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Biomedical subjects

David Stroncek

Publications and source records attributed to David Stroncek.

At least 19 recordsLinked to original sources
PubMed2006

Molecular signatures induced by interleukin-2 on peripheral blood mononuclear cells and T cell subsets.

Experimentally, interleukin-2 (IL-2) exerts complex immunological functions promoting the proliferation, survival and activation of T cells on one hand and inducing immune regulatory mechanisms on the other. This complexity results from a cross talk among immune cells which sways the effects of IL-2 according to the experimental or clinical condition tested. Recombinant IL-2 (rIL-2) stimulation of peripheral blood mononuclear cells (PBMC) from 47 donors of different genetic background induced generalized T cell activation and anti-apoptotic effects. Most effects were dependent upon interactions among immune cells. Specialized functions of CD4 and CD8 T cells were less dependent upon and often dampened by the presence of other PBMC populations. In particular, cytotoxic T cell effector function was variably affected with a component strictly dependent upon the direct stimulation of CD8 T cells in the absence of other PBMC. This observation may provide a roadmap for the interpretation of the discrepant biological activities of rIL-2 observed in distinct pathological conditions or treatment modalities.

Journal Article↗
PubMed2006

Inflammatory protein profile during systemic high dose interleukin-2 administration.

Systemic interleukin-2 (IL-2) administration induces an assortment of downstream effects whose biological and therapeutic significance remains unexplored mostly because of the methodological inability to globally address their complexity. Protein array analysis of sera from patients with renal cell carcinoma obtained prior and during high-dose IL-2 therapy had previously revealed extensive alterations in expression of the soluble factors analyzed, whose discovery was limited by the number of capture antibodies selected for protein detection. Here, we expanded the analysis to SELDI-TOF-MS and quantitative protein analysis (nephelometry). All cytokines/chemokines detected by protein arrays were below the SELDI detection limit, while novel IL-2-specific changes in expression of acute-phase reactants and high-density lipoprotein metabolites could be identified. Serum amyloid protein A (SAA) and C-reactive protein expression were consistently up-regulated after four doses of IL-2, while other proteins were down-regulated. These findings were confirmed by SELDI immunoaffinity capture and nephelometry. Immunoaffinity capture revealed different, otherwise undetectable, isoforms of SAA. A linear correlation between peak area by SELDI and protein concentration by nephelometry was observed. Overall distinct yet complementary information was obtained using different platforms, which may better illustrate complex phenomena such as the systemic response to biological response modifiers.

Biomarkers↗
PubMed2006

Identification of HLA-A33-restricted CMV pp65 epitopes as common targets for CD8(+) CMV-specific cytotoxic T lymphocytes.

OBJECTIVE: To identify an immunodominant HLA-A33-restricted epitope within the CMV matrix phosphoprotein 65 (pp65) that could be used to elicit CMV-specific CTLs. METHODS: A computer algorithm was used to identify pp65 peptides that were expected to bind to HLA-A33. The peptides were screened for their ability to reactivate memory T lymphocytes from CMV-seropositive HLA-A33 donors by using quantitative real-time RT-PCR. The most promising peptides were then tested for their ability to expand a CD8(+) population of HLA-A33 CTLs that produced interferon-gamma (IFN-gamma) and were cytotoxic to either peptide-loaded or CMV-infected target cells. RESULTS: Sixteen out of 250 peptides were selected using a computer algorithm and peptide stimulation by 8 out of the 16 induced a significant quantity of IFN-gamma mRNA transcript from CMV-seropositive HLA-A33 peripheral blood mononuclear cells. All the eight peptides induced consistent T-cell reactivation. One specifically, the peptide pp65(91-100) (SVNVHNPTGR), proved to be more active. T cells in vitro sensitized for 2 or 3 weeks with pp65(91-100) were cytotoxic to both HLA-A33 peptide-loaded and CMV-infected target cells. CONCLUSIONS: CMV pp65(91-100) is a new HLA-A33-restricted peptide that broadens the list of antigenic determinants that can be used for CMV adoptive immunotherapy.

