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Biomedical subjects

Peter Stastny

Publications and source records attributed to Peter Stastny.

18 recordsLinked to original sources
PubMed2006

A high degree of HLA disparity arises from limited allelic diversity: analysis of 1775 unrelated bone marrow transplant donor-recipient pairs.

The allelic diversity and associated human leukocyte antigen (HLA) disparity of 1775 bone marrow recipients and their unrelated donors, matched for six of six (1361/1775,77%), five of six (397/1775, 22%), or four of six (17/1775, 1%) HLA-A, -B, -DR antigens, were retrospectively evaluated. The comprehensive HLA analysis included the class I (A, B, C) and II (DRB1, DQA1, DQB1, DPA1, DPB1) loci. Most (>66%) of the predominantly Caucasian study population carried one or two of five to seven common alleles at each HLA locus. In spite of this limited diversity, 29% of the six of six antigen-matched transplants carried allele mismatches at HLA-A, -B, and/or -DRB1, and 92% carried at least one allele mismatch at one of the eight HLA loci tested. Of the 968 HLA-A,-B,-DRB1 allele-matched pairs, 89% carried mismatches at other HLA loci, predominantly at DP loci. The substantially greater than expected HLA allelic disparity between donor and recipient suggests extensive haplotypic diversity and underscores the importance of enhancing approaches to mitigate the deleterious effect of HLA mismatches.

Alleles↗
PubMed2006

MICA antigens stimulate T cell proliferation and cell-mediated cytotoxicity.

In previous experiments we have found that transplant recipients had specific antibodies against MICA. In the present study, we measured T cell proliferation, cytokine production, and cytotoxicity to investigate whether immunization with MICA can produce a specific cellular immune response. BALB/c mice were immunized with recombinant MICA (rMICA). Lymphoid cell suspensions obtained after immunization were used to measure T cell proliferation. We detected a robust proliferative response in MICA-stimulated cultures as determined by [3H]thymidine uptake. Using carboxyfluorescein diacetate succinimidyl ester (CFSE) to measure proliferation, we found that in MICA-stimulated cultures, 21% of the CD3+ T cells were CD4+ and CFSE-low and 3% of the T cells were proliferating CD8+ T cells. Among CFSE-low CD4+ spleen cells, 25% secreted IL-4 and only 2% produced IFN-gamma, suggesting a predominant Th2-type response. Blocking of MHC class I or class II molecules with monoclonal antibodies resulted in prominent inhibition of CD8+ or CD4+ T cell proliferation, respectively. In addition, we found that blocking the NKG2D receptor did not cause inhibition of the T cell response. MICA-stimulated CD8+ T lymphocytes exerted cytotoxicity against a BALB/c monocyte cell line (RAW 267.4) primed with soluble rMICA. Our results suggested that MICA-activated T cells may have a role in a cellular component of transplant rejection.

Animals↗
PubMed2006

MICA allele-level typing by sequence-based typing with computerized assignment of polymorphic sites and short tandem repeats within the transmembrane region.

MICA genes are located close to, but are structurally distinct from, HLA class I genes and have been found to be associated with some diseases and with immune responses to transplants. We have developed a MICA typing method based on polymerase chain reaction (PCR)/sequence-based typing and a computer program that determines the polymorphisms and distinguishes the GCT repeats in exon 5. One PCR amplification was performed to obtain templates of 2.2 kb including exons 2, 3, 4, and 5 of MICA to be sequenced with two forward and two reverse primers. Overlay of nucleotide sequencing signals resulting from presence of different GCT repeats in exon 5 antisense from two different MICA alleles can be identified by a computer-based analysis of the combined signal string containing the 35 bases. Eighteen reference samples from the 10th International Histocompatibility Workshop with known MICA alleles, as more recently determined, were tested and the concordance was 100% with the previous typing. In addition, 46 samples from kidney or heart transplant recipients and donors were analyzed for their MICA typing by this approach. Results demonstrated that the majority of samples were MICA heterozygous. The most common allele was MICA*00801/A5.1 (44.7%), which was consistent with previous reports. Three samples manifested ambiguous results, either because of polymorphism in exon 6 which was not tested or because the combination of two alleles gives the same pattern as the other two. The procedure was relatively simple and fast and is presently our method of choice for high-resolution clinical MICA typing.

