Microsatellite typing for DRB1 alleles: application to the analysis of HLA associations with rheumatoid arthritis.

The current methods for molecular typing of HLA-DR alleles incur a substantial financial burden when performing large population studies. In the current study, we aimed to provide much less expensive typing approach with high predictability for DRB1 genotype. We have used a panel of three microsatellite markers in the class II region (D6S2666, D6S2665 and D6S2446) for genotyping and haplotype reconstruction in a total of 1687 Caucasian (1313 RA patients and 374 controls) and 1364 Korean individuals (744 RA patients and 620 controls), all of whom were previously genotyped for DRB1. We found that a total of 88.4 and 87.4% of all observed three-marker haplotypes could determine the DR type with a positive predictive value >0.8 with high sensitivity and specificity. There was a high degree of haplotype conservation when comparing Caucasian and Asian populations. Interestingly, we found that the majority of DRB1*09 and DRB1*10 alleles share a common three-marker haplotype in both Caucasian and Asian populations. This is unexpected, since these two alleles are found on very different haplotype families. In addition, these two alleles are both associated with rheumatoid arthritis, making the elucidation of these haplotype relationships potentially important for understanding disease susceptibility.

High-density SNP analysis of 642 Caucasian families with rheumatoid arthritis identifies two new linkage regions on 11p12 and 2q33.

We have completed a genome wide linkage scan using >5700 informative single-nucleotide polymorphism (SNP) markers (Illumina IV SNP linkage panel) in 642 Caucasian families containing affected sibling pairs with rheumatoid arthritis (RA), ascertained by the North American Rheumatoid Arthritis Consortium. The results show striking new evidence of linkage at chromosomes 2q33 and 11p12 with logarithm of odds (LOD) scores of 3.52 and 3.09, respectively. In addition to a strong and broad linkage interval surrounding the major histocompatibility complex (LOD>16), regions with LOD>2.5 were observed on chromosomes 5 and 10. Additional linkage evidence (LOD scores between 1.46 and 2.35) was also observed on chromosomes 4, 7, 12, 16 and 18. This new evidence for multiple regions of genetic linkage is partly explained by the significantly increased information content of the Illumina IV SNP linkage panel (75.6%) compared with a standard microsatellite linkage panel utilized previously (mean 52.6%). Stratified analyses according to whether or not the sibling pair members showed elevated anticyclic citrullinated peptide titers indicates significant variation in evidence for linkage among strata on chromosomes 4, 5, 6 and 7. Overall, these new linkage data should reinvigorate efforts to utilize positional information to identify susceptibility genes for RA.

Peripheral blood gene expression profiling in rheumatoid arthritis.

We carried out gene expression profiling of peripheral blood mononuclear cells (PBMCs) in 29 patients with active rheumatoid arthritis (RA) and 21 control subjects using Affymetrix U95Av2 arrays. Using cluster analysis, we observed a significant alteration in the expression pattern of 81 genes (P<0.001) in the PBMCs of RA patients compared with controls. Many of these genes correlated with differences in monocyte counts between the two study populations, and we show that a large fraction of these genes are specifically expressed at high levels in monocytes. In addition, a logistic regression analysis was performed to identify genes that performed best in the categorization of RA and control samples. Glutaminyl cyclase, IL1RA, S100A12 (also known as calgranulin or EN-RAGE) and Grb2-associated binding protein (GAB2) were among the top discriminators. Along with previous data, the overexpression of S100A12 in RA patients emphasizes the likely importance of RAGE pathways in disease pathogenesis. The altered expression of GAB2, an intracellular adaptor molecule involved in regulating phosphatase function, is of particular interest given the recent identification of the intracellular phosphatase PTPN22 as a risk gene for RA. These data suggest that a detailed study of gene expression patterns in peripheral blood can provide insight into disease pathogenesis. However, it is also clear that substantially larger sample sizes will be required in order to evaluate fully gene expression profiling as a means of identifying disease subsets, or defining biomarkers of outcome and response to therapy in RA.

Expression levels for many genes in human peripheral blood cells are highly sensitive to ex vivo incubation.