Amino Acid Sequence↗
PubMed2006

The gene overexpressed in polycythemia rubra vera, PRV-1, and the gene encoding a neutrophil alloantigen, NB1, are alleles of a single gene, CD177, in chromosome band 19q13.31.

BACKGROUND: PRV-1 mRNA is overexpressed by neutrophils from polycythemia vera patients and is homologous to NB1 a gene overexpressed in reactive neutrophilia. STUDY DESIGN AND METHODS: These investigations were designed to confirm searches of genome databases suggesting that PRV-1 and NB1 are alleles of the same gene, CD177, and confirm a pseudogene adjacent to CD177. Methods included polymerase chain reaction (PCR), cloning, sequencing, and fluorescent hybridization studies. RESULTS: The coding region of PRV-1 was PCR-amplified from human fetal RNA, cloned, and used to screen the RPCI-11 bacterial artificial chromosome (BAC) library. Five BACs were reactive with the PRV-1 probe. PCR analysis of the BACs with primers encompassing PRV-1 exons, containing four known single-nucleotide polymorphisms, followed by sequencing rendered amplicons identical to PRV-1 in all five BACs. Analysis of all five by restriction digestion yielded fragments possible only if both the gene and the pseudogene are present. End sequencing of the BACs localized them to the same chromosome region. G-banding and fluorescence in situ hybridization at the 400- and 850-band levels of resolution mapped one BAC to chromosome band 19q13.2 and sublocalized the BAC to band 19q13.31, respectively. CONCLUSION: PRV-1 and NB1 are alleles of the same gene now referred to as CD177. Changes in CD177 expression may be a marker of increased or decreased myelopoiesis and are therefore an effect of, rather than a cause of, myeloproliferative disorders.

Alleles↗
PubMed2005

HLAMatchmaker-driven analysis of responses to HLA-typed platelet transfusions in alloimmunized thrombocytopenic patients.

This study describes a novel application of HLAMatchmaker to determine platelet compatibility in 16 alloimmunized patients with aplastic anemia refractory to random donor platelet transfusions. HLAMatchmaker is a software algorithm that predicts HLA compatibility by identifying immunogenic epitopes represented by amino acid triplets in antibody-accessible regions of human leukocyte antigen (HLA) molecules and determines the number of triplet mismatches (TMMs) and highly immunogenic triplet mismatches (HIMMs). Corrected count increments (CCIs) and molecular HLA typing were available for 523 transfusions. Conventional compatibility assessment based on cross-reactive group (CREG) determination was not predictive of transfusion outcome. Low HIMMs and TMMs numbers were associated with a higher likelihood of satisfactory (CCIs > or = 8) compared with unsatisfactory (CCIs < 8) outcomes (median HIMMs = 4 vs 6, p2 value < .001; median TMMs = 11 vs 13, p2 value < .001). Although receiver operator characteristic curves revealed that HIMMs or TMMs number are not powerful predictors of individual transfusion outcome, a threshold of at least 3 HIMMs or at least 9 TMMs appeared to be associated with successful transfusions. Triplet-matched transfusions were successful, regardless of CREG matching. Our data validate HLAMatchmaker for platelet transfusions and demonstrate its potential to refine and expand donor selection for HLA-alloimmunized patients.

Cross Reactions↗
PubMed2005

HLA class I and II genotype of the NCI-60 cell lines.

Sixty cancer cell lines have been extensively characterized and used by the National Cancer Institute's Developmental Therapeutics Program (NCI-60) since the early 90's as screening tools for anti-cancer drug development. An extensive database has been accumulated that could be used to select individual cells lines for specific experimental designs based on their global genetic and biological profile. However, information on the human leukocyte antigen (HLA) genotype of these cell lines is scant and mostly antiquated since it was derived from serological typing. We, therefore, re-typed the NCI-60 panel of cell lines by high-resolution sequence-based typing. This information may be used to: 1) identify and verify the identity of the same cell lines at various institutions; 2) check for possible contaminant cell lines in culture; 3) adopt individual cell lines for experiments in which knowledge of HLA molecule expression is relevant. Since genome-based typing does not guarantee actual surface protein expression, further characterization of relevant cell lines should be entertained to verify surface expression in experiments requiring correct antigen presentation.