Alleles↗
PubMed2006

Detection of anti-MICA antibodies in patients awaiting kidney transplantation, during the post-transplant course, and in eluates from rejected kidney allografts by Luminex flow cytometry.

Previously we have reported on the development of antibodies against MICA alleles in kidney transplant recipients. These alloantibodies have now been determined using a new assay using Luminex beads bound to soluble recombinant MICA antigens produced in insect cells. In the present study we have analyzed sera from 85 kidney transplant recipients on the waiting list and 66 patients transplanted within the last 4 years and 59 acid eluates obtained from allograft nephrectomy specimens. Many of the patients in those groups were sensitized and some had previous transplants (waiting list: 15%; post-tx: 7.6%; eluates 22%) and their sera were found to contain anti-human leukocyte antigen (HLA) and anti-MICA antibodies. Anti-MICA antibodies were detected in 21/85 (24.7%) of the waiting list patients and in 15/66 (22.7%) of the transplanted recipients; 11 of the eluates (18.6%) were found to have MICA-specific antibodies (6 of them also had anti-HLA antibodies and 5 did not). These data suggested that immunization against mismatched MICA alleles induces development of anti-MICA antibodies. The finding of MICA allele-specific antibodies in eluates of kidney transplants suggests that anti-MICA antibodies can be involved in the pathogenesis of kidney allograft rejection. Further studies will be required to determine whether patients who produce alloantibodies against MICA alleles are at risk for transplant rejection even when no HLA antibodies are detected.

Adult↗
PubMed2006

Contact inhibition causes strong downregulation of expression of MICA in human fibroblasts and decreased NK cell killing.

The polymorphic MICA gene encodes glycoproteins that activate T cells and NK cells through the NKG2D receptor and may costimulate immune functions. We found that MICA was expressed on freshly isolated human fibroblasts and was markedly decreased when fibroblasts were grown to confluency in culture dishes. MICA surface protein was measured by flow cytometry with the MICA-specific monoclonal antibody (mAb) 6B3, and HLA class I-specific protein was determined with mAb w6/32. In these experiments, after culture for 120 hours, the staining for MICA in fibroblasts decreased to about 20% of the initial amount and MICA mRNA fell in parallel, while HLA class I staining was maintained or even became somewhat stronger. In other experiments, MICA expression was not decreased when fibroblast contact was prevented by the addition of 1 muM Rottlerin, a specific inhibitor of protein kinase C delta known to prevent contact inhibition of fibroblasts. In the NK cell cytotoxicity assay, blocking MICA by antibody or downregulation by cell contact resulted in a decrease of specific killing by 30%. Increased MICA expression during proliferation of fibroblasts may support the host response to injury.

Acetophenones↗
PubMed2005

Effect of human cytomegalovirus on expression of MHC class I-related chains A.

The MHC-encoded MHC class I-related chains A (MICA) glycoproteins are known to enhance the functions of NK and T cells by ligating the stimulating receptor NKG2D and appear to play an important role in host defense. Human CMV (HCMV) evades the immune response in many different ways, but has not previously been found to down-regulate MICA. We have found that a common form of MICA, which has a nucleotide insertion in exon 5 corresponding to the transmembrane region and no cytoplasmic tail, was increased on the surface of fibroblasts HFS-13 compared with the mock-infected sample of the same cells that had been cultured to confluence. However, an astrocytoma cell line, U373, which has a full-length variant of MICA, showed that the expression of MICA was decreased after HCMV infection. Retroviral transduction of different MICA alleles into fibroblasts HFF-D, which express no MICA of their own, established that full-length MICA was down-regulated by HCMV, and the truncated form was not. Fibroblasts with decreased MICA due to HCMV infection were found to be protected from NK cell killing, whereas in the presence of the truncated form of MICA, the virus-infected cells were destroyed. Thus, the truncated form of MICA, which is the most common, has a mutation that allows it to persist on the surface and hinder efforts of the virus to evade the immune response.

Astrocytoma↗
PubMed2005

Genetic interaction among three genomic regions creates distinct contributions to early- and late-onset type 1 diabetes mellitus.