Monitoring of gene and protein expression in peripheral blood cells has significant potential for improving the diagnosis and therapy of many human diseases. As genomic-scale microarray and proteomic technologies are applied to peripheral blood, it is important to consider the variables that may affect interpretation of data. Here we report experiments performed to identify genes that are particularly sensitive to ex vivo handling prior to RNA extraction for gene expression microarrays or quantitative real-time RT-PCR assays. We examined Affymetrix gene expression in samples from eight normal individuals where blood was processed for RNA either immediately after blood draw or the next day following overnight incubation. These studies identified hundreds of genes that are sensitive to ex vivo handling of blood, and suggest that this is an important variable to consider when designing and interpreting human PBMC experiments.

Simultaneous flow cytometric analysis of cell surface markers and telomere length: analysis of human tonsilar B cells.

Telomere Flow FISH is a recently developed method which allows the measurement of telomere length in purified subsets of cells using flow cytometry. However, the harsh conditions required for flow FISH have precluded its use with conventional cell surface staining, thus limiting its utility for large scale clinical studies. We have now developed a method which permits simultaneous analysis of cell surface markers along with telomere length estimation by flow cytometry. This new assay employs the covalent crosslinking of monoclonal antibodies conjugated with a heat stable fluorochrome to the cell surface prior to flow FISH. Using this technique we have confirmed that human germinal center B cells (IgD(-)/CD38(+)) have dramatically longer telomeres than pre-germinal center founder B cells (IgD(+)/CD38(+)). This approach simplifies the analysis of complex cell populations and will facilitate widespread investigation of telomere length in health and disease states.

Oligoclonal expansions in the CD8(+)CD28(-) T cells largely explain the shorter telomeres detected in this subset: analysis by flow FISH.

We have previously reported that CD8(+)CD28(-) T cells have relatively shorter telomeres compared with CD8(+)CD28(+) T cells. Oligoclonal expansion is a common feature of CD8(+) T cells in human peripheral blood, and these expansions predominantly occur in the CD57(+)/CD28(-) population. We studied the telomere length in subsets of CD8(+) T cells using quantitative fluorescence in situ hybridization and flow cytometry (flow FISH). Our results confirm that CD8(+)CD28(-) T cells have shorter telomeres as compared with their CD28(+) counterpart cells. In addition, the oligoclonally expanded cells within the CD8(+)CD28(-) T cell subset generally have even shorter telomeres than the CD28(-) subset as a whole. We conclude that the presence of clonal expansions in the CD8(+)CD28(-) T cell population largely explain the shorter telomeres in this subset. These clonally expanded CD8(+)CD28(-) T cells generally have characteristics of terminally differentiated effector cells. Nevertheless, there is considerable individual variation in the degree of telomere shortening in these cells, which may reflect host genetic factors as well as the type and timing of the antigenic exposure.

Apoptosis of CD8+ T cells is mediated by macrophages through interaction of HIV gp120 with chemokine receptor CXCR4.

CD8-positive T cells are thought to play an important role in the control of infection by human immunodeficiency virus (HIV) as a result of their cytotoxic activity and by releasing soluble factors. In AIDS patients, the absolute number of CD8+ T lymphocytes is decreased in peripheral blood and their turnover rate is increased, suggesting that there is more cell renewal and cell death occurring. Anti-retroviral therapy raises CD8+ T-cell counts in HIV-infected patients. Here we report that the death rate of CD8+ T cells by apoptosis increased markedly during HIV infection of peripheral blood mononuclear cells in vitro. Apoptosis is induced in a dose-dependent manner by recombinant envelope glycoprotein gp120 from HIV strain X4, or by stromal-derived factor-1 (SDF-1), the physiological ligand of the chemokine receptor CXCR4. Apoptosis is mediated by the interaction between tumour-necrosis factor-alpha bound to the membrane of macrophages (mbTNF) and a receptor on CD8+ T cells (TNF-receptor II, or TNFRII). The expression of both of these cell-surface proteins is upregulated by HIV infection or by treatment with recombinant gp120 or SDF-1. Apoptosis of CD8+ T cells isolated from HIV-infected patients is also mediated by macrophages through the interaction between mbTNF and TNFRII. These results indicate that the increased turnover of CD8+ T cells in HIV-infected subjects is mediated by the HIV envelope protein through the CXCR4 chemokine receptor.

