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Hintergrund: Der Stoffwechsel maligner Tumoren unterscheidet sich von dem gesunder Gewebe oftmals durch eine gesteigerte Rate an Glukoseumsatz. In den letzten Jahren stieg das Interesse an einem diatischen Therapieansatz, der das Ziel verfolgt, mithilfe einer Kohlenhydrateinschrankung diese metabolische Besonderheit von Tumorzellen auszunutzen. Praklinische Studien, bei denen tumortragende Tiere eine kohlenhydratbeschrankte Diat – entweder in Form von Fasten/Kalorienrestriktion oder einer kohlenhydratarmen, fettreichen ketogenen Diat – verabreicht bekamen, konnten wachstumshemmende Effekte gegenuber Tumoren nachweisen. Dabei wirken diese Diaten uber mehrere verschiedene und sich teilweise uberlappende Signalwege, die hauptsachlich uber 4 Stoffwechselveranderungen induziert werden. Dies sind eine Absenkung des Blutzucker- und damit Insulinspiegels und eine Erhohung des Ketonkorper- sowie freien Fettsaurespiegels. Die Ubertragbarkeit dieser zumeist an Mausen gewonnenen Ergebnisse auf den Menschen erscheint zunachst fraglich. Deshalb werden in dieser Arbeit die bisher publizierten klinischen Daten in Bezug auf eine mogliche Wachstumshemmung durch ketogene Diaten untersucht. Material und Methodik: Es wurden alle bisher erschienenen relevanten klinischen Studien ausgewertet, die tumorwachstumshemmende Effekte untersuchten. Ergebnisse: Bisher existieren nur Studien mit kleinen Fallzahlen. Deshalb lassen sich derzeit nur ein paar vorsichtige Schlusse ziehen. Zum einen scheint es so, dass eine ketogene Ernahrung als Monotherapie keine tumorhemmenden Effekte auf das Wachstum fortgeschrittener Tumoren hat. Zum anderen scheint es aber einen messbaren Einfluss auf den Tumorzellstoffwechsel zu geben, wie in FDG-PET-Studien und anhand von intratumoralen Laktatmessungen bei HNO-Tumoren gezeigt werden konnte. Wahrscheinlich eignet sich eine ketogene Ernahrung am besten als Unterstutzung von Standardtherapien. Schlussfolgerung: Eine ketogene Diat scheint den Tumorstoffwechsel beeinflussen zu konnen und kame damit als Unterstutzung zytotoxischer Standardtherapien infrage.

Fragestellung: Die Auswirkungen einer ketogenen Diat auf das Wachstum solider Tumoren werden aktuell kontrovers diskutiert. Aus diesem Grund wurde in vitro untersucht, bei welchen Sauerstoffkonzentrationen Tumorzellen Ketonkorper (β-Hydroxybutyrat) und die beiden Metabolite Glukose und Laktat, die auch bei einer ketogenen Diat vorkommen, verstoffwechseln. Zudem wurde exemplarisch das Tumorwachstum unter einer standardisierten ketogenen Diat im Tiermodell uberpruft. Material und Methodik: Konfluente Kulturen von 6 humanen gastrointestinalen Tumorzelllinien wurden in vitro bei 21 % und 1 % Sauerstoff untersucht. Glukose, Laktat und β-Hydroxybutyrat wurden mit Standardenzymassays bestimmt. Mit einer standardisierten ketogenen Diat wurde in Kleinnagern eine physiologische Ketose ausgelost und durch subkutane Injektion von Tumorzellen das Wachstum solider Tumoren induziert. Ergebnisse: Kulturen der 6 Tumorzelllinien verstoffwechseln Ketonkorper und Laktat bei 21 %, nicht aber bei 1 % Sauerstoff; dagegen wird Glukose bei beiden Sauerstoffkonzentrationen metabolisiert. Stehen Tumorzellen bei 21 % Sauerstoff die beiden Energietrager Glukose und Laktat gleichzeitig zur Verfugung, wird zuerst Glukose und anschliesend Laktat verstoffwechselt. Unter einer ketogenen Diat entwickeln Mause eine physiologische Ketose mit signifikant erhohten Ketonkorperspiegeln. Die Tumoren dieser Mause weisen verringerte intratumorale Laktatspiegel und ein verzogertes Wachstum auf. Schlussfolgerung: Die In-vitro-Daten dieser experimentellen Arbeit unterstutzen das Konzept, dass in vivo der Stoffwechsel solider Tumoren durch die lokale Sauerstoffversorgung wesentlich beeinflusst wird. Sauerstoffkonzentrationen von 1 %, die regelhaft in soliden Tumoren vorzufinden sind (Tumorhypoxie), reichen in vitro nicht aus, damit gastrointestinale Tumorzellen Ketonkorper und Laktat verstoffwechseln; im Gegensatz zur Glukose. Um Tumorzellen in Hypoxie zum Absterben zu bringen, sollte bei ihnen daher der Glukosestoffwechsel selektiv gehemmt und zusatzlich das Glukoseangebot durch diatetische Masnahmen eingeschrankt werden.

