Jutta Jahns

Low-dose radiotherapy (LD-RT) and the modulation of iNOS expression in adjuvant-induced arthritis in rats

Purpose: Low‐dose radiotherapy (LD‐RT) of arthritic joints applied during the peak of the acute inflammatory response improves the clinical and histomorphological development of adjuvant arthritis. The study was undertaken to investigate the cellular composition of the inflammatory infiltrate and the expression of the pro‐inflammatory and anti‐inflammatory enzymes, inducible nitric oxide synthase (iNOS), cyclo‐oxygenase 2 (COX‐2) and haem‐oxygenase 1 (HO‐1), in response to LD‐RT. Materials and methods: Adjuvant arthritis in female Lewis rats was induced by intradermal injection of heat‐inactivated mycobacterium tuberculosis on day 0. Both arthritic hind paws were sham irradiated (group 1) or X‐irradiated with either 5×1.0 Gy (group 2) or 5×0.5 Gy (group 3) from days 15 to 19 after induction (15 animals/group). On days 21 (n=12 joints/group) and 30 (n=18 joints/group), cryostat sections were analysed histologically and immunohistologically after specific staining for macrophages, iNOS, COX‐2 and HO‐1. Results: A total of 5×1.0 Gy or 5×0.5 Gy led to a significant reduction of clinical symptoms from days 21 to 29, and a highly significant reduction of cartilage and bone destruction on day 30. Macrophage‐positive areas could be detected continuously throughout the periarticular infiltrate, and were slightly reduced after LD‐RT on days 21 and 30. This reduction was more pronounced after 5×1.0 Gy. Following LD‐RT, the iNOS score was reduced by about 45–50% on days 21 (p<0.05) and 30 (p<0.001). In contrast, the HO‐1 score was increased by about 50% on days 21 (p=0.08) and 30 (p=0.03). Conclusions: The clinically and histologically observed prevention of the pro‐gression of adjuvant arthritis after LD‐RT given during the peak of the acute inflam‐matory response and the reduction of cartilage and bone destruction in the chronic phase appears to be related to the modulation of iNOS activity by low X‐ray doses.

Radiation treatment of acute inflammation in mice.

Purpose: Low-dose radiotherapy (RT) has often been used effectively for the treatment of a variety of benign diseases, particularly those with acute inflammatory features. Here we report findings on radiation treatment of acute inflammation using a murine carrageenin air pouch model. Materials and methods: Air pouches raised on the dorsal surface of mice were injected with λ carrageenin and were irradiated 6 h later with doses ranging from 0 – 5 Gy. Treatment success was evaluated at various times thereafter by volume of exudate and number of inflammatory cells, and levels of inflammation-related cytokines tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and transforming growth factor beta-1 (TGFβ-1), and expression of inducible nitric oxide synthase (iNOS), heme oxygenase-1 (HO-1), cyclooxygenase-2 (COX-2) and inducible heat shock protein 70 (HSP70) as determined by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. Results: Crude inflammatory parameters such as the amount of exudates and number of inflammatory cells remained largely unaffected by radiation or were even a slightly and transiently increased. However, the expression of iNOS was attenuated by radiation concomitant with an increase in the levels of HO-1 and HSP70. Cytokine levels varied with the radiation dose and the time point. Conclusions: Ionizing radiation, even at low doses, functionally modulates inflammatory cells. Our findings indicate possible mechanisms as to how low-dose radiation may exert anti-inflammatory effects and provide the first evidence that heat shock proteins may be involved in this response.

Effects of low dose radiation therapy on adjuvant induced arthritis in rats.

