Demonstration of delta rec-pseudo J alpha rearrangement with deletion of the delta locus in a human stem-cell leukemia.

It has been hypothesized that a rearrangement between the delta recombining element (delta Rec) and a pseudo J alpha gene serves to delete the TCR-delta locus before rearrangement of the TCR-alpha genes. We have now sequenced a direct, site-specific rearrangement between the delta Rec element and a pseudo J alpha gene in a human leukemic stem-cell line. Putative "N-sequence" addition was noted at the site of recombination, suggesting that this event occurred at a time when the enzyme(s) involved in N-region addition were active in this cell. This provides support for the view that deletion of the TCR-delta locus is required before rearrangement of the TCR-alpha chain genes.

Activation of NOD2 in vivo induces IL-1beta production in the eye via caspase-1 but results in ocular inflammation independently of IL-1 signaling.

Nucleotide-binding and oligomerization domain 2 (NOD2) belongs to the emerging Nod-like receptor (NLR) family considered important in innate immunity. Mutations in NOD2 cause Blau syndrome, an inherited inflammation of eye, joints, and skin. Mutations in a homologous region of another NLR member, NALP3, cause autoinflammation, wherein IL-1beta plays a critical role. Here, we tested the hypothesis that IL-1beta is a downstream mediator of NOD2-dependent ocular inflammation. We used a mouse model of NOD2-dependent ocular inflammation induced by muramyl dipeptide (MDP), the minimal bacterial motif sensed by NOD2. We report that MDP-induced ocular inflammation generates IL-1beta and IL-18 within the eye in a NOD2- and caspase-1-dependent manner. Surprisingly, two critical measures of ocular inflammation, leukocyte rolling and leukocyte intravascular adherence, appear to be completely independent of IL-1 signaling effects, as caspase-1 and IL-1R1-deficient mice still developed ocular inflammation in response to MDP. In contrast to the eye, a diminished neutrophil response was observed in an in vivo model of MDP-induced peritonitis in caspase-1-deficient mice, suggesting that IL-1beta is not essential in NOD2-dependent ocular inflammation, but it is involved, in part, in systemic inflammation triggered by NOD2 activation. This disparity may be influenced by IL-1R antagonist (IL-1Ra), as we observed differential IL-1Ra levels in the eye versus plasma at baseline levels and in response to MDP treatment. This report reveals a new in vivo function of NOD2 within the eye yet importantly, distinguishes NOD2-dependent from NALP3-dependent inflammation, as ocular inflammation in mice occurred independently of IL-1beta.

Anterior uveitis accompanies joint disease in a murine model resembling ankylosing spondylitis.

BACKGROUND: Uveitis is often associated with a systemic inflammatory disease such as ankylosing spondylitis. Our understanding of the eye's susceptibility to immune-mediated uveitis as in the apparent absence of infection has been limited by a relative lack of experimental models. Here we sought to assess whether ocular inflammation occurs in a previously described murine model of proteoglycan-induced spondylitis, wherein mice develop progressive spondylitis, sacroiliitis and peripheral arthritis--features common to the clinical presentations of ankylosing spondylitis. METHODS: Using intravital microscopy we examined the ocular inflammatory response after the onset of arthritis in mice that overexpressed the T cell receptor (TCR) specific for a dominant arthritogenic epitope of cartilage proteoglycan [TCR-Tg (transgenic) mice] or BALB/c controls. RESULTS: Immunized TCR-Tg mice showed a significant increase in the number of rolling and adhering cells within the iris vasculature compared to adjuvant control mice. Cellular infiltration within the iris tissue, as assessed by intravital microscopy and histology, was also increased. Our initial temporal analysis has revealed that immunized TCR-Tg mice show a significant increase in intravascular inflammation by 2 weeks after immunization, but it diminishes at 4 weeks after immunization. CONCLUSIONS: Although these data are preliminary, this model has the potential to clarify the mechanisms accounting for the coexistence of eye and sacroiliac inflammation as occurs in patients with ankylosing spondylitis.

A solid-phase assay for circulating immune complexes using monoclonal rheumatoid factors and peroxidase-linked protein A.

