Mirko Nitsche

Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions

Microglia are crucially important myeloid cells in the CNS and constitute the first immunological barrier against pathogens and environmental insults. The factors controlling microglia recruitment from the blood remain elusive and the direct circulating microglia precursor has not yet been identified in vivo. Using a panel of bone marrow chimeric and adoptive transfer experiments, we found that circulating Ly-6ChiCCR2+ monocytes were preferentially recruited to the lesioned brain and differentiated into microglia. Notably, microglia engraftment in CNS pathologies, which are not associated with overt blood-brain barrier disruption, required previous conditioning of brain (for example, by direct tissue irradiation). Our results identify Ly-6ChiCCR2+ monocytes as direct precursors of microglia in the adult brain and establish the importance of local factors in the adult CNS for microglia engraftment.

In vitro response of human dermal fibroblasts to X-irradiation: relationship between radiation-induced clonogenic cell death, chromosome aberrations and markers of proliferative senescence or differentiation.

Purpose : To analyse the relationship between radiation-induced clonogenic cell death, chromosome aberrations and markers of proliferative senescence or differentiation. Materials and methods : Plateau-phase human dermal fibroblasts from 18 donors were irradiated with graded doses of 1-6 Gy 200kV X-rays. Cell survival was determined by a colony-forming assay. Markers of differentiation or senescence were: spontaneous and radiation-induced clonal differentiation, which was determined morphologically and by the cellular potential to proliferate in clonal culture, also single-cell g -galactosidase (g -gal) staining at pH 6.0; and the secretion of transforming growth factor- g (TGF- g 1) into the culture medium. Chromosome aberrations were determined as genomic yields of dicentric chromosomes and the excess acentric fragments, scored in Giemsa-stained metaphases, and as partial yields of reciprocal translocations for chromosomes 4, 7 and 9 using the FISH method. Results : A broad spread was found in the shapes of the survival curves, with SF2 ranging from 0.041 - 0.015 to 0.63 - 0.05. Radiation-induced clonal differentiation as well as the secretion of TGF- g 1 was elevated in radiosensitive samples. With respect to chromosome aberrations, a significant correlation was found between clonogenic survival and radiation-induced excess acentric fragments. Conclusions : In the fibroblast cell system, in vitro radiosensitivity is determined not only by processes directly involved in DNA-damage recognition and repair, but also by intracellular signalling cascades, which will lead to differentiation processes.

Neck lymph node metastases from an unknown primary tumor retrospective study and review of literature.

