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R J Hartzman

Publications and source records attributed to R J Hartzman.

At least 55 records · Page 3Linked to original sources
PubMed1996

Large-scale DRB and DQB1 oligonucleotide typing for the NMDP registry: progress report from year 2.

DNA typing of HLA class II alleles of the DRB1/3/4/5 and DQB1 loci using sequence-specific oligonucleotide probes and polymerase chain reaction amplified DNA has been used for the large-scale typing of donors for the National Marrow Donor Program unrelated donor registry. The results of quality control analysis for the second year of the project (10/1/939/30/94) show the typing continues to be highly accurate, specific, and reliable. The average percent of correctly classified HLA oligotypes (groups of alleles defined by a hybridization pattern with a panel of sequence-specific oligonucleotide probes) based on 9,244 DRB1 and 7,244 DQB1 assignments was 99.8% (range 99.4%100.0%) for DRB1/DRB3/DRB4/DRB5 and 99.8% (range 99.6%100.0%) for DQB1. This level of accuracy is particularly remarkable because the 4,636 DRB quality control samples were tested blindly and could not be distinguished from 57,580 donor samples tested at the same time by the laboratories.

Alleles↗
PubMed1996

Novel HLA-B alleles, B*8201, B*3515 and B*5106, add to the complexity of serologic identification of HLA types.

Three class I alleles, B*8201, B*3515 and B*5106, have been described using DNA and cDNA sequencing. The B*8201 allele is most structurally related to B*5602, differing from it by 14 nucleotide substitutions resulting in 5 amino acid differences. The other two alleles, B*3515 and B*5106, differ from their most closely related HLA-B alleles by 2-3 nucleotide substitutions resulting in 1-2 amino acid substitutions, respectively. The majority of nucleotide substitutions marking these new alleles are observed in other HLA-B alleles suggesting that gene conversion and/or reciprocal recombination have created this diversity. All of the amino acid substitutions are predicted to alter the antigen binding site of the HLA-B molecule. The newly defined HLA-B allelic products were originally defined by their unusual serologic reactivity patterns. The B*8201 allelic product is serologically typed as a B "blank" or as a variant of B22 or B45. These patterns and the serologic reactivity of the other newly described allelic products are consistent with the protein sequence homology among specific HLA-B molecules. While serology remains a powerful tool for detecting HLA diversity, alleles generated by events resulting in the sharing of HLA sequence polymorphisms among alleles at a locus will continue to create complexity in the interpretation of typing results.

Alleles↗
PubMed1996

Typing the HLA-B locus by a nested primer approach and oligonucleotide hybridization.

A system for intermediate level identification of the HLA-B locus alleles was devised. This system can be extended to identify individual alleles in any sample. The first step used primers which amplify all HLA-B alleles. This amplicon was subjected to SSOP hybridization to allow intermediate level typing of samples. In the second step, group-specific primers were utilized to obtain specific amplification of groups consisting of a few alleles. The oligotypes within each group were identified by the use of SSOP. The separation of groups of alleles by amplification allowed the use of a limited number of probes to identify oligotypes present in a sample. Additional probes can be added as new alleles are identified, increasing the flexibility of the system. HLA typing software was developed to determine the resolution of the system and to identify HLA oligotypes. PCR-SSOP methods are in wide use and have been extensively validated. The procedures reported here will be relatively easy to implement for large-scale DNA-based typing of the HLA-B locus.

Base Sequence↗
PubMed1995

Direct sequencing of SSP-PCR-amplified cDNA to identify new alleles in the DR52-associated DRB1 group: identification of DRB1*1115, DRB1*1117 and DRB1*1319.

Low and high resolution sequence specific oligonucleotide probe hybridization patterns were used to design an approach to direct sequencing of allele specific amplified cDNA. Several PCR amplifications were used to derive overlapping sequence fragments to define complete first domain sequences for a single allele. This method has been used to characterize three new DRB1 alleles in the DR52 family, DRB1*1115, DRB1*1117, and DRB1*1319. All three alleles carry polymorphisms previously observed in other DRB alleles and underscore the importance of utilizing a directed sequencing approach for obtaining unambiguous typing results in matching for bone marrow transplantation between unrelated donor and recipient.

Alleles↗
PubMed1994

Melanoma-specific CD4+ T lymphocytes recognize human melanoma antigens processed and presented by Epstein-Barr virus-transformed B cells.