There are two peaks in the distribution of the age of onset of type 1 diabetes mellitus (T1DM)--the first in early childhood and the second at the time of puberty. Although T1DM results from the interaction of genetic and non-genetic factors, it has not been established which factors contribute to the bimodal distribution. The genetic component of T1DM is in large part related to genes from the human leukocyte antigen (HLA) complex (IDDM1); however, loci from the variable nucleotide tandem repeat (VNTR) region of the insulin (INS) gene (IDDM2) and more recently, the cytotoxic T-lymphocyte-associated protein-4 region (CTLA4, IDDM12) have also been implicated. Therefore, we examined the potential interaction between these loci through the influence of the age of onset of T1DM in diabetic and control Caucasian individuals. We discovered that younger individuals with HLA-DRB1*0301/DRB1*04 and INS I/I genotypes exhibited increased susceptibility to T1DM, whereas the interaction of INS I/I and CTLA4 G/G genotypes was more common in older children with T1DM. Combining the age of onset of T1DM with specific genotypes may operate to produce a single disease through different underlying causes.

Adolescent↗
PubMed2005

Psychiatric advance directives: qualitative study of informed deliberations by mental health service users.

BACKGROUND: Established legal mandates and high expectations for psychiatric advance directives are not matched by empirical evidence documenting their actual implementation. AIMS: To explore the interests, concerns and planning activities of informed mental health service users contemplating such directives. METHOD: Standard qualitative research techniques were used: field observations, interviews, focus groups, archival research and key informant interviews; 33 persons participated in the interviews and focus groups. Transcripts were coded and analysed for thematic content, and results were member-checked. RESULTS: Training set in motion labour-intensive projects: conceptualising how a psychiatric advance directive would work in one's life, mobilising resources, reviewing past experiences and assessing risks. Especially meaningful was the prospect of being treated as a responsible agent in future interactions with the mental health system. CONCLUSIONS: Advance directives are best thought of as complex planning tools for future psychiatric crisis management, rather than focal interventions to enhance compliance. Research is needed to explore the institutional response to this prospective decision-sharing initiative.

Adult↗
PubMed2004

High prevalence of autoreactive, neuroantigen-specific CD8+ T cells in multiple sclerosis revealed by novel flow cytometric assay.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) with features suggestive of T-cell-mediated pathology. Most prior reports have focused on CD4(+) T cells with the underlying assumption that MS is predominantly a CD4(+) T helper 1 (Th1)-mediated disease. In this report, we used a novel flow cytometric approach to evaluate autoreactive T-cell responses against a large variety of neuroantigenic targets. We found that both CD4(+) and CD8(+) T cells targeted against several CNS autoantigens were widely prevalent in patients with MS and healthy individuals. Whereas the distribution of CD4(+) responses was similar in different groups, patients with relapsing-remitting MS showed a higher proportion of CNS-specific CD8(+) responses. Autoreactive CD4(+) T cells from patients with MS exhibited a more differentiated Th1 phenotype compared with healthy subjects. Similarly, CNS-specific CD8(+) T-cell responses from patients with MS were functionally distinct from those in healthy individuals. Collectively, these studies reveal the high prevalence of class I-restricted autoreactive CD8(+) T-cell responses in MS that has been underappreciated thus far. The results emphasize the need to evaluate both CD4(+) and CD8(+) T-cell responses in MS and to make both subsets a consideration in the development of novel therapeutic strategies.

Adult↗
PubMed2004

[Empowerment, quality of life and participation in neurological rehabilitation. Empirical study with stroke patients and their relatives].

The active role (participation) that patients with chronic conditions are able to achieve has increasingly been recognised as a measure for the effectiveness of prevention- and rehabilitation strategies. An empowerment scale is an especially effective instrument for measuring social participation, and was applied to stroke patients in neurological rehabilitation for the first time. 26 stroke survivors and 26 informal carers, who participated in self-help groups in Lower Austria, were surveyed. The mean age was 63.9 (+/- 10.4) (stroke survivors) and 61.9 (+/- 9.6) years (informal carers). The mean duration of disease was 7.3 (+/- 3.2) years and the mean length of self-help group participation approximately 4 years. Every other stroke survivor and every fifth informal carer had to give up their professional life because of the stroke. Financial burden, reduction of vacations and social activities was found for both groups. Informal carers more frequently reported an increased fear of a relapse and generally of the future. One third of the stroke survivors had insomnia, depression, and nervousness. Reduced mobility, memory impairment, and increased sensitivity to temperature-changes were seen as the largest burdens. In spite of the large overlap in many domains of empowerment- and quality-of-life measures, empowerment measures also seem to reflect aspects of social participation. Therefore, measures of empowerment should be included in long-term outcome measurements following stroke.

