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Biomedical subjects

R L Griffith

Publications and source records attributed to R L Griffith.

18 recordsLinked to original sources
PubMed1991

Rapid typing of DNA sequence polymorphism at the HLA-DRB1 locus using the polymerase chain reaction and nonradioactive oligonucleotide probes.

An HLA-DR typing system that uses sequence-specific oligonucleotide (SSO) probes conjugated to horseradish peroxidase (HRP) for analyzing DRB alleles amplified by the polymerase chain reaction has been developed. Using 25 HRP-SSO probes and two primer pairs for generic and for DRB1 locus-specific amplification, we can distinguish 31 of 34 HLA-DRB1 alleles. This procedure is suitable for typing heterozygous samples from a variety of sources, including cDNA templates, and can detect in a simple and rapid dot-blot format allelic variants not distinguishable by serological methods. It should prove valuable for tissue typing, determining individual identity, and studies of disease susceptibility.

Amino Acid Sequence↗
PubMed1991

DNA amplification for DQ typing as an adjunct to serological prenatal HLA typing for the identification of potential donors for umbilical cord blood transplantation.

It has recently been demonstrated that umbilical cord blood from genotypically human leukocyte antigen (HLA)-matched donors can provide sufficient numbers of progenitor cells for hematopoietic reconstitution. This technique has been successfully applied in the treatment of two children affected with Fanconi anemia (FA). Fetal cells from the potential sibling donors were first tested to determine that the fetus was not affected with FA. Unaffected fetal cells were then tested for HLA type. Cord blood from compatible donors can be harvested at birth and used immediately or frozen for subsequent use in hematopoietic reconstitution. We now show that fetal cell DNA amplification and hybridization for DQ typing can be an important adjunct procedure to verify serologically determined HLA class II types and/or to establish class II haplotype identity with the affected sibling.

Blood Transfusion↗
PubMed1991

Implication of specific DQB1 alleles in genetic susceptibility and resistance by identification of IDDM siblings with novel HLA-DQB1 allele and unusual DR2 and DR1 haplotypes.

Genetic susceptibility to insulin-dependent diabetes mellitus (IDDM) is associated with the HLA-DR3 and DR4 haplotypes. The HLA-DR2 haplotype is negatively associated with IDDM, an association that has been interpreted as dominant protection. Here, we describe the molecular analysis of the HLA class II genes in an unusual family with three HLA-DR1/2 siblings, all of whom have IDDM. With polymerase chain reaction amplification and sequence analysis to characterize the class II alleles, we identified a novel DQB1 allele on the DR1 haplotype and an unusual DQB1 allele on the DR2 haplotype. However, the DRB1 alleles on these DR1 and DR2 haplotypes are the conventional alleles (*0101 and *1501, respectively). These results suggest that it is the conventional DQB1 allele (*0602) not the DRB1 allele (*1501) on the protective DR2 haplotype that confers protection in the general population and, furthermore, that these unusual DQB1 alleles may confer susceptibility to IDDM in this family. The unusual DQB1 allele on this DR2 haplotype encodes Asp at position 57, indicating that it is the allele DQB1*0602 and not simply the presence of this residue that is responsible for the protective effect.

Adolescent↗
PubMed1990

Unique sequences for two HLA-DRB1 genes expressed on distinct DRw6 haplotypes.

We have used restriction fragment length polymorphism (RFLP) analysis and DNA sequencing to characterize two distinct DRB1 alleles expressed on DRw52 and DQw7-associated haplotypes but not readily defined by conventional DR serology. These two haplotypes, designated HLA-D "HAG" and "PEV", react variably with DRw13(w6), DRw14(w6), and the more broad DR"3 + 6" antisera. Analysis of RFLP revealed that HLA-D "HAG" and "PEV" are associated with different DRw52 variants, and that "HAG" is indistinguishable from DRw18(3) haplotypes. Sequencing of the "HAG" and "PEV" DRB1 genes showed each to represent novel alleles. Nevertheless, these sequences show similarities with the other alleles of the DR5,w6, and w8 family. "HAG" (DRB1*1303) appears to have arisen either from two recombinational events involving at least three DRB1 sequences (DRB1*1101, DRB1*0803, DRB1*0401) or from a single recombinational event together with multiple point mutational events. "PEV" appears to represent a DRB1*1301-1302/DRB1*1101 recombinant allele, with recombination having occurred in the region of bases 175-198. The results of this study suggest that the DRw52 family of haplotypes is derived from a relatively restricted number of ancestral sequences, with diversity among DRB1 alleles within this family arising through gene conversion or recombination events.