Journal Article↗
PubMed2005

Proteomic signature of myeloproliferation and neutrophilia: analysis of serum and plasma from healthy subjects given granulocyte colony-stimulating factor.

OBJECTIVE: Proteomic analysis could improve our understanding of the mechanisms and consequences of myeloproliferation. Healthy subjects treated with granulocyte colony-stimulating factor (G-CSF) were used as a model of myeloproliferation. METHODS: Levels of 80 soluble factors were measured by enzyme-linked immunosorbent assay before and after 5 days of G-CSF. Both serum and plasma levels were measured to generate a comprehensive profile and determine whether serum or plasma best portrays biological and physiological changes. RESULTS: Comparison of samples collected prior to G-CSF demonstrated that 44 factors differed between serum and plasma. Concentrations of several growth factors and chemokines were greater in serum than in plasma, while the opposite was true for several interleukins. Following G-CSF serum levels of 14 factors and plasma levels of 15 factors changed. Eleven increased in both serum and plasma, including cell adhesion molecules (vascular cell adhesion molecule-1, E-selectin, and L-selectin), matrix metalloproteases (MMP-1, -8, and -13), cytokine receptors (tumor necrosis factor receptor 1 and 2, and interleukin-2 receptor), the acute phase reactant, serum amyloid A, and a growth factor, hepatocyte growth factor. CONCLUSION: Some protein levels differ markedly in serum and plasma. Myeloproliferation is associated with changes in the levels of several proteases, adhesion molecules, and cytokines.

Blood Proteins↗
PubMed2005

Transfusion-related acute lung injury: definition and review.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is now the leading cause of transfusion-associated mortality, even though it is probably still underdiagnosed and underreported. NATIONAL HEART, LUNG, AND BLOOD INSTITUTE ACTION: The National Heart, Lung, and Blood Institute convened a working group to identify areas of research needed in TRALI. The working group identified the immediate need for a common definition and thus developed the clinical definition in this report. MAJOR CONCEPTS IN THE DEFINITION: The major concept is that TRALI is defined as new acute lung injury occurring during or within 6 hrs after a transfusion, with a clear temporal relationship to the transfusion. Also, another important concept is that acute lung injury temporally associated with multiple transfusions can be TRALI, because each unit of blood or blood component can carry one or more of the possible causative agents: antileukocyte antibody, biologically active substances, and other yet unidentified agents. RECOMMENDATION: Using the definition in this report, clinicians can diagnose and report TRALI cases to the blood bank; importantly, researchers can use this definition to determine incidence, pathophysiology, and strategies to prevent this leading cause of transfusion-associated mortality.

Blood Transfusion↗
PubMed2004

Cell processing: current status and future directions.

Specialized clinical cell processing began in the Department of Transfusion Medicine at the National Institutes of Health in 1984. The number and complexity of procedures performed increased quickly and in 1997 a highly specialized cell processing laboratory was opened. The laboratory has approximately 3,000 square feet, specialized air handing, a highly trained staff, and written laboratory procedures. In addition to standard laboratory equipment, the laboratory has numerous cell isolation instruments, flow cytometers, and automated cell counting instruments. The laboratory supports blood and bone marrow transplant protocols by isolating CD34+ stem cells, removing T lymphocytes, culturing lymphocytes to eliminate donor lymphocytes that are reactive with recipient alloantigens, and stimulating lymphocytes to induce Th2 type cells to reduce graft versus host disease. The laboratory has also been preparing dendritic cells to support protocols using immune therapy to treat cancer. In addition, pancreatic islet cells are isolated from organ donors for transplantation to treat type I diabetes mellitus.

Antigens, CD34↗
PubMed2004

Adoptive immunotherapy for cytomegalovirus (CMV) disease in immunocompromised patients.

