Immunization with syngeneic interferon-gamma (IFN-g) secreting tumour cells enhance the Therapeutic effect and Abscopal effect from combined treatment of subcutaneously implanted contra-lateral N29 tumours on Fischer rats with Pulsed electric fields (PEF) and 60Co-gamma radiation.

The aim of the present study is to study the Abscopal regression of subcutaneously implanted N29 rat glioma after immunization with syngeneic IFNg secreting cells and treatment of contra-lateral tumours with pulsed electric fields (PEF) and/or radiation therapy (RT). The study was performed on rats of the Fischer-344 strain with rat glioma N29 tumours implanted subcutaneously on the flank or on both the right treated hind leg and the left untreated hind leg. Once weekly for three weeks, the animals were given intra-peritoneal injections of irradiated, modified N29 tumour cells, secreting interferon-gamma (IFN-g). PEF was given with 16 exponentially decaying pulses at a maximum electric fields strength of 1400 V/cm and t1/e= 1 ms. RT was given with 60Co gamma radiation at daily fractions of 5 Gy, to a total absorbed dose of 20 Gy. The animals were arranged into controls and groups of various treatments: PEF, RT, PEF+RT and immunization (IFNg). Fitting the data obtained from consecutive measurements of tumour volume (TV) of each individual tumour to an exponential model TV = TV0*exp[TGR*t] estimated the tumours growth rate (TGR %per day) after the day of treatment (t = 0). TGR of the right-lateral treated tumour was significantly decreased for independent treatments with PEF and RT and with the combined treatment PEF+RT. With immunization (IFNg) alone and in combination with PEF there was, however, no significant decrease of the TGR of the right-lateral tumours. But in the combination of immunization with RT or PEF+RT there was a highly significant decrease of the TGR values. The Abscopal effect was evaluated by comparing the growth rate of the untreated contra lateral tumours with the treated tumours. TGR of the left-lateral untreated tumour in the groups with independent treatment of right-lateral tumours with PEF, was not significantly reduced. But the TGR values are significantly reduced in the group of rats treated with RT and the combination PEF + RT. With IFNg alone and in combinations with PEF or RT there was no significant decrease of the TGR in the left lateral tumours. But in the combination of IFNg with PEF+RT there was a highly significant decrease of the TGR values in the left lateral tumours. The specific therapeutic effect (STE = 1 - TGRExposed/ TGRCtrl ) after treatments with PEF was 0.30±0.01 and after RT 0.46±0.04 and after the combination PEF+RT 0.36+/- 0.08. After immunization with IFNg secreting tumour cells the STE 0.09+/- 0.07 is not significantly different from zero. Also for the combination of immunization and PEF the STE value of 0.07+/- 0.07 is not significantly different from zero. In the combination of immunization with RT the STE value was 0.32+/- 0.01 that is significantly different from zero and only slightly lower than for RT alone. The STE of the combination of immunization with (PEF+RT) resulted in an unexpectedly high STE value of 0.70+/- 0.08 that is highly significantly different from zero (p < 0.0001). The specific Abscopal effect (SAE = 1 - TGRUn-Exposed/ TGRCtrl ) of the contra lateral unexposed tumours in rats treated with PEF or RT are both significantly different from zero. For RT the average SAE value is 0.33+/- 0.04 and for PEF it is 0.11+/- 0.05. The SAE value for the combined treatment with PEF + RT is 0.26+/- 0.02 that is about the same as for RT alone. For immunization with IFNg secreting tumour cells only and IFNg +PEF the SAE values were not significantly different from zero. But IFNg combined with RT result in a SAE value of 0.18±0.12 and the combination of IFNg with PEF+RT results in an improved abscopal effect with the SAE value of 0.33+/- 0.06. After combined treatment with PEF + RT the average of the therapeutic enhancement ratio (TER = STEExperimental / STEIndependent) is 0.47 +/- 0.12 and the abscopal enhancement ratio (AER = SAEExperimental / SAEIndependent) is 0.61 +/- 0.1 respectively. With all three treatment modalities combined IFNg + PEF + RT and all combinations of independent treatments with PEF, RT or IFNg are considered, the average of the TER is 1.20+/- 0.15 and AER is 1.22+/- 0.20. This might indicate that there is a synergism on the tumours on both sides by combining PEF, RT and immunization with IFNg secreting cells. These results were first presented Nov 21-24, 2002, as Poster at Society of Neuro-Oncology (SNO) Annual Meeting, San Diego, USA (Persson et al 2002)

Mörka Neuron och Mobiltelefoner : Dedicerad till en 90-årig man, Arne Brun i Lund