Purpose : Substantial clinical evidence shows the efficacy of low dose radiotherapy (RT) in the treatment of painful osteoarthritis. Experimental investigations into these empirically clinical observations remain scarce. This study investigated in vivo the effects of daily 5 1.0Gy versus 5 0.5Gy on adjuvant induced arthritis in rats in order to explore whether there is a dose dependence of anti-inflammatory efficacy. Materials and methods : Adjuvant arthritis in female Lewis rats was induced by intradermal injection of heat inactivated Mycobacterium tuberculosis on day 0. Both hind paws were X-irradiated daily from days 15 to 19 after induction according to four protocols (15 animals/group): group 1, 5 1.0Gy (non-arthritic animals); group 2, sham-irradiated control; group 3, 5 1.0Gy; group 4, 5 0.5Gy. The clinical parameters arthritis score (AS), hind paw volume (HPV), body weight, and erythrocyte sedimentation rate (ESR) were determined. On days 21 and 30 histological sections of at least 12 ankle joints per group were analysed semi-quantitatively. Results : Local irradiation of non-arthritic rats (group 1) with 5 1 Gy did not induce any arthritic signs. Sham-irradiated arthritic rats (group 2) showed a full-blown arthritic syndrome. Treatment of arthritic rats with 5 1 Gy (group 3) or 5 0.5Gy (group 4) led to a reduction of mean AS from day 21 to 29 compared with group 2 (days 27-29-group 3: p =0.037; group 4: p =0.034), with no differences in efficacy between groups 3 and 4. Concurrently, following radiation treatment there was no further increase in HPV. At the end of the observation period, this effect demonstrated a dose-dependent level of significance (days 27-29-group 3: p =0.0036; group 4: p =0.039). A significant decrease in the ESR was noted in both irradiated arthritic groups on day 21 (group 3: p =0.015; group 4: p =0.006). The histopathological analysis revealed a highly significant reduction of cartilage and bone destruction on day 30 in both irradiated groups. Conclusions : This study confirms by objective criteria the anti-iinflammatory efficacy of low dose RT and gives some indication for a dose dependence of its efficacy.PURPOSE Substantial clinical evidence shows the efficacy of low dose radiotherapy (RT) in the treatment of painful osteoarthritis. Experimental investigations into these empirically clinical observations remain scarce. This study investigated in vivo the effects of daily 5 x 1.0 Gy versus 5 x 0.5 Gy on adjuvant induced arthritis in rats in order to explore whether there is a dose dependence of anti-inflammatory efficacy. MATERIALS AND METHODS Adjuvant arthritis in female Lewis rats was induced by intradermal injection of heat inactivated Mycobacterium tuberculosis on day 0. Both hind paws were X-irradiated daily from days 15 to 19 after induction according to four protocols (15 animals/group): group 1, 5 x 1.0 Gy (non-arthritic animals); group 2, sham-irradiated control; group 3, 5 x 1.0 Gy; group 4, 5 x 0.5 Gy. The clinical parameters arthritis score (AS), hind paw volume (HPV), body weight, and erythrocyte sedimentation rate (ESR) were determined. On days 21 and 30 histological sections of at least 12 ankle joints per group were analysed semi-quantitatively. RESULTS Local irradiation of non-arthritic rats (group 1) with 5 x 1 Gy did not induce any arthritic signs. Sham-irradiated arthritic rats (group 2) showed a full-blown arthritic syndrome. Treatment of arthritic rats with 5 x 1 Gy (group 3) or 5 x 0.5 Gy (group 4) led to a reduction of mean AS from day 21 to 29 compared with group 2 (days 27-29--group 3: p=0.037; group 4: p=0.034), with no differences in efficacy between groups 3 and 4. Concurrently, following radiation treatment there was no further increase in HPV. At the end of the observation period, this effect demonstrated a dose-dependent level of significance (days 27-29--group 3: p=0.0036; group 4: p=0.039). A significant decrease in the ESR was noted in both irradiated arthritic groups on day 21 (group 3: p=0.015; group 4: p=0.006). The histopathological analysis revealed a highly significant reduction of cartilage and bone destruction on day 30 in both irradiated groups. CONCLUSIONS This study confirms by objective criteria the anti-inflammatory efficacy of low dose RT and gives some indication for a dose dependence of its efficacy.

Inhibition of the iNOS pathway in inflammatory macrophages by low-dose X-irradiation in vitro. Is there a time dependence?