Cytomegalovirus (CMV) reactivation in immune compromised patients such as those undergoing hematopoietic progenitor cell transplantation (HPCT) and those with HIV infections can cause severe morbidity and mortality despite treatment with appropriate antiviral agents. The recovery of Cytomegalovirus (CMV) specific cytotoxic T lymphocytes (CTL) plays an important role in the reconstitution of CMV specific immunity in immunocompromised patients. Recent studies have reported that CMV reactivation can be successfully treated by adoptive transfer of CMV-specific T cell clones from CMV seropositive donors expanded in vitro with CMV infected fibroblasts or lysates of CMV infected cells. Other studies have used immune dominant CMV proteins or peptides to expand CMV-specific cytotoxic T lymphocytes. This review describes the clinical manifestations of CMV disease in immunocompromised patients, recent advances of antiviral therapy for CMV disease, the principals of the induction of cellular immune response to CMV, and the clinical application of CMV immunotherapy.

Cytomegalovirus Infections↗
PubMed2004

CD177 polymorphisms: correlation between high-frequency single nucleotide polymorphisms and neutrophil surface protein expression.

BACKGROUND: Human neutrophil antigen-2a (NB1) is located on NB1 glycoprotein, which is expressed on subpopulations of neutrophils. PRV-1 is a gene that is over-expressed in neutrophils from patients with polycythemia rubra vera. The gene encoding NB1 differs from PRV-1 at four nucleotides. The purpose of this study was to determine if PRV-1 and NB1 are alleles of the same gene or two separate genes; and, moreover, if they are alleles, to explore potential correlations to NB1 glycoprotein expression. STUDY DESIGN AND METHODS: Primer pairs were used to amplify WBC DNA in the regions surrounding the four NB1 polymorphic sites. The four resulting amplicons were sequenced. The size of the neutrophil population in each donor that stained brightly with CD177 antibody was assessed by flow cytometry. RESULTS: If PRV-1 and NB1 are separate genes, then all people should be heterozygous for the PRV-1/NB1 polymorphisms. Because 11 of 23 donors were homozygous for PRV-1 polymorphisms at all four sites, PRV-1 and NB1 are alleles of the same gene, CD177. When the sequenced exons were compared with PRV-1, 13 single nucleotide polymorphisms (SNPs) that result in amino acid changes were found. The G42C NB1 polymorphism, found in 10 donors, was the most common SNP. The size of the CD177 bright-staining neutrophil population was greater in 42C/C donors than in 42G/G donors (66 +/- 20% vs. 41 +/- 21%, p = 0.004). CONCLUSIONS: PRV-1 and NB1 are alleles of the polymorphic gene CD177. The most common SNP in bp 42 predicted an amino acid change that may have an effect on protein expression.

Black or African American↗
PubMed2003

Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR).

The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158-66 and CMVpp65495-503 peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-gamma (IFN-gamma), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-gamma mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization.

Journal Article↗
PubMed2003

Expression of human neutrophil antigen-2a (NB1) is increased in pregnancy.

BACKGROUND: The expression of human neutrophil antigen-2a (HNA-2a) or NB1 is highly variable. This study investigated variations in neutrophil expression of HNA-2a by comparing the expression of epitopes recognized by three HNA-2a-specific CD177 antibodies among healthy adults and pregnant women. STUDY DESIGN AND METHODS: Flow cytometry was used to compare the neutrophil reactions of three CD177 antibodies (MEM-166, TAG4, and 7D8) among 52 healthy adults. Because HNA-2a expression is greater in women than men, neutrophils were studied from 21 women early in pregnancy and 23 women late in pregnancy. RESULTS: All three CD177 antibodies reacted with two populations of neutrophils: a brightly staining population and a dimly staining population. Among the 52 healthy adults, there was no difference in the mean size of the brightly reactive population of neutrophils recognized by each antibody (46 +/- 23% for MEM-166, 49 +/- 23% for TAG4, and 49 +/- 23% for 7D8), and all three antibodies were specific for HNA-2a. For all three antibodies the size of the antigen bright neutrophil population was greater on neutrophils collected both early and late in pregnancy than in healthy adults. There was no difference in the size of the antigen bright population among neutrophils tested early and late in pregnancy. CONCLUSION: HNA-2a antigen expression increases in pregnancy. Some of the variations in neutrophil expression of HNA-2a among individuals is likely due to differences in gene regulation or differences in post-translational protein modifications rather than gene polymorphisms.

Adult↗
PubMed2003

G-CSF-induced spleen size changes in peripheral blood progenitor cell donors.