While much emphasis has been placed on the role of MHC class I-restricted CD8+ T cells in the recognition of tumor-specific antigens (Ag), evidence has accumulated that CD4+ T cells also play a critical role in the anti-tumor immune response. However, little information exists on the nature of MHC class II-restricted human tumor Ag. In an attempt to develop in vitro systems to characterize such Ag, we examined the ability of Epstein-Barr virus (EBV)-transformed B cells to present melanoma-associated Ag to melanoma-specific CD4+ cells. CD4+ T cells cultured from lymphocytes infiltrating a s.c. melanoma metastasis secreted TNF-alpha and GM-CSF specifically in response to autologous cultured melanoma cells expressing MHC class II molecules. These CD4+ cells also recognized MHC class II-compatible EBV-B cells pulsed with extracts of autologous melanoma cells, but failed to recognize EBV-B cells pulsed with autologous non-transformed cells or a variety of allogeneic tumors or normal cells. B cells pre-fixed with paraformaldehyde were incapable of Ag presentation, suggesting that intracellular processing events were occurring. Antibody-blocking studies defined HLA-DR as the dominant if not exclusive restriction locus in this T-B interaction, and HLA-DR genotyping revealed DRBI*0404 to be the probable restriction element. In a second patient, a CD4+ T-cell clone cultured from a melanoma lesion recognized autologous tumor Ag presented by autologous EBV-B; no corss-reactivity was observed with the other tumor system investigated, nor with autologous CD4+ T cells specific for tetanus toxoid. These findings demonstrate that tumor Ag can be processed and presented by EBV-transformed B cells to MHC class II-restricted tumor-specific CD4+ T cells. They also provide a model system for direct identification of these tumor-derived antigens.

Antigen Presentation↗
PubMed1992

Video analysis of DNA sequence homologies.

A method for the rapid quantitative analysis of dot blot assays is presented. A video camera, an NTSC compatible frame grabber board, and an AT personal computer are used to read photographic exposures of the assay plate. Image processing and image analysis techniques are used to calculate the orientation of the dot raster and then to compensate for the effect of variations in field illumination on measurements of local contrast. Local contrast (between dots and background) is an exponential function of the amount of hybridization between blotted DNA and complimentary oligonucleotide probes. The amount of hybridization between blotted DNA and oligonucleotide probes of known sequence is the criteria used to establish HLA-DR tissue types. Although the assay described here utilizes a chemiluminescent reaction, this algorithm may be used to read any assay that produces a rectangular raster of dots.

Calibration↗
PubMed1992

Calibration of a resonance energy transfer imaging system.

A quantitative technique for the nondestructive visualization of nanometer scale intermolecular separations in a living system is described. A calibration procedure for the acquisition and analysis of resonance energy transfer (RET) image data is outlined. The factors limiting RET imaging of biological samples are discussed. Measurements required for the calibration include: (a) the spectral sensitivity of the image intensifier (or camera); (b) the transmission spectra of the emission filters; and (c) the quantum distribution functions of the energy transfer pair measured in situ. Resonance energy transfer imaging is demonstrated for two DNA specific dyes. The Förster critical distance for energy transfer between Hoechst 33342 (HO) and acridine orange (AO) is 4.5 +/- 0.7 nm. This distance is slightly greater than the distance of a single turn of the DNA helix (3.5 nm or approximately 10 base pairs), and is well below the optical diffraction limit. Timed sequences of intracellular energy transfer reveal nuclear structure, strikingly similar to that observed with confocal and electron microscopy, and may show the spatial distribution of eu- and hetero- chromatin in the interphase nuclei.

Acridine Orange↗
PubMed1990

Analysis of HLA-DP in HLA-DR/GLO recombinant families and in the population of south-western France.

The existing estimates of the recombination fraction between DR and DP are quite variable and often based on anecdotal observations. We have estimated the DR/DP crossover frequency on the basis of families typed for HLA markers and GLO. The frequency of DR/GLO crossing over was 8.7% (23/264 informative meioses), maternal recombinations being about twice as frequent as paternal ones. Of 17 DR/GLO recombinant families typed for DPw1-6, DP was informative in 11 (13 recombinations) but only one of these gave rise to a DR/DP crossover. According to these data the DR/DP recombination fraction is below 1%, in contrast to some earlier published materials. HLA-DR/DP haplotypic associations on 127 informative Caucasoid haplotypes have been evaluated. In agreement with previous studies, DR3 was positively associated with DPw1 and, in addition, DR7 was found to be positively associated with DP-blank (not DPw1-6). The rare DPw6 allele is possibly associated with the DR4, Dw14 allele. The DR-DP haplotype profiles suggest other associations which might become significant if larger materials are tested. The frequency of DP alleles in a random material (N = 201) was found to be in accordance with most of the previously published frequences on European Caucasoids with DPw4 as the predominating frequency (gene frequency 40%) and a blank frequency of 27%.