Cerebral ischemia enhances polyamine oxidation: identification of enzymatically formed 3-aminopropanal as an endogenous mediator of neuronal and glial cell death.

To elucidate endogenous mechanisms underlying cerebral damage during ischemia, brain polyamine oxidase activity was measured in rats subjected to permanent occlusion of the middle cerebral artery. Brain polyamine oxidase activity was increased significantly within 2 h after the onset of ischemia in brain homogenates (15.8 +/- 0.9 nmol/h/mg protein) as compared with homogenates prepared from the normally perfused contralateral side (7.4 +/- 0.5 nmol/h/mg protein) (P <0.05). The major catabolic products of polyamine oxidase are putrescine and 3-aminopropanal. Although 3-aminopropanal is a potent cytotoxin, essential information was previously lacking on whether 3-aminopropanal is produced during cerebral ischemia. We now report that 3-aminopropanal accumulates in the ischemic brain within 2 h after permanent forebrain ischemia in rats. Cytotoxic levels of 3-aminopropanal are achieved before the onset of significant cerebral cell damage, and increase in a time-dependent manner with spreading neuronal and glial cell death. Glial cell cultures exposed to 3-aminopropanal undergo apoptosis (LD50 = 160 microM), whereas neurons are killed by necrotic mechanisms (LD50 = 90 microM). The tetrapeptide caspase 1 inhibitor (Ac-YVAD-CMK) prevents 3-aminopropanal-mediated apoptosis in glial cells. Finally, treatment of rats with two structurally distinct inhibitors of polyamine oxidase (aminoguanidine and chloroquine) attenuates brain polyamine oxidase activity, prevents the production of 3-aminopropanal, and significantly protects against the development of ischemic brain damage in vivo. Considered together, these results indicate that polyamine oxidase-derived 3-aminopropanal is a mediator of the brain damaging sequelae of cerebral ischemia, which can be therapeutically modulated.

A 15-year follow-up of AJCC stage III malignant melanoma patients treated postsurgically with Newcastle disease virus (NDV) oncolysate and determination of alterations in the CD8 T cell repertoire.

BACKGROUND: The development of effective adjuvant therapies for the treatment of high-risk melanoma patients is critical for the prevention of metastatic disease and improvement of patient survival. Active specific immunotherapy has been tested as an adjuvant treatment in numerous clinical trials with overall limited, but occasionally promising, success rates. Newcastle disease virus (NDV) oncolysate has been utilized as an adjunctive immunotherapeutic agent in the postsurgical management of these patients. A phase II study initiated in 1975 using adjuvant vaccine therapy composed of allogeneic and autologous human melanoma cells infected with live NDV (NDV oncolysate) in patients with AJCC stage III melanoma following therapeutic lymph node dissection has shown >60% survival rate at 10 years with no adverse effects. Continued long-term analysis of trials with promising early results as well as assessment of immunologic responses generated in these patients may result in improved therapeutic decisions for clinical trials in the future. MATERIALS AND METHODS: We analyzed the 15-year survival of patients treated postsurgically with NDV oncolysate in the phase II study described above. In an attempt to understand the immunological effects of this treatment, we have also carried out a comprehensive analysis of the peripheral blood T cell repertoire in these patients. RESULTS: The overall 15-year survival of this group of patients is 55%. Previous studies have suggested that improved outcome in patients undergoing immunotherapy is correlated with increased numbers of CD8(+)CD57(+) cells. In surviving patients, we observed a striking oligoclonality in the CD8(+) T cell population in peripheral blood, which reflects clonal expansions in the CD8(+)CD57(+) subset. CONCLUSIONS: The data suggest that adjuvant vaccination with NDV oncolysates is associated with prolonged survival of patients with lymph node-positive malignant melanoma and that CD8(+) T cells may be an important component of therapeutic efficacy.

Oligoclonality of CD8+ T cells in health and disease: aging, infection, or immune regulation?