BACKGROUND: PBPC donors given G-CSF experience splenic enlargement and, rarely, spontaneous rupture of the spleen. This study evaluated the incidence and time course of splenic enlargement in PBPC concentrate donors and assessed factors affecting size changes. STUDY DESIGN AND METHODS: Twenty healthy adults were given G-CSF (10 microg/kg/day) for 5 days and a PBPC concentrate was collected by apheresis. Ultrasound was used to assess craniocaudal spleen length before giving G-CSF, on the day of apheresis and 3 or 4 days after apheresis. The effects of donor age, gender, race, and changes in blood chemistries, blood counts, and CD34+ cell counts on spleen length change were assessed. RESULTS: Spleen length increased in 19 of 20 donors. Mean length changed from 10.9 +/- 2.0 cm before G-CSF to 12.3 +/- 2.1 cm on the day of apheresis (p < 0.001). The mean increase in length was 1.5 +/- 0.9 cm or 13.8 +/- 9.1 percent. Spleen length increased 20 percent or more in six subjects. The spleen length fell to 11.3 +/- 1.8 cm (p < 0.001) 3 or 4 days after apheresis, but it remained greater than baseline levels (p = 0.03). Spleen length change was not affected by donor gender, race, or age. There was no relationship between changes in spleen length and 1) baseline and apheresis-day blood counts and chemistries, or 2) changes in blood counts and chemistries. CONCLUSIONS: Spleen size increases in almost all PBPC donors. Enlargement is transient but may be marked in some donors and may place them at risk for splenic rupture.

Adult↗
PubMed2002

The use of bioinformatics to identify the genomic structure of the gene that encodes neutrophil antigen NB1, CD177.

The gene encoding neutrophil alloantigen NB1, CD177, is highly homologous to a gene overexpressed in polycythemia vera neutrophils, polycythemia rubra vera-1 (PRV-1). The cDNAs of both genes have been cloned, but their genomic structure is unknown. The purpose of this study was to determine the intron-exon organization of NB1 and PRV-1 and discern if they are separate members of a homologous gene family or alleles of the same gene. GenBank's human genome sequences were probed in silico with PRV-1's 1605-nucleotide coding sequence. Searches identified two adjacent bacterial artificial chromosomes (BACs) on chromosome 19q13.2: BC338531 and BC52850. BC338531 contained sequences 100% homologous to the first 654 nucleotides of PRV-1. Comparison of coding and genomic sequences allowed us to separate this region into exons 1 through 5, interrupted by five introns. BC52850 contained sequences 95% homologous to nucleotides 413 through 1605 of PRV-1, organized into exons 4 through 9. However, the orientation of PRV-1-homologous in the first BAC was plus-plus and of the second was plus-minus, indicating they could not be portions of the same gene. The GenBank sequence of BC338531 was incomplete, creating a sequence gap in chromosome 19q13.2. Evaluation of BC338531's unfinished sequences in Joint Genome Institute public databases allowed us to complete the gap and revealed that BC338531 contained sequences 98% homologous to all nine PRV-1 exons followed by a second gene consisting of exons 9 through 4. Most likely, NB1 and PRV-1 are alleles of the same gene, CD177, and the duplication of exons 4 through 9 is a pseudo gene.

Amino Acid Sequence↗
PubMed2002

Neutrophil alloantigens.

Antibodies to neutrophil antigens can cause neonatal alloimmune neutropenia, autoimmune neutropenia, febrile transfusion reactions, and transfusion-related acute lung injury. Several neutrophil antigen systems have been described serologically, but only the human neutrophil antigen-1 (HNA-1) or NA and HNA-2 or NB systems have been well characterized biochemically and molecularly. HNA-1 antigens are located on FcgammaRIIIb, CD16. HNA-2 antigens are located on 58- to 64-Kd glycoprotein, CD177, and are encoded by a gene on chromosome 19 that belongs to the Ly-6 family. The function of the CD177 is not known, but the CD177 gene is highly homologous to a gene overexpressed in neutrophils from patients with polycythemia rubra vera called PRV-1. New polymorphisms in these antigen systems are still being described, but the complete understanding of these neutrophil antigen systems has been slow because of the complexity of these genes.

Amino Acid Substitution↗