Alleles↗
PubMed1989

Heterogeneity of CD4-positive human T-cell clones which recognize the surface protein antigen of Rickettsia typhi.

Immunity to the typhus group of rickettsiae is largely dependent on the effector function of several classes of T lymphocytes, including those which produce gamma interferon. Since the surface protein antigen (SPA) derived from typhus group rickettsiae has been shown to be an effective immunogen in animal models, human T-cell clones specific for the SPA of Rickettsia typhi were isolated and tested for their antigenic specificity, as well as for their ability to produce gamma interferon. Eighteen CD4-positive clones specific for the SPA of R. typhi exhibited considerable diversity in their response to the SPAs derived from two strains of Rickettsia prowazekii and from Rickettsia canada. The vast majority of clones also recognized the SPAs from R. prowazekii but not from R. canada. Two heteroclitic clones demonstrated significantly higher proliferative responses to the SPAs derived from one or both of the R. prowazekii strains than to the SPA of R. typhi, and one clone demonstrated a significantly higher response to the SPA of R. typhi than to the other SPAs. All 18 clones produced gamma interferon in response to SPA stimulation. We conclude that the SPAs from typhus group rickettsiae can elicit both a diverse T-cell response in humans and the efficient stimulation of gamma interferon-mediated immunity.

Antigens, Bacterial↗
PubMed1988

HIV-1-infected T cells show a selective signaling defect after perturbation of CD3/antigen receptor.

The binding of antigen or monoclonal antibody to the T cell receptor for antigen or the closely associated CD3 complex causes increases in the concentration of intracellular ionized calcium and subsequent cell proliferation. By measuring second messenger production in primary cultures of human immunodeficiency virus (HIV-1)--infected T cells stimulated with monoclonal antibodies specific for either CD3 or CD2, a specific impairment of membrane signaling was revealed. The HIV-1--infected T cells were unable to mobilize Ca2+ after stimulation with anti-CD3, whereas CD2-induced calcium mobilization remained intact. Furthermore, the HIV-1--infected cells proliferated poorly after CD3 stimulation, although the cells retained normal DNA synthesis in response to interleukin-2 stimulation. These results show that the signals initiated by CD2 and CD3 can be regulated independently within the same T cell; uncoupling of signal transduction after antigen-specific stimulation provides a biochemical mechanism to explain, in part, the profound immunodeficiency of patients with HIV-1 infection.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Human allospecific TLCs generated against HLA antigens associated with DR1 through DRw8. III. Family segregation analyses.

We have studied the complexity and fine specificity of the HLA-D region using a panel of T lymphocyte clones generated against alloantigens associated with HLA-DR1 through DRw8. After extensive testing in population studies, 89 clones were tested in proliferation assays with 14 families. Segregation patterns were analyzed for haplotype associations by calculating sequential lod scores to test the likelihood that genes encoding epitopes detected by TLCs were linked to HLA genes. Four general categories were identified: (1) clonal responses that segregated with the same HLA-D region haplotype in all informative pedigrees; (2) clonal responses that segregated with HLA in all pedigrees but not always with the same haplotype; (3) clonal responses that segregated with HLA in some families but failed to segregate in others or produced equivocal results; (4) clonal responses that did not segregate with HLA haplotypes.

Cell Line↗
PubMed1988

Measles virus-specific human T cell clones: studies of alloreactivity and antigenic cross-reactivity.

Cross-reactivity between altered self and foreign major histocompatibility complex (MHC) may be of etiologic importance in autoimmune disease. We have studied 29 measles virus-specific cloned and uncloned T cell lines from a patient with multiple sclerosis (MS) and from a normal subject. Two of the T cell clones derived from the normal subject reacted with foreign MHC determinants. No cross-reactivity between measles virus and either myelin basic protein (BP) or galactocerebroside (GC) was detected. T cell clones which are specific for nominal antigen and which also recognize alloantigen were detected with much smaller frequency than that reported in murine systems. Our data do not support a role for alloreactive measles-specific T cells, nor for cross-reactivity between measles virus and either BP or GC, in the pathogenesis of MS.

Antigens, Viral↗
PubMed1987

Lysis of cells infected with typhus group rickettsiae by a human cytotoxic T cell clone.

