement not accompanied by increased microvascular volume. CONCLUSION: 1) Elevated rCBV on perfusion MRI corresponding to the contrast-enhancing lesion supports the diagnosis of recurrent malignant tumor. 2) A mismatch showing a volume of rCBV elevation smaller than that of contrast enhancement can be seen in particularly aggressive tumor growth and is thus not always a sign of reactive non-tumor changes. 3) The combination of perfusion MRI and conventional contrast MRI provides useful information in the follow-up of glioblastoma multiforme treatment.

Immunotreatment in patients with glioblastoma multiforme--a histopathological evaluation of reactive and inflammatory changes.

Glioblastoma multiforme (GBM) is the most common highly malignant brain tumor and is also one among the most therapy-resistant human neoplasias. At the University Hospital in Lund, a group of patients with GBM were treated with a new therapy form attempting immunization by glioma cells transfected to produce interferon-gamma. The purpose of this report was to evaluate tumor material from the first nine patients treated with this therapy, assessing the levels of inflammatory/reactive cells (lymphocytes and macrophages). Tumor biopsies from surgery performed at different time points during treatment were analyzed with conventional histotechnical methods and immunohistochemistry. A post-mortem neuropathological investigation with a whole brain assessment was achieved in 5 immunized patients. The results show that cytotoxic T lymphocytes exhibited a mild increase during immunotreatment. This increase indicates an invoked stimulation of a cytotoxic T cell reaction, which may prove beneficial when immunization is adequately manipulated in dosage and timing.

Radiation sterilisation of cultured human brain tumour cells for clinical immune tumour therapy.

The aim is to investigate the radiosensitivity of noninfected cultured human glioma cells to ascertain that intracutaneously administered cells are viable enough to produce interferon-gamma but not able to proliferate. Cell cultures were established from five patients undergoing brain tumour surgery. By karyotyping, we found four malignant (three glioblastoma multiforme (GBM), one giant cell glioma) and one normal. The cells were irradiated with (137)Cs-gamma rays at absorbed dose levels of 0, 20, 40, 60, 80, 100 and 120 Gy. The fraction of viable cells was examined by MTT incorporation assay. The average of the data obtained from three GBM cell cultures was fitted to an exponential model. The parameters were: extrapolation number n=0.85+/-0.10, mean lethal dose D(0)=12.4+/-3.2 Gy and an additional uncertainty parameter deltaS=0.14+/-0.03. By setting deltaS=0, the corresponding values of the parameters were n=0.86+/-0.16 and D(0)=30.0+/-8.1 Gy. The rate of proliferation was examined by (3)H-thymidine incorporation. The average of the proliferation data obtained from three GBM cell cultures was fitted to an exponential model yielding n=0.943+/-0.005 and D(0)=5.8+/-0.5 Gy for deltaS=0.057+/-0.005, and by setting deltaS=0, n=1.00+/-0.02 and D(0)=8.4+/-1.6 Gy. No outgrowth of plated cells was observed after 4 weeks at an absorbed dose of 100 Gy. This absorbed dose is recommended for irradiation of 2 x 10(6) glioma cells used for clinical immunisation.

A model for evaluating therapeutic response of combined cancer treatment modalities: applied to treatment of subcutaneously implanted brain tumors (N32 and N29) in Fischer rats with pulsed electric fields (PEF) and 60Co-gamma radiation (RT).