Med denna svenska översikt av våra egna och andra forskares observationer av mörka neuron vid mikrovågs exponering från mobiltelefoner, som lite senkommet tillägnas Arne Brun på hans 90 års-dag, vill vi att hans insatser blir uppmärksammade och inte faller i glömska.Kring 2000 millennium skiftet pågick ett intensivt arbete i Lund med att sammanfatta och bekräfta effekterna av exponering med GSM-900 MHz mikrovågor på blod-hjärna barriären och hjärnans neuroner. Leif G. Salford, Arne Brun och medarbetare presenterade år 2003 i tidskriften Environmental Health Perspectives resultaten från en undersökning av skador på nervcellerna i råtthjärna efter exponering för mikrovågor från GSM Mobiltelefoner. Kontroller och testdjur visade alla tecken på närvao av albumin i hypotalamus, vilket år normalt och indikerar att albumin infärgningen av BBB läckaget också fungerar. Cresylviolettfärgningen avslöjade förekomst av spridda och grupperade mörka nervceller, som ofta var skrumpna och mörkt homogent färgade utan urskiljbara interna cellstrukturer. Några av dessa mörka nervceller var också albuminpositiva eller visade cytoplasmatiska mikrovakuoler som indikerar en aktiv patologisk process. År 2008 presenterades resultaten av ytterligare undersökningar av blod-hjärn barriärens permeabilitet och nervcellsskador i råtthjärnan efter en återhämtningstid på antingen 14 och 28 dagar efter 2 timmas exponering för mikrovågor från GSM-mobiltelefoner i 900 MHz-bandet. Efter 14 dagars återhämtningstid observerades albumin-läckage i BBB och albumin upptag i neuroner. Mörka neuron observerades endast hos råttor som exponerats med det lägsta SAR-värdet, 0,12 mW/kg. Efter 28 dagars återhämtnings period observerades läckage av albumin endast hos råttor som exponerats med det högsta SAR-värdet, 100 mW/kg. Däremot observerades efter 28 dagar förekomst av mörka neuron i råtthjärnor hos alla grupperna vilket korrelerade väl med neuronernas albumin upptag.I studien observeras neuro-patologiska förändringar redan vid SAR-värden så låga som 0,12 mW/kg vilket överensstämmer med våra tidigare resultat. Speciellt iögonfallande är att det högsta albumin upptaget i neuroner observeras vid den lägsta SAR nivån på 0,12 mW/kg. Frekvensen hos förekomsten av mörka nervceller ökade, jämfört med kontrollerna både efter 14 och 28 dagars återhämtning, men var endast signifikant vid 28 dagar efter exponering. Inga signifikanta tecken på förekomsten av mörka neuron observerades emellertid efter 7 dagars återhämtning.I en Fransk studie redovisad av Poulletier de Gannes och medarbetare 2009 exponerades enbart huvudet hos 16 st. Fischer 344-råttor (14 veckor gamla) för GSM-900 under 2 timmar vid SAR värden 0,14 och 2,0 W/kg. Fjorton alternatvt 50 dagar efter GSM-900 exponeringen kunde varken BBB-läckage eller förekomst av mörka nervceller upptäckas i rått hjärnorna. Deras resultat indikerar att det föreligger en väsentlig skillnad i resultaten vid helkropp exponering jämfört med exponering av endast huvudet.År 2015 presenterades en studie, stödd av Nationella Vetenskaps Akademin i Kina (NSFC), avseende albumin-läckage i blod-hjärnbarriären efter exponering med kontinuerliga mikrovågor på 900 MHz med SARvärden mellan 0,016 (hela kroppen) och 2 W/kg (lokalt i huvudet). Hos råttor som exponerats under 28 dagar observerades cellulärt ödem och neuronal cellorganell degeneration hos råttorna. Dessutom observerades med immun-färgning BBB-läckage av albumin i hippocampus och cortex. Efter exponering för 900 MHz mikrovågor under 14 respektive 28 dagar hade serum albumin diffunderat in i neuropilen mellan cellkropparna, som omger neuronerna. Upptag av Albumin i hippocampus neuron hos råttor exponerade under 28 dagar, visar förekomst av mörka neuron. Deras resultat är i linje med Lunda-resultaten som publicerades 2003 och 2008