Background: Low radiation doses (≤ 1.25 Gy), if applied 6 h before or after stimulation, are known to inhibit the inducible nitric oxide synthase (iNOS) pathway in inflammatory macrophages in vitro. We therefore investigated the time dependence and the underlying molecular mechanism of this effect, since it may be involved in the clinically observed anti-inflammatory and analgesic efficacy of low-dose radiotherapy. Material and Methods: Metabolic activity, nitric oxide (NO) production, iNOS- and hemoxygenase 1-(HO-1-)protein and -mRNA expression by macrophages in vitro after stimulation with LPS/IFN-γ (0.1 μg ml−1/100 U ml−1) were investigated. Irradiation was performed at 6, 4, 2 h before and 0, 2, 4, 6 h after stimulation with doses ranging from 0.3 to 10 Gy. For each group, three independent experiments were performed over a period of 30 h with sampling intervals of 3 h. Results: In stimulated macrophages, metabolic activity was not affected by radiation doses up to 10 Gy. A dose-dependent modulation of the cumulative NO production was observed with significant inhibition by low radiation doses ≤ 1.25 Gy) and return to control level and even higher concentrations by higher doses (≤ 5 Gy). The degree of inhibition did not show any significant time dependence within the experimental time window used. The iNOS-mRNA expression 3–18 h following stimulation and subsequent irradiation was not affected by doses ≤ 1.25 Gy. The iNOS-protein expression 6–24 h following stimulation and subsequent irradiation was reduced by doses ≤ 1.25 Gy. By contrast, neither HO-1-protein nor HO-1-mRNA expression at the same time points was influenced by these low doses. Conclusion: The inhibitory interference of low radiation doses with the iNOS pathway in inflammatory macrophages appears to be based on radiation effects on the translational and posttranslational control mechanisms of iNOS activity. However, contrary to our working hypothesis this is not related to radiation-induced induction of HO-1 expression and thereby increased degradation of heme which is essential for iNOS activity. Thus, other posttranslational modifications such as the proteasome degradation pathway might be involved.Hintergrund: Niedrige Strahlendosen (≤ 1,25 Gy), wenn 6 h vor oder nach Zellstimulation appliziert, können die Aktivität der induzierbaren Stickoxidsynthase (iNOS) inflammatorischer Makrophagen in vitro inhibieren. Wir untersuchten die Zeitabhängigkeit und mögliche molekulare Wirkmechanismen dieses Effekts, da er in die klinisch beobachtete antiinflammatorische und analgetische Wirksamkeit der niedrig dosierten Strahlentherapie involviert sein könnte. Material und Methodik: Metabolische Aktivität, kumulative Stickoxid-(NO-)Produktion, iNOS- und Hämoxygenase-1-(HO-1-)Protein- und -mRNA-Expression durch Makrophagen wurden nach Stimulation mit LPS/IFN-γ (0,1 μg ml−1/100 U ml−1) in vitro untersucht. Die Bestrahlung erfolgte entweder 6, 4, 2 h vor oder 0, 2, 4, 6 h nach Stimulation mit Dosen von 0,3–10,0 Gy. Für jede Gruppe wurden drei unabhängige Experimente über eine Zeitraum von 30 h mit Zeitintervallen von 3 h durchgeführt. Ergebnisse: Die metabolische Aktivität stimulierter Makrophagen blieb nach Stahlendosen ≤ 10 Gy unbeeinflusst. Es wurde eine dosisabhängige, diskontinuierliche Modulation der kumulativen NO-Produktion mit signifikanter Inhibition durch niedrige Strahlendosen (≤ 1,25 Gy) und Rückkehr zu Kontrollwerten oder höheren Konzentrationen nach Dosen ≤ 5 Gy beobachtet. Das Ausmaß der Inhibition zeigte innerhalb des untersuchten experimentellen Zeitfensters keine signifikante Zeitabhängigkeit. Die iNOSmRNA-Expression blieb 3–18 h nach Stimulation und unmittelbar anschließender Bestrahlung mit Dosen ≤ 1,25 Gy unbeeinflusst. Die iNOS-Protein-Expression war 6–24 h nach Stimulation und unmittelbar anschließender Bestrahlung mit Dosen ≤ 1,25 Gy vermindert. Im Gegensatz dazu war weder die HO-1-Protein- noch die HO-1-mRNA-Expression zu den gleichen Zeitpunkten nach diesen niedrigen Dosen beeinflusst. Schlussfolgerung: Die inhibitorische Interferenz niedriger Strahlendosen mit dem iNOS-Pathway inflammatorischer Makrophagen scheint auf Strahleneffekten auf die translationellen und posttranslationellen Kontrollmechanismen der iNOS-Aktivität zu beruhen. Im Gegensatz zu unserer Arbeitshypothese ist dies nicht durch eine Induktion des Stressproteins HO-1 durch niedrige Dosen und die dadurch bedingte gesteigerte Degradation von Häm als essentiellem oxidativem Kofaktor der iNOS-Aktivität zu erklären. Andere posttranslationelle Modifikationen wie der Proteasom-Degradations-Pathway könnten involviert sein.