The aim of the present study is to develop a mathematical model for evaluating therapeutic response of combined treatment modalities. The study was performed in rats of the Fischer-344 strain with rat glioma N32 or N29 tumors implanted subcutaneously on the thigh of the hind leg. Pulsed electric fields, PEF, with 16 exponentially decaying pulses with a maximum electric field strength of 140 V/mm and t(1/e)= 1 ms were first applied to the tumors. Then within 5 min radiation therapy with (60)Co-gamma radiation, RT, was given in daily fractions of 5 Gy. The animals were arranged into one group of controls and 3 groups of different kind of treatments: PEF only, RT only or combination of PEF + RT. At about 4 weeks after inoculation, the tumors were given the treatment sessions during one week. In 2 experimental series with totally 52 rats with N32 tumors, of which 16 were controls, were given 4 sessions of PEF treatments and RT (totally 20 Gy). In a special experimental series with totally 56 rats with N32 tumors, of which 10 were controls, the different groups were given 1, 2, 3 or 4 treatment sessions respectively. Another strain of glioma tumor, N29 with 62 tumors of which 14 were controls was studied in 2 series given 4PEF + 4RT and 2PEF + 4RT respectively. Fitting the data obtained from consecutive measurements of tumor volume (TV) of each individual tumor to an exponential model TV = TV(0). exp[TGR.t] estimated the tumor growth rate (TGR % per day) after the first day of treatment (t = 0). The TGR of N32 tumors treated with the combination of 4PEF + 4RT are significantly decreased compared to the controls (p < 0.0001), compared to RT alone (p < 0.0001) and compared to PEF alone (p < 0.001). The combined treatment of N32 gives significant effect on the tumor growth rate after 2, 3 and 4 treatment session while RT alone seems to be most efficient after one treatment of 5 Gy and PEF alone is most efficient after 2 treatments at 2 consecutive days. The TGR of N29 tumors treated with the combination of 4PEF + 4RT are significantly decreased compared to the controls (p < 0.05) but the combination of 2PEF + 4RT was more effective (p < 0.005). The specific therapeutic effect STE is defined as the difference between the average tumor growth rates of controls and exposed tumors divided by the average tumor growth rate of the controls. With 4PEF treatments alone the average STE value was 0.32 for N32 tumors and 0 for N29; for 4RT alone the STE values were 0.29 and 0.42 respectively, and for combined treatments 4PEF + 4RT 0.67 and 0.17 respectively. For the N29 tumors treated with 2PEF + 4RT the STE value was 0.53. The therapeutic enhancement ratio, TER, value increase with the number of treatment sessions and the TER of the combined treatments is above 1 in two of the N32 series, which indicates a synergistic effect of 4PEF + 4RT. This work demonstrate the use of our model for analyzing the combination PEF + RT, but it can also be used for evaluation the therapeutic effects of combining RT with chemotherapy or immunogenetic therapy.

Interaction between weak low frequency magnetic fields and cell membranes.

The question of whether very weak low frequency magnetic fields can affect biological systems, has attracted attention by many research groups for quite some time. Still, today, the theoretical possibility of such an interaction is often questioned and the site of interaction in the cell is unknown. In the present study, the influence of extremely low frequency (ELF) magnetic fields on the transport of Ca(2+) was studied in a biological system consisting of highly purified plasma membrane vesicles. We tested two quantum mechanical theoretical models that assume that biologically active ions can be bound to a channel protein and influence the opening state of the channel. Vesicles were exposed for 30 min at 32 degrees C and the calcium efflux was studied using radioactive (45)Ca as a tracer. Static magnetic fields ranging from 27 to 37 micro T and time varying magnetic fields with frequencies between 7 and 72 Hz and amplitudes between 13 and 114 micro T (peak) were used. We show that suitable combinations of static and time varying magnetic fields directly interact with the Ca(2+) channel protein in the cell membrane, and we could quantitatively confirm the model proposed by Blanchard.

Electrically mediated drug delivery for treatment of an adenocarcinoma transplanted into rat liver.

BACKGROUND: In this study, electrochemotherapy (ECT), i.e. tumour treatment based on local augmentation of intracellular drug delivery from short, intense electric pulses, was evaluated in rats with an adenocarcinoma implanted into the liver. Tumour response and concentrations of macrophages and T-lymphocytes (CD4 and CD8) in and around the tumour were measured. MATERIALS AND METHODS: Rats were treated with permeabilizing electric pulses, bleomycin, or both, eight days after implantation of the tumour, while one group received sham treatment. RESULTS: Treatment with electric pulses and bleomycin resulted in a significantly reduced lesion volume and 92% cure rate (12 out of 13, p<0.0002 compared to the other treatment groups). The highest concentration of CD8 lymphocytes was found in tumours treated with electric pulses and bleomycin. Macrophages were found mainly in tumours treated with electric pulses, with or without bleomycin. CONCLUSION: Electrochemotherapy using millisecond exponential pulses and bleomycin is efficient in a rat liver tumour model and appears to stimulate the host's immune system.

A new antitumour treatment combining radiation and electric pulses.

AIM: To investigate the antitumour effect of radiation in combination with electropermeabilization on subcutaneous rat glioma tumours. MATERIALS AND METHODS: Sub-optimal radiation treatment was administered separately or in combination with electric pulses of high voltage to subcutaneous rat brain tumours. The treatment was repeated on four consecutive days and evaluated by TGD and microscopical examination. The tumours were stained for Factor VlII/von Willebrand Factor to investigate the effects on the tumour vasculature. RESULTS: Radiation and electric pulses applied concomitantly resulted in a cure rate of 67% (tumour free >80 days after treatment). Radiation-treated animals showed progressive disease. Histological and immunohistochemical examination of electric impulse-treated tumours showed instant and severe deteriorating effects on tumour vasculature. CONCLUSION: A distinct antitumour effect of the combined treatment of electric pulses and radiation treatment was observed. We believe that the tumouricidal effect arises from destruction of the tumour vasculature but also from DNA related damage from reactive oxygen formed by the electric pulses and the radiation treatment.

Expression of the IL-6 family cytokines in human brain tumors.