A solid-phase monoclonal rheumatoid factor (mRF) assay for circulating immune complexes (IC) is described. Microtiter plates are coated with mRF, serum diluted 1 : 40 is added, and complexed IgG is detected with protein A coupled to peroxidase. Selective binding was obtained with aggregated human gamma-globulin and soluble IC prepared in vitro. Sucrose density gradient ultracentrifugation studies indicate that mRF binds intermediate-size IC (between 7S and 19S). Abnormal levels of IC were found in 78% of sera from patients with rheumatoid arthritis, 67% with systemic lupus erythematosus, and 87% with subacute bacterial endocarditis.

TCRB clonotypes are present in CD4+ T cell populations prepared directly from rheumatoid synovium.

The identification of clonal T cells at sites of inflammation is hampered by the large number of polyclonal T cells that nonspecifically accumulate. In this report, we combine the use of T cell sorting with spectratyping of the third complementarity determining region (CDR3) and direct sequence analysis to rapidly screen for and identify clonal expansions of T cells from synovial tissue specimens from patients with rheumatoid arthritis (RA). Initially, we used a polymerase chain reaction specific for the variable region gene of the T cell receptor beta chain (TCRBV) to compare the TCRBV repertoire expressed by CD4+ T cells from the peripheral blood and synovium of five patients with long-standing RA. Each patient had several TCRBV genes that were amplified to a greater degree from synovium. Extensive sequence analysis (n > 170) showed that each patient contained junctional sequences that occurred more than once, implying the presence of T cell clones within the starting CD4+ T cell population. To assess a more straightforward approach to identifying clones, six additional patients were recruited and CD4+, TCRBV2+ synovial T cells were positively selected and analyzed by CDR3 spectratyping. Bands deviating from a normal distribution were excised from the gel and sequenced directly. Clones were detected in half of the patients. These data are consistent with the possibility of an antigen-driven T cell response in RA that remains present in the setting of advanced disease.

T-cell receptor variable beta genes show differential expression in CD4 and CD8 T cells.

Studies in transgenic and inbred strains of mice have shown that the critical molecular interactions controlling positive selection involve major histocompatibility complex (MHC), T-cell receptor (TCR), and CD4 or CD8 coreceptor molecules. Correlations have been established between MHC gene products and the percentage of CD4 or CD8 T cells that express specific variable (V) beta-gene products as part of the alpha beta heterodimer. These studies have important implications regarding potential mechanisms of HLA-linked autoimmune diseases in humans. If similar interactions are required for positive selection in humans, one would predict that the TCR repertoire expressed by mature, peripheral blood CD4 and CD8 T cells would vary. To test this hypothesis the expression of specific TCR V beta-region genes by CD4 and CD8 T cells from healthy individuals was compared using both triple-color flow cytometry and polymerase chain reaction based experimental approaches. The results show that the TCR repertoire does vary as a function of CD4 and CD8 T-cell subsets. Among unrelated individuals certain V beta genes were consistently overrepresented in the CD4 population (V beta-5.1, -6.7a, and -18); some were skewed to the CD8 population (V beta-14) while others showed variable patterns (V beta-12 and -17). Deletion of entire V beta gene families was not observed suggesting that this is a rare event in humans. Attempts to correlate the expressed TCR repertoire in humans with HLA alleles will require consideration of these differences in expression as a function of subset.

Power spectra and coherence in the EEG of a vegetative patient with severe asymmetric brain damage.

OBJECTIVES: To examine differences in power spectra and intra-hemispheric coherence between the left and right hemispheres in the presence of severe asymmetric brain damage. METHODS: Power spectra and coherence functions were computed for a patient with severe damage to subcortical gray matter structures on the right side but relative preservation on the left. RESULTS: Power spectra differed modestly over the hemispheres, with greater low frequency power and less high frequency power over the more damaged right hemisphere. Coherence differed dramatically, with marked reduced coherence over the right hemisphere, particularly frontally where the damage was most extensive. CONCLUSIONS: Damage to subcortical structures of one hemisphere may result in a marked reduction in coherence in the ipsilateral EEG with only a modest change in the power spectrum. We speculate that the physiologic basis of this selective change is damage to structures mediating communication between cortical areas.