Inhibition of the iNOS Pathway in Inflammatory Macrophages by Low-Dose X-Irradiation In Vitro

Background: Low radiation doses (≤ 1.25 Gy), if applied 6 h before or after stimulation, are known to inhibit the inducible nitric oxide synthase (iNOS) pathway in inflammatory macrophages in vitro. We therefore investigated the time dependence and the underlying molecular mechanism of this effect, since it may be involved in the clinically observed anti-inflammatory and analgesic efficacy of low-dose radiotherapy. Material and Methods: Metabolic activity, nitric oxide (NO) production, iNOS- and hemoxygenase 1-(HO-1-)protein and -mRNA expression by macrophages in vitro after stimulation with LPS/IFN-γ (0.1 μg ml−1/100 U ml−1) were investigated. Irradiation was performed at 6, 4, 2 h before and 0, 2, 4, 6 h after stimulation with doses ranging from 0.3 to 10 Gy. For each group, three independent experiments were performed over a period of 30 h with sampling intervals of 3 h. Results: In stimulated macrophages, metabolic activity was not affected by radiation doses up to 10 Gy. A dose-dependent modulation of the cumulative NO production was observed with significant inhibition by low radiation doses ≤ 1.25 Gy) and return to control level and even higher concentrations by higher doses (≤ 5 Gy). The degree of inhibition did not show any significant time dependence within the experimental time window used. The iNOS-mRNA expression 3–18 h following stimulation and subsequent irradiation was not affected by doses ≤ 1.25 Gy. The iNOS-protein expression 6–24 h following stimulation and subsequent irradiation was reduced by doses ≤ 1.25 Gy. By contrast, neither HO-1-protein nor HO-1-mRNA expression at the same time points was influenced by these low doses. Conclusion: The inhibitory interference of low radiation doses with the iNOS pathway in inflammatory macrophages appears to be based on radiation effects on the translational and posttranslational control mechanisms of iNOS activity. However, contrary to our working hypothesis this is not related to radiation-induced induction of HO-1 expression and thereby increased degradation of heme which is essential for iNOS activity. Thus, other posttranslational modifications such as the proteasome degradation pathway might be involved.Hintergrund: Niedrige Strahlendosen (≤ 1,25 Gy), wenn 6 h vor oder nach Zellstimulation appliziert, können die Aktivität der induzierbaren Stickoxidsynthase (iNOS) inflammatorischer Makrophagen in vitro inhibieren. Wir untersuchten die Zeitabhängigkeit und mögliche molekulare Wirkmechanismen dieses Effekts, da er in die klinisch beobachtete antiinflammatorische und analgetische Wirksamkeit der niedrig dosierten Strahlentherapie involviert sein könnte. Material und Methodik: Metabolische Aktivität, kumulative Stickoxid-(NO-)Produktion, iNOS- und Hämoxygenase-1-(HO-1-)Protein- und -mRNA-Expression durch Makrophagen wurden nach Stimulation mit LPS/IFN-γ (0,1 μg ml−1/100 U ml−1) in vitro untersucht. Die Bestrahlung erfolgte entweder 6, 4, 2 h vor oder 0, 2, 4, 6 h nach Stimulation mit Dosen von 0,3–10,0 Gy. Für jede Gruppe wurden drei unabhängige Experimente über eine Zeitraum von 30 h mit Zeitintervallen von 3 h durchgeführt. Ergebnisse: Die metabolische Aktivität stimulierter Makrophagen blieb nach Stahlendosen ≤ 10 Gy unbeeinflusst. Es wurde eine dosisabhängige, diskontinuierliche Modulation der kumulativen NO-Produktion mit signifikanter Inhibition durch niedrige Strahlendosen (≤ 1,25 Gy) und Rückkehr zu Kontrollwerten oder höheren Konzentrationen nach Dosen ≤ 5 Gy beobachtet. Das Ausmaß der Inhibition zeigte innerhalb des untersuchten experimentellen Zeitfensters keine signifikante Zeitabhängigkeit. Die iNOSmRNA-Expression blieb 3–18 h nach Stimulation und unmittelbar anschließender Bestrahlung mit Dosen ≤ 1,25 Gy unbeeinflusst. Die iNOS-Protein-Expression war 6–24 h nach Stimulation und unmittelbar anschließender Bestrahlung mit Dosen ≤ 1,25 Gy vermindert. Im Gegensatz dazu war weder die HO-1-Protein- noch die HO-1-mRNA-Expression zu den gleichen Zeitpunkten nach diesen niedrigen Dosen beeinflusst. Schlussfolgerung: Die inhibitorische Interferenz niedriger Strahlendosen mit dem iNOS-Pathway inflammatorischer Makrophagen scheint auf Strahleneffekten auf die translationellen und posttranslationellen Kontrollmechanismen der iNOS-Aktivität zu beruhen. Im Gegensatz zu unserer Arbeitshypothese ist dies nicht durch eine Induktion des Stressproteins HO-1 durch niedrige Dosen und die dadurch bedingte gesteigerte Degradation von Häm als essentiellem oxidativem Kofaktor der iNOS-Aktivität zu erklären. Andere posttranslationelle Modifikationen wie der Proteasom-Degradations-Pathway könnten involviert sein.

