Analysis ofras mutations in human melanocytic lesions: activation of theras gene seems to be associated with the nodular type of human malignant melanoma

We have analyzed the Ha-ras, Ki-ras and N-ras gene for point mutations at codons 12, 13 and 61 via restriction fragment length polymorphism/polymerase chain reaction analysis and subsequent direct sequencing in non-cultured fresh-frozen tissues of 16 superficial spreading melanomas (SSM), 13 nodular malignant melanomas (NMM), 2 lentigo malignant melanomas (LMM), 1 dysplastic nevus, 1 congenital nevus and 5 normal nevi from 38 patients. Mutations were found in 4 melanoma samples, all belonging to the nodular malignant type. Three of them were mutated in N-ras and one in the Ha-ras gene. Mutation in N-ras was also detected in the congenital nevus. All mutations were exclusively located at the first two base pairs of codon 61. No Ki-ras mutation was detected in any lesion. No mutation could be found in SSM and LMM in addition to dysplastic and normal nevi. The frequency ofras mutation in NMM was 31%, whereas in SSM it was 0%. Our study suggests (a) an association betweenras mutations (mainly N-ras) and the NMM as a subgroup of human melanoma; (b) that activation of Ki-ras is not involved in the pathogenesis of melanoma. The role of UV radiation in point mutations ofras genes in human melanoma is discussed. This investigation was supported by grant EV5V-CT92-0096     

Positively selected autologous blood CD34+ cells and unseparated peripheral blood progenitor cells mediate identical hematopoietic engraftment after high-dose VP16, ifosfamide, carboplatin, and epirubicin

To investigate the feasibility of peripheral blood CD34+ cell selection and to analyze CD34+ cell-mediated engraftment after high-dose chemotherapy, we performed a phase I/II trial in 21 patients with advanced malignancies. The rationale for the selection of CD34+ cells from peripheral blood progenitor cell (PBPC) collections is based on the observation that contaminating tumor cells can be depleted approximately 3 logs using this procedure. CD34+ cells from chemotherapy+granulocyte colony-stimulating factor-mobilized PBPCs were positively selected with an avidin-biotin immunoadsorption column (CEPRATE SC system). One leukapheresis product with a median number of 2.8 x 10(6) CD34+ cells/kg was labeled with a biotinylated anti-CD34 monoclonal antibody and subsequently processed over the column. The yield of selected CD34+ cells was 73% +/- 24.6%. The purity of the CD34+ cell fraction was 61.4% +/- 19.7%. CD34+ cells were shown to represent predominantly committed progenitors coexpressing CD33, CD38, and HLA-DR molecules (lin+). They gave rise to myeloid as well as erythroid and multilineage colonies in vitro. In addition, positively selected CD34+ cells also comprised early hematopoietic progenitor cells, as shown by the presence of CD34+/lin- cells. Transfusion of positively selected CD34+ cells (2.5 x 10(6) CD34+/kg; range, 0.45 to 5.1) after high-dose VP16 (1,500 mg/m2), ifosfamide (12 g/m2), carboplatin (750 mg/m2), and epirubicin (150 mg/m2) (VIC-E) in 15 patients resulted in a rapid and stable engraftment of hematopoiesis without any adverse events. As compared with 13 historical control patients reconstituted with a comparable number of unseparated PBPCs, time to neutrophil and platelet recovery was identical in both groups (absolute neutrophil count > 500/microL, day + 12; platelet count > 50,000/microL, day + 15). These data indicate that autologous peripheral blood CD34+ cells and unseparated PBPCs mediate identical reconstitution of hematopoiesis after high-dose VIC-E chemotherapy. Because positive selection of CD34+ cells from mobilized blood results in a median 403-fold depletion of T cells, allogeneic CD34+ cells from mobilized blood should be investigated as an alternative to bone marrow cells for allotransplantation.     

LD50 versus acute toxicity

Conclusions Based on today's extensive experience on acute toxicity testing of chemical substances, and on the outcome of specific studies (Schütz and Fuchs 1982; Müller and Kley 1982; Lorke in preparation; Günzel et al. in preparation), it is now possible to conduct acute toxicity studies with the sacrifice of fewer animals and even to increase at the same time, the quality of the data obtained. Whereas for drugs, data on acute toxicity usually represents only part of the information available, for other chemical substances, acute toxicity may be the only data available, thus asking for more extensive examination of the animals used 'for this kind of study.     