Activities of Daily Living↗
PubMed2003

Multiplex single nucleotide extension: a robust and high throughput method for HLA-A locus typing.

This report describes a typing method that can identify all known human leukocyte antigen A (HLA-A) alleles by determining nucleotides present at polymorphic sites using single nucleotide extension. Allele specific primers are bound to capture oligonucleotides which allows for a multiplex approach during single nucleotide extension (SNE) reactions. Eleven group-specific polymerase chain reaction amplifications were performed to obtain the templates to be analyzed with sets of primers designed to investigate the polymorphisms. Extension of biotin-labeled ddNTPs onto allele-specific primers was catalyzed by a DNA polymerase and each primer was hybridized to a specific capture oligonucleotide covalently bound to a bead. After staining with streptavidin-PE, incorporated fluorescence was determined with a flow cytometer. Fluorescence intensities were interpreted by computer and the nucleotide sequence was translated into HLA-A genotypes. Group-specific amplification reactions and primer sets for SNE were validated with 42 reference samples of known HLA-A alleles. In addition, 296 samples from three populations (N. A. Caucasian, African-American, Terena S. A. Indian) were analyzed and results compared to previous typing by SSOP. Reproducibility between repeated typings was 100% and ambiguities were quite rare. The method has been found to be accurate, relatively simple to perform and fast. It is our method of choice for high resolution clinical HLA-A typing.

Alleles↗
PubMed2003

Study of MICA alleles in 201 African Americans by multiplexed single nucleotide extension (MSNE) typing.

We have developed a method for major histocompatibility complex class I chain-related gene A (MICA) genotyping using multiplexed single nucleotide extension (MSNE) and flow cytometric analysis of an array of fluorescent microspheres. This technique employs a polymerase chain reaction-derived target DNA containing all the polymorphic sites of MICA, synthetic complementary primers, biotinylated dideoxynucleotide triphosphate, fluorescent reporter molecules (streptavidin-phycoerythrin), and thermophilic DNA polymerase. Genomic DNA was amplified by MICA locus-specific primers and the MSNE reactions were carried out in the presence of 30 MSNE primers used to assay polymorphisms in exons 2, 3, and 4 of the MICA genes. Thirty-two previously typed cell lines were used as reference material. The MICA gene frequencies among 201 African-American unrelated donors were determined. Of 51 previously known alleles, 18 were observed in African-Americans, compared to 16 that were found in North American Caucasians and 9 in South American Indians, suggesting a more diversified allelic distribution in African-Americans. MICA*00201 and MICA*00801 were the two most frequent alleles in African-Americans. We observed a high degree of linkage disequilibrium between certain alleles of MICA and of human leukocyte antigen-B in the African-American population. The methodology described here offers a powerful new approach to DNA typing of the MICA alleles.

Black or African American↗
PubMed2002

Alternatively spliced forms of MICA and MICB lacking exon 3 in a human cell line and evidence of presence of similar RNA in human peripheral blood mononuclear cells.

MICA and MICB genes encode MHC class I chain-related proteins, which are polymorphic, do not appear to present peptides or associate with beta(2)-microglobulin, and are expressed predominantly in epithelial cells, endothelial cells, fibroblasts and several cultured cell lines. Alternatively spliced isoforms are known to exist for HLA-A and B, as well as HLA-G and the MHC class I-related gene, MR1. In the course of cloning MICA and MICB cDNA from the colon carcinoma cell line HCT 116, it was observed that two kinds of cDNAs were obtained: a 1161-bp cDNA, representing full-length MICA or MICB, and a shorter variant of 873 bp. The sequences of these short cDNAs were those of the correct MICA or MICB alleles but lacking exon 3. They were found in 7 of 72 clones examined or about 10% and were called MICA2 and MICB2. MICA1 and MICA2 were transfected into Chinese hamster ovary cells and found to be expressed both in the cells and on their surface. PCR with a primer based on a sequence formed by the joining of exons 2 and 4 allowed detection of the isoform RNA in different cells including freshly prepared normal PBMC.