In our RT-PCR screen for cytokine expression in human brain tumors we discovered increased levels of oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), all belonging to the interleukin-6 (IL-6) cytokine family, in most of the tumors. The expression of these cytokines in normal adult brain tissue was found to be very low or below detection limit. OSM expression was elevated in most of the tumors and immunohistochemistry analysis showed that the tumor cells contained OSM in their cytoplasm, suggesting they produce this factor. Overexpression of OSM has not previously been reported in primary human brain tumors. The IL-6 cytokine family acts through a common gp130 receptor subunit that activates the JAK/STAT signaling pathway and therefore they have been suggested to have overlapping effects. Tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase 1 (MMP-1) and MMP-3 and IL-6 have been reported to be regulated by OSM. IL-6 was low or absent in the tumors. TIMP-1, MMP-1 and MMP-3 was expressed in most tumors but with no strict correlation to OSM levels.

Expression of TGF-beta isoforms, TGF-beta receptors, and SMAD molecules at different stages of human glioma.

Human gliomas express TGF-beta but, so far the expression of downstream mediators has been investigated in only a few cell lines. We have examined tissue specimens of 23 gliomas: 3 astrocytomas grade II (AST), 8 anaplastic astrocytomas grade III (AAST), and 12 glioblastoma multiforme grade IV (GBM). We analyzed the mRNA expression of TGF-beta1, TGF-beta2, TGF-beta3, the TGF-beta receptors type I (TbetaR-I) and type II (TbetaR-II), Smad2, Smad3, and Smad4. mRNA expression of IL-10 and CD95 (FAS/APO-1) were also studied. We detected increased mRNA levels of the 3 TGF-beta isoforms, correlating with the degree of malignancy. TGF-beta3 mRNA was increased, particularly in AST and AAST, while TGF-beta1 and TGF-beta2 mRNAs were strongly expressed in GBM. TGF-beta normally up-regulates the TGF-beta receptors, and TbetaR-I and TbetaR-II showed stronger expression in all gliomas when compared to normal tissues. However, the mRNA expression of Smad2, Smad3, and Smad4 was decreased in GBM. IL-10 mRNA expression was detected in glioma tissues but not in glioma cell lines. No marked increase in the expression of soluble CD95 splicing variants was found in the gliomas compared with normal tissue. However, total CD95 mRNA was elevated among GBM tissues.

Distribution of bleomycin in a rat model.

Bleomycin has, in the years of developing electrochemotherapy (ECT), proven to be an extremely potent drug for this cancer treatment modality and is also the most frequently applied chemical agent. It is of importance to investigate the pharmacokinetics of bleomycin under normal conditions and particularly in combination with ECT.

Treatment of rat glioma with electrochemotherapy.

The first attempt to apply electrochemotherapy (ECT) to the brain was reported in 1993 by Salford et al. 1993 (1). They managed to significantly prolong the survival of RG2 glioma bearing Fischer-344 rats by 200% by iv administration of bleomycin followed by intracranial electrochemotherapy with exponential decaying pulses. In collaboration with the department of tumor immunology, Lund University, an ethyl-nitroso-urea induced rat glioma cell line (N32) is developed that produces glioma of malignant astrocytoma type with only half the growth rate of the RG2 cells (2). The N32 tumor implanted in rats was treated with intracranial electrochemotherapy and enhanced uptake of [111In]bleomycin from intracranial ECT was also demonstrated with a scintillation camera (3). (111)In-labeled bleomycin has been used to investigate the uptake and retention after ECT treatment of subcutaneous N32 tumors (4).

Studies of in vivo electropermeabilization by gamma camera measurements of (99m)Tc-DTPA.

A protocol was developed to study the drug uptake from in vivo electropermeabilization at different settings of parameters influencing the uptake efficiency. Radiolabelled diethylenetriaminepentaacetic acid (DTPA) was used to trace the distribution and internalization of a hydrophilic drug after in vivo electropermeabilization. Skeletal muscle tissue in rat was treated with permeabilizing electric pulses before or after intravenous administration of (99m)Tc-DTPA. The drug accumulation in the treated volume was subsequently evaluated with a scintillation camera. The dependence of uptake on field strength and duration of the applied electric pulses was investigated for exponentially decaying pulses and square wave pulses. Further, the uptake dependence on time interval between injection and pulsation was studied as well as the uptake dependence on the number of pulses applied in a single electropermeabilization treatment. Dynamic gamma camera studies were performed to quantify the time scale of the drug uptake in electropermeabilized tissue.

Frontal lobe dysfunction in patients with non-frontal malignant gliomas: a monoaminergic dysregulation?