Low-dose X-irradiation of adjuvant-induced arthritis in rats: Efficacy of different fractionation schedules

Background and Purpose:Low-dose radiotherapy is widely accepted as a very effective treatment option for inflammatory symptoms associated with painful degenerative joint disorders. Radiation doses and fractionation schedules in practical use are empirical and mainly based on clinical observations. Experimental data are rare. The efficacy of low-dose X-irradiation on adjuvant induced arthritis in rats using different fractionation schemes was investigated in vivo, in order to explore whether there is a dose and fractionation dependence.Material and Methods:Adjuvant arthritis in female Lewis rats (n = 128) was induced by intradermal injection of heat-inactivated Mycobacterium tuberculosis on day 0. Both arthritic hind paws were sham-irradiated (group 1: days 10–14; group 2: days 15–19; group 3: days 22–26) or X-irradiated with either 5 × 1.0 Gy (group 4: days 10–14; group 6: days 15–19; group 8: days 22–26; group 10: days 10, 12, 14, 16, and 18) or 5 × 0.5 Gy (group 5: days 10–14; group 7: days 15–19; group 9: days 22–26; group 11: days 10, 12, 14, 16, and 18; group 12: days 10–14 and 22–26). The clinical parameters arthritis score (AS), hind paw volume (HPV), and body weight were determined.Results:A significant decrease of the clinical arthritis parameters was observed following 5 × 0.5 Gy or 5 × 1.0 Gy during the acute maximum of the inflammatory response (days 15–19). The most pronounced treatment effect was reached after two daily fractionated series of 5 × 0.5 Gy with an early treatment onset (days 10–14) and repetition in interval (days 22–26). After the application of 5 × 1.0 Gy on days 10–14 or in a protracted scheme (days 10, 12, 14, 16, and 18), only a nonsignificant positive trend could be detected. Daily fractionated X-irradiation in the chronic phase of adjuvant arthritis (days 22–26) did not show any positive clinical effect.Conclusion:Low-dose radiotherapy is able to prevent a full-blown arthritic reaction if given during the florid phase of adjuvant arthritis. Two series of 5 × 0.5 Gy with an early treatment onset (days 10–14) and repetition in interval (days 22–26) were the most effective treatment schedule in this experimental study.Hintergrund und Ziel:Die niedrig dosierte Strahlentherapie wird als effektive Behandlungsoption bei schmerzhaften, entzündlich reaktivierten degenerativen Gelenkerkrankungen angesehen. Die verwendeten Einzeldosen und Fraktionierungen basieren im Wesentlichen auf klinisch-empirischen Beobachtungen. Experimentelle Untersuchungen sind sehr selten. Das Ziel dieser Studie war die Beurteilung der klinischen Wirksamkeit verschiedener Fraktionierungsschemata und Einzeldosen in einem experimentellen Arthritismodell der Ratte, um zu überprüfen, ob eine Dosis- oder Fraktionierungsabhängigkeit vorliegt.Material und Methodik:Bei weiblichen Lewis-Ratten (n = 128) wurde durch intradermale Injektion von hitzeinaktiviertem Mycobacterium tuberculosis am Tag 0 eine Adjuvansarthritis induziert. Beide Hinterpfoten arthritischer Versuchstiere wurden scheinbestrahlt (Gruppe 1: Tage 10–14; Gruppe 2: Tage 15–19; Gruppe 3: Tage 22–26) oder lokal bestrahlt mit entweder 5 × 1,0 Gy (Gruppe 4: Tage 10–14; Gruppe 6: Tage 15–19; Gruppe 8: Tage 22–26; Gruppe 10: Tage 10, 12, 14, 16 und 18) oder 5 × 0,5 Gy (Gruppe 5: Tage 10–14; Gruppe 7: Tage 15–19; Gruppe 9: Tage 22–26; Gruppe 11: Tage 10, 12, 14, 16 und 18; Gruppe 12: Tage 10–14 und 22–26). Die klinischen Parameter Arthritisscore (AS), Hinterpfotenvolumen (HPV) und Körpergewicht wurden im Verlauf bestimmt.Ergebnisse:Nach 5 × 0,5 Gy oder 5 × 1,0 Gy während des akuten Entzündungsmaximums (Tage 15–19) konnte eine signifikante Reduktion der klinischen Arthritisparameter beobachtet werden. Der günstigste Behandlungseffekt wurde mit zwei täglich fraktionierten Serien von 5 × 0,5 Gy bei frühzeitigem Behandlungsbeginn (Tage 10–14) und Wiederholung im Intervall (Tage 22–26) erreicht. Nach 5 × 1,0 Gy an den Tagen 10–14 oder in einem protrahierten Schema (Tage 10, 12, 14, 16 und 18) war lediglich ein nichtsignifikanter positiver Trend nachweisbar. Die tägliche fraktionierte Bestrahlung in der chronischen Phase der Adjuvansarthritis (Tage 22-26) zeigte keinen positiven klinischen Effekt.Schlussfolgerung:Eine niedrig dosierte Strahlentherapie ist in der Lage, die volle Ausprägung einer arthritischen Reaktion zu verhindern, wenn sie während der floriden Phase der Adjuvansarthritis appliziert wird. In dieser Studie waren zwei Bestrahlungsserien von 5 × 0,5 Gy mit frühzeitigem Behandlungsbeginn (Tage 10–14) und Wiederholung im Intervall (Tage 22–26) das effektivste Behandlungsschema.