OBJECTIVE Preferential secretion of Th1-like cytokine is mainly a property of monocyte derived dendritic cells (DC). Since normal early pregnancy is characterized by a shift towards a Th2-like cytokine pattern, it may be assumed that cytokine secretion by DC during early pregnancy could be modulated by the non-classical HLA molecules G and E present on invasive trophoblast. MATERIAL AND METHODS DC were cultivated from monocytes isolated from peripheral blood mononuclear cells. DC were cocultured with K-562 leukemia cells lacking the class I and II HLA antigens transfected with either HLA-G or HLA-E or ultratransfected cells (controls) and the concentrations of IL-8, IL-10, IL-12p70, IL-18 and TNF-alpha were measured in the supernatants by ELISA. RESULTS Coculture with ultratransfected cells resulted in a significant increase of the production of IL-8 and TNF-alpha by mature and immature DC and of IL-10 by immature DC (p < 0.01). When cocultured with HLA-G and HLA-E transfected K-562 cells, the secretion of IL-8 by immature and mature DC and that of IL-10 and TNF-alpha by immature DC was significantly (p < 0.01) decreased. The contact with HLA-G and HLA-E transfected cells had no effect on the production of IL-12p70 and IL-18 by DC. CONCLUSIONS These results show that DC react with an increased cytokine release upon contact with cells lacking HLA class I and II antigens. The suppressive effect of HLA-G and HLA-E on the secretion of TNF-alpha (Th1 cytokine), IL-10 (Th2 cytokine) and IL-8 (chemokine) by immature DC could be interpreted as further evidence for the central immunotolerance role of HLA-G and HLA-E during early pregnancy.

Im Gegensatz zu Frauen mit ausgetragenen Schwangerschaften finden sich bei Frauen mit wiederholten Aborten typische Veranderungen des zellularen und humoralen Immunsystems, die in erster Linie das uterine Kompartiment betreffen, jedoch in der peripheren Zirkulation nur zum Teil nachweisbar sind. Manche dieser Phanomene sind wahrscheinlich kausal am ungunstigen Ausgang der Schwangerschaft beteiligt, andere durften auf eine veranderte endokrine und parakrine Regulation im Verlauf des Abortgeschehens zuruckzufuhren sein. Zum Zeitpunkt der Implantation, in der Lutealphase des Konzeptionszyklus, findet man bei Frauen mit wiederholten Aborten, im Vergleich zu fertilen Frauen ohne Aborte, eine veranderte Population von Lymphozyten im Endometrium, eine erhohte Zahl und Aktivitat von CD16 + CD56 + -naturlichen Killerzellen (NK) und eine Verschiebung der von den T-Helferzellen (Th) produzierten Zytokine in Richtung der inflammatorischen Th-1-Zytokine (IL-2, IL-12, Interferon-γ). In der peripheren Zirkulation ist zum Zeitpunkt der Konzeption ebenfalls die Aktivitat der NK-Zellen und die Produktion von Th-1-Zytokinen durch T-Effektorzellen erhoht. Organspezifische, nichtorganspezifische und gerinnungsaktive Antikorper weisen auf eine generelle Aktivierung des humoralen Immunsystems hin. Zum Zeitpunkt eines erneuten Abortes ist die Tiefe der Trophoblastinvasion in das Myometrium meist ungenugend. Die Implantationsstelle enthalt eine erhohte Zahl von NK-Zellen, und die Produktion der antiinflammatorischen und protektiven Th-2-Zytokine (z. B. LIF, IL-4, IL-10) durch deziduale T-Zellen ist reduziert. Die genannten Phanomene sind zum Teil durch Immunisierung mit allogenen Lymphozyten, Gabe von Immunglobulinen oder Medikamente (Acetylsalicylsaure, Heparin, Prednison) therapeutisch beeinflussbar.