Radiobiological studies with low energy ionizing radiation - dosimetric aspects

Background: During the last 20 years both in vitro and in vivo models have been developed for studies of the cell killing characteristics of low energy electrons, e.g. Auger electrons. This thesis deals with radiobiological effects of radiation from indium isotopes in V79 cells in vitro (Paper I-III) and the rat testis in vivo (Paper IVV). The focus has mainly been on studies of the accuracy and limitations of the radiation dosimetry in both models. Material and Methods: Thermo luminescent dosimetry (TLD) was used for measurement of the absorbed dose to cells exposed to photon induced Auger electrons or X-ray photons at ultrahigh dose rates. A rat testis model was developed and intratesticularly exposed to 110In, 111In and X-ray photons. The biokinetics and dosimetry were carefully evaluated by use of a scintillation camera, HPGe semiconductor detector and TL Dosimetry. Results: In none of the in vitro experiments could an enhanced biological effect attributed to Auger electrons be proved. It was concluded early in the work that the dosimetry was a matter of decisive importance for assessment of radiobiological results and that every single irradiation occasion needs to be scrutinized. Although the rat testis model was demonstrated to be an appropriate model for radiobiological studies in vivo, there was no difference in the sperm count survival when exposed to the Auger emitting radionuclide 111In compared to 110In and X-ray photons. Discussion and Conclusion: Radiobiology must rely on a radiation quantity such as absorbed dose to compare the effects between different experiments and laboratories. This investigation has shown the complexity of determining the absorbed dose in radiobiological experiments. The accuracy depends on several factors, especially the experimental setup and the accurate calibrations of the TLDs. For the testis, the kinetics, mass, volume and radionuclide localization influence the accuracy of the absorbed dose and thus the RBE. The overall question raised by this thesis is the concept of absorbed dose and its use in diagnostic and therapeutic nuclear medicine. Many of the results point in the direction of the inadequacy of using the mean absorbed dose concept for radionuclides

Histopathological examinations of rat brains after long-term exposure to GSM-900 mobile phone radiation.

In order to mimic the real life situation, with often life-long exposure to the electromagnetic fields emitted by mobile phones, we have investigated in a rat model the effects of repeated exposures under a long period to Global System for Mobile Communication-900MHz (GSM-900) radiation. Out of a total of 56 rats, 32 were exposed once weekly in a 2-h period, for totally 55 weeks, at different average whole-body specific absorption rates (SAR) (of in average 0.6 and 60mW/kg at the initiation of the experimental period). The animals were exposed in a transverse electromagnetic transmission line chamber (TEM-cell) to radiation emitted by a GSM-900 test phone. Sixteen animals were sham exposed and eight animals were cage controls, which never left the animal house. After behavioural tests, 5-7 weeks after the last exposure, the brains were evaluated for histopathological alterations such as albumin extravasation, dark neurons, lipofuscin aggregation and signs of cytoskeletal and neuritic neuronal changes of the type seen in human ageing. In this study, no significant alteration of any these histopathological parameters was found, when comparing the GSM exposed animals to the sham exposed controls

(99m)Tc-DTPA Uptake and Electrical Impedance Measurements in Verification of In Vivo Electropermeabilization Efficiency in Rat Muscle.

Objective: In vivo electropermeabilization of cell membranes in rat muscle tissue cause a significant decrease of the electrical impedance, in the frequency region of 1-10 kHz. We aimed to study how the Tc-99m-DTPA uptake in the electropermeabilized region correlates to the change of admittance Y = 1/absZ, where Z is the measured impedance. Methods: The electropermeabilization was performed in vivo by applying high-voltage (0.5-2 kV) short (0.1-2 ms) pulses through gold-plated needle electrodes in skeletal muscle. The impedance was measured before and after each electropermeabilization pulse. The uptake of Tc-99m-DTPA uptake in the electropermeabilized region was measured after 6 and 24 hours with a gamma camera. Results: The pulse shape (square and exponential), duration, and amplitude of the applied electric field were varied, and electropermeabilization efficiency was evaluated using the various measurement modalities. Good correlations were found (correlation coefficient approximate to 0.9) between the Tc-99m-DTPA uptake in the electropermeabilized and control "region of interest" the admittance ratio Y (post-treatment)/Y (pretreatment), and charge displacement parameter Q. Conclusion: The electrical impedance measurements method can be utilized in clinical settings to verify the efficiency of electropermeabilization applied to chemotherapy and to power RNAi (RNA-interference) and DNA-plasmid transfection in vaccination, immunization, and gene-therapy

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 (60)Co-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 t1/e = 1 ms were first applied to the tumors. Then within 5 min radiation therapy with 60Co-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 immunogene-therapy

Cell survival after Auger electron emission from stable intracellular indium exposed to monochromatic synchrotron radiation

