Replication of the BANK1 genetic association with systemic lupus erythematosus in a European-derived population.

Systemic lupus erythematosus (SLE) is an autoimmune disease with highly variable clinical presentation. Patients suffer from immunological abnormalities that target T-cell, B-cell and accessory cell functions. B cells are hyperactive in SLE patients. An adapter protein expressed in B cells called BANK1 (B-cell scaffold protein with ankyrin repeats) was reported in a previous study to be associated with SLE in a European population. The objective of this study was to assess the BANK1 genotype-phenotype association in an independent replication sample. We genotyped 38 single nucleotide polymorphisms (SNPs) in BANK1 on 1892 European-derived SLE patients and 2652 European-derived controls. The strongest associations with SLE and BANK1 were at rs17266594 (corrected P-value=1.97 x 10(-5), odds ratio (OR)=1.22, 95% CI 1.12-1.34) and rs10516487 (corrected P-value=2.59 x 10(-5), OR=1.22, 95% CI 1.11-1.34). Our findings suggest that the association is explained by these two SNPs, confirming previous reports that these polymorphisms contribute to the risk of developing lupus. Analysis of patient subsets enriched for hematological, immunological and renal ACR criteria or the levels of autoantibodies, such as anti-RNP A and anti-SmRNP, uncovers additional BANK1 associations. Our results suggest that BANK1 polymorphisms alter immune system development and function to increase the risk for developing lupus.

Multiplex serum cytokine monitoring as a prognostic tool in rheumatoid arthritis.

OBJECTIVE: Early optimized therapy of rheumatoid arthritis (RA) results in improved outcomes. The initiation of optimized therapy is hindered by the difficulty of early diagnosis and the limitations of current disease activity and therapeutic response assessment tools. Identifying patients requiring early combination DMARD/biologic therapy is currently a significant clinical challenge given the lack of definitive prognostic criteria. Since cytokines are soluble intracellular signaling molecules that modulate disease pathology in RA, we tested the recent conjecture that en mass serum cyto-kine measurement and monitoring will provide a useful tool for effective therapeutic management in RA. METHODS: We assayed the levels of 16 serum cytokines in 18 RA patients treated prospectively with methotrexate and from 18 unaffected controls. Specific mechanistic aspects of inflammatory pathology in the periphery could be discerned on a patient-specific basis from patients' serum cytokine profiles, information that may aid in the design of anti-cytokine biologic therapy. A serum Cytokine Activity Index (CAI) was also created using multi-variant analysis methods. RESULTS: Distinct cytokines were significantly elevated in RA patients relative to controls, and three distinct clusters with correlations to disease activity were identified. The Cytokine Activity Index correlated well with the therapeutic res-ponse; responders and non-responders in this cohort were distinguishable as early as one month post initiation of methotrexate therapy, well before clinical assessments of response are commonly completed. CONCLUSION: Clinical assessment tools could be derived from this approach that may provide a means to continually track patients, allowing intervention strategies to be better evaluated on a patient-specific basis and to identify residual cytokine activity that could be used to guide combination therapy.

Circulating cytokines in Norwegian patients with psoriatic arthritis determined by a multiplex cytokine array system.

OBJECTIVES: Serum cytokines play an important role in the pathogenesis of psoriatic arthritis (PsA) by initiating and perpetuating various cellular and humoral autoimmune processes. The aim of this study was to describe a broad spectrum of T- and B-cell cytokines, growth factors and chemokines in patients with PsA and healthy individuals. METHODS: A novel protein array system, denoted as multiplex cytokine assay was utilized to measure simultaneously the levels of 23 circulating cytokines of patients with PsA and healthy individuals. Additionally, correlational clustering and discriminant function analysis (DFA), two multivariate, supervised analysis methods were employed to identify a subset of biomarkers in order to describe potential functional inter-relationships among these pathological cytokines and identify biomarkers with prognostic and diagnostic utility. RESULTS: Univariate analysis demonstrated that serum levels of a complex set of immune and inflammatory modulating cytokines are significantly up-regulated in patients with PsA relative to unaffected controls including interleukin (IL)-10, IL-13, interferen (IFN)-alpha, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor [CCL3 macrophage inflammatory protein (MIP)-1alpha], CCL4 (MIP-1beta) and CCL11 (Eotaxin), while granulocyte-colony stimulating factor was significantly reduced in PsA patients. Correlational clustering was able to discriminate among, and hence subclassify, patients with varying levels of disease activity, which may prove useful in guiding therapy in these apparently phenotypically distinct disease subsets. DFA identified EGF, IFN-alpha, VEGF, CCL3 (MIP-1alpha) and IL-12p40 as analytes with the strongest discriminatory power among various PsA patients and controls. CONCLUSIONS: Our findings suggest that these factors modulate PsA pathology and the articular involvement in a synergistic manner. Identifying factors could be used in the development of clinical diagnostic tests, which are valuable to guide evidence-based diagnosis and disease management of PsA.

