The identification of HL-A antigens on fresh and cultured human endothelial cells.

Human endothelial cells were obtained from the umbilical cord veins of 16 newborns by methods previously described and tested for HL-A antigens by a microcytotoxicity method. HL-A antigens were present on all endothelial cell lines tested. When the HL-A phenotypes of fresh endothelial cells and autologous fetal lymphocytes were compared, a concordance of 70% was observed. When the HL-A phenotypes of maternal lymphocytes and fresh endothelial cells were compared, a maternal contribution to the endothelial cell phenotype was evident in 72% of the possible commmon antigens. Some HL-A antigens were deleted from 11 of 16 endothelial cell lines that were re-typed after 2 weeks in tissue culture. The majority (90%) of deleted antigens were from the second HL-A locus. When three lines of endothelials cells were again re-typed after 6 weeks in culture, no further changes in antigenicity were noted. These findings: a) demonstrate that HL-A antigens are present on human endothelium and suggest that endothelial cells are actively involved in establishing the immunogenicity of a graft, and b) demonstrate that the HL-A antigens on human endothelial cells may be modulated by in vitro culture.     

Enhancement of pentobarbital effect by continuous administration of morphine in the mouse

These studies demonstrated that continuous morphine treatment from implantation of a 75 mg morphine pellet for 3 days potentiated pentobarbital narcosis and enhanced pentobarbital hypothermia. In the morphine implant mice, sleeping time after two different doses of pentobarbital was greater than 2.5 × the sleeping time in placebo pellet implant animals and also greater than sleeping time in animals treated acutely with morphine prior to pentobarbital. Moreover, in the morphine implant mice both the degree and duration of pentobarbital induced hypothermia were enhanced. The above findings were due to slower rate of metabolism of pentobarbital as evidenced by inhibition of hepatic N-demethylation, and higher levels of brain and serum pentobarbital in the morphine implant mice compared to both placebo and acute morphine mice.     

Expression of a functional chimeric Ig-MHC class II protein.

We have generated a chimeric protein molecule composed of the alpha- and beta-chains of the MHC class II I-E molecule fused to antibody V regions derived from anti-human CD4 mAb MT310. Expression vectors were constructed containing the functional, rearranged gene segments coding for the V region domains of the antibody H and L chains in place of the first domains of the complete structural genes of the I-E alpha- and beta-chains, respectively. Cells transfected with both hybrid genes expressed a stable protein product on the cell surface. The chimeric molecule exhibited the idiotype of the antibody MT310 as shown by binding to the anti-idiotypic mAb 20-46. A protein of the anticipated molecular mass was immunoprecipitated with anti-mouse IgG antiserum. Furthermore, human soluble CD4 did bind to the transfected cell line, demonstrating that the chimeric protein possessed the binding capacity of the original mAb. Thus, the hybrid molecule retained: 1) the properties of a MHC class II protein with regard to correct chain assembly and transport to the cell surface; as well as 2) the Ag binding capacity of the antibody genes used. The generation of hybrid MHC class II molecules with highly specific, non-MHC-restricted binding capacities will be useful for studying MHC class II-mediated effector functions such as selection of the T cell repertoire in thymus of transgenic mice.     

Synthetic probes for the alpha-factor receptor

The binding of the tridecapeptide yeast mating pheromone, alpha-factor, to its receptor represents an excellent model for the investigation of peptide hormone-receptor interactions. In this paper we present a number of strategies to probe the binding site of the alpha-factor receptor, and discuss the synthesis of probes containing radioactive and affinity tags. Preferential acylation of the alpha- or epsilon-amine in [Nle12]-alpha-factor was accomplished using 3-[3,5-diiodo-4-hydroxyphenyl] propanoic acid hydroxysuccinimide ester (diiodo Bolton-Hunter reagent). At pH 8.0 in a N-N-dimethylformamide/water mixture the ratio of epsilon- to alpha-acylation was 2.15 to 1, whereas at pH 6.5 in a 1,2-dimethoxyethane/water mixture alpha-acylation was favored by more than 3 to 1. The product distribution was found to depend on pH, organic cosolvent, and the ratio of organic solvent and aqueous buffer. Product distributions were followed using analytical high performance liquid chromatography and the products were characterized enzymatically and by mass spectrometry. Citraconic anhydride preferentially alpha-acylated [Nle12]-alpha-factor and served as a temporary masking group during the synthesis of epsilon-Bolton-Hunter acylated pheromone. Biotin or diiodo Bolton-Hunter reagents were also directly incorporated into [Nle12]-alpha-factor or Lys[Nle12]-alpha-factor during peptide synthesis. The peptides were assembled on a chloromethyl polystyrene resin or on a (phenylacetamido)methyl resin, and cleaved using anhydrous hydrogen fluoride (HF). Probes were inserted on amino groups either prior (biotin) or subsequent (Bolton-Hunter reagent) to HF cleavage. The biological activity of the synthetic peptides was characterized using growth arrest assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipopolysaccharide and serum cause the translocation of G-protein to the membrane and prime neutrophils via CD14.

Lipopolysaccharide (LPS) in combination with human serum, in the absence of a second stimulus, causes an increase in the amount of the alpha -subunit (Gi alpha 2) of the guanine nucleotide binding protein Gi2 associated with the membrane. The LPS-serum complex also primes human neutrophils for O2- production in response to stimulation by the chemotactic factor fMet-Leu-Phe. Added serum factor is essential for priming at low concentrations of LPS. In the presence of serum, significant potentiation can be observed at LPS concentration as low as 0.1 ng/ml. The priming is dose and time dependent. Furthermore, the observed actions of the LPS-serum complex are not reversible since they cannot be overcome by washing. Monoclonal antibody against CD14 inhibits both the direct and priming actions of the LPS-serum complex. On the other hand, neither the antibody against CD11b nor the antibody against TNF-alpha inhibits the action of this complex.     

Alcohol and Drug Use—Is There a `Safe' Amount?

All human endeavors are associated with quantifiable risks. Knowledge of the risk is essential for personal health maintenance. Nontherapeutic use of psychoactive drugs poses an important danger to individual persons and society. What are the quantitative estimates of these risks? Are they acceptable?Because the basic mechanism of the toxic effect of alcohol or other drugs is unknown, deciding on acceptable risks is difficult. Based on current information, the recreational abuse of inhalants, hallucinogens, stimulants, narcotics and sedative-hypnotic drugs poses unacceptable individual and societal risks. Groups at special risk should not consume alcohol or any drug unless they are under medical supervision. The threshold for increased morbidity from the regular use of alcohol in adults is in the range of three to five drinks per day; this rises sharply after six drinks per day. The apparent “safe” level of alcohol consumption appears to be one to two drinks per day. Further basic studies are required to refine these risk estimates.