Amino Acid Sequence↗
PubMed1990

HLA-DQ beta sequence polymorphism and genetic susceptibility to IDDM.

The analysis of HLA-DQ beta nucleotide sequence polymorphism in insulin-dependent diabetes mellitus (IDDM) patients and control subjects suggests a role for the DQ beta-chain in genetic susceptibility. Sequence determination and oligonucleotide hybridization was carried out on enzymatically amplified DNA from various HLA-DR-typed individuals, including the rare class of DR2+ patients. In the analysis of DQ beta variation in DR4, DRw6, and DR2 haplotypes, a correlation was observed between the presence of the negatively charged residue Asp at position 57 and low susceptibility and the presence of an Ala (DR4), Val (DRw6), or Ser (DR2) and higher susceptibility. However, important exceptions to this pattern have been identified in the analysis of heterozygous DR1/4 IDDM patients. In these individuals, susceptibility appears to correlate with specific DR beta l alleles (Dw4) on the DR4 haplotype, rather than with the DQ beta allele (DQB3.2) that contains Ala at position 57. The DQ beta alleles found in some Chinese IDDM patients also proved discordant with the position-57 correlations. Thus, although there is a general correlation between the residue at position 57 of the DQ beta-chain and IDDM susceptibility, these data do not support the notion that Asp 57 confers complete resistance or protection to IDDM. In general, these results suggest that IDDM susceptibility is conferred by specific combinations of DQ beta and DR beta sequences.

Alleles↗
PubMed1988

Low density lipoprotein metabolism by human macrophages activated with low density lipoprotein immune complexes. A possible mechanism of foam cell formation.

Human macrophages play a key role in atherogenesis and are believed to be the progenitors of the cholesteryl ester (CE)-laden foam cells present in early atherosclerotic lesions. Several mechanisms by which macrophages accumulate CE have been recently described. One involves a perturbation in LDL metabolism subsequent to macrophage activation. Thus, we decided to study the effect of macrophage activation by immune complexes on N-LDL metabolism. Initially, LDL-containing immune complexes (LDL-IC) were chosen, since increased plasma levels of these IC have been reported in patients with coronary heart disease. Human macrophages stimulated for 22 h with LDL-IC (250 micrograms/ml) and incubated afterwards for 20 h with 10 micrograms/ml 125I-N-LDL showed a six- and fourfold increase in the accumulation and degradation, respectively, of 125I-N-LDL over the values observed in nonstimulated cells. Scatchard analysis of 125I-N-LDL-specific binding suggests an increase (20-fold) in the number of LDL receptors in macrophages stimulated with LDL-IC. We studied other immune complexes varying in size and antigen composition. Some of the IC were able to stimulate, although to a lesser degree, the uptake of N-LDL by macrophages. Lipoprotein IC are more efficient and have the greatest capacity to increase N-LDL uptake and CE accumulation. We conclude that human macrophage activation by LDL-IC leads to an increase in LDL receptor activity and promotes in vitro foam cell formation.

Antigen-Antibody Complex↗
PubMed1988

Loss of human immunodeficiency virus type 1 (HIV-1) antibodies with evidence of viral infection in asymptomatic homosexual men. A report from the Multicenter AIDS Cohort Study.

Four asymptomatic homosexual men reverted from positive to negative serologic results for the human immunodeficiency virus, type 1 (HIV-1) over 2.5 years, as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot. Antibody bands in the Western blot from three men were undetectable 6 to 12 months after being positive; gradual fading of the number and intensity of bands was seen in the other man. No HIV-1-p24 antigenemia was detected; cryopreserved peripheral blood mononuclear cells were negative for HIV-1 by standard culture assay. Polymerase chain reaction (gene amplification) assays were done on peripheral blood mononuclear cells and showed the HIV-1 provirus in all subjects 6 to 18 months after the last positive antibody test. Serum specimens from each participant were genetically identical. Polymerase chain reaction showed that peripheral blood mononuclear cells from one subject at different times matched by HLA DNA typing. Clinical and laboratory features of these four men were similar to those of other seronegative subjects. Rare, asymptomatic persons seropositive for HIV-1 may not remain seropositive, but may remain latently infected with HIV-1.