Cytolytic human T cell clones generated in response to the intracellular bacterium Rickettsia typhi were characterized. Growing clones were tested for their ability to proliferate specifically in response to antigens derived from typhus group rickettsiae or to lyse targets infected with R. typhi or Rickettsia prowazekii. Two clones were able to lyse targets infected with typhus group rickettsiae. One of these clones was more fully characterized because of its rapid growth characteristics. This cytolytic clone was capable of lysing an autologous infected target as well as a target matched for class I and II histocompatibility leukocyte antigens (HLA). It was not capable, however, of lysing either a target mismatched for both class I and II HLA or a target partially matched for class I HLA. In addition, the clone exhibited specificity in that it was able to lyse an autologous target infected with typhus group rickettsiae, but did not lyse an autologous target infected with an antigenically distinct rickettsial species, Rickettsia tsutsugamushi. These results demonstrate, for the first time, that cells infected with intracellular bacteria can be lysed by human cytotoxic T lymphocytes.

Antigens, Bacterial↗
PubMed1987

Unique HLA-D region heterogeneity in American blacks.

American blacks and whites show heterogeneity in serologically detected and lymphocyte-defined HLA-D region antigen frequencies. Also, alterations in linkage relationships are seen in DR and DQ specificities. Since homozygous typing cells probably identify a cluster of specificities that are usually found in linkage disequilibrium in a given population, it is not surprising that blacks have unique HLA-D types. The significance of the observed differences in the function of class II molecules is being investigated.

Black People↗
PubMed1986

Polypeptide specificities of measles virus-reactive T cell lines and clones derived from a patient with multiple sclerosis.

Eleven cloned and uncloned measles virus-specific T cell lines were generated from peripheral blood lymphocytes obtained from a patient with multiple sclerosis and were assayed for measles polypeptide specificity. Three clones reacted specifically with the fusion (F) protein and one recognized the hemagglutinin (HA). Two reacted with whole virus but not with any of the purified proteins. Five cell lines proliferated in response to multiple measles polypeptides. The addition of anti-HA or anti-F monoclonal antibodies to two of the multispecific cell lines each resulted in partial suppression of the proliferative response to whole virus by the cell lines. Two of the three F-reactive clones recognized antigen in association with a subgroup of HLA-DR4; the third responded to F only in the presence of autologous antigen-presenting cells. Of the two clones that reacted only with whole virus, one was restricted to DP3 and one to autologous cells. The HA-specific clone was DP3 restricted. Several cell lines recognized multiple measles polypeptides in association with a single HLA antigen. Recognition of individual measles polypeptides does not segregate with specific genetic restriction elements.

Antigens, Viral↗
PubMed1986

Recognition of a hybrid HLA-DP-associated determinant by a human T lymphocyte clone.

Alloreactive, human T cell clones were derived from an HLA-DPw1-specific primed lymphocyte typing cell line by limiting dilution. The specificities of the clones were analyzed with allogeneic stimulator cells and in family segregation studies. One clone, TLC 56.94, recognized some, but not all, DPw1-positive stimulator cells and in two families, failed to proliferate in response to stimulatory cells from DPw1-homozygous individuals. The simplest explanation for these results is that TLC 56.94 recognizes a hybrid alloantigen produced by transcomplementation or transassociation between an element of DPw1 and some other gene product.

Antibodies, Monoclonal↗
PubMed1986

Human allospecific TLCs generated against HLA antigens associated with DR1 through DRw8. I. Growth and specificity analysis.

To study the fine specificity of the HLA-D region, a panel of human T-lymphocyte clones (TLCs) was generated against alloantigens associated with HLA-DR1 through DRw8. HLA-DR-homozygous peripheral blood lymphocytes (PBLs) were stimulated with DR-heterozygous PBLs in primary mixed lymphocyte cultures for 4 days. Blasts were cloned by limiting dilution at 0.3 cells/well in the presence of 20% T-cell growth factor and irradiated stimulator cells. Viable clones were subsequently tested in proliferation assays against the original stimulator and a limited panel of stimulators bearing relevant DR specificities. Initial primings produced approximately 800 clones; some recognized DR-associated antigens, 70 recognized only their original stimulator, and approximately 50% were nonresponsive. Analysis on extended stimulator panels revealed alloantigenic complexity within similar DR-associated antigens as recognized by TLCs. The data are consistent with evidence that extreme heterogeneity exists within the HLA-D region.

Clone Cells↗