The human leukocyte antigen complex and chronic ocular inflammatory disorders.

PURPOSE: To review the role of gene products from the human leukocyte antigen (HLA) complex in the normal functioning of the immune system, ocular inflammation, and models of autoimmunity. METHOD: A review of recently published reports. RESULTS: Many chronic ocular inflammatory diseases are associated with specific alleles of the HLA complex. Understanding how HLA gene products function normally provides clues to the mechanism of disease associations. In the thymus, these molecules control the shape of the developing T-cell repertoire, leading to self-tolerance. In the periphery, HLA molecules bind and present peptide fragments to T cells, leading to a variety of effector functions. Although effector functions are for the most part beneficial, models are reviewed in which peptide-HLA interactions lead to T-cell responses with pathologic consequences. Herpes stromal keratitis is an informative animal model highlighting the role of self-tolerance, infection, and molecular mimicry in the development of autoimmunity. CONCLUSIONS: Human leukocyte antigen gene products may be associated with chronic inflammatory disorders through the unique presentation of "disease-inducing" peptides or the development of a T-cell repertoire prone to autoreactivity and molecular mimicry.

The spatiotemporal properties of the Craik-O'Brien-Cornsweet effect are consistent with 'filling-in'.

The Craik-O'Brien-Cornsweet effect (COCE) is an illusion in which luminance discontinuities give rise to illusory brightness. One hypothesised mechanism for the induction of illusory brightness is that the cortex constructs a brightness percept from edge information by a lateral 'filling-in' process. A requirement for the filling-in hypothesis is that ability of the illusion to form would be limited by the speed of propagation of the filling-in. The results presented here from three methods indicate that in the case of COCE gratings brightness information propagates at a fixed speed across the central visual field of about 19 degrees/s, and across visual areas V1 or V2 at 155 or 205 (+/- 20) mm/s, respectively.

The role of T cells in autoimmune uveitis.

Autoimmune diseases result from the activation of self-reactive T cells recognizing autoantigens or foreign antigens cross-reactive with an autoantigen. T cells are thought to play a major role in autoimmune diseases in different organs, including the eye. This review focuses on the role of T cells in autoimmune uveitis in humans and in animal models of experimental autoimmune uveitis. Since rheumatoid arthritis is an autoimmune disease that has been studied far more extensively than uveitis, we have also included a review of clinical and experimental observations relevant to that disease.

Activation of nucleotide oligomerization domain 2 exacerbates a murine model of proteoglycan-induced arthritis.

In addition to its role in innate immunity, nucleotide oligomerization domain 2 (NOD2) has been shown to play a suppressive role in models of colitis. Notably, mutations in NOD2 cause the inherited granulomatous disease of the joints called Blau syndrome, thereby linking NOD2 with joint disease as well. However, the role of NOD2 in joint inflammation has not been clarified. We demonstrate here that NOD2 is functional within the mouse joint and promotes inflammation, as locally or systemically administered muramyl dipeptide (MDP; the NOD2 agonist) resulted in significant joint inflammation that was abolished in NOD2-deficient mice. We then sought to investigate the role of NOD2 in a mouse model of inflammatory arthritis dependent on adaptive immunity using TCR-transgenic mice whose T cells recognized the dominant epitope of proteoglycan (PG). Mice immunized with PG in the presence of MDP developed a more severe inflammatory arthritis and histopathology within the joints. Antigen-specific activation of splenocytes was enhanced by MDP with respect to IFN-gamma production, which would be consistent with the Th1-mediated disease in vivo. Intriguingly, NOD2 deficiency did not alter the PG-induced arthritis, indicating that NOD2 does not play an essential role in this model of joint disease when it is not activated by MDP. In conclusion, we demonstrate that in a model of inflammatory arthritis dependent on T and B cell priming, NOD2 activation potentiates disease. However, the absence of NOD2 does not alter the course of inflammatory arthritis, in contrast to models of intestinal inflammation.

Patterns of T-cell receptor variable beta gene expression by synovial fluid and peripheral blood T-cells in rheumatoid arthritis.