Adult↗
PubMed1985

The proliferation and function of human mononuclear leukocytes and natural killer cells in serum-free medium.

We recently developed a serum-free (SF) culture medium that supports the growth of several established lymphoid cell lines. In an effort to develop a standardized medium for assay of human natural killer (NK) cell activity, we compared the cytotoxic activity of peripheral blood mononuclear leukocytes (PBL) and purified large granular lymphocytes (LGL) cultured in SF medium containing interleukin 2 (IL-2) or medium containing 10% fetal bovine serum (FBS) plus IL-2. The results indicated that PBL had a 30% increase in cumulative net cell growth and had as high or higher cytotoxic activity after growth in SF medium than in medium containing FBS. Purified LGL had a 50% increase in cumulative net cell growth and persisted approximately 2 weeks longer in culture in medium containing FBS than in SF medium. However, the cytotoxic activity of cells grown in SF medium persisted during the initial 3 weeks of culture. Purified LGL that were maintained and were subcultured at cell densities of 10(6) cells or greater per milliliter of either SF or FBS-containing medium had equivalent levels of cytotoxicity over a 44-day period in either medium compared with cells subcultured at a density of 5 X 10(5) cells per milliliter of medium. NK cells produced a cytotoxic factor (NKCF) in SF medium, and its cytotoxic activity was blocked by 10% FBS. We conclude that the SF medium supplemented with IL-2 can be used as an alternative to FBS-containing medium with IL-2 for the growth of NK cells and is advantageous for the production of NKCF.

Cell Division↗
PubMed1985

Modulation of interleukin 2 release from a primate lymphoid cell line in serum-free and serum-containing media.

The ability to grow a clone of the cell line, MLA144, which is a constitutive producer of interleukin 2 (IL-2), in serum-free medium permitted the study of the direct effect of various agents on cell growth and IL-2 production in a homogeneous population. Bovine serum albumin (BSA) at 4 mg/ml was optimal for cell growth and IL-2 production. Selenium at 10 ng/ml enhanced IL-2 production nearly twofold and lithium at 42 ng/ml also enhanced IL-2 production by nearly twofold. Neither compound at these levels altered cellular proliferation. Two other compounds, iron and zinc, known to be associated with cellular proliferation and/or immunoregulation did not alter IL-2 production. Catalase or horseradish peroxidase was able to substitute for BSA and maintain the long-term growth of the MLA144 clone with only a 30% decrease in the rate of cellular proliferation and a 50% decrease in IL-2 production compared to cells maintained in the serum-free formulation with BSA. Addition of 0.5 mg of BSA to the catalase serum-free formulation increased the production of IL-2 to 70% of that of cells cultured in the BSA-containing serum-free formulation. The catalase-containing serum-free formulation has the advantage of consisting of only three proteins, catalase, insulin, and transferrin, at a very low protein content. The catalase-containing serum-free medium also supported the long-term growth of a human T-cell line, HSB-2.

Animals↗
PubMed1979

The CSF pressure-volume relationship before and after cardiac arrest in the cat.

The CSF pressure-volume (P-V) function was evaluated before and after cardiac arrest in 15 cats. The CSF volume change was produced by bolus loading (loading rate, greater than 0.1 mL/s) of the CSF space. Comparison of the CSF P-V function before and after cardiac arrest was made over a CSF pressure range of 5 to 46 mm Hg and for a CSF volume change of up to 9% of total CSF volume. After cardiac arrest, all CSF P-V curves were well described by the mathematical model konwn to be valid under normal physiological conditions. In eight animals, there was no significant difference between the prearrest and postarrest P-V functions. For the seven animals demonstrating a significant difference between prearrest and postarrest P-V data, all but one of the postarrest P-V curves were within the normal range. These results suggest that the shape of the CSF P-V curve is not substantially altered by cardiac arrest. We conclude that under normal circumstances material properties of brain tissue are the most important factors in determining the configuration of the CSF P-V curve and that under normal circumstances cerebral hemodynamic factors do not affect the shape of this curve.

