3H]-flunitrazepam binding after morphine treatment and under abstinence syndrome.

Chronic morphine treatment produced increases in [3H]-flunitrazepam binding in some hippocampal areas of the rat brain. The differences in binding were statistically significant in some cases. Both morphine-dependent and morphine-deprived (abstinence syndrome) animals showed an identical response in binding, which confirms a real, although small, increase in benzodiazepine binding sites in the hippocampus after morphine treatment, that is not affected by a naloxone-induced abstinence syndrome under the conditions studied. These findings support the hypothesis of a morphine-induced up-regulation of benzodiazepine binding sites in the hippocampus. A possible different response in benzodiazepine binding sites 1 and 2 could explain the different findings reported in the literature. Our data suggest that the detected increase in benzodiazepine binding would be mainly due to type 2 binding sites, since the hippocampus has a higher density of this type of benzodiazepine binding sites.     

The plasminogen-activation system in ovarian tumors

We studied the plasminogen activation system in tumor tissue by measuring the antigen level of the 2 plasminogen activators, tissue-type (t-PA) and urokinase-type (U-PA) and their inhibitors, plasminogen-activator inhibitors type-1 (PAI-1) and type-2 (PAI-2) in the tissue extracts of 43 human benign and malignant ovarian tumors. U-PA levels were significantly higher in malignant than in benign tumors. In addition, U-PA antigen levels were higher in the metastatic tissue of advanced disease (FIGO stage III) than in the primary localized tumor (FIGO stage I/II). Also PAI-1 concentrations tended to be higher in malignant than in benign tumors, but this difference was not statistically significant. In contrast, t-PA levels were lower in metastatic than in non-metastatic tumors, whereas PAI-2 levels were unrelated to the stage of ovarian malignancy. These results were integrated in a plasminogen-activation-dependent malignancy index (U-PA × PAI-1/t-PA). This index distinguished the different groups of benign ovarian tumors, localized and metastatic ovarian carcinomas better than U-PA levels. It could be useful as a prognostic indicator in ovarian cancer © 1993 Wiley-Liss, Inc.     

B-B10 (anti-CD25)-saporin immunotoxin--a possible tool in graft-versus-host disease treatment.

An immunotoxin containing the B-B10 MoAb, directed against the CD25 determinant, and the ribosome-inactivating protein saporin, inhibits 3H-TdR incorporation in phytohemagglutin, allogeneic-stimulated lymphocytes (primary and secondary mixed-lymphocyte reaction), and in an alloreactive T cell clone. A lower degree of inhibition was obtained with the B-B10 MoAb, which is known to inhibit IL-2 activity, as well as with the unconjugated compounds. These results suggest that the in vivo administration of the conjugate might be a more effective tool in the treatment of patients affected by graft-versus-host disease than B-B10 alone, by inducing an efficient killing of allogeneic-reacting T lymphocytes.     

A comprehensive survey for polymorphisms in the bovine IFN-gamma gene reveals a highly polymorphic intronic DNA sequence allowing improved genotyping of Bovinae.

This study aimed to identify interferon-gamma (IFN-gamma) gene variants in cattle for diagnostic purposes. Therefore, the entire bovine IFN-gamma gene (BoIFNG) and 2605 bp of its promoter DNA were sequenced. The BoIFNG DNA sequence conforms to the published part of Bo-IFN-gamma cDNA. Primer extension experiments show the presence of a 5' extension of exon 1 by 42 nucleotides (nt). One SINE element (Bov-A2) is located in the 5'-region, and two SINE elements (Bov-tA, Bov-B) are contained in the 3'-region of BoIFNG. The variants were detected by comparative sequence analysis of PCR amplicons from different bovine species. Four polymorphic mononucleotide repeats are situated in the promoter and in intron 1. Four distinct series of single nucleotide polymorphisms (SNP) were found in functionally important regions of BoIFNG. The region between the two intron 1 microsatellites contains the highest density of SNPs in Bos taurus breeds. One G-T transversion in the coding region of exon 1 causes a Gly(14) to Val(14) exchange in the BoIFNG signal peptide of different bovine species. A G-A transition in exon 2 encodes a Ser(19) to Asn(19) change in the mature protein of the Tibetan yak. Genotyping of randomly sampled Holstein Friesian cows at selected SNPs and of both intron 1 microsatellites revealed two dominant BoIFNG microhaplotypes. The detected SNPs improve the recently reported genotyping system of cattle.     

New Potent Azomethine Prodrugs of the Histamine H3-Receptor Agonist (R)-α-Methylhistamine Containing a Heteroarylphenyl Partial Structure

The therapeutic value of histamine H₃-receptor ligands is under current investigation. On the basis of recently described diaryl imine prodrugs of the histamine H₃-receptor agonist (R)-α-methyl-histamine ( 1 ) a series of new azomethine prodrugs containing five- and six-membered heterocycles were synthesized and tested for their in vitro hydrolysis rates and in vitro activity after oral application. It was found that electron-deficient six-membered heterocycles drastically destabilized the imine double bond so that these prodrugs decomposed unsuitably fast. On the contrary, prodrugs containing five-membered heterocycles appeared to be highly effective for the CNS delivery of 1 , and a remarkable correlation between chemical structure and pharmacokinetic profile was observed. Particularly (R)-4-fluoro-2-[[N-[1-(1H-imidazol-4-yl)-2-propyl]imino](1H-pyrrol-2-yl)methyl]phenol ( 8c ), the 2-furanyl analogue 8d , and its 3-furanyl isomer 8e proved to be equipotent to the most potent of recently described halogenated diaryl imine prodrugs of 1. However, in contrast to any other azomethine prodrug, 8c exhibited an incomparably long lasting delivery of 1 in the CNS and can thus be regarded as a ‘retard’ prodrug. Assuming that a therapeutic indication of histamine H₃-receptor agonists will soon be established, these highly potent heteroarylphenyl azomethine prodrugs, which already serve as valuable pharmacological tools, may also become potential drugs in clinical use.     

