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.

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.

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.

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.

Antagonistic interactions among T cell subsets of old mice revealed by limiting dilution analysis.

When CD4 spleen cells from old (but not young) mice are tested for Con A-induced proliferation in limiting dilution assays, the dose response curve shows a nonlinear relationship. We interpret these observations using a two-cell model, in which proliferation of one cell type (LPC1) can be blocked by a second cell type (LPC2), which can itself generate detectable proliferation only at high multiplicities. The two-cell model accounts for several observations: 1) the variation in curve shape as a function of incubation time; 2) the skewed distribution of wells scored as "negative" in cultures of old splenocytes; and 3) the initially antagonistic effects of old splenocytes titrated into cultures containing fixed numbers of young responders. To provide a further test of the two-cell model, ionomycin-resistant (CaR) and ionomycin-sensitive (CaS) cells were separated using a Percoll/ionomycin gradient. The CaR preparation, shown previously to consist largely of memory T cells, showed the dose curve predicted for the LPC2 cell type, whereas the CaS (naive) cells showed the single-hit kinetics postulated for LPC1 cells. Furthermore, mixtures of CaR and CaS cells from young mice reproduced the zigzag dose curve characteristically produced by unseparated cells from old mice. These data suggest that the spleens of both young and old mice contain two kinds of Con A-responsive CD4 cell: one that proliferates vigorously, and a second, calcium ionophore-resistant type that proliferates less well, that can interfere with proliferation of the first cell type, and whose frequency increases with age.

The growth of hemopoietic stem cells is inhibited by natural killers only in the nonsyngeneic microenvironment.

The growth of hemopoietic cells of some inbred animal lines is repressed in hybrids of the first generation (F1)--the phenomenon of hybrid resistance. We have investigated the mechanism of hybrid resistance by studying the growth of hemopoietic cells of C57BL/6 mice in syngeneic and semisyngeneic heterotopic hemopoietic foci formed under the kidney capsule in (CBA*C57BL/6)F1 recipient mice. We found that hybrid resistance depended upon the joint involvement of NK cells and semisyngeneic hemopoietic stroma. It is concluded that NK cells recognize antigenic markers appearing on target cells located in a nonsyngeneic microenvironment.

Comparative study of immunomodulatory properties of muramyl peptides on immune system cells of young and old mice.

The immunomodulatory effects of two synthetic muramyl peptides (MP): muramyl dipeptide and glucosaminyl- muramyl dipeptide have been compared. It was shown, that MP effects on immune response are a consequence of the alteration in T lymphocyte regulators balance. MP action on old mice immune response and lymphocyte function was stimulating only: increasing of T helper precursors frequency and IL-1 production by macrophages. In the latter both MPs acted as correctors, recovering the decreased IL-1 production by old mice macrophages to young control level.

Quantitative analysis of interleukin-2-induced proliferation in the presence of inhibitors using a mathematical model.

The proliferative response of CTLL-2 cells to human recombinant interleukin-2 (IL-2) can be modeled mathematically using enzyme kinetic equations. This approach has been used to analyze dose-response curves (IL-2 concentration vs. level of proliferation) measured by MTT and [3H]TdR assays. The values of functional dissociation constants, equivalent to IL-2 concentrations giving 50% of the maximal response, depended on the cell concentration and increased from 4 to 60 pM for the [3H]TdR assay and from 40 to 140 pM for the MTT assay when the cell concentration was increased from 2 x 10(3) to 4 x 10(4) cells/well. The types of inhibition and dissociation constants for various inhibitors of IL-2-dependent proliferation such as mAbs against IL-2 receptor (7D4 and AMT13) and normal mouse serum (NMS) were also analyzed. Both mAbs exhibited competitive mechanisms of inhibition whereas NMS inhibited IL-2-driven proliferation in a mixed manner. Two gel-filtration fractions of NMS with inhibitory activity manifested different types of inhibition: purely competitive type of inhibition in the case of a 10-15 kDa fraction and a mixed type of inhibition for a 100-150 kDa fraction. The proposed model can also be used for quantitative analysis of the influence of various factors (pH, temperature, cultivation condition) on the level of proliferation.