Template-driven gene selection procedure.

The hierarchical clustering and statistical techniques usually used to analyse microarray data do not inherently represent the underlying biology. Herein, a hybrid approach involving characteristics of both supervised and unsupervised learning is presented. This approach is based on template matching in which the interaction of the variables of inherent malignancy and the ability to express the malignant phenotype are modelled. Immortalised normal urothelial cells and bladder cancer cells of different malignancy were grown in conventional two-dimensional tissue culture and in three dimensions on extracellular matrices (ECMs) that were either permissive or restrictive for expression of the malignant phenotype. The transcriptome represents the effects of two variables--inherent malignancy and the modulatory effect of ECM. By assigning values to each of the biological variables of inherent malignancy and the ability to express the malignant phenotype, a template was constructed, which encapsulated the interaction between them. Gene expression correlating both positively and negatively with the template was observed, but when iterative correlations were carried out, the different models for the template converged on the same actual template. A subset of 21 genes was identified, which correlated with two a priori models or an optimised model above the 95% confidence limits identified in a bootstrap resampling with 5000 permutations of the data set. The correlation coefficients of expression of several genes were > 0.8. Analysis of upstream transcriptional regulatory elements (TREs) confirmed that these genes were not a randomly selected set of genes. Several TREs were identified as significantly over-expressed in the sample of 20 genes for which TREs were identified, and the high correlations of several genes were consistent with transcriptional co-regulation. The authors suggest that the template method can be used to identify a unique set of genes for further investigation.

A genome-scale assessment of peripheral blood B-cell molecular homeostasis in patients with rheumatoid arthritis.

OBJECTIVE: While rheumatoid arthritis (RA) is considered a prototypical autoimmune disease, the specific roles of B-cells in RA pathogenesis is not fully delineated. METHODS: We performed microarray expression profiling of peripheral blood B-cells from RA patients and controls. Data were analysed using differential gene expression analysis and 'gene networking' analysis (characterizing clusters of functionally inter-relelated genes) to identify both regulatory genes and the pathways in which they participate. Results were confirmed by quantitative real-time polymerase chain reaction and by measuring the levels of 10 serum cytokines involved in the pathways identified. RESULTS: Genes regulating and effecting the cell-cycle, proliferation, apoptosis, autoimmunity, cytokine networks, angiogenesis and neuro-immune regulation were differentially expressed in RA B-cells. Moreover, the serum levels of several soluble factors that modulate these pathways, including IL-1beta, IL-5, IL-6, IL-10, IL-12p40, IL-17 and VEGF were significantly increased in this cohort of RA patients. CONCLUSIONS: These results outline aspects of the multifaceted role B-cells play in RA pathogenesis in which immune dysregulation in RA modulates B-cell biology and thereby contributes to the induction and perpetuation of a pathogenic humoral immune response.

Transcriptional modulation of TCR, Notch and Wnt signaling pathways in SEB-anergized CD4+ T cells.

Gene expression changes in CD4 + Vbeta8+ T cells energized by in vivo exposure to staphylococcal enterotoxin B (SEB) bacterial superantigen compared to CD4 + Vbeta8+ non-energic T cells were assessed using DNA microarrays containing 5184 murine complementary DNAs. Anergy in splenic T cells of SEB-immunized BALB/c mice was verified by dramatically reduced proliferative capacity and an 8 x overexpression of GRAIL mRNA in CD4 + Vbeta8+ T cells taken from mice 7 days after injection. At an Associative t-test threshold of P<0.0005, 96 genes were overexpressed or detected only in anergic T cells, while 256 genes were suppressed or not detected in anergic T cells. Six of eight differential expressions tested using real-time quantitative PCR were validated. Message for B-Raf was detected only in non-anergic cells, while expression of the TCR signaling modulator Slap (Src-like adapter protein) and the TCR zeta-chain specific phosphatase Ptpn3 was enhanced. Modulation of multiple genes suggests downregulation of Wnt/beta-catenin signaling and enhanced Notch signaling in the anergic cells. Consistent with previous reports in a non-superantigen in vivo anergy model, mRNA for CD18 and the transcription factor Satb1 (special AT-rich-binding protein 1) was increased in SEB-energized T cells. This is the first report of global transcriptional changes in CD4+ T cells made anergic by superantigen exposure.