Alternative Splicing↗
PubMed2002

MICA polymorphism in South American Indians.

We have studied the MICA alleles of 196 unrelated subjects from three South American Indian tribes (Toba, Wichi and Terena). They are members of isolated tribes located in the Gran Chaco area in northeastern Argentina and in Mato Grosso do Sul in South Central Brazil. Of 55 previously known alleles, nine were observed in South American Indians, compared with 16 that were found in North American Caucasians, suggesting a more restricted allelic distribution of MICA in these tribes. In South American Indians, MICA*00201 was the most frequent allele, with a gene frequency of 33% in Toba, 47% in Wichi and 44% in Terena. MICA*00201, MICA*027 (external domain sequence like MICA*008/TM allele A5) and MICA*010 accounted for more than 90% of all the MICA genes in South American Indians. In North American Caucasians, MICA*00801 (*008/A5.1) accounted for 42% of the genes and was the most common allele. We observed a high degree of linkage disequilibrium between certain alleles of MICA and of HLA-B in the South American Indian populations. Phylogenetic trees constructed using gene frequencies of the transmembrane short tandem repeats in the populations reported here, and in other populations taken from published reports, suggest that South American Indians are more closely related to Asians than to Europeans.

Adult↗
PubMed2002

MICA is a target for complement-dependent cytotoxicity with mouse monoclonal antibodies and human alloantibodies.

The highly polymorphic major histocompatibility class I related chain A (MICA) gene encodes glycoproteins that have been shown to be expressed in epithelial cells, endothelial cells, keratinocytes, monocytes, and tumor cells. In previous experiments, we have studied MICA antigens using rabbit sera obtained by immunization with MICA peptides. We also found that several transplant recipients had specific antibodies against MICA in an ELISA assay with recombinant of MICA (r-MICA). In the present work we produced monoclonal antibodies by immunization of mice with recombinant MICA*008. Based on the different patterns of reactivity observed in ELISA, Western blot, and flow cytometry, mAbs 1.9C2, 2.4F5, 1.7AD, and 2.3D4 only reacted with denatured MICA and mAb 1.7A8 and 3.2H3 reacted also with native MICA as illustrated by flow cytometry with live cells. These monoclonal antibodies were postulated to bind to different sites of the MICA molecule. In order to investigate whether MICA expressed on the cell surface is able to mediate cell killing, antibody absorption, flow cytometry and complement-dependent cytotoxicity (CDC) were performed. We found that mouse monoclonal antibody 3.2H3 was able to kill 70% of HeLa cells. Absorption of a patient serum with pooled human platelets to remove antibodies against class I HLA resulted in a small shift of fluorescence and reduced killing from 100% to 70-75%. Absorption with the platelets and r-MICA produced a remarkable reduction in fluorescence staining and virtually reduced complement-dependent killing to the level of the negative controls. The results suggested that MICA alloantigens may be more immunogenic than could have been previously suspected.

Animals↗
PubMed2002

Glatiramer acetate (Copaxone) therapy induces CD8(+) T cell responses in patients with multiple sclerosis.

Glatiramer acetate (GA; Copaxone) is a random copolymer of glutamic acid, lysine, alanine, and tyrosine that is used therapeutically in patients with multiple sclerosis (MS). To investigate the mechanism of the drug's immunomodulatory effect, we used immunophenotypic approaches to characterize the precise nature of GA-induced T cell responses. We demonstrate here that healthy individuals and untreated MS patients exhibit prominent T cell proliferative responses to GA. However, these responses are different in distinct subsets of T cells. Whereas GA-induced CD4(+) T cell responses are comparable in healthy individuals and MS patients, CD8(+) T cell responses are significantly lower in untreated MS patients. Treatment with GA results in upregulation of these CD8(+) responses with restoration to levels observed in healthy individuals. Both CD4(+) and CD8(+) GA-specific responses are HLA-restricted. GA therapy also induces a change in the cytokine profile of GA-specific CD4(+) and CD8(+) T cells. This study provides the first direct immunophenotypic evidence, to our knowledge, of GA-specific CD8(+) T cell responses and their upregulation during the course of therapy, which may suggest a role for these responses in the immunomodulatory effects of the drug.

Adjuvants, Immunologic↗