OBJECTIVE:Bruton's tyrosine kinase (BTK) plays a critical role in B cell development and function. We recently described a selective BTK inhibitor, RN486, that blocks B cell receptor (BCR) and Fcγ receptor signaling and is efficacious in animal models of arthritis. The aim of this study was to examine the potential efficacy of BTK in systemic lupus erythematosus (SLE), using an NZB × NZW mouse model of spontaneous SLE.METHODS:Mice received RN486 or its vehicle (administered in chow) at a final concentration of 30 mg/kg for 8 weeks, starting at 32 weeks of age.RESULTS:The administration of RN486 completely stopped disease progression, as determined by histologic and functional analyses of glomerular nephritis. The efficacy was associated with striking inhibition of B cell activation, as demonstrated by a significant reduction in CD69 expression in response to BCR crosslinking. RN486 markedly reduced the secretion of IgG anti-double-stranded DNA (anti-dsDNA) secretion, as determined by enzyme-linked immunosorbent and enzyme-linked immunospot assays. Flow cytometric analysis demonstrated depletion of CD138(high) B220(low) plasma cells in the spleen. RN486 inhibited secretion of IgG anti-dsDNA but not IgM anti-dsDNA, suggesting that pharmacologic blockade of BTK resembles the reported transgenic expression of low levels of endogenous BTK in B cells. In addition, RN486 may also impact the effector function of autoantibodies, as evidenced by a significant reduction in immune complex-mediated activation of human monocytes in vitro and down-regulation of the expression of macrophage-related and interferon-inducible genes in both the kidneys and spleens of treated mice.CONCLUSION:Collectively, our data suggest that BTK inhibitors may simultaneously target autoantibody-producing and effector cells in SLE, thus constituting a promising therapeutic alternative for this disease.

Background: A unique feature of juvenile idiopathic arthritis (JIA) is that a subgroup of children can spontaneously enter prolonged remission, suggesting auto-regulation of the chronic inflammatory response.T cells are thought to play an important role in the pathogenesis of JIA, and the abundant synovial T cells are highly activated.Despite evidence for chronic persistence of these T cells, only a small proportion show signs of recent division when analysed ex vivo, and they proliferate poorly in vitro.We have investigated this by analysing the response of synovial T cells to mitogens, cytokines and antigens.We suggest that a subpopulation of synovial T cells, defined by the phenotype CD4+CD25+ are responsible for the poor proliferation, and that these cells may represent regulatory T cells (Treg) within the joint Methods: Paired samples of peripheral blood (PBMC) and synovial fluid mononuclear cells (SFMC) were prepared form 10 children with oligoarticular JIA (4 persistent, 6 extended).Cells were analysed directly ex vivo by 3-colour flow cytometry.For some experiments cells were sorted by magnetic bead separation, or on the FACS, before use.PBMC or SFMC were labelled with CFSE, stimulated for 5 days in the presence of mitogen (PHA), with or without cytokines (IL-2 or IL15), or antigen, and then analysed by flow cytometry using conventional monoclonal antibodies and labelled tetrameric MHC/peptide complexes.Results: Synovial T cells were hyporesponsive to PHA compared to blood T cells and this effect was overcome by addition of IL-2 or IL-15.Stimulation by either cytokine alone led to enhanced division of SF T cells compared to blood.This property was maintained when CD3+ cells were stimulated alone.Tetramer analysis after proliferation to the influenza antigenic peptide HA307-319 showed that SF T cells responding to this memory epitope are present in SF, but also that a large number of SF T cells divide in an antigen non-specific manner.After sorting of SF T cells into CD4+CD25+ and CD4+CD25-populations, the 'anergic' phenotype was shown to be associated specifically with the CD25+ population of synovial T cells.In contrast, CD25-cells alone had a strong proliferative response to mitogen. Conclusions:The inflamed joints of children with JIA contain a population of T cells which are hyporesponsive to mitogen but are highly sensitive to cytokine driven proliferation.This phenotype is a property of the CD4+CD25+ cells within the joint.We suggest that these are regulatory T cells (Treg): functional studies of these cells are in progress.

Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells. Type II cell proliferation after lung injury from 85% oxygen is regulated, in part, by a fall in lung PTHrP. In this study, we investigated lung PTHrP after injury induced by >95% oxygen in rats and rabbits. In adult rats, lung PTHrP rose 10-fold over controls to 6,356 +/- 710 pg/ml (mean +/- SE) at 48 h of hyperoxia. Levels fell to 299 +/- 78 pg/ml, and staining for PTHrP mRNA was greatly reduced at 60 h (P < 0.05), the point of most severe injury and greatest pneumocyte proliferation. In adult rabbits, lung PTHrP peaked at 3,289 +/- 230 pg/ml after 64 h of hyperoxia with 24 h of normoxic recovery and then dropped to 1,629 +/- 153 pg/ml at 48 h of recovery (P < 0.05). Type II cell proliferation peaked shortly after the fall in PTHrP. In newborn rabbits, lavage PTHrP increased by 50% during the first 8 days of hyperoxia, whereas type II cell growth decreased. PTHrP declined at the LD50, concurrent with increased type II cell division. In summary, lung PTHrP initially rises after injury with >95% hyperoxia and then falls near the peak of injury. Changes in PTHrP are temporally related to type II cell proliferation and may regulate repair of lung injury.

