Validation and population studies of the loci LDLR, GYPA, HBGG, D7S8, and Gc (PM loci), and HLA-DQ alpha using a multiplex amplification and typing procedure.

Studies were performed to evaluate the forensic applicability of multiplex amplification of the loci low density lipoprotein receptor, glycophorin A, hemoglobin G gammaglobin, D7S8, and group-specific component (PM loci) and simultaneous typing of these loci using a reverse dot blot approach where allele specific oligonucleotide probes are immobilized on a nylon membrane strip. These results were obtained by using the AmpliType PM PCR Amplification and Typing Kit. The experiments included: mixed body fluid studies; chemical contaminant effects on the DNA in body fluid samples; the effect of typing DNA from body fluid samples deposited on various substrates; the effect of microorganism contamination on typing DNA derived from blood and semen; the effect of sunlight and storage conditions on DNA typing; determination of the sensitivity of detection of the PM test kit; determination of cross-reactivity of DNA from species other than human; typing DNA derived from various tissues from an individual; and an evaluation of the hybridization temperature of the assay. The data demonstrate that DNA exposed to a variety of environmental insults yields reliable PM typing results. Allele and genotype frequencies for six loci (PM loci and HLA-DQ alpha) were determined in African Americans. Caucasians, southeastern Hispanics, and southwestern Hispanics. All loci meet Hardy-Weinberg expectations and there is little evidence for association of alleles between the loci. The frequency data can be used in forensic analyses and paternity tests to estimate the frequency of a multiple locus DNA profile in various general United States populations.

PCR amplification and typing of the HLA DQ alpha gene in forensic samples.

The polymerase chain reaction (PCR) was used to amplify the HLA DQ alpha gene using DNA recovered from evidentiary samples. Amplified HLA DQ alpha DNA was then typed using sequence-specific oligonucleotide probes. Slight modifications of previously published DNA extraction methods improved typing success of bloodstains and semen-containing material. Evidentiary samples, consisting of 206 known bloodstains, 26 questioned bloodstains, and 123 questioned semen-containing evidentiary materials were analyzed from 96 cases previously analyzed by restriction fragment length polymorphism (RFLP) typing in the FBI Laboratory. Of the known bloodstains, 98.5% yielded DQ alpha typing results. Of the questioned samples, 102 of 149 (24/26 bloodstains and 78/123 semen-containing materials), or 68%, produced typing results. Of the 78 cases that were RFLP inclusions, 59 yielded interpretable DQ alpha results and these were all inclusions. The remaining 19 cases could not be interpreted for DQ alpha. Of the 18 RFLP exclusions, eleven were DQ alpha exclusions, four were DQ alpha inclusions, and three could not be interpreted for DQ alpha. It is expected that because of the difference in discrimination potential of the two methods, some RFLP exclusions would be DQ alpha inclusions. Some samples that failed to produce typing results may have had insufficient DNA for analysis. Employment of a human DNA quantification method in DQ alpha casework would allow the user to more consistently use sufficient quantities of DNA for amplification. It also could provide a guide for determining if an inhibitor of PCR is present, thus suggesting the use of a procedure to improve amplification. This study provides support that the HLA DQ alpha typing procedure is valid for typing forensic samples.

Interactions between the monocyte/macrophage and the vascular smooth muscle cell. Stimulation of mitogenesis by a soluble factor and of prostanoid synthesis by cell-cell contact.

The effect of soluble factors from the monocyte/macrophage (M phi) on cell proliferation and the functional effects of cell-cell contact on the arachidonic acid (AA) cascade were studied with vascular smooth muscle cells (SMCs). Peripheral blood M phi s were isolated by adherence or in a Percoll gradient, and alveolar M phi s were obtained by lavage. Conditioned medium (CM) was prepared by preincubating M phi s with medium alone or by separating SMC and M phi cocultures by a membrane insert. Cell proliferation (image analysis) and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha, radioimmunoassay) were measured in SMCs. Labeled prostanoids and other eicosanoid metabolites were isolated by high-performance liquid chromatography from SMCs prelabeled with 14C-AA. M phi s did not synthesize 6-keto-PGF1 alpha. The CM enhanced proliferation but did not stimulate 6-keto-PGF1 alpha synthesis in SMCs. However, cell-cell contact in cocultures of SMCs with the same concentration of M phi s used to generate CM resulted in increased 6-keto-PGF1 alpha synthesis by SMCs. Since the stimulatory effect of cell contact was not blocked by butylated hydroxytoluene, it could not be attributed to an oxidative burst from M phi s. Functional studies showed that the stimulatory effect of cell contact was enhanced by exogenous free AA and by endogenous AA release through A23187. Release of total radioactivity from prelabeled SMCs was enhanced by cell contact, and this effect was blocked by indomethacin (IM). Cell contact did not increase the release of free AA from prelabeled SMCs, even in the presence of IM. Finally, cell contact only stimulated the formation of prostanoids (IM-sensitive eicosanoid metabolites) from prelabeled SMCs. Lipoxygenase and other products of AA were not formed through cell-cell contact. These data showed that M phi s express a soluble factor that enhances SMC proliferation without affecting prostanoid synthesis. Subsequent cell contact between SMCs and M phi s stimulates prostanoid synthesis, which may possibly serve as a local and focal homeostatic mechanism for the regulation of uncontrolled SMC proliferation in atherogenesis.