Mobile classification in microarray experiments.

In a homogeneous group of samples, there are genes whose expression variations can be attributed to factors other than experimental errors. These factors can include natural biological oscillations or metabolic processes. These genes are rarely classified as 'interesting' based on their variability profile. However, their dynamic behaviour can tease out important clues about naturally occurring biological processes in the organism under study and can be used for group classification. Dynamical discriminate function analysis was developed on the concept that stable classification parameters (roots) can be derived from highly variable gene-expression data. Stability of these combinations implies a strongly compensatory relationship that may divulge functional interconnections.

5alpha-Androstane-3alpha,17beta-diol activates pathway that resembles the epidermal growth factor responsive pathways in stimulating human prostate cancer LNCaP cell proliferation.

5alpha-Androstane-3alpha,17beta-diol (3alpha-diol) is considered to have no androgenic effects in androgen target organs unless it is oxidized to 5alpha-dihydrotestosterone (5alpha-DHT). We used microarray and bioinformatics to identify and compare 3alpha-diol and 5alpha-DHT responsive gene in human prostate LNCaP cells. Through a procedure called 'hypervariable determination', a similar set of 30 responsive genes involving signal transduction, transcription regulation, and cell proliferation were selected in 5alpha-DHT-, 3alpha-diol-, and epidermal growth factor (EGF)-treated samples. F-means cluster and networking procedures showed that the responsive pattern of these genes was more closely related between 3alpha-diol and EGF than between 5alpha-DHT and 3alpha-diol treatments. We conclude that 3alpha-diol is capable of stimulating prostate cell proliferation by eliciting EGF-like pathway in conjunction with androgen receptor pathway.

Renal transplant patients show variations in their self-reactive repertoires: a serial study.

We addressed the question of whether allo-transplantation (Tx) induces breakdown of tolerance to self-antigens or alteration of the autoreactive T cell repertoire in humans. The serial variation of T cell autoreactivity was studied in the peripheral blood of 12 renal transplant patients, by autologous limiting dilution assay and autologous mixed lymphocyte reaction. Ten of 12 patients presented a positive response in autologous peripheral blood mononuclear cells in the post-Tx period, in contrast to four of 12 patients before Tx (P = 0.038). Multi-hit kinetics was found in 57% of the assays analyzed, indicating frequent regulatory control of the autologous response. Quantitative analysis performed in eight patients showed an increase in precursor frequency at >1 year post-Tx in five patients. These data indicate that autoreactivity increases or develops following Tx, in humans. Post-Tx events such as alloreactivity, infections or immunosuppression could interfere with the balance of autoreactive and regulatory cells, leading to changes in the T cell repertoires to self-antigens and eventually breakdown of self-tolerance. Further investigation is needed to elucidate whether post-Tx autoreactivity contributes to rejection, plays a regulatory role over alloreactivity or both, at separate times.

Array-based expression analysis of mouse liver genes: effect of age and of the longevity mutant Prop1df.

Ames dwarf mice, homozygous for the df allele at the Prop1 locus, live 40% to 70% longer than nonmutant siblings and represent the first single-gene mutant that extends life span in a mammal. To gain insight into the basis for the longevity of the Ames dwarf mouse, we measured liver mRNA levels for 265 genes in a group of 11 df/df mice, (three to four mice per age group), at ages 5, 13, and 22 months, and in 13 age- and sex-matched control mice. The analysis showed seven genes where the effects of age reach p < .01 in normal mice and six others with possible age effects in dwarf mice, but none of these met Bonferroni-adjusted significance thresholds. Thirteen genes showed possible effects of the df/df genotype at p < .01. One of these, insulin-like growth factor 1 (IGF-1), was statistically significant even after adjustment for multiple comparisons; and genes for two IGF-binding proteins, a cyclin, a heat shock protein, p38 mitogen-activated protein kinase, and an inducible cytochrome P450 were among those implicated by the survey. In young control mice, half of the expressed genes showed SDs that were more than 58% of the mean, and a simulation study showed that genes with this degree of interanimal variation would often produce false-positive findings when conclusions were based on ratio calculations alone (i.e., without formal significance testing). Many genes in our data set showed apparent young-to-old or normal-to-dwarf ratios above 2, but the large majority of these proved to be genes where high interanimal variation could create high ratios by chance alone, and only a few of the genes with large ratios achieved p < .05. The proportion of genes showing relatively large changes between 5 and 13 months, or from 13 to 22 months of age, was not diminished by the df/df genotype, providing no support for the idea that the dwarf mutation leads to global delay or deceleration of the pace of age-dependent changes in gene expression. These survey data provide the foundation for replication studies that should provide convincing proof for age- and genotype-specific effects on gene expression and thus reveal key similarities among the growing number of mouse models of decelerated aging.