Background and Purpose:Up to 10% of all neck lymph node metastases present without a known primary site. The optimal treatment strategy for these patients is still undefined. The purpose of this retrospective analysis is to assess the outcome in patients with neck metastases from an unknown primary tumor (CUP). Furthermore, prognostic factors and treatment modalities are discussed.Patients and Methods:From 1984 to 2003, 28 patients with squamous cell neck metastases from a CUP were treated at the authors’ institution. In 17 patients, neck dissection (twelve radical, five modified radical) was performed. In that case, adjuvant radiotherapy was carried out with a mean of 56.7 Gy. In eleven patients, only biopsies were done. These patients received definitive radiotherapy with a mean of 66.8 Gy. In summary, 25 patients received extended radiotherapy including both sides of the neck and potential mucosal primary sites. Additional chemotherapy was administered to five patients.Results:The duration of follow-up was 4.1–189.5 months (median 45.1 months). After this period of time, ten patients (36%) remained alive. 5-year overall survival was 40.1%, neck control rate 72.7%. No subsequent primary could be detected. Extracapsular extension and surgery had significant influence on prognosis. Grade 3 toxicity (mucositis or skin reactions) was seen in three patients; no hematologic toxicity > grade 2 was observed. 19 patients suffered from grade 2 xerostomia.Conclusion:With radical surgery followed by radiotherapy good survival rates in patients with neck metastases from a CUP can be obtained. Whether limited radiotherapy might be equal to extended irradiation and can reduce side effects, must be shown in ongoing clinical trials.Hintergrund und Ziel:Bei bis zu 10% aller Patienten mit zervikalen Lymphknotenmetastasen wird der Primärtumor nicht gefunden. Die Behandlungskonzepte für diese Patienten sind bisher noch uneinheitlich. Die vorliegende retrospektive Analyse zeigt die Therapiemodalitäten und Behandlungsergebnisse in der Universitätsklinik Göttingen. Außerdem werden Therapieoptionen und prognostische Faktoren diskutiert.Patienten und Methodik:Von 1984 bis 2003 wurden 28 Patienten mit zervikalen Lymphknotenmetastasen eines Plattenepithelkarzinoms mit unbekanntem Primärtumor in der Universitätsklinik Göttingen behandelt. 17 der Patienten wurden zervikal lymphdisseziert (zwölf radikal, fünf modifiziert radikal). Sie erhielten anschließend eine adjuvante Strahlentherapie mit durchschnittlich 56,7 Gy. Elf Patienten wurden biopsiert und mit primärer Radiotherapie behandelt (durchschnittliche Dosis: 66,8 Gy). Die Bestrahlung umfasste bei insgesamt 25 Patienten die zervikalen Lymphabflussgebiete beidseits sowie den gesamten Rachenraum, vier Patienten wurden lediglich ipsilateral zervikal bestrahlt.Ergebnisse:Die Nachbeobachtungszeit betrug median 45,1 Monate (4,1–189,5 Monate). Nach 5 Jahren betrugen das Gesamtüberleben 40,1% und die lokoregionäre Kontrollrate am Hals 72,7%. Ein Primärtumor konnte im Verlauf bei keinem Patienten identifiziert werden. Patienten, die radikal lymphdisseziert wurden, zeigten signifikant bessere Überlebens- sowie lokoregionäre Kontrollraten als Patienten, bei denen eine Biopsie durchgeführt wurde. Die lokoregionäre Kontrolle war bei Lymphknoten mit Kapseldurchbruch signifikant schlechter als ohne Kapseldurchbruch.Schlussfolgerung:Mit radikaler Operation und anschließender adjuvanter Radiotherapie können bei Patienten mit zervikalen Lymphknotenmetastasen eines unbekannten Primärtumors gute Überlebens- und lokoregionäre Kontrollraten erzielt werden. Ob durch eine—weniger toxische—auf das ipsilaterale zervikale Lymphabflussgebiet limitierte Bestrahlung dieselben Ergebnisse wie durch eine ausgedehnte Bestrahlung der gesamten Rachenregion erreicht werden können, muss durch laufende klinische Studien gezeigt werden.

Plasminogen Activator Inhibitor-1 From Bone Marrow–Derived Cells Suppresses Neointimal Formation After Vascular Injury in Mice

Objective—To investigate the ability of bone marrow (BM)–derived cells to modulate neointimal growth after injury by expressing plasminogen activator inhibitor-1 (PAI-1). Methods and Results—We performed BM transplantation (BMT) in lethally irradiated wild-type (WT) and PAI-1−/− mice. Three weeks after carotid injury with ferric chloride, analysis of Y-chromosome DNA expression in the vessel wall of female hosts revealed that 20.8±6.0% of the cells in the neointima and 37.6±5.7% of those in the media were of BM origin. Lack of PAI-1 in either the host or the donor cells did not affect recruitment of BM-derived cells into sites of vascular injury. The neointima consisted predominantly of smooth muscle cells, and a proportion of these cells expressed PAI-1. Overall, lack of PAI-1 was associated with enhanced neointimal formation. However, importantly, BMTWT→PAI-1−/− mice exhibited reduced neointimal area (P=0.05) and luminal stenosis (P=0.04) compared with BMTPAI-1−/−→PAI-1−/− mice. Although PAI-1–expressing cells were shown to be present in BMTWT→PAI-1−/− lesions, these mice did not exhibit detectable levels of the inhibitor in the circulation, suggesting that local production of PAI-1 by cells in the neointima and media was sufficient to reduce luminal stenosis. Conclusions—PAI-1 from BM-derived cells appears capable of suppressing neointimal growth after vascular injury.