A nested PCR for the detection and rapid identification of human picornaviruses is described. Enteroviruses and rhinoviruses were amplified with the same set of four primers from the 5'-noncoding region. The nested primers allowed the detection of far less than 1 PFU in diluted virus stocks without Southern blot hybridization. In patients with neurological disorders (mainly aseptic meningitis), 43% of 37 specimens (11 of 21 cerebrospinal fluid specimens, 2 of 10 serum specimens, and 3 of 6 stool specimens) were positive by PCR. A total of 21% (10 of 47 specimens) of heart biopsy specimens from patients with dilative cardiomyopathy were PCR positive, whereas 3% (2 of 70 specimens) of control biopsy specimens from patients with coronary artery disease were PCR positive. PCR-amplified fragments from 27 of 29 clinical isolates and 14 of 28 patient samples were successfully serotyped by restriction enzyme digestion. Two specimens were further investigated by direct sequencing of PCR products, leading to the identification of a poliovirus type 3 isolate with a sequence that was highly divergent from previously published sequences.

Fifteen-30% of breast cancer patients develop central nervous system (CNS) metastases. The most potent drugs for the treatment of breast cancer like taxanes, anthracyclines and trastuzumab have limited efficacy for brain metastases. No standardized therapy has yet been established for this condition. Drugs with proven efficacy in the CNS and which are commonly used for primary brain tumors were applied. We evaluated the capacity of these drugs to inhibit breast tumor cell growth in vitro.Twelve primary cell cultures of pulmonary/pleural metastases of breast cancer and 3 commercially available cell lines were used for non-radioactive cytotoxicity assays to evaluate the efficacy of 3 different concentrations of Topotecan, Cisplatin, Nimustine, Vincristine, Irinothecan, Caelyx (pegylated liposomal Doxorubicin) and Etoposide.Topotecan, Cisplatin, Caelyx and Vincristine showed significantly higher cytostatic activity in vitro than Irinotecan, Etoposide and Nimustine. With regard to the median cytotoxicity, the order of drugs in our assays was Topotecan, Cisplatin, Vincristine, Caelyx, Irinotecan, Etoposide and Nimustine. Nimustine showed almost no efficacy against breast cancer cells.Topotecan, Cisplatin, Vincristine and Caelyx seem to be suitable candidates for further clinical evaluation. The data and the "liposomal packaging" suggest that Caelyx might be effective in the CNS. Since pulmonary metastases are often associated with brain metastases, evaluatingprimary cell cultures from malignant pleural effusions could be a valuable approach for the testing of new cytostatic drugs for brain metastases.

Human endometrium and early pregnancy decidua harbor a considerable and diverse population of antigen-presenting cells (APC). Changes in the number and distribution of macrophages and dendritic cells (DC) could point to a possible role of these immunocompetent cells in implantation and success of early pregnancy.Uterine tissue was obtained from 22 women undergoing hysterectomy for bleeding disorders or dysmenorrhea and from 11 women undergoing legal abortion. Tissue was investigated with antibodies against CD14, CD68, CD83, DC-SIGN, Ki-67, and human leukocyte antigen (HLA)-DR using single and double immunohistochemical staining techniques.The number of CD14(+) cells was stable during all phases of the menstrual cycle and early pregnancy. In comparison to nonpregnant endometrium, DC-SIGN(+) cells showed a higher proliferation rate and were found associated in clusters with CD56(+) natural killer (NK) cells in early pregnancy. In the late secretory phase of the menstrual cycle, numbers of CD83(+) (P.01) cells were significantly higher than in other endometrial phases and early pregnancy. HLA-DR(+) expression was significantly increased in early pregnancy but remained unchanged throughout the menstrual cycle.The presence of DC-SIGN(+) cells during the menstrual cycle and their proliferation in early pregnancy suggests an important role of these cells with regard to the balance between defense against pathogens and tolerance of the fetal allograft. Whether the increase of CD83(+) mature DC and CD68(+) macrophages in the late secretory phase is caused by hormonal stimuli and/or is due to changes of the cytokine/chemokine micromilieu remains to be investigated.

Preferential secretion of Th1-like cytokine is mainly a property of monocyte derived dendritic cells (DC). Since normal early pregnancy is characterized by a shift towards a Th2-like cytokine pattern, it may be assumed that cytokine secretion by DC during early pregnancy could be modulated by the non-classical HLA molecules G and E present on invasive trophoblast.DC were cultivated from monocytes isolated from peripheral blood mononuclear cells. DC were cocultured with K-562 leukemia cells lacking the class I and II HLA antigens transfected with either HLA-G or HLA-E or ultratransfected cells (controls) and the concentrations of IL-8, IL-10, IL-12p70, IL-18 and TNF-alpha were measured in the supernatants by ELISA.Coculture with ultratransfected cells resulted in a significant increase of the production of IL-8 and TNF-alpha by mature and immature DC and of IL-10 by immature DC (p0.01). When cocultured with HLA-G and HLA-E transfected K-562 cells, the secretion of IL-8 by immature and mature DC and that of IL-10 and TNF-alpha by immature DC was significantly (p0.01) decreased. The contact with HLA-G and HLA-E transfected cells had no effect on the production of IL-12p70 and IL-18 by DC.These results show that DC react with an increased cytokine release upon contact with cells lacking HLA class I and II antigens. The suppressive effect of HLA-G and HLA-E on the secretion of TNF-alpha (Th1 cytokine), IL-10 (Th2 cytokine) and IL-8 (chemokine) by immature DC could be interpreted as further evidence for the central immunotolerance role of HLA-G and HLA-E during early pregnancy.