Animals↗
PubMed1979

Bolous versus steady-state infusion for determination of CSF outflow resistance.

For rapid changes in cerebrospinal fluid volume an exponential relationship was demonstrated between CSF pressure and CSF volume in 15 cats. This relationship was valid over a CSF pressure range from 7 to 50 mm Hg and for acute increases of up to 9% to total CSF volume (approximately 13 ml for humans). Our data agree well with previous reports for the cat. A similar relationship has been shown in the dog and in humans. It has been claimed that, given the equations for CSF bulk flow and the exponential relationship between CSF pressure and CSF volume, one can calculate CSF outflow resistance by observing the decay of CSF pressure after a bolus injection into the CSF space. This claim was evaluated in an additional 18 cats. In these animals CSF outflow resistance calculated by the bolus method was compared with resistance calculated by a steady-state infusion method over the CSF outflow resistance range of 74 to 293 mm Hg/ml min-1. Resistance calculated by the bolus method underestimated resistance calculated by the steady-state method, and this underestimate grew larger with increasing resistance. The bolus technique is therefore not a valid method for determining CSF outflow resistance. The explanation offered for these results is that the decay of CSF pressure after a bolus injection into the CSF space occurs not only because of runoff of the injected volume of CSF but also because of "pressure relaxation" of the brain parenchyma around the CSF space. The phenomenon of pressure relaxation was not considered in developing the equation for calculation of CSF outflow resistance by the bolus technique. The time dependency of pressure relaxation allows for a fundamental element of hysteresis within the CSF space. A method of quantifying this element of hysteresis is suggested.

Animals↗
PubMedDate not supplied

Enhanced uptake and impaired intracellular metabolism of low density lipoprotein complexed with anti-low density lipoprotein antibodies.

We have previously shown that incubation of human macrophages with antigen-antibody complexes prepared with native human low density lipoprotein (LDL) and rabbit anti-LDL antibodies (LDL-ICs) results in an increased intracellular accumulation of cholesteryl esters (CEs) and induces a marked increase in the number of LDL receptors. To determine whether the increased CE accumulation in these cells occurred during incubation of the cells with LDL-ICs or whether it was secondary to the uptake of LDL by overexpressed LDL receptors, we incubated human macrophages with LDL-ICs for 22 hours, followed by incubation with native LDL for another 20 hours. We found that about 90% of the accumulated CEs could be accounted for by the first incubation with LDL-ICs. We then proceeded to show that the CEs accumulated during incubation of cells with LDL-ICs was secondary to enhanced uptake and impaired degradation of the LDL complexed with immunoglobulin G (IgG) (LDL-IC), which led to a marked intracellular accumulation of undergraded LDL (levels 199-fold higher than those obtained when the cells were incubated with the same concentration of native LDL not complexed with IgG). We have also shown that not all CEs accumulated in these cells were derived from accumulation of undegraded LDL and that some of them were derived from the reesterification of free cholesterol released during hydrolysis of LDL. LDL-ICs promoted increased CE accumulation and foam cell formation at concentrations as low as 25 micrograms/ml. To determine which receptors were involved in the uptake of LDL-ICs, we performed experiments in which the uptake of LDL-ICs was competitively inhibited with heat-aggregated gamma globulin, native LDL, beta-very low density lipoprotein, or acetylated LDL. Our results demonstrated that LDL-IC uptake was most effectively inhibited by heat-aggregated gamma globulin, partially inhibited by native LDL or by a monoclonal antibody to the LDL receptor, and not inhibited by acetylated LDL or beta-very low density lipoprotein. Thus, we conclude that the majority of LDL-ICs are taken up through Fc gamma receptors. Finally, we investigated whether the increase in LDL receptor expression was dependent on the receptor pathway used by the LDL-ICs, and we were able to demonstrate that when macrophages were incubated with LDL-ICs prepared with F(ab')2 fragments of the anti-LDL antibody, LDL receptor expression was not enhanced. Therefore, we postulate that the uptake of LDL-ICs through Fc gamma receptors results in an uncoupling of the normal regulation of the LDL receptor expression.

Antibodies↗