The impaired ability of human monocytes to stimulate autologous and allogeneic mixed lymphocyte reactions after exposure to cyclosporine. Associated alterations of HLA-DR expression and physical characteristics of monocytes.

Human monocytes (M phi) preexposed to cyclosporine (CsA) concentrations ranging between 1.0 and 10.0 micrograms/ml were impaired in their ability to stimulate autologous and allogeneic mixed lymphocyte reactions (MLR) when they were compared with control M phi unexposed to CsA. M phi preexposed to CsA and M phi preexposed to PGE2 displayed reduced expression of HLA-DR. Indomethacin protected M phi from decreased HLA-DR expression at lower CsA concentrations, but was unable to prevent the decrease of HLA-DR with higher concentrations of CsA. CsA appeared capable of perturbing M phi membranes because decreases in the indirect light scattering properties of M phi were detected with the various CsA concentrations tested. Higher CsA concentrations significantly reduced the cellular volumes of M phi. The reductions of cellular volume were considerably less than the decreases in indirect light scatter. These data show that CsA interacts directly with M phi, reducing their functional ability to trigger MLR responses and their phenotypic expression of HLA-DR. The decreased HLA-DR expression is mediated via prostaglandins at low CsA concentrations and the decreased HLA-DR expression is mediated via membrane perturbations unrelated to prostaglandins at high CsA concentrations.

Fatty acid metabolism and cell proliferation. VII. Antioxidant effects of tocopherols and their quinones.

The antioxidant capacities of alpha- and gamma-tocopherols (alpha-E and gamma-E) and their quinones (alpha-EQ and gamma-EQ) were determined in non-biological and biological systems. The non-biological system consisted of arachidonic acid [20:4 (n-6)], the oxidant cumene hydroperoxide, and a Fe3+ catalyst to facilitate malondialdehyde (MDA) formation from lipid peroxides. alpha-E and gamma-E had similar antioxidant capacities in this system. alpha-EQ also functioned as an antioxidant, while gamma-EQ exhibited a crossover effect by functioning as an antioxidant at low concentrations and a prooxidant at high concentrations. Biological lipid peroxidation in smooth muscle cells challenged with 20:4 (n-6) was measured both by MDA formation in confluent cultures and by cell growth in proliferating cultures. alpha-E, gamma-E and alpha-EQ had similar antioxidant capacities, but gamma-EQ was highly cytotoxic for cells in both confluent and proliferating cultures. Cellular retention of antioxidants was estimated indirectly from MDA formation when cells were loaded with an antioxidant (preincubation) and then incubated for varying periods of time in fresh media containing 20:4 (n-6). Cellular retention also was measured directly with tritiated alpha-E and tritiated alpha-EQ. These studies showed that cellular retention decreased in the sequence gamma-E greater than alpha-E greater than alpha-EQ. Thus, cellular retention does not explain the enhanced antioxidant capacity of alpha-E compared to gamma-E that has been reported for animal systems. The antioxidant capacity of alpha-E evidently is enhanced by its metabolism to a quinone which, unlike the quinone from gamma-E, functions as a biological antioxidant.

Characteristics of cyclosporine induction of increased prostaglandin levels from human peripheral blood monocytes.

Human monocytes (M phi) exposed to 0.5-20 micrograms/ml of cyclosporine (CsA) produced levels of prostaglandins of the E series (PGE) that were 2-3-fold greater than control M phi cultured in medium alone. Maximal PGE levels were obtained at 24-48 hr incubation, and the failure to observe a linear increase of PGE levels at higher CsA concentrations appeared partially related to cytotoxic effects. CsA was considerably less effective than phorbol myristate acetate or bacterial lipopolysaccharide in increasing PGE production, but the PGE levels achieved with CsA approximated those known to suppress immune responsiveness. Other experiments showed that, although the increased PGE production with CsA was indomethacin-sensitive, CsA mostly functioned to increase the availability of free arachidonic acid (AA) instead of accelerating AA conversion by the cyclooxygenase pathway. Thus CsA can alter M phi physiology, and these alterations might inhibit quite early events during the induction phase of immune responses.