The biological effect of Auger electrons emitted from indium in V79 cells was investigated. K-shell vacancies were induced by synchrotron x-rays. Two energies, 100 eV above and below the K-edge of indium, were used. The cell survival for controls was similar to that which has been reported by others, with D37 = 4.4 Gy. Indium-oxine-labelled cells exhibited a survival clearly below that of the controls, D37 = 3.2 Gy, but no significant difference in survival between irradiations above and below the K-edge could be observed. The explanation is, inter alia, that the number of photons interacting with indium atoms incorporated into the cell, is small compared with the number of photons interacting with other atoms in the cell. The toxicity of indium oxine made it impossible to incorporate a sufficient number of indium atoms into the cells to observe a difference in this study. However, monoenergetic irradiation above and below the K-edge, provides a technique for the investigation of basic biological effects of Auger processes

Treatment of tumour cell with 5-aza-2-deoxycytidine (DAC) for immune tumour therapy of Glioma in Fischer-344 rats

Fisher 344 rats with implanted N29 glioma tumours were treated with Pulsed Electric Fields (PEF) in combination with immunization using either IFN-gamma-gene-transfected syngeneic tumour cells or IFN-gamma transfected N29 cells treated with 10 micro-M 5-aza-2-deoxycytidine (DAC). Tumours (N29) were inoculated subcutaneously on both thighs of female F- 344 syngeneic rats. The left tumour was treated once with 16 exponential pulses with an electric field strength of 1400 V/cm, and 1.0 ms duration (time constant). No anticancer drugs were given at any time. The following day and then once weekly for three weeks, the animals were given intra-peritoneal injections of irradiated, modified N29 tumour cells. The results were evaluated by daily measuring the size of tumour on both sides of the animals. Treatment with solely PEF in 32 animals resulted in a specific growth rate decrease of 20±6 % on the PEF exposed tumour. The effect at the non targeted tumour was negligible (0±4 %). Treatment with IFN-gamma secreting tumour cells resulted in a significant decrease of tumour growth rate on the right tumour of 20± 2 % (p< 0.05) and no significant effect (3±0.3% ) was observed on the left tumour. Immunization with DAC treated IFN-gamma secreting cells in 12 animals showed no significant decreased growth rate, on neither the left nor the right tumours. By combining PEF+IFN-gamma no significant decrease in growth rate was achieved. But in the combination of PEF and IFN-gamma secreting cells grown in DAC medium the tumour growth rate decreased by about 50 % at the PEF treated tumour and there was a decrease of about 20% in tumour growth at the non-PEF treated tumour rate which is about the same as for PEF treatment alone. Immune therapy of rats with intracranial N32 tumours by immunization with IFN-gamma secreting syngeneic cells treated with DAC resulted in a slight (3%) but not significant increase in survival time. With a single RT fraction of 15 Gy there was, however, a significant increase of 32% in the length of survival time of the rats with N32 tumours (p<0.02). Radiation therapy with a single fraction of 15 Gy combined with immunization with IFN-gamma secreting syngeneic cells treated with DAC resulted in significant (p<0.01) 34% increased length of survival time for the N32 tumours although there were no complete remissions

Rat testis as a radiobiological in vivo model for radionuclides.

The radiobiological effect of intracellularly localised radionuclides emitting low energy electrons (Auger electrons) has received much attention. Most in vivo studies reported have been performed in the mouse testis. We have investigated the rat testis as an in vivo radiobiological model, with sperm-head survival, testis weight loss and also alteration in the blood plasma hormone levels of FSH and LH as radiobiological endpoints. Validation of the rat testis model was evaluated by using mean absorbed doses of up to 10 Gy from intratesticularly (i.t.) injected In-111 oxine or local X-ray irradiation. Biokinetics of the i.t. injected radionuclide was analysed by scintillation camera imaging and used in the absorbed dose estimation. By the analysis of the autoradiographs, the activity distribution was revealed. Cell fractionation showed In-111 to be mainly associated with the cell nuclei. External irradiations were monitored by thermoluminescence dosimeters. The sperm-head survival was the most sensitive radiobiological parameter correlated to the mean absorbed dose, with a D-37 of 2.3 Gy for In-111 oxine and 1.3 Gy for X rays. The levels of plasma pituitary gonadal hormones FSH and LH were elevated for absorbed doses > 7.7 Gy. This investigation shows that the radiobiological model based on the rat testis has several advantages compared with the previously commonly used mouse testis model. The model is appropriate for further investigations of basic phenomena such as radiation geometry, intracellular kinetics and heterogeneity, crucial for an understanding of the biological effect of low-energy electrons