Stickstoffdioxydwirkungen an der Lungenalveole der Maus

Zusammenfassung Wiederholte Einwirkung des lungenödemerzeugenden Reizstoffes Stickstoffdioxyd führt zur Ausbildung einer Toleranz, deren Mechanismus in früheren Untersuchungen in einer ödematösen Durchtränkung des Alveolarseptums und einer damit verbundenen Hemmung der Diffusion der schädlichen Gase gesehen worden ist. Zur Klärung der Zusammenhänge werden morphometrische Untersuchungen an den Lungen zweier normaler Mäuse und zweier Tiere, die 36 Std zuvor mit 35 ppm Stickstoffdioxyd 6 Std lang begast worden waren, durchgeführt. Nach lichtmikroskopischer Vorprüfung identischer Partien der rechten Unterlappen, die eine Auswahl nach dem Grad der Entfaltung des Lungenparenchyms gestatten, werden Schnittserien in mit Epon eingebettetem Material durchgeführt und in Anlehnung an die Methode vonWeibel u.Knight (1964) morphometrisch ausgewertet. Dabei ergibt sich bei den mit Stickstoffdioxyd vorbehandelten und tolerantgemachten Tieren ein hochsignifikanter Anstieg des arithmetischen Dickenmittels (¯) auf das 1 1/2 fache. Diese Meßstrecke repräsentiert vorwiegend die dicken Partien der zwischen Alveole und Capillare gelegenen Gewebselemente. Das harmonische Dickenmittel ( _h), das vorwiegend die dünnsten, zellkörperfreien Abschnitte der Diffusionsstrecke erfaßt, ist hingegen unverändert. Eine Häufigkeitsanalyse der im Lungenparenchym gefundenen drei Zelltypen ergibt keine deutlichen Verschiebungen durch die NO₂-Behandlung. Da auch für ein Ödem im Interstitialraum kein Anhalt gegeben ist, spricht der morphometrische Befund für eine Volumenzunahme der am Aufbau der Alveolarsepten beteiligten Zellelemente. Art und Ursache dieser Größenzunahme bedürfen weiterer Klärung.     

Carcinogenicity of dichloroacetylene: an inhalation study

Dichloroacetylene (DCA) is a by-product of the synthesis ofcertain chlorinated, aliphatic hydrocarbons (e.g., vinylidenechloride). In a long-term carcinogenicity inhalation study,groups of 30 male and 30 female rats and mice were exposed toDCA vapour under the following conditions: mice, group I: 9p.p.m. 6 h/day, 1 day/week for 12 months; group II: 2 p.p.m.6 h/day, 1 day/week for 12 months; group II: 2 p.p.m. 6 h/day,2 days/week for 18 months; rats: 14 p.p.m. 6 h/day, 2 days/weekfor 18 months; controls: under identical conditions except foraddition of DCA to breathing air. The most important resultis a striking increase in the formation of kidney cystadenomasof the proximal tubuli in all DCA exposed animals. The medianlatency time of this tumor varied widely in both mice and rats.Renal cystic adenmrcinomas were found in statistically significantnumbers in male mice. In addition to kidney tumors, the developmentof cystadenomas of the Harderian gland in mice and of livercholangiomas in rats were highly significant findings. DCA failedto induce liver tumors in mice. Our results confirm that DCApossesses a high carcinogenic potential, pronounced organotropicproperties and distinct species differences referring to thenumber and nature of the induced tumors. The risk associatedwith handling chlorinated aliphatic hydrocarbons is essentialtyincreased by the decomposition product DCA.     

Metabolism of hexachloro-1,3-butadiene in mice: in vivo and in vitro evidence for activation by glutathione conjugation

1. The metabolism of 14C-hexachloro-1,3-butadiene (HCBD) was studied in mice and in subcellular fractions from mouse liver and kidney. 2. In the presence of glutathione (GSH), liver microsomes and cytosol transformed HCBD to S-(pentachlorobutadienyl)glutathione (PCBG). PCBG formation in subcellular fractions from mouse kidney was very limited. Oxidative metabolism of HCBD by cytochrome P-450 could not be demonstrated. 3. Cysteine conjugate beta-lyase was present in mitochondria and cytosol from mouse liver and kidney. 4. After an oral dose of 30 mg/kg 14C-HCBD, mice eliminated 67.5-76.7% of dose in faeces; urinary elimination accounted for 6.6-7.6%. 5. Metabolites of HCBD identified are: S-(pentachlorobutadienyl)glutathione in faeces; S-(pentachlorobutadienyl)-L-cysteine, N-acetyl-S-(pentachlorobutadienyl)-L-cysteine and 1,1,2,3-tetrachlorobutenoic acid in urine. 6. The results suggest that conjugation of HCBD with GSH in liver, followed by renal processing of the glutathione S-conjugates and beta-lyase-catalysed formation of reactive intermediates, accounts for the organ specific toxicity of HCBD in mice.