Neck Lymph Node Metastases from an Unknown Primary Tumor

Background and Purpose:Up to 10% of all neck lymph node metastases present without a known primary site. The optimal treatment strategy for these patients is still undefined. The purpose of this retrospective analysis is to assess the outcome in patients with neck metastases from an unknown primary tumor (CUP). Furthermore, prognostic factors and treatment modalities are discussed.Patients and Methods:From 1984 to 2003, 28 patients with squamous cell neck metastases from a CUP were treated at the authors’ institution. In 17 patients, neck dissection (twelve radical, five modified radical) was performed. In that case, adjuvant radiotherapy was carried out with a mean of 56.7 Gy. In eleven patients, only biopsies were done. These patients received definitive radiotherapy with a mean of 66.8 Gy. In summary, 25 patients received extended radiotherapy including both sides of the neck and potential mucosal primary sites. Additional chemotherapy was administered to five patients.Results:The duration of follow-up was 4.1–189.5 months (median 45.1 months). After this period of time, ten patients (36%) remained alive. 5-year overall survival was 40.1%, neck control rate 72.7%. No subsequent primary could be detected. Extracapsular extension and surgery had significant influence on prognosis. Grade 3 toxicity (mucositis or skin reactions) was seen in three patients; no hematologic toxicity > grade 2 was observed. 19 patients suffered from grade 2 xerostomia.Conclusion:With radical surgery followed by radiotherapy good survival rates in patients with neck metastases from a CUP can be obtained. Whether limited radiotherapy might be equal to extended irradiation and can reduce side effects, must be shown in ongoing clinical trials.Hintergrund und Ziel:Bei bis zu 10% aller Patienten mit zervikalen Lymphknotenmetastasen wird der Primärtumor nicht gefunden. Die Behandlungskonzepte für diese Patienten sind bisher noch uneinheitlich. Die vorliegende retrospektive Analyse zeigt die Therapiemodalitäten und Behandlungsergebnisse in der Universitätsklinik Göttingen. Außerdem werden Therapieoptionen und prognostische Faktoren diskutiert.Patienten und Methodik:Von 1984 bis 2003 wurden 28 Patienten mit zervikalen Lymphknotenmetastasen eines Plattenepithelkarzinoms mit unbekanntem Primärtumor in der Universitätsklinik Göttingen behandelt. 17 der Patienten wurden zervikal lymphdisseziert (zwölf radikal, fünf modifiziert radikal). Sie erhielten anschließend eine adjuvante Strahlentherapie mit durchschnittlich 56,7 Gy. Elf Patienten wurden biopsiert und mit primärer Radiotherapie behandelt (durchschnittliche Dosis: 66,8 Gy). Die Bestrahlung umfasste bei insgesamt 25 Patienten die zervikalen Lymphabflussgebiete beidseits sowie den gesamten Rachenraum, vier Patienten wurden lediglich ipsilateral zervikal bestrahlt.Ergebnisse:Die Nachbeobachtungszeit betrug median 45,1 Monate (4,1–189,5 Monate). Nach 5 Jahren betrugen das Gesamtüberleben 40,1% und die lokoregionäre Kontrollrate am Hals 72,7%. Ein Primärtumor konnte im Verlauf bei keinem Patienten identifiziert werden. Patienten, die radikal lymphdisseziert wurden, zeigten signifikant bessere Überlebens- sowie lokoregionäre Kontrollraten als Patienten, bei denen eine Biopsie durchgeführt wurde. Die lokoregionäre Kontrolle war bei Lymphknoten mit Kapseldurchbruch signifikant schlechter als ohne Kapseldurchbruch.Schlussfolgerung:Mit radikaler Operation und anschließender adjuvanter Radiotherapie können bei Patienten mit zervikalen Lymphknotenmetastasen eines unbekannten Primärtumors gute Überlebens- und lokoregionäre Kontrollraten erzielt werden. Ob durch eine—weniger toxische—auf das ipsilaterale zervikale Lymphabflussgebiet limitierte Bestrahlung dieselben Ergebnisse wie durch eine ausgedehnte Bestrahlung der gesamten Rachenregion erreicht werden können, muss durch laufende klinische Studien gezeigt werden.

Radiosensitization dependent on p53 function in bronchial carcinoma cells by the isoflavone genistein and estradiol in vitro.