Muramyl dipeptide-induced changes in murine splenocyte responses to concanavalin A.

The effect of muramyl dipeptide (MDP) on Con A-stimulated activation of murine spleen cells was studied. MDP was found to enhance or suppress the proliferative response of splenocytes when different concentrations of Con A were used. MDP was shown to change the IL-2 content in culture supernatants of stimulated cells and to influence IL-2-dependent proliferation of Con A-blasts. A high degree of correlation was found between the proliferation of Con A-blasts and the expression of IL-2 receptors on Con A-blasts. This correlation, however, disappeared in the presence of MDP. The effects of MDP were shown to depend on the level of initial cell activity or rather on conditions leading to a given initial activity of cells.

Target cells for immunomodulatory action of muramyl dipeptide.

Identification of the target cells for the immunomodulatory action of muramyl dipeptide (MDP) was addressed by investigation of various B-cell and T-cell lines. The lines used were: IM-9, a human lymphoblastoid B-cell line that spontaneously produces IgG; EL-4, a murine T-cell line that produces interleukin-2 (IL-2) on stimulation with phorbol myristate acetate; and CTLL-2, an IL-2-dependent murine T-cell line. MDP was shown to modulate such T-cell and B-cell functions as cell proliferation and secretion of IL-2 and IgG, respectively, in vitro. The effect of MDP in vitro was determined by both MDP dose and the control level of cell activity. The evidence obtained supports the possibility of the direct action of MDP on T and B lymphocytes.

Study of immunomodulatory properties of N-acetylmuramyl-L-alanyl-D-isoglutamine and N-acetylglucosaminyl-(beta 1----4)-N-acetylmuramyl-L-alanyl-D-isoglutamine.

The immunomodulatory activities of two synthetic muramylpeptides (MP), a muramyl dipeptide and a glucosaminyl-muramyl dipeptide, have been compared and have been found to exhibit many common features in their effects. In addition, the differential effects of low and high concentrations of MP on the primary humoral immune response in vitro were examined in detail. At high concentrations MP augmented the frequency of induced T-suppressor cells, while at low concentrations the primary immune response was stimulated by enhancement of the antigen-presenting function of accessory cells and by increasing the frequency of induced T-helper cells.

[The participation of oxygen in the process of the interphase death of T-lymphocytes following irradiation in vivo]. / Uchastie kisloroda v protsesse interfaznoi gibels T-limfotsitov posle oblucheniia in vivo.

The ability of lymphocytes to inhibit proliferation of non-syngeneic stem cells decreases differently after exposure in vivo and in vitro. The causes of the observed differences and the mechanism of radiation impairment of this function under different irradiation conditions have been investigated. Cells exposed in vivo die in the interphase irreversibly. The newly formed lymphocytes start the repair process as late as one month after irradiation. The injury to in vivo exposed cells is severer due to the presence of oxygen in tissues. A definite time interval is needed for the damaging effect of oxygen radicals to be implemented: the effect is maximum as early as 4 h following irradiation. With in vivo exposure under hypoxic conditions the functional activity of lymphocytes is the same as that of lymphocytes irradiated in vitro with the same dose. In vitro irradiation of lymphocytes at a high oxygen content causes a decrease in the functional activity of cells.

[The role of the serum interleukin-2 inhibitor in the rejection of allogeneic hematopoietic tissue in sublethally irradiated mice]. / Rol' syvorotochnogo ingibitora interleikina-2 v protsesse ottorzheniiaallogennoi krovetvornoi tkani v organizme subletal'no obluchennykh myshei.

The influence of ionizing radiation (5 Gy) on the interleukin-2 inhibitor in mouse serum has been investigated. It has been shown that the concentration of IL-2 inhibitor decreases on days 3-6 and increases considerably on days 10-15 after irradiation. A correlation has been found between the number of T-helpers in spleens of exposed allogenic chimeras and low IL-2 inhibitor content of serum. An attempt has been made to use the increased IL-2 inhibitor level for improving the acceptance of allogenic cells in the sublethally exposed mice.