Age-associated decline in responses of naïve T cells to in vitro immunization reflects shift in glucocorticoid sensitivity.

Naïve T lymphocytes from young mice can be immunized to protein antigens in vitro if the initial exposure to antigen is followed by a brief period of clonal expansion in the presence of both the glucocorticoid dexamethasone (at 10(-8) M) and antibodies to Interleukin-10 (IL-10). These cultures produce cell lines that respond to antigen rechallenge by proliferation and cytokine secretion. T cells from older mice, however, do not respond under these conditions unless the dexamethasone concentration is raised to levels (10(-7) M) that are inhibitory for T cells of young mice. Suitably timed exposure to dexamethasone can also increase proliferative responses to polyclonal activation via the CD3 component of the T cell receptor, and again optimal responses are obtained from old mice only at steroid concentrations that are super-optimal for young T cells. Diminished sensitivity to glucocorticoid effects may contribute to the poor responses of aged mice to novel immunogens.

Generation of antigen-specific Th2 cells from unprimed mice in vitro: effects of dexamethasone and anti-IL-10 antibody.

We describe a system for the in vitro production of Ag-specific mouse CD4 cell lines from unprimed mice. Purified CD4+ CD45RB(high) T cells were exposed to Ag-pulsed accessory cells in serum-free medium for 24 h; cultured in the absence of Ag and in the presence of serum, IL-2, dexamethasone, and Abs to IL-10 for an additional 4 days; and then re-exposed to the original sensitizing Ag. The presence of dexamethasone and Abs to IL-10 during the initial expansion stage appeared to be critical for the ability of the stimulated and expanded T cells to respond to restimulation with the same Ag. Repeated cycles of in vitro stimulation led to increased specificity for the sensitizing Ag (in the current case, pigeon cytochrome c), a decline in production of IL-2 and IFN-gamma, and increased production of IL-4, IL-5, and IL-10. This culture protocol provides a test system for exploration of factors that regulate the conversion of naive cells to memory cells and the development of specific immune responses to protein Ags. The data are consistent with models that implicate glucocorticoids as regulators of immune response specificity.

In vitro production of antigen-specific T cells from unprimed mice: role of dexamethasone and anti-IL-10 antibodies.

We describe here a culture system for studying the development, in vitro, of antigen-specific CD4 T cells from unprimed mice. T cells from young mice are initially exposed to antigen, such as pigeon cytochrome C or keyhole limpet hemocyanin, in the presence of adherent accessory cells and then allowed to proliferate in the absence of antigen but in the presence of IL-2, 10(-8) M dexamethasone, and antibodies to IL-10. Proliferation and IL-2 production by T cells harvested from such expansion cultures are antigen-dependent but not antigen-specific and at different doses can be either stimulated or inhibited both by the priming antigen and by irrelevant proteins. Antigen-specific T cell reactions can be elicited by any of three modifications of the culture protocol: (a) absorption of nonspecific cells on accessory cell monolayers bearing irrelevant proteins; (b) increased doses of dexamethasone during the expansion phase; or (c) a second cycle of antigen activation and antigen-free expansion. These observations provide a foundation for further analysis of in vitro maturation of primary immune responses and suggest an important role for IL-10 and glucocorticoids in regulating the early stages of activation and proliferation by naive T cells.

Nanomolar concentrations of gangliosides stimulate primary humoral response.

Exogenous gangliosides act as immunosuppressors when applied at micromolar concentrations corresponding to their average level in human plasma. Here we show that at nanomolar concentrations the gangliosides GD3, GD1a and GM1 can act as immunostimulators markedly enhancing the number of plaque-forming cells in mouse splenocyte culture responding to sheep erythrocytes. At such low concentration these gangliosides as well as GM3 were not able to influence significantly proliferative responses of splenic B and T lymphocytes or of cytotoxic T-cells. Neither did they change significantly the production of IL-1 by antigen- representing cells, or of IL-2 by Con A-induced blasts in the splenocyte culture. It is suggested that the stimulatory effect of low ganglioside concentrations on humoral response is due to their influence on cooperative cell-cell interactions required for the differentiation of B-cells into Ig-secreting cells.