Low-Dose X-Irradiation of Adjuvant-Induced Arthritis in Rats

Background and Purpose:Low-dose radiotherapy is widely accepted as a very effective treatment option for inflammatory symptoms associated with painful degenerative joint disorders. Radiation doses and fractionation schedules in practical use are empirical and mainly based on clinical observations. Experimental data are rare. The efficacy of low-dose X-irradiation on adjuvant induced arthritis in rats using different fractionation schemes was investigated in vivo, in order to explore whether there is a dose and fractionation dependence.Material and Methods:Adjuvant arthritis in female Lewis rats (n = 128) was induced by intradermal injection of heat-inactivated Mycobacterium tuberculosis on day 0. Both arthritic hind paws were sham-irradiated (group 1: days 10–14; group 2: days 15–19; group 3: days 22–26) or X-irradiated with either 5 × 1.0 Gy (group 4: days 10–14; group 6: days 15–19; group 8: days 22–26; group 10: days 10, 12, 14, 16, and 18) or 5 × 0.5 Gy (group 5: days 10–14; group 7: days 15–19; group 9: days 22–26; group 11: days 10, 12, 14, 16, and 18; group 12: days 10–14 and 22–26). The clinical parameters arthritis score (AS), hind paw volume (HPV), and body weight were determined.Results:A significant decrease of the clinical arthritis parameters was observed following 5 × 0.5 Gy or 5 × 1.0 Gy during the acute maximum of the inflammatory response (days 15–19). The most pronounced treatment effect was reached after two daily fractionated series of 5 × 0.5 Gy with an early treatment onset (days 10–14) and repetition in interval (days 22–26). After the application of 5 × 1.0 Gy on days 10–14 or in a protracted scheme (days 10, 12, 14, 16, and 18), only a nonsignificant positive trend could be detected. Daily fractionated X-irradiation in the chronic phase of adjuvant arthritis (days 22–26) did not show any positive clinical effect.Conclusion:Low-dose radiotherapy is able to prevent a full-blown arthritic reaction if given during the florid phase of adjuvant arthritis. Two series of 5 × 0.5 Gy with an early treatment onset (days 10–14) and repetition in interval (days 22–26) were the most effective treatment schedule in this experimental study.Hintergrund und Ziel:Die niedrig dosierte Strahlentherapie wird als effektive Behandlungsoption bei schmerzhaften, entzündlich reaktivierten degenerativen Gelenkerkrankungen angesehen. Die verwendeten Einzeldosen und Fraktionierungen basieren im Wesentlichen auf klinisch-empirischen Beobachtungen. Experimentelle Untersuchungen sind sehr selten. Das Ziel dieser Studie war die Beurteilung der klinischen Wirksamkeit verschiedener Fraktionierungsschemata und Einzeldosen in einem experimentellen Arthritismodell der Ratte, um zu überprüfen, ob eine Dosis- oder Fraktionierungsabhängigkeit vorliegt.Material und Methodik:Bei weiblichen Lewis-Ratten (n = 128) wurde durch intradermale Injektion von hitzeinaktiviertem Mycobacterium tuberculosis am Tag 0 eine Adjuvansarthritis induziert. Beide Hinterpfoten arthritischer Versuchstiere wurden scheinbestrahlt (Gruppe 1: Tage 10–14; Gruppe 2: Tage 15–19; Gruppe 3: Tage 22–26) oder lokal bestrahlt mit entweder 5 × 1,0 Gy (Gruppe 4: Tage 10–14; Gruppe 6: Tage 15–19; Gruppe 8: Tage 22–26; Gruppe 10: Tage 10, 12, 14, 16 und 18) oder 5 × 0,5 Gy (Gruppe 5: Tage 10–14; Gruppe 7: Tage 15–19; Gruppe 9: Tage 22–26; Gruppe 11: Tage 10, 12, 14, 16 und 18; Gruppe 12: Tage 10–14 und 22–26). Die klinischen Parameter Arthritisscore (AS), Hinterpfotenvolumen (HPV) und Körpergewicht wurden im Verlauf bestimmt.Ergebnisse:Nach 5 × 0,5 Gy oder 5 × 1,0 Gy während des akuten Entzündungsmaximums (Tage 15–19) konnte eine signifikante Reduktion der klinischen Arthritisparameter beobachtet werden. Der günstigste Behandlungseffekt wurde mit zwei täglich fraktionierten Serien von 5 × 0,5 Gy bei frühzeitigem Behandlungsbeginn (Tage 10–14) und Wiederholung im Intervall (Tage 22–26) erreicht. Nach 5 × 1,0 Gy an den Tagen 10–14 oder in einem protrahierten Schema (Tage 10, 12, 14, 16 und 18) war lediglich ein nichtsignifikanter positiver Trend nachweisbar. Die tägliche fraktionierte Bestrahlung in der chronischen Phase der Adjuvansarthritis (Tage 22-26) zeigte keinen positiven klinischen Effekt.Schlussfolgerung:Eine niedrig dosierte Strahlentherapie ist in der Lage, die volle Ausprägung einer arthritischen Reaktion zu verhindern, wenn sie während der floriden Phase der Adjuvansarthritis appliziert wird. In dieser Studie waren zwei Bestrahlungsserien von 5 × 0,5 Gy mit frühzeitigem Behandlungsbeginn (Tage 10–14) und Wiederholung im Intervall (Tage 22–26) das effektivste Behandlungsschema.