Conflicting data have been reported regarding the presence or absence of a predominant variable (V) region T-cell receptor (TCR) gene in the peripheral blood or synovial fluid of patients with rheumatoid arthritis (RA). In this study we have used the polymerase chain reaction (PCR) to compare the level of TCR V beta gene expression by peripheral blood mononuclear cells (PBMC) and by synovial fluid cells obtained from HLA DRB1 *0401 and *0404 RA patients. PCR was performed using cDNA synthesized from freshly obtained cells (not stimulated in vitro). The pattern of expression observed for most of the V beta genes studied showed either preferential expression by PBMC or similar levels of expression between PBMC and synovial fluid T-cells. However, among individual patients (N = 5), several V beta genes were identified that were expressed to a significantly greater degree by synovial fluid cells. V beta 14 expression was detected in PBMC of all patients and the level of transcripts encoding V beta 14 increased following stimulation with immobilized anti-CD3 and IL-2. In vitro manipulation of populations of T-cells was found to alter the level of expressed V beta gene products. A V beta gene common to all patients that was consistently deleted from PBMC or expressed to a greater degree by resident, unsorted synovial fluid cells compared to PBMC was not identified.

Detecting the orientation of short lines in the periphery.

PURPOSE: Visual information processing in the human cortex is based on a highly ordered representation of the surrounding world. In addition to the retinotopic mapping of the visual field, systematic variations of the orientation tuning of neurons have been described in the primary visual cortex. As a step to understanding the relationship between position and orientation representation, we investigated psychophysically the minimum spatial requirements for the determination of orientation at various positions across the visual field. We know that the shortest line whose orientation can be resolved varies with eccentricity, such that its length corresponds to slightly less than 0.2 mm projected onto the cortical surface. Along the horizontal meridian horizontal lines are detected with higher precision than vertical or oblique lines. In the present experiments, we tested whether this is a preference for horizontal lines or for lines that are orientated radially away from the fovea. METHODS: Human observers were tested with lines positioned at one vertical, two horizontal and two oblique meridians at eccentricities between 5 and 25 degrees. RESULTS/CONCLUSION: Three of the four subjects were most sensitive for targets aligned with the meridian of presentation. This suggests that the visual system has the highest resolution in directions radiating from the fovea, which may be particularly useful for the analysis of flow fields resulting from forward translation.

Association of anti-F (ab')2 antibodies (pepsin agglutinators) with immune complexes as determined by enzyme-linked immunosorbent assays.

A solid-phase assay for the detection of anti-F(ab')2 antibodies is described. Wells of microtiter plates are coated with F(ab')2, dilutions of sera are added, and IgG bound to the solid phase is detected using peroxidase-labeled Protein A. Anti-F(ab')2 antibodies were found in 61% of sera from patients with rheumatoid arthritis, 77% with subacute bacterial endocarditis , and 34% with systemic lupus erythematosus. Simultaneous analysis of these sera for immune complexes (IC), using modified Clq and monoclonal rheumatoid factor assays, showed that a correlation existed between anti-F(ab')2 antibodies and the levels of IC. Characterization of anti-F(ab')2 antibodies by inhibition and absorption experiments and by immunological and physical means indicated that they were similar to serum proteins described in the 1960s and designated pepsin agglutinators.

T cell receptor V beta gene expression in monozygotic twins. Discordance in CD8 subset and in disease states.

The peripheral T cell repertoire is shaped by positive and negative selection. These intrathymic events are dependent on the direct interaction of MHC and TCR molecules. Inasmuch as one possible mechanism for HLA-linked disease involves the role that these molecules play in shaping the peripheral T cell repertoire, an understanding of how stable the repertoire remains is an important question that will influence future studies. The purpose of this study was to analyze the stability of the T cell repertoire in monozygotic twins. To investigate this question the percentage of CD4 and CD8 T cells expressing TCR V beta gene products was determined for seven sets of healthy monozygotic twins ages 2 through 44. V beta expression was determined by three-color flow cytometric analysis using antibodies to V beta-5.1, -5.2, -5.3, -6.7, -8, and -12. The percentage of CD4 cells expressing each V beta gene was highly concordant between twins. In contrast, differences were noted for V beta expression within the CD8 subset. This was especially marked when sets of twins were studied (n = 3) where one individual had an underlying disease. Although expression in the CD4 subset was again concordant, significant differences were noted within the CD8 subset compared to the healthy twin. These data indicate that in both health and disease, the CD4 T cell repertoire is tightly regulated although often sizable differences have developed in the CD8 compartment.