The Use of Spectral Analysis of Limiting Dilution for Investigating the Immune Response.

In this study we present a method for characterizing a spectrum of cells engaged in the humoral response by two parameters: the frequency and potential activity of antigen specific precursor cells. Spleen cells from mice immunized with ovalbumin were placed in microcultures in a limiting dilution assay. The values derived from positive cultures in an activity/cell number plot were compatible with Poisson distributions for distinct families of precursor cells displaying either positive or negative (suppressor-like) activities. Proposed limiting dilution spectral analysis combines the potentialities of the standard limiting dilution analysis with those provided by histograms of distribution according to the activity of positive cultures. This method opens new opportunities in detecting and characterizing distinct groups of limiting precursor cells.

Regulatory interactions between virgin and memory CD4 T lymphocytes.

Naive and memory CD4 T cells from mouse spleen, alone or in a 1:1 mixture, were tested for Con A-induced proliferation in limiting dilution cultures. Dose-response curves for naive cells were linear, but curves for memory cells were hyperbolic, suggesting that positive responses required the activation of several cells of the memory type. Mixtures (1:1) gave zig-zag curves, consistent with a previously described quantitative model in which memory cells block naive cell proliferation at low multiplicities and generate their own positive responses at higher multiplicities. Inhibition of naive cell proliferation by memory cells could be mimicked by IL-10 and blocked by anti-IL-10 antibody. IL-2 addition converted the multihit dose curves of memory T cells to single-hit curves, suggesting that poor IL-2 production limits growth in memory cell cultures. Surprisingly, IL-2 addition to cultures of naive cells led to a decrease in proliferation at high cell input doses. This inhibitory effect of IL-2 could be blocked by antibody to IL-10, and may reflect the presence of contaminating memory cells in the naive cell preparations. These models for analysis of interaction between naive and memory T cells in limiting dilution conditions point to a series of reciprocal interactions between IL-10 and IL-2 producing cells.

Analysis of cellular interactions in limiting dilution cultures.

Limiting dilution (LD) cultures are often used to study cellular heterogeneity in responses of murine splenocytes to specific or polyclonal activation. LD titration curves often reveal a nonlinear dependence of response on input cell dose. Although 'zigzag' shaped curves of this kind are often interpreted and analyzed as resulting from interactions among three distinct cell types, we observe that a more parsimonious two cell model, including a cell type that can generate both positive and negative effects, provides better fit to a wide range of experimental data. We have developed mathematical models for the accurate estimation of the frequencies of both interacting cell types and of the parameters for their multi-hit interaction. We show examples of LD cultures in which specific experimental manipulations alter the frequency of only one of the two cell types, or alter the interaction parameters without a change in responder frequency. We also provide a simplified method for approximation of the model parameters using graphical approaches and simple algebra. Lastly, we present an improved method for calculation of the effect generated per responder cell in microclonal cultures.

Effects of retinoids on regulatory cellular interactions in primary mixed lymphocyte reaction (MLR).

The effects of two retinoids, all-trans-retinoic acid and 13-cis-retinoic acid on murine splenic lymphocyte proliferative response in mixed culture were evaluated. In contrast with previously reported absence of retinoic acid (RA) effect on mixed lymphocyte reaction (MLR) the conditions for a strong potentiation of proliferative response of murine lymphocytes with RA were obtained. Stimulatory cells were determined to be the main targets for RA. The data suggest that the RA potentiating effect is the result of an increase in stimulator cell immunogenicity after their pre-treatment with RA before use in MLR. Optimal potentiation by retinoids of proliferative response was found at non-optimal conditions of mixed culture.

Joint influence of natural killers (NK) and hemopoietic stroma on rejection of parent stem cells.

The growth of hemopoietic stem cells of C57BL/6 mice was explored in syngeneic and semi-syngeneic foci of heterotopic hemopoiesis, formed under the kidney capsule of (CBA x C57BL/6)F1 recipients with and without prior treatment with cyclophosphamide or fractionated irradiation, which is known to abrogate hybrid resistance and NK cell activity. Our results show that the suppression of the growth of parent stem cells in semi-syngeneic F1-recipients is due to NK cell participation. However, no inhibition was found in the growth of identical cells in the foci of syngeneic hemopoietic stroma in the same recipients, despite the presence of NK cell activity. The experimental model used shows clearly that hybrid resistance is the result of the joint involvement of NK cells and the semi-syngeneic stroma.