Previous investigations concerned with the neuropsychological function of patients with intracerebral supratentorial malignant gliomas has revealed the frequent occurrence of signs suggestive of an inhibitory frontal lobe dysfunction regardless of the intracerebral localization of the tumor and before the diagnosis was known to either the investigator or the patient. Upon closer analysis, the frontal lobe dysfunction has been verified by the demonstration of reduced blood flow in frontal areas in these patients. Since many of the findings can be related to a dysfunction of dopaminergic neurotransmission, we hypothesize that abnormal astrocytes interfere with the metabolism, transport and release of various neurotransmitters of which dopamine may be the one responsible for the most striking neuropsychological abnormalities in patients with malignant gliomas.

HERV-F (XA34) is a full-length human endogenous retrovirus expressed in placental and fetal tissues.

The complete sequence of XA34 was identified from a 107 kb genomic clone originating from the human chromosome 7q31.1-q31.3. The 7.1 kb human endogenous retrovirus (HERV) contains LTR's, gag, pol and env, and a pol sequence which is identical to the 2.3 kb XA34 cDNA clone which we previously isolated from a human glioma cDNA library (Widegren et al., 1996). The HERV is located in a reversed orientation within an intron-sequence of a gene similar to mouse adseverin(D5). The gag and protease regions are intact. However, the pol and env regions are truncated by a deletion which removes the C-terminal end of the integrase and the complete surface protein. The HERV sequence is bordered by a five base-pair direct repeat and has the TG...CA structure. Over the complete HERV genome, XA34 is very similar to members of the HERV-F family and shares the same primer binding site which is homologous to phenylalanine (F) tRNA. Therefore, XA34 is termed HERV-F(XA34). HERV-F(XA34) has an open reading frame (ORF) of 1000 bp in the gag region which starts with Met-Gly in a favorable context and stops in the capsid protein. A strong mRNA expression of HERV-F(XA34) is demonstrated in placental tissue, mainly residing in two transcripts of approximately 7.5 and 8.5-9 kb respectively. Analyses of expressed sequence tags (ESTs) have identified the expression of HERV-F(XA34) sequences in placental tissue, fetal liver/spleen, olfactory epithelium and in an epithelial skin tumor. EST analysis has also identified splice variants of HERV-F(XA34), in which the gag, pol and most of the env regions are spliced out. These splice variants contain a short ORF encoded in the region from the C-terminal portion of env to the 3'-LTR. In addition, ESTs identical to HERV-Fb have been identified in retinal, fetal liver/spleen and brain tissue as well as Jurkat cells. The analyses indicate that the 5'-LTR of HERV-Fb may function as an alternative poly A site of a Krüppel related zinc finger gene (ZNF195).

Psychological profile related to malignant tumours of different histopathology.

In a previous preoperative study of patients with gliomas, we made the original observation that patients with high grade as opposed to those with low-grade gliomas have a psychological profile marked by extreme emotional reactivity. In this postoperative study of the psychological profiles of patients with breast cancer, the main funding was unexpectedly analogous with the findings in the brain tumour study. The patients with poorly differentiated ductal carcinomas showed a specific and, compared to the patients with well differentiated carcinomas, outstanding psychological profile marked by extreme emotional reactivity as well as by genuine creativity. Some of the present patients with well differentiated carcinomas showed personality profiles marked by compulsive inhibition, also described earlier in the literature of patients with breast cancer. The psychobiological relations between emotional reactivity and aggressiveness of tumour growth are discussed.

Attitude towards aggression and creative functioning in patients with breast cancer.

Three projective personality tests were used to assess attitude to aggression (The Identification Test), anxiety and defenses (The Meta-Contrast Technique) and creative functioning (The Creative Functioning Test) in 70 patients with breast cancer. Discriminant analyses were applied pro primo to characterize psychologically patients with a better prognosis and patients with a poorer prognosis. A second aim was to characterize psychologically older (postmenopausal) and younger (premenopausal) women. Generally, high scores on the Identification Test indicated maladaptive attitudes towards aggression among all the patients. Patients with a poorer prognosis showed responses that in healthy subjects indicate acknowledgement of aggressive impulses, perhaps suggesting lack of "defenses" against such impulses among those patients. Another way to describe it would be that patients with a better prognosis seem to have (normally nonadaptive) "defenses" against aggressive impulses while those with poorer prognosis have not. Surprisingly, the patients with a better prognosis (but not those with a poorer prognosis) gave responses classified as depression in the Meta-Contrast Technique. Typical of premenopausal patients were responses classified as anxiety as well as reaction formation on the Identification Test. Responses classified as adaptive defenses (isolation) were seen in the Meta-Contrast Technique. A surprising finding was that many of these patients were characterized by high scores on the creativity test. These original statistically significant findings of attitudes towards aggression and creative functioning in breast cancer patients are discussed in relation to the underlying nature of aggression and creativity.