CD3+ leukemic large granular lymphocytes utilize diverse T-cell receptor V beta genes.

CD3+ large granular lymphocyte (LGL) leukemia is a disease of unknown etiology characterized by clonal proliferation of T cells that usually express T-cell receptor (TCR) alpha beta heterodimers. The purpose of this study was to identify the variable (V), joining (J), and diversity (D) region TCR beta-chain genes expressed by CD3+ LGL leukemic cells in an attempt to gain insights into the etiology of this disorder. Twelve patients with LGL leukemia were studied, including seven with both LGL leukemia and rheumatoid arthritis (RA). RA is also a disease of unknown etiology that occurs frequently in patients with LGL leukemia. Clonally expanded T cells that express specific TCR V beta genes have been identified in fluid and tissue specimens from the joints of patients with RA. In this study, V beta expression was determined by PCR using a panel of 22 unique V beta primers to amplify cDNA prepared from peripheral blood mononuclear cells (PBMC). A dominant V beta gene product was readily apparent in all patients. To confirm that the dominant V beta gene originated from a clonal expansion, DNA fragments corresponding to the dominant V beta genes were subcloned into plasmids and independently isolated recombinants were sequenced. V-D-J region sequences that occurred repeatedly indicated clonality. The V beta and J beta genes expressed by the leukemic cells showed a pattern of distribution that followed the frequency with which these genes are represented in the peripheral blood. The residues corresponding to the third complementarity-determining region of the TCR beta chain were different in all cases. A specific pattern of VDJ usage was not identified for those patients with both LGL leukemia and RA; however, utilization of V beta-6 by LGL clones (N = 3) was observed only in the setting of RA. These data suggest that leukemic CD3+ LGL cells have been clonally transformed in a random fashion with respect to the TCR beta chain.

Release of interleukin-1 by peripheral blood mononuclear cells in patients with tuberculosis and active inflammation.

Peripheral blood monocytes from patients with active tuberculosis and acute inflammatory disease showed spontaneous interleukin-1 production when compared with those from control patients or healthy controls. Moreover, interleukin-1 production appeared to be a more specific indicator of active disease than were other commonly used indices, such as the erythrocyte sedimentation rate and serum C-reactive protein levels.

Protein kinase A-anchoring inhibitor peptides arrest mammalian sperm motility.

Cyclic AMP-dependent protein kinase (PKA) is anchored at specific subcellular sites through the interaction of the regulatory subunit (R) with protein kinase A-anchoring proteins (AKAPs) via an amphipathic helix binding motif. Synthetic peptides containing this amphipathic helix domain competitively disrupt PKA binding to AKAPs and cause a loss of PKA modulation of cellular responses. In this report we use S-Ht31, a cell-permeant anchoring inhibitor peptide, to study the role of PKA anchoring in sperm. Our analysis of three species of mammalian sperm detected three isoforms of PKA (RIIalpha, RIIbeta, and RIbeta) and one 110-kDa AKAP. The addition of S-Ht31 to bovine caudal epididymal sperm inhibits motility in a time- and concentration-dependent manner. A control peptide, S-Ht31-P, identical to S-Ht31 except for a proline for isoleucine substitution to prevent amphipathic helix formation, had no effect on motility. The inhibition of motility by S-Ht31 is reversible but only if calcium is present in the suspension buffer, suggesting a role for PKA anchoring in regulating cellular calcium homeostasis. Surprisingly, inhibition of PKA catalytic activity had little effect on basal motility or motility stimulated by agents previously thought to work via PKA activation. These data suggest that the interaction of the regulatory subunit of PKA with sperm AKAPs, independent of PKA catalytic activity, is a key regulator of sperm motility and that disruption of this interaction using cell-permeable anchoring inhibitor peptides may form the basis of a sperm-targeted contraceptive.