Oligoclonality of the CD8+ T cell subset is a common and characteristic feature of the normal human peripheral T cell repertoire. These clonally expanded populations are predominantly found in a CD57+ or CD28- CD8+ T cell subset. While CD8 oligoclonality is somewhat more common in the older age group, it is also very prevalent in young to middle-aged adults. Recent experiments have also demonstrated that the clonally expanded populations may actually occur in two distinct subpopulations of CD8+ CD28- cells, distinguished by the expression of the CD57 surface marker. A major difficulty with studies involving CD8+ CD28- CD57+ T cells is their relative lack of proliferative capacity. We have recently investigated the possibility that this phenotype may be due to a state of "replicative senescence" in some cases. In this regard, we have demonstrated that the telomere lengths of CD8+ CD28- T cells are generally shorter than that of their CD8+ CD28+ counterparts, consistent with a distinct replicative history for the CD8+ CD28- population. Additional studies of the normal biology of clonally expanded CD8+ T cells are likely to yield important insights into immune function in health and disease.

Shortened telomeres in clonally expanded CD28-CD8+ T cells imply a replicative history that is distinct from their CD28+CD8+ counterparts.

Long term in vitro culture of clonally expanded CD8+T cells, generally found within the CD57+ or CD28-subset, has generally been unsuccessful, suggesting that these cells may have a limited replicative potential. Telomeric shortening may reflect the action of a "mitotic clock" regulating the number of divisions a cell can undergo. In this study, we have compared the telomeric lengths of CD28-CD8+ and CD28+CD8+ T cells in 10 normal individuals to assess their replicative history. Overall, the telomeric lengths were found to be significantly shorter in the CD28-CD8+ T cell subset compared with the CD28+CD8+ subset. Furthermore, clonally expanded TCRBV11+CD8+ T cells from an individual exhibited telomeric lengths that were 2.9 kb shorter than those found in the polyclonal CD28+CD8+ T cell subset. These findings indicate that clonally expanded CD28-CD8+ T cells have undergone many more rounds of replication than CD28+CD8+ T cells, and consistent with the loss of CD28 expression, they may have reached a state of replicative senescence.

Oligoclonal CD8+ T cells are preferentially expanded in the CD57+ subset.

A number of recent reports have established that oligoclonality and/or clonal expansion is a common feature of the CD8+ T cell population. Oligoclonal expansion has also been observed in bone marrow transplant recipients and rheumatoid arthritis patients, disease states in which CD57+CD8+ T cells are occasionally elevated. In this study we have compared the TCR repertoire of the CD57+ and CD57- subsets of CD8+ T cells in normal persons by using three-color FACS analysis with a panel of 16 mAbs specific for TCR V segments. The CD57 surface marker was highly variable in frequency but generally present on a minority of CD8+CD3+ T cells (mean 16.3%, SD 12.7) in a group of 41 normal volunteers. Dramatic oligoclonal expansion was present in the CD57+CD8+ T cell population in 15 of 41 (37%) of our study population and thus is a characteristic feature of the normal immune system. No such prominent oligoclonal expansions were observed in the CD57-CD8+ subset, although preliminary experiments suggest that oligoclonality per se is occasionally present at a lower frequency in CD57- cells. The reasons for this persistent accumulation of oligoclonal CD8+CD57+ T cells and their function in immune homeostasis are unclear.

Differential phosphorylation in normal and leukemic granulocytes in response to phorbol 12-myristate 13-acetate.

Granulocytes from the peripheral blood of patients with chronic myeloid leukemia (CML) exhibit a number of functional defects. To explore the relationship of these aberrations to signal transduction, granulocytes from normal subjects and CML patients were labelled with 32Pi, stimulated with phorbol myristate acetate (PMA) and the phosphoproteins (Pps) in the unstimulated and stimulated cells analyzed by 2D-SDS-PAGE followed by autoradiography. Results show that there are six distinct reproducibly phosphorylated proteins referred to as Pp1-Pp6 identifiable in the basal patterns of the resting granulocytes. Amongst these, Pp1 and Pp5 are more intensely phosphorylated and Pp3 is very faint or absent in unstimulated CML cells, relative to the normal granulocytes. On stimulation of normal cells with PMA, Pp1, Pp3, Pp4 and Pp6 exhibit distinct patterns of phosphorylation-dephosphorylation. In the CML cells, however, Pp1 and Pp4 are unresponsive to PMA. We conclude that PKC-mediated functions involving Pp1, Pp3 and Pp4 are most probably defective in CML cells.