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.

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.

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.

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.

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.

[Adaptive T-lymphocyte differentiation in an allogeneic environment]. / Adaptivnaia differentsirovka T-limfotsitov v allogennom okruzhenii.

Some patterns of adaptive changes of the hemopoietic stem cells in allogenic environment and the functional activity and specificity of cytotoxic T-lymphocytes developing from them and responsible for the inactivation of allogenic hemopoietic stem cells were studied on the radiation chimaeras. The allogenic environment did not prevent the functional maturation of these T-lymphocytes. The phenotype of T-lymphocytes matured under the conditions of allogenic recipient is similar but not identical with that of lymphocytes of the recipient mouse strain.

[Regulating role of the B-lymphocytes in lymphocyte interaction with allogeneic hematopoietic stem cells]. / Reguliruiushchaia rol' B-limfotsitov vo vzaimodeistvii limfotsitov s allogennymi krovetvornymi stvolovymi kletkami.

The preparative separation of lymph-node cells according to their charge and the study of the activity of each fraction have revealed that B-lymphocytes in the lymph nodes of mice have a regulating function in the phenomenon of the inactivation of allogeneic hematopoietic stem cells. The analysis of the dependence of effects, obtained in various experiments, on the ratio of B-lymphocytes and hematopoietic stem cells indicates that stem cells serve as targets for B-lymphocytes and the most probable mechanism of the above-mentioned effects is the direct interaction of the cells, one regulatory B-lymphocyte being capable of interacting with one target cell. Depending on the ratio of B-lymphocytes and stem cells, the inactivating effect may be either suppressed (the suppressor activity of B-lymphocytes) or enhanced (the helper activity of B-lymphocytes). The quantitative characteristics of the regulating activity of B-lymphocytes have been introduced.

[Characteristics of the B lymphocytes participating in the reaction of allogeneic stem cell inactivation]. / Kharakteristika B-limfotsitov, uchastvuiushchikh v reaktsii inaktivatsii allogennykh stvolovykh kletok.

In this work the B-cells of mouse lymph nodes are characterized. B-cells produce a helper effect on the capacity of the T-lymphocytes of the lymph nodes for inactivating nonsyngeneic stem cells. The study has revealed that the genetic heterogeneity of the B-lymphocytes does not lead to the abolition of their helper activity. B-lymphocytes of "B-mice" have also been shown to be capable of enhancing the inactivating activity of T-cells.

[Two-dimensional size-charge distribution of bone marrow cells in children with chronic immune neutropenia]. / Dvukhmernoe raspredelenie razmer-zariad kletok kostnogo mozga u detei s kronicheskoi immunnoi neitropeniei.

Physical characteristics of bone marrow cells of normal donors were comparatively studied with those of children with immune neutropenia. As a result of the bone marrow cell separation in the density gradient according to their sedimentation rate, fractions enriched with cells of one histogenetic series (lymphoid, erythroid and granulocytic) were obtained. Electrophoretic mobility of immature granulocytes in normal donors differed from that in children with immune neutropenia.

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.

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.

[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.

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.

[Effect of administration of allogeneic lymphocytes on the survival of sublethally irradiated mice]. / Vliianie vvedeniia allogennykh limfotsitov na vyzhivanie subletal'no obluchennykh myshei.

The administration of allogenic (CBA----C57B1/6) and semi-allogenic (CBA----F1) lymphocytes to sublethally exposed recipient mice either stimulates or inactivates endogenous colony-formation depending on the dose of lymphocytes administered. The stimulation of endogenous colony-formation correlates with the increased survival rate after radiation doses that decrease the survival rate of the control recipients.

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.

[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.