Mast cells are now known to derive from CD34+ haemopoietic stem cells in the bone marrow. However, it has not yet been established whether the various types of mastocytosis, which involve tumour-like proliferation of mast cells, are true neoplastic disorders or reactive/hyperplastic conditions. In this study, tissue specimens (five bone marrow, two spleen, one skin) from female patients with histologically confirmed mastocytosis were investigated with a recently developed polymerase chain reaction assay for the determination of clonality of female cells using the human androgen receptor gene (HU-MARA). Mast cells purified to near homogeneity from hysterectomy specimens served as a control. The findings in bone marrow and skin either were not reproducible, or indicated polyclonality. However, both spleen specimens exhibited monoclonality. In addition, DNA analysis by flow cytometry was performed and revealed a diploid chromosome content with proliferation indices of under 8% in all the specimens. This is the first molecular biological study to indicate that mastocytosis is indeed neoplastic in nature.

Enteroviruses of the human Picornaviridae are common agents of viral myocarditis. Various members of the enterovirus group, e.g. coxsackieviruses and echoviruses, have been associated with viral heart disease. Human enterovirus myocarditis is most often caused by group B coxsackieviruses (CVB) [1–3]. The genetic material of CVB is encoded in a single-stranded RNA molecule of positive polarity and about 7500 nucleotides in length [4, 5]. During replication, the genomic viral plus-strand RNA serves as a template for transcription of minus-strand RNA, an intermediate in the life cycle of enteroviruses, which is transcribed again into large amounts of infectious plus-strand RNA [6].

Interactions of leukocytes with vascular endothelium are important components of inflammation tissue reactions and have been implicated in cardiac transplant rejection and demonstrated to be mediated by cell adhesion molecules (CAM's). The expression of ICAM-1, VCAM-1 and E-selectin in human myocardium is variable and little is known about the expression of LFA-1 and Mac-1 during allograft rejection. This study investigated these CAM's in myocardial biopsies of transplanted hearts (HTX) and of coronary artery disease eligible for coronary artery bypass grafting (CABG) as non-inflammatory controls and explicitly examines vascular endothelium, cardiomyocytes and infiltrating cells. Immunohistochemistry was performed using the APAAP-method and directing specific mouse anti-human monoclonal antibodies against ICAM-1 (CD54), VCAM-1 (CD106), E-selectin (CD62E), alpha-LFA-1 (CD11a), alpha-Mac-1 (CD11b), alpha-p150/95 (CD11c) and the beta2-integrin chain (CD18). CD18, LFA-1 (CD11a), Mac-1 (CD11b) and p150/95 (CD11c) were markedly expressed on infiltrating immunocytes in HTX compared to CABG where no expression of beta2-integrins was observed. Cardiac allografts demonstrated a strong expression of ICAM-1 on vascular endothelium and on infiltrating cells. ICAM-1 was not detected on cardiomyocytes. In CABG a weak expression of ICAM-1 was observed on endothelial cells but not on myocytes. VCAM-1 was expressed on vascular endothelium and perivascular infiltrating cells in HTX but not in CABG. VCAM-1 was not found to be expressed on myocytes. There was no evidence for the presence of E-selectin in any of our biopsy specimens. Our study shows that the study of cell adhesion molecules adds to the pathophysiological understanding of inflammation after transplantation in cardiac disease. This offers a potential for the development of diagnostic tools and new therapeutic strategies.

A nested PCR was developed that allows the specific amplification of enteroviruses as well as rhinoviruses with one set of four primers from the 5’-noncoding region. For rapid identification of the respective serotypes, restriction enzyme (RE) digestion of PCR products was done. Experiments with serotyped isolates showed a sensitivity of 10 genome equivalents or less. RE digestion could predict the serotype correctly in almost all cases investigated, minimizing the need for sequencing of PCR products. Application to clinical samples is demonstrated for cases of aseptic meningitis and other neurological diseases and for myocardial biopsies from experimentally infected animals as well as from patients with dilative cardiomyopathy.