Fatty acid metabolism and cell proliferation. V. Evaluation of pathways for the generation of lipid peroxides.

Primary cultures of smooth muscle cells were established from the medial layer of guinea pig aorta. Confluent cells at passage level 4-6 were challenged with arachidonic acid and treated with a number of antioxidants and inhibitors of specific lipid peroxidation pathways. Lipid peroxidation was measured by the thiobarbituric acid test for malondialdehyde (MDA) and the isolation of hydroperoxy fatty acids (HPETE) by high performance liquid chromatography (HPLC). Prostanoids were measured by radioimmunoassay and the separation of labeled compounds by HPLC. MDA, 6-keto-PGF1 alpha, and PGE2 were formed when cells were challenged with arachidonic acid and these cells synthesized small amounts of one HPETE isomer, 15-HPETE. The HPETE isomers characteristic of the lipoxygenase pathway, 12-HPETE and 5-HPETE, were not detected. Furthermore, the lipoxygenase inhibitors, eicosatetraynoic acid (ETYA) and 6,7-dihydroxycoumarin (Esculetin), did not block MDA formation. These data show that MDA is not generated in the cells by a lipoxygenase pathway. The cyclooxygenase inhibitors, indomethacin and ETYA, did not block MDA formation but these agents blocked the formation of 15-HPETE. These data show both that 15-HPETE is generated by a cooxidation pathway and that 15-HPETE and cooxidation are not involved in MDA formation. Three inhibitors of cytochrome P450 linked lipid peroxidation, 2-amino-3-ethoxycarbonyl-6-benzyl-4, 5,6,7-tetrahydrothieno-[2,3-C]-pyridine (Tinoridine), 3-methyl-1,2-di-3-pyridyl-1-propanone (Metyrapone) and phenobarbital, did not block MDA formation. These data support earlier studies that indicated that MDA is not generated by a P450 pathway. Cells contained a bound precursor that decomposed to MDA when cells were treated with Fe3+. The cells exhibited autofluorescence and concentric lamellae in lipid droplets that are characteristic of ceroid-lipofuscin. These observations are consistent with lipid peroxidation through increased peroxisomal activity leading to the generation of MDA and the accumulation of ceroid-lipofuscin. The natural antioxidants, vitamin E and vitamin E quinone (EQ), and the synthetic antioxidants, butylated hydroxytoluene and nordihydroguaiaretic acid (NDGA), alpha-naphthol (alpha-N) and propyl gallate (PrGa), all blocked MDA formation in confluent smooth muscle cells, showing that these antioxidants did not function solely as specific inhibitors of lipoxygenase, cooxidation or P450 pathways. Cell proliferation was measured in cells challenged with arachidonic acid and treated with antioxidants and other inhibitors. The least cytotoxic and most potent antioxidant, EQ, blocked MDA formation in confluent cells and promoted grow

Fatty acid metabolism and cell proliferation: IV. Effect of prostanoid biosynthesis from endogenous fatty acid release with cyclosporin-A.

Cyclosporin-A (Cyc-A) stimulates prostanoid (PGI2) synthesis in confluent smooth muscle cells from guinea pig aorta through the release of endogenous fatty acid. Cyc-A, like other stimulatory agents for prostanoids, promotes smooth muscle cell proliferation and prostanoid synthesis in these proliferating cells. Indomethacin, a cyclooxygenase inhibitor, and exogenous arachidonic acid block the Cyc-A effect on cell proliferation.

Fatty acid metabolism and cell proliferation. III. Effect of prostaglandin biosynthesis either from exogenous fatty acid or endogenous fatty acid release with hydralazine.

Primary cultures of smooth muscle cells were established from the medial layer of guinea pig aorta. Cells were seeded at from 40 to 80 cells per cm2 and cloned for 8 days. Media were analyzed for PGI2 (6-keto-PGF1 alpha) using radioimmunoassay. Prostanoids were synthesized when cells were grown in media alone. Arachidonic acid stimulated prostanoid synthesis and promoted cell proliferation. Indomethacin blocked prostanoid synthesis and abolished the stimulatory effect of arachidonic acid on cell proliferation. Hydralazine stimulated fatty acid release and prostanoid synthesis in confluent cells. Hydralazine also stimulated prostanoid synthesis and promoted proliferation in growing cells. Indomethacin blocked prostanoid synthesis and abolished the stimulatory effect of hydralazine on cell proliferation.