Influence of low dose irradiation on differentiation, maturation and T-cell activation of human dendritic cells

Ionizing irradiation could act directly on immune cells and may induce bystander effects mediated by soluble factors that are released by the irradiated cells. This is the first study analyzing both the direct effect of low dose ionizing radiation (LDIR) on the maturation and cytokine release of human dendritic cells (DCs) and the functional consequences for co-cultured T-cells. We showed that irradiation of DC-precursors in vitro does not influence surface marker expression or cytokine profile of immature DCs nor of mature DCs after LPS treatment. There was no difference of single dose irradiation versus fractionated irradiation protocols on the behavior of the mature DCs. Further, the low dose irradiation did not change the capacity of the DCs to stimulate T-cell proliferation. But the irradiation of the co-culture of DCs and T-cells revealed significantly lower proliferation of T-cells with higher doses. Summarizing the data from approx. 50 DC preparations there is no significant effect of low dose ionizing irradiation on the cytokine profile, surface marker expression and maturation of DCs in vitro although functional consequences cannot be excluded.

Additive effects of 5-aza-2'-deoxycytidine and irradiation on clonogenic survival of human medulloblastoma cell lines.

Background and Purpose:In recent years, epigenetic modulators were introduced into tumor therapy. Here, the authors investigated the antitumor effect of 5-aza-2’-deoxycytidine-(5-aza-dC-)induced demethylation combined with irradiation on human medulloblastoma (MB) cells, which form the most common malignant brain tumor in children.Material and Methods:Three MB cell lines were treated with 5-aza-dC in a low-dose (0.1 μM, 6 days) or high-dose (3/5 μM, 3 days) setting and irradiated with 2, 4, 6, or 8 Gy single dose on an X-ray unit. Methylation status and mRNA expression of three candidate genes were analyzed by methylation-specific PCR (polymerase chain reaction) and quantitative real-time RT-PCR. Cell survival and mortality were determined by trypan blue exclusion test. Proliferation was analyzed by BrdU incorporation assay, and long-term cell survival was assessed by clonogenic assay.Results:5-aza-dC treatment resulted in partial promoter demethylation and increased expression of hypermethylated candidate genes. A significant decrease of vital cell count, proliferation inhibition and increase of mortality was observed in 5-aza-dC-treated as well as in irradiated MB cells, whereby combination of both treatments led to additive effects. Although high-dose 5-aza-dC treatment was more effective in terms of demethylation, clonogenic assay revealed no differences between high- and low-dose settings indicating no relevance of 5-aza-dC-induced demethylation for decreased cell survival. MB cells pretreated with 5-aza-dC showed significantly lower plating efficiencies than untreated cells at all irradiation doses investigated. Analysis of surviving curves in irradiated MB cells, however, revealed no significant differences of α-, β-values and 2-Gy surviving fraction with or without 5-aza-dC