Background and Purpose:Simultaneous radiotherapy with chemotherapy is a standard treatment for inoperable non-small cell lung cancer (NSCLC), but the clinical outcome still remains poor. To further intensify treatment, substances need to be identified, which increase the effect of radiation on tumor cells without further enhancing toxicity to normal tissue. Hormones have a different toxicity profile than radiation or cytostatic drugs. As NSCLC often express estrogen receptors (ERs), the combination of genistein or estradiol and radiation in vitro was investigated.Material and Methods:A549 NSCLC cells with an inducible expression of a mutated TP53 and fibroblasts of a male donor (DF-18) were examined. ER expression was immunocytologically confirmed in all studied cell lines. Clonogenic survival was measured after incubation of the cells with genistein or estradiol (0.01 μM and 10 μM as maximum clinically applicable dose) and irradiation with different doses (0–4 Gy). The differentiation state of fibroblasts after combined therapy was analyzed.Results:A549 cells expressing mutated TP53 were more radioresistant than TP53 wild-type cells. Incubation of nonfunctional TP53 cells with genistein or estradiol increased radiosensitivity in both tested concentrations. By contrast, radiosensitivity of A549 with wild-type TP53 and DF-18 was not altered by hormonal incubation. In DF-18 radiation induced growth arrest that was not increased by additional hormonal incubation.Conclusion:NSCLC cells with nonfunctional TP53 might be sensitized against radiation by genistein or estradiol. As genistein is better tolerable than estradiol in patients, additional studies are warranted to assess potential gains of this combination therapy.Hintergrund und Ziel:Etablierte Standardtherapie für inoperable nichtkleinzellige Bronchialkarzinome ist die simultane Radiochemotherapie. Trotz dieser intensiven Therapie ist die Prognose schlecht. Um die Therapie zusätzlich zu intensivieren, müssen Substanzen gefunden werden, die die Bestrahlungseffekte an den Tumorzellen verstärken, ohne die toxischen Nebenwirkungen an Normalgeweben zu erhöhen. Da nichtkleinzellige Bronchialkarzinome oft Estrogenrezeptoren (ERs) exprimieren, wurde die Kombination von Genistein oder Estradiol und Bestrahlung in vitro untersucht.Material und Methodik:A549-Bronchialkarzinomzellen mit induzierbarer Expression von mutiertem TP53 und Fibroblasten eines männlichen Spenders (DF-18) wurden untersucht. Die Expression von ER-β und ER-β wurde immunzytologisch in allen untersuchten Zelllinien bestätigt. Das klonogene Zellüberleben wurde nach Inkubation der Zellen mit Genistein oder Estradiol (0,01 μM und 10 μM [klinisch maximal applizierbare Dosis]) und Bestrahlung mit verschiedenen Dosen (0–4 Gy) gemessen. Der Differenzierungsstatus der Fibroblasten wurde nach Einwirken der Kombinationstherapie analysiert.Ergebnisse:A549-Zellen mit Expression von mutiertem TP53 waren strahlenresistenter als TP53-Wildtyp-Zellen. Inkubation der TP53-mutierten Zellen mit Genistein oder Estradiol erhöhte die Radiosensibilität in beiden getesteten Hormonkonzentrationen. Die Radiosensibilität der A549-Zellen mit funktionellem p53 und der DF-18-Fibroblasten wurde durch die Hormoninkubation nicht beeinflusst. Bei den Fibroblasten zeigte sich kein Einfluss der begleitenden Hormontherapie auf die strahleninduzierte proliferative Seneszenz.Schlussfolgerung:Bronchialkarzinome mit nichtfunktionellem TP53 scheinen durch die Inkubation mit Genistein oder Estradiol radiosensibilisiert zu werden. Da Genistein von den Patienten besser vertragen wird als Estradiol, sollten weitere Studien einen etwaigen klinischen Nutzen durch die Kombinationstherapie untersuchen.

Concomitant radiochemotherapy in primary inoperable advanced head and neck cancer with 5-fluorouracil and mitomycin-C

The purpose of this study was to evaluate the efficacy and toxicity of radiotherapy and concomitant 5‐fluorouracil (5‐FU) and mitomycin‐C infusion in inoperable head and neck cancer.

