Recombinant human stem cell factor stimulates growth of a human glioblastoma cell line expressing c-kit protooncogene.

The growth of a panel of eight different human glioblastoma cell lines was examined in a human tumor cloning assay in agar, a tritiated thymidine uptake assay, and by counting cell numbers, in cultures performed in the absence or presence of increasing concentrations (1 to 100 ng/ml) of recombinant human stem cell factor (SCF). Growth of 7 of 8 cell lines was not significantly and reproducibly affected by recombinant human SCF. However, growth of the CRL 1620 cell line could be stimulated up to 5-fold by the cytokine. In contrast to the other cell lines investigated, CRL 1620 expressed the c-kit protooncogene assessed on the mRNA and protein level. Furthermore, SCF-induced proliferation of CRL 1620 cells was sensitive to the tyrosine kinase inhibitor erbstatin. Our data suggest that SCF can be operative in growth modulation of malignant cells outside the hematopoietic system, and this finding should be further studied for its possible clinical implications.     

Detection of the Arg702Trp, Gly908Arg and Leu1007fsinsC polymorphisms of the NOD2/CARD15 gene by real-time PCR with melting curve analysis.

Crohn's disease is a complex disorder, with multiple genetic traits. A frameshift mutation (Leu1007fsinsC) and two missense mutations (Gly908Arg and Arg702Trp) in the NOD2/CARD15 gene are strongly associated with susceptibility to Crohn's disease. The presence of one of these risk alleles confers a 2- to 4-fold increase in the risk of developing Crohn's disease, and the presence of two mutant alleles increases the risk over 20-fold. To facilitate the analysis of these polymorphisms, we developed three LightCycler assays to detect the missense mutations Arg702Trp and Gly908Arg and the frameshift mutation Leu100fsinsC in the NOD2/ CARD15 gene. All three assays can be run simultaneously on one LightCycler using identical cycling parameters. Analysis of 53 DNAs from Crohn's patients helped to identify carriers at allele frequencies similar to other Caucasian populations. The sequencing of such DNAs confirmed the accuracy of the assays. In conclusion, we present three rapid and robust assays to detect the Arg702Trp, the Gly908Arg and the Leu1007fsinsC ins mutations in the NOD2/CARD15 gene [corrected]     

Depressed antigen presentation function and membrane interleukin-1 activity of peritoneal macrophages after laparotomy

Although major tissue trauma produces profound depression of cell-mediated immunity, it is not known whether surgical trauma (i.e., midline laparotomy) has any adverse effect on the antigen presentation function and membrane interleukin-1 (IL-1) activity of peritoneal macrophages. To study this, C3H/HEJ (endotoxin-tolerant) mice were anesthetized. An approximately 1-inch midline abdominal incision was made, followed by abdominal closure. On days 1, 3, 5, and 7, peritoneal macrophages were harvested by means of peritoneal lavage, and antigen presentation capability was tested by incubating various numbers of peritoneal macrophages with 2 X 10(4) D10.G4.1 cells per well in the presence of conalbumin (400 micrograms/ml). The T helper cell clone (D.10.G4.1) proliferates on recognition of conalbumin in the context of Iak and also proliferates in the presence of membrane-bound IL-1 plus concanavalin A. To measure membrane IL-1 expression in peritoneal macrophages, Concanavalin A (10 micrograms/ml) was substituted for conalbumin. Cultures were incubated for 72 hours, pulsed with tritiated thymidine, and harvested. Peritoneal macrophages from laparotomized mice induced significantly less T helper cell proliferation on days 1 and 3 in the antigen presentation assay (37% and 30%, respectively; p less than 0.05) and in the membrane IL-1 assay (14% and 10%, respectively; p less than 0.05) as compared with the control. This difference was not detectable on day 5. More effective antigen presentation capability (167% of control; p less than 0.05) was seen on day 7. Thus laparotomy by itself produces marked depression of both antigen presentation function and membrane IL-1 activity of peritoneal macrophages, which may enhance susceptibility to intra-abdominal sepsis.     

Antigenic structure of the hexacosapeptide melittin: evidence for three determinants, one with a helical conformation.

ELISA-based epitope analysis was performed using rabbit polyclonal antisera against melittin. Antigenic sites were found at the C-terminus, in the middle section and within the N-terminal helix. Antibodies against the helical segment could discriminate between two faces of the amphiphilic helix. The antigenic sites include the bulk of the melittin hexacosapeptide, which is synonymous with a very high epitope density.     

Mycetoma-like manifestations in invasive pulmonary aspergillosis

Invasive pulmonary aspergillosis is characterized by radiological signs allowing a correct diagnosis, including differentiation from pulmonary candidiasis, when they are associated with appropriate clinical symptoms (neutropenia and fever persisting despite broad-spectrum antibiotics). In particular the formation of a pulmonary mycetoma in a previously normal lung is one of these signs. Unlike a simple fungus ball (the saprophytic form of aspergillosis), the rounded density of invasive pulmonary aspergillosis consists of sequestrum of devitalized lung tissue owing to blood vessel invasion by Aspergillus hyphae. This morphologic phenomenon is demonstrated in the present case report and is discussed together with the other roentgenological signs of the invasive aspergillosis.     

Prenatal nicotine exposure selectively affects perinatal forebrain aromatase activity and fetal adrenal function in male rats.

Previous studies have revealed effects of prenatal nicotine treatment on fetal plasma testosterone and perinatal sexual brain differentiation in the rat. In an attempt to further elucidate the processes underlying this action of nicotine, we studied the effect of the drug on brain steroid aromatase which converts androgens to estrogens and is known to be important in sexual brain differentiation. Aromatase activity (AA) was measured by the conversion of [1 beta-3H]-androstenedione to estrone in a brain region comprising preoptic, hypothalamic and amygdaloid areas. In untreated animals, the development of AA between gestational day (GD) 18 and postnatal day (PN) 15 was similar in both sexes, except for a significant drop of AA in female brain at PN6, i.e., during the later part of the critical period for sexual brain differentiation. When time-pregnant rats were treated with nicotine delivered by an osmotic minipump for either one week (2 mg/kg/d or 6 mg/kg/d from GD12) or two weeks (6 mg/kg/d from GD8), their male offspring showed a decrease of AA to female levels at PN6, the sex difference existing at this stage thus being abolished. AA of offspring from dams bearing tartaric acid-containing minipumps or sham-operated at GD8 or GD12 was identical to that of untreated controls. No drug effect was seen in female fetuses and offspring. Sex differences in the developmental effect of nicotine may thus involve brain aromatase. An additional sex-dependent effect of nicotine was observed in the male fetal adrenal axis at GD18. Whether the drug effects on the two steroid hormone systems are interrelated, remains to be elucidated.