Class I MHC-peptide oligomers (MHC tetramers) have become popular reagents for the detection and characterization of antigen-specific CD8(+) T cells. Class II MHC proteins can be produced by expression in Escherichia coli followed by in vitro folding, or by native expression in insect cells; biotin can be introduced by site-specific chemical modification of cysteine, or by enzymatic modification of a peptide tag; and a variety of fluorescent streptavidin preparations can be used for oligomerization. Here we review methodologies for production of fluorescent oligomers of soluble class II MHC proteins and discuss their use in analysis of antigen-specific CD4(+) T cells. We explore the experimental conditions necessary for efficient staining of CD4(+) T cells using oligomers of class II MHC proteins, and we establish a standard protocol. Finally, we consider complications and challenges associated with these reagents, discuss the interpretation of staining results, and suggest future directions for investigation, in particular the use of MHC oligomers for the study of T cell avidity modulation.

While the T-cell receptor (TCR) repertoire of the newborn is highly diverse, a gradual alteration in diversity of the expressed TCR repertoire, in particular the oligoclonality of CD8+ T cells, occurs with increasing age. The timing of the initiation of this process is unknown. These changes are associated with an accumulation of T-cell expansions, thought to be in response to chronic antigen stimulation, frequently by persistent viruses such as Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Using reverse transcription-polymerase chain reaction heteroduplex analysis we have characterized the TCR expression of CD4 and CD8 cells from healthy young children and adults in order to delineate the age range at which these oligoclonal populations appear. We demonstrate that considerable oligoclonality can occur, even in healthy young children, and also that these expanded clonotypes persist. These are shown by heteroduplex to be exclusively within the CD28- subpopulation. The presence of such oligoclonal expansions correlates closely with the percentage of CD8+ cells that have the CD28- phenotype. However, we also show that control of chronic infection with EBV or CMV may coexist with a highly diverse, polyclonal TCR repertoire well into adulthood. These studies suggest that many factors affect the overall regulation of clone size in response to chronic antigens during the development of the immune system.

Clinically distinct forms of childhood arthritis are associated with different risk alleles of polymorphic loci within the MHC, which code for the antigen-presenting class I or class II molecules. We have compared the TCR diversity of synovial T cells from children with enthesitis-related (HLA-B27(+)) arthritis and oligoarticular arthritis (with class II MHC risk allele associations) in parallel with peripheral blood T cells from each child, using a high-resolution heteroduplex TCR analysis. We demonstrate that multiple clonal T cell expansions are present and persistent within the joint in both groups, but that there is disease-specific divergence in the dominant T cell subset containing these expansions. Thus, the largest clonotypes within the inflamed joints of children with class II-associated arthritis are within the CD4(+) synovial T cell population, while the dominant clones from children with enthesitis-related arthritis (associated with a class I allele) are within the CD8(+) synovial T cell population. These data provide powerful data to support the concept that recognition of MHC-peptide complexes by T cells plays a role in the pathogenesis of juvenile arthritis.

A Cycling Probe Technology (CPT) assay was developed for the detection of the mecA gene from methicillin resistant staphylococcal cultures. The assay is based on a colorimetric enzyme–immuno–assay (EIA) and uses a mecA probe (DNA–RNA–DNA) labeled with fluorescein at the 5′-terminus and biotin at the 3′-terminus. The reaction occurs at a constant temperature that allows the target DNA to anneal to the probe. RNase H cuts the RNA portion, allowing the cut fragments to dissociate from the target, making it available for further cycling. CPT–EIA uses streptavidin-coated microplate wells to capture uncut probe followed by detection with horseradish-peroxidase conjugated anti-fluorescein antibody. The assay was compared to PCR and shown to accurately detect the presence or absence of the mecA gene in 159 staphylococcal clinical isolates. The CPT-EIA assay takes two hours starting from cultured cells compared with the 24–48 h required for detection of methicillin resistance by conventional susceptibility tests.

Background/Purpose: Improved outcomes of preterm infants born to mothers treated prenatally with corticosteroids have been documented. The authors investigated the role of prenatal maternal corticosteroid therapy in congenital diaphragmatic hernia (CDH). Methods: Five CDH lambs of ewes given 0.5 mg/kg betamethasone intravenously 24 hours before delivery (single-dose), four CDH lambs of ewes similarly dosed at 48 and 24 hours before delivery (double-dose), five untreated CDH lambs and five control lambs were studied. After 2 hours of ventilation, compliance, arterial oxygen (po2) and carbon dioxide (pco2) concentrations were recorded. Lavage protein and phospho-lipid levels were measured, and lung tissue was analyzed for antioxidant enzyme activity (AOE). Results: No improvement in gas exchange was noted in either treatment group. Significant increases in compliance (P = .02) were noted in the double-dose steroid group, which were different from that of untreated CDH lambs or controls. Minimal changes in AOE activities were seen with steroid administration. Conclusions: Although the metabolic changes were not significant, the marked improvement in compliance seen in the double-dosed steroid group suggests a potential role for prenatal maternal corticosteroids in CDH. Further timing and dosage studies are warranted in this model.