Rationale for Individualized Therapy in Sinonasal Teratocarcinosarcoma (SNTC): Case Report

Background: Sinonasal Teratocarcinosarcoma (SNTC) is a very unusual and aggressive neoplasm characterized by the combination of malignant teratoma and carcinosarcoma features. We present the first case of malignant SNTC treated with individualized multimodal therapy including a histology-specific chemotherapy. Case Report: A 31-year-old man presented with an obstruction of the right pansinus. Histology showed an SNTC with major parts of small cell, poorly differentiated carcinoma and a small proportion of highly differentiated embryonal rhabdomyosarcoma. An operation was performed followed by intraoperative application of a 5-FU ointment. Adjuvant chemotherapy with cisplatin, etoposid and ifosfamid were given in regard to the major components of this heterogeneous tumor. Radiotherapy up to 59.4 Gy was applied. Conclusion: 36 months after the end of therapy, there is no sign of tumor recurrence or metastasis in our patient. We suggest that surgery, radiotherapy and a histology-specific multidrug chemotherapy seems to be a therapeutic approach that is appropriate for this heterogeneous tumor.

The combined effect of fludarabine monophosphate and radiation as well as gemcitabine and radiation on squamous carcinoma tumor cell lines in vitro.

Purpose: Despite proven antitumor activity of gemcitabine in chemoradiotherapy of advanced head and neck cancer, many authors refer to severe acute and late local and haematological toxicity. Fludarabine does imply nearly the same mechanisms of action as gemcitabine, inhibiting various enzymes involved in DNA replication. This investigation focuses on the combined effect of either fludarabine or gemcitabine and radiation on human squamous carcinoma cell lines in vitro, providing data for future decisions on head and neck chemoradiotherapy regimen. Materials and methods: ZMK-1, A549, BW-225, GR-145, OH-65 and CaSki cell lines were incubated with either drug at defined schedules and irradiated at a single fraction dose of 2 Gy every 24 hours up to 8 Gy. Cytotoxic effects were measured by colony-forming assays, quantitative determination of apoptosis and isobologram analysis. Results: Incubation of fludarabine led to a radiosensitizing effect in the A549, CaSki and ZMK-1 cell lines and an additive effect in the BW-225, GR-145 and OH-65 cell lines. Treatment with gemcitabine only indicated significant radiosensitization in the CaSki cell line in combination with augmented resistance against gemcitabine application alone. Conclusions: Our results reveal a potential radiosensitizing effect of fludarabine and its possible application in chemoradiotherapy of advanced head and neck carcinoma and possibly other tumor entities.

No supra-additive effects of goserelin and radiotherapy on clonogenic survival of prostate carcinoma cells in vitro

BackgroundOncological results of radiotherapy for locally advanced prostate cancer (PC) are significantly improved by simultaneous application of LHRH analoga (e.g. goserelin). As 85% of PC express LHRH receptors, we investigated the interaction of goserelin incubation with radiotherapy under androgen-deprived conditions in vitro.MethodsLNCaP and PC-3 cells were stained for LHRH receptors. Downstream the LHRH receptor, changes in protein expression of c-fos, phosphorylated p38 and phosphorylated ERK1/2 were analyzed by means of Western blotting after incubation with goserelin and irradiation with 4 Gy. Both cell lines were incubated with different concentrations of goserelin in hormone-free medium. 12 h later cells were irradiated (0 – 4 Gy) and after 12 h goserelin was withdrawn. Endpoints were clonogenic survival and cell viability (12 h, 36 h and 60 h after irradiation).ResultsBoth tested cell lines expressed LHRH-receptors. Changes in protein expression demonstrated the functional activity of goserelin in the tested cell lines. Neither in LNCaP nor in PC-3 any significant effects of additional goserelin incubation on clonogenic survival or cell viability for all tested concentrations in comparison to radiation alone were seen.ConclusionThe clinically observed increase in tumor control after combination of goserelin with radiotherapy in PC cannot be attributed to an increase in radiosensitivity of PC cells by goserelin in vitro.