Association between coagulation factors VII and X with triglyceride rich lipoproteins.

The association between the concentration of different plasma lipoproteins and plasma factor VII (F VII) was analysed by isolating plasma very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) lipoproteins and assessing their in vitro interaction with F VII by immunoenzyme assay using peroxidase labelled anti-factor VII immunoglobulins to determine whether F VII coagulant activity is prognostic for cardiovascular mortality. F VII bound to triglyceride rich lipoproteins, the fixation being stronger on chylomicrons and VLDL fractions than on LDL fractions. In our experiments HDL did not bind to F VII. The fixation of coagulation factor X (FX) tested by the same method is comparable with that of F VII. The nature of this fixation seemed to arise from hydrophobic interaction as calcium was not necessary and the use of Tween 20 inhibited the interaction. The binding of factors VII and X was increased when lipids were previously treated by phospholipase C and the interaction seemed to be completely dependent on the lipid part of the lipoproteins. Hyrophobic fixation is a possible mechanism of interaction of plasma lipoproteins and F VII and X, and it may be of importance in the covariance of triglyceride concentrations and the activity of vitamin K dependent coagulation factors.     

No association between schizophrenia and homozygosity at the D3 dopamine receptor gene

The D₃ dopamine receptor gene is an important candidate gene for schizophrenia, since (because of its almost exclusive expression in the limbic system) it combines the dopamine receptor hypothesis with the limbic system hypothesis of schizophrenia. A BalI restriction fragment length polymorphism of the D₃ dopamine receptor gene has been typed in 107 schizophrenic patients and 98 normal controls from Sichuan (China). With regard to alleles or genotypes, no significant differences were obtained between controls from Europe and China, between patients and controls, and between patient subgroups and controls. These results indicate a lack of association between schizophrenia and the D₃ dopamine receptor gene in our sample. Our findings are at variance with reports of a significant excess of homozygosity at the D₃ dopamine receptor gene in schizophrenic patients from Wales (United Kingdom) and Alsace (France). In conclusion, further studies will be needed with larger samples of patients from Wales and Alsace as well as with samples of different racial groups to prove or disprove the initial positive association between schizophrenia and genotypes of the D₃ dopamine receptor gene. © 1993 Wiley-Liss, Inc.     

Chemokine receptors that mediate B cell homing to secondary lymphoid tissues are highly expressed in B cell chronic lymphocytic leukemia and non-Hodgkin lymphomas with widespread nodular dissemination

B cell neoplasms present heterogeneous patterns of lymphoid organ involvement, which may be a result of the differential expression of chemokine receptors. We found that chemokine receptor (CCR)7, CXC chemokine receptor (CXCR)4, or CXCR5, the main chemokine receptors that mediate B cell entry into secondary lymphoid tissues and their homing to T cell and B cell zones therein, were highly expressed in B malignancies with widespread involvement of lymph nodes. Conversely, those pathologies with little or no nodular dissemination showed no expression to very low levels of CCR7 and CXCR5 and low to moderate levels of CXCR4. These findings provide evidence for the role of CCR7, CXCR4, and CXCR5 in determining the pattern of lymphoid organ involvement of B tumors. Functional studies were performed on B malignancies expressing different levels of CCR7, CXCR5, and CXCR4. Multiple myeloma (MM) cells did not express CCR7 nor CXCR5 and did not migrate in response to their ligands; a moderate expression of CXCR4 on MM cells was accompanied by a migratory response to its ligand, CXCL12. By contrast, cells from B cell chronic lymphocytic leukemia (B-CLL) expressed the highest levels of these chemokine receptors and efficiently migrated in response to all ligands of CCR7, CXCR4, and CXCR5. In addition, the migration index of B-CLL cells in response to both of the CCR7 ligands correlated with the presence of clinical lymphadenopathy, thus indicating that the high expression of functional chemokine receptors justifies the widespread character of B-CLL, representing a clinical target for the control of tumor cell dissemination.     

Stimulation and proliferation of CD4+ peripheral blood T lymphocytes induced by an anti-CD4 monoclonal antibody

There is experimental evidence that the CD4 molecule participates in the antigen-driven activation of T cells expressing this surface glycoprotein. Whether CD4, a member of the immunoglobulin supergene family, acts as a ligand-binding molecule and/or is directly involved in the activation pathway has yet to be established. In this study, we show that human CD4⁺ lymphocytes can be activated by exposure to the anti-CD4 monoclonal antibody (mAb) B66. Normal peripheral blood CD4⁺ cells were induced to proliferate and to synthesize interleukin 2 (IL2) by the antibody. The specificity of the antibody stimulatory activity was tested by using IL2-producing clones bearing either CD4 or CD8 on their surface. IL2 production was induced by mAb B66 in CD4+, but not CD8⁺, clones, whereas both types of clones responded to stimulation by the anti-CD3 mAb Leu-4. Despite its unique stimulatory activity, mAb B66 shared with other anti-CD4 antibodies the ability to inhibit the specific cytolytic activity of CD4⁺ effector cells. These results clearly indicate that cross-linking of surface CD4 molecules with appropriate antibodies can fully activate CD4⁺ lymphocytes. Whether the natural ligand for CD4 can trigger this activation pathway remains to be defined.