treatment.Conclusion:5-aza-dC did not enhance radiation sensitivity of MB cells but significantly reduced the clonogenicity versus irradiation alone, which merits further investigation of its potential clinical application in MB possibly by combination with other chemotherapeutic agents.Hintergrund und Ziel:In den letzten Jahren wurden epigenetische Modulatoren in die Tumortherapie eingeführt. In dieser Arbeit untersuchten die Autoren den Antitumoreffekt der 5-Aza-2’-deoxycytidin-(5-aza-dC-)induzierten Demethylierung in Kombination mit Bestrahlung auf humane Medulloblastom-(MB-)Zellen, welche die häufigsten malignen Hirntumoren im Kindesalter bilden.Material und Methodik:Drei MB-Zell-Linien wurden mit 5-aza-dC in Niedrigdosis (0,1 μM, 6 Tage) oder Hochdosis (3/5 μM, 3 Tage) behandelt und mit 2, 4, 6 oder 8 Gy Einzeldosis bestrahlt. Die Untersuchung des Methylierungsstatus und der mRNA-Expression von drei Kandidatengenen erfolgte durch methylierungsspezifische PCR (Polymerase-Kettenreaktion) und quantitative Real-Time-RT-PCR. Lebendzellraten und Mortalität wurden durch Trypanblau-Ausschlusstest und die Proliferationsrate im BrdU-Assay bestimmt. Das Langzeitüberleben wurde durch einen klonogenen Assay ermittelt.Ergebnisse:Die 5-aza-dC-Behandlung führte zur partiellen Promotordemethylierung und zu einem Anstieg der Expression hypermethylierter Kandidatengene. Eine signifikante Verminderung der Lebendzellzahl und Proliferation bei Zunahme der Mortalität wurde sowohl in 5-aza-dC-behandelten als auch in bestrahlten MB-Zellen beobachtet. Bei kombinierter Behandlung summierten sich die Effekte der Einzelbehandlungen. Während die Inkubation mit 5-aza-dC in Hochdosis hinsichtlich der Demethylierung effektiver war als in Niedrigdosis, ergaben sich im klonogenen Assay keine Unterschiede zwischen beiden Behandlungsschemata, was darauf hinweist, dass die 5-aza-dC-induzierte Demethylierung nicht relevant für die Verminderung des Zellüberlebens ist. Mit 5-aza-dC vorbehandelte Zellen zeigten im untersuchten Bestrahlungsdosisbereich eine signifikant niedrigere „plating efficiency“ als unbehandelte Zellen. Die Überlebenskurven bestrahlter MB-Zellen wiesen jedoch keine signifikanten Unterschiede der α/β-Werte und der Überlebensfraktion nach 2 Gy in 5-aza-dC-behandelten gegenüber unbehandelten Zellen auf.Schlussfolgerung:5-aza-dC erhöhte die Strahlensensitivität von MB-Zellen nicht, reduzierte jedoch die Klonogenität signifikant gegenüber alleiniger Bestrahlung, was weitere Untersuchungen zur potentiellen klinischen Anwendung von 5-aza-dC bei MB, möglicherweise in Kombination mit anderen Chemotherapeutika, rechtfertigt.

Allogeneic Non-Adherent Bone Marrow Cells Facilitate Hematopoietic Recovery but Do Not Lead to Allogeneic Engraftment

Background Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4+, murine CD4−/−, HLA-DR3+ mice. Methodology/Principal Findings Bone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45+ showed depletion of CD4+, CD8+, CD19+, and CD117+ cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4+ donor chimerism of 5.8±2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death. Conclusions/Significance NA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies.