Stephen P. Young

Nailfold capillary microscopy in healthy children and in childhood rheumatic diseases: a prospective single blind observational study

Objectives: To develop an objective method of nailfold capillaroscopy (NFC), applicable to a wide age range of paediatric patients. To compare the morphological characteristics of the nailfold capillaries in different rheumatology patient groups and controls. Methods: A colour digital video camera attached to a stereomicroscope was used to capture nailfold capillary images. Computerised image processing was used to analyse and store data. Subsequent quantitative and qualitative morphological analysis was performed in the following paediatric patient and control groups: 18 children with connective tissue diseases (CTD: juvenile dermatomyositis, systemic sclerosis, and undifferentiated connective tissue disease), eight with systemic lupus erythematosus, nine with primary Raynaud’s disease, three with primary vasculitis, 15 with juvenile idiopathic arthritis, 17 healthy children and 20 healthy adults. Images were analysed by a single assessor who was unaware of the patient details. Results: The NFC technique was simple to perform and gave reproducible results, although some intra- and intersubject variation was noted. Capillary density and width was age related, with younger children having fewer and wider capillaries than older children and adults. Linear capillary density was significantly higher in healthy adults (mean (SD) 8.6 (1.6) capillaries/mm) compared with healthy children (HC 6.9 (0.9) capillaries/mm). The group with CTD had the most abnormal findings, with lower linear density (4.9 (1.7) capillaries/mm) and increased capillary loop width (10.7 (7.3) mm) compared with HC (3.5 (1.7) mm). In addition, 11/18 (61%) patients in the CTD group had more than two definitely abnormal capillaries in at least two nailfolds, an abnormality not seen in other subjects. Two qualitative measures, the degree of avascularity and general disarrangement of capillary pattern, were more commonly observed in the CTD group than in HC. The proportion of tortuous capillaries did not differ significantly between study groups. Conclusions: This study is unique in measuring objective quantitative and qualitative parameters of the nailfold vasculature across a wide spectrum of age and disease. Differences in capillary morphology and frequency in children with CTD compared with other paediatric diseases and healthy controls were demonstrated. In the clinical situation, an assessment of the general degree of disarrangement may offer a fast tool for assessment of the nailfold vasculature which correlates well with NFC data.

NMR‐based metabolomic analysis of cerebrospinal fluid and serum in neurological diseases – a diagnostic tool?

We sought to evaluate the diagnostic accuracy of metabolomic biomarker profiles in neurological conditions (idiopathic intracranial hypertension (IIH), multiple sclerosis (MS) and cerebrovascular disease (CVD) compared to controls with either no neurological disease or mixed neurological diseases).

Differential association of cytoplasmic signalling molecules SHP-1, SHP-2, SHIP and phospholipase C-γ1 with PECAM-1/CD31

Recent studies have shown that, in addition to its role as an adhesion receptor, platelet endothelial cell adhesion molecule 1/CD31 becomes phosphorylated on tyrosine residues Y663 and Y686 and associates with protein tyrosine phosphatases SHP‐1 and SHP‐2. In this study, we screened for additional proteins which associate with phosphorylated platelet endothelial cell adhesion molecule 1, using surface plasmon resonance. We found that, besides SHP‐1 and SHP‐2, platelet endothelial cell adhesion molecule 1 binds the cytoplasmic signalling proteins SHIP and PLC‐γ1 via their Src homology 2 domains. Using two phosphopeptides, NSDVQpY663TEVQV and DTETVpY686SEVRK, we demonstrate differential binding of SHP‐1, SHP‐2, SHIP and PLC‐γ1. All four cytoplasmic signalling proteins directly associate with cellular platelet endothelial cell adhesion molecule 1, immunoprecipitated from pervanadate‐stimulated THP‐1 cells. These results suggest that overlapping immunoreceptor tyrosine‐based inhibition motif/immunoreceptor tyrosine‐based activation motif‐like motifs within platelet endothelial cell adhesion molecule 1 mediate differential interactions between the Src homology 2 containing signalling proteins SHP‐1, SHP‐2, SHIP and PLC‐γ1.

Metabolic Profiling Predicts Response to Anti–Tumor Necrosis Factor α Therapy in Patients With Rheumatoid Arthritis

Objective Anti–tumor necrosis factor (anti-TNF) therapies are highly effective in rheumatoid arthritis (RA) and psoriatic arthritis (PsA), but a significant number of patients exhibit only a partial or no therapeutic response. Inflammation alters local and systemic metabolism, and TNF plays a role in this. We undertook this study to determine if the patients metabolic fingerprint prior to therapy could predict responses to anti-TNF agents. Methods Urine was collected from 16 RA patients and 20 PsA patients before and during therapy with infliximab or etanercept. Urine metabolic profiles were assessed using nuclear magnetic resonance spectroscopy. Discriminating metabolites were identified, and the relationship between metabolic profiles and clinical outcomes was assessed. Results Baseline urine metabolic profiles discriminated between RA patients who did or did not have a good response to anti-TNF therapy according to European League Against Rheumatism criteria, with a sensitivity of 88.9% and a specificity of 85.7%, with several metabolites contributing (in particular histamine, glutamine, xanthurenic acid, and ethanolamine). There was a correlation between baseline metabolic profiles and the magnitude of change in the Disease Activity Score in 28 joints from baseline to 12 months in RA patients (P = 0.04). In both RA and PsA, urinary metabolic profiles changed between baseline and 12 weeks of anti-TNF therapy. Within the responders, urinary metabolite changes distinguished between etanercept and infliximab treatment. Conclusion The clear relationship between urine metabolic profiles of RA patients at baseline and their response to anti-TNF therapy may allow development of novel approaches to the optimization of therapy. Differences in metabolic profiles during treatment with infliximab and etanercept in RA and PsA may reflect distinct mechanisms of action.

Ceruloplasmin, transferrin and apotransferrin facilitate iron release from human liver cells

The rate of iron release from HepG2 liver cells was increased not only by extracellular apotransferrin, but also by diferric transferrin, in a non‐additive, concentration‐dependent manner and to a similar magnitude. This suggests that rapid equilibration between receptor‐mediated uptake and the release process determines net iron retention by the liver. Release was also accelerated by ceruloplasmin; most importantly, the effect of this protein was greatest when iron release was occurring rapidly, stimulated by apotransferrin, or under conditions of limited oxygen. Thus iron release involves both apotransferrin and ferrotransferrin, with ceruloplasmin playing a role in tissues with limited oxygen supply, as in the liver in vivo.

Old drug, new tricks: haloperidol inhibits secretion of proinflammatory cytokines

OBJECTIVES It was noted that treatment of a patient with acute mania by haloperidol was associated with marked improvement in activity of rheumatoid arthritis. The objective of this study was to examine the effects of haloperidol on inflammatory cytokine release in vitro, as a potential mechanism to explain the in vivo anti-inflammatory effects of haloperidol. METHODS The effect of haloperidol on the production of inflammatory cytokines interleukin 1β (IL1β) and tumour necrosis factor α (TNFα) was measured in bacterial lipopolysaccharide stimulated whole blood cultures and on the promonocyte cell line THP-1, using commercial and in house enzyme linked immunosorbent assays to measure cytokine concentrations. RESULTS Haloperidol inhibited lipopolysaccharide stimulated production of both IL1β and TNFα in vitro in a dose dependent manner and over a prolonged time period. Marked inhibition was seen over a range of concentrations of haloperidol from 0.5 μg/ml to 50 μg/ml, including those predicted to occur in the patient’s blood. CONCLUSIONS Haloperidol treatment seemed to alleviate inflammation in rheumatoid arthritis. In vitro experiments would suggest that the mechanism is by direct inhibition of proinflammatory cytokine release. This phenomenon requires further investigation and may potentially lead to the development of novel treatment.

TNFR1-induced sphingomyelinase activation modulates TCR signaling by impairing store-operated Ca2+ influx

Tumor necrosis factor α (TNF‐α) is a potent, pleiotrophic cytokine, which is proinflammatory but can also suppress T lymphocyte function. In chronic inflammatory disease such as rheumatoid arthritis, exposure of T cells to TNF‐α alters their ability to mount a response by modulating the T cell receptor (TCR) signaling pathway, but the mechanisms involved remain obscure. Here, we investigated the specific role of TNF receptor 1 (TNFR1) signaling in the modulation of the TCR signaling pathway. We observed a down‐regulation of the intracellular calcium ([Ca2+]i) signal in Jurkat T cells after just 30 min exposure to TNF‐α, and maximum suppression was reached after 3 h. This effect was transient, and signals returned to normal after 12 h. This depression of [Ca2+]i was also observed in human CD4+ T lymphocytes. The change in Ca2+ signal was related to a decrease in the plasma membrane Ca2+ influx, which was apparent even when the TCR signal was bypassed using thapsigargin to induce a Ca2+ influx. The role of TNF‐α‐induced activation of the sphingolipid cascade in this pathway was examined. The engagement of TNFR1 by TNF‐α led to a time‐dependent increase in acid sphingomyelinase (SMase; ASM) activity, corresponding with a decrease in cellular sphingomyelin. In parallel, there was an increase in cellular ceramide, which correlated directly with the decrease in the magnitude of the Ca2+ response to phytohemagglutinin. Exogenous addition of SMase or ceramide mimicked the effects of TNFR1 signals on Ca2+ responses in Jurkat T cells. Direct evidence for the activation of ASM in this pathway was provided by complete abrogation of the TNF‐α‐induced inhibition of the Ca2+ influx in an ASM‐deficient murine T cell line (OT‐II+/+ASM−/−). This potent ability of TNF‐α to rapidly modulate the TCR Ca2+ signal via TNFR1‐induced ASM activation can explain its suppressive effect on T cell function. This TNFR1 signaling pathway may play a role as an important regulator of T cell responses.

Metabolomics--a novel window into inflammatory disease.

Inflammation is an important component of normal responses to infection and injury. However, chronic activation of the immune system, due to aberrant responses to normal stimuli, can lead to the establishment of a persistent inflammatory state. Such inflammatory conditions are often debilitating, and are associated with a number of important co-morbidities including cardiovascular disease. Resting non-proliferative tissues have distinctive metabolic activities and requirements, which differ considerably from those in infiltrating immune cells, which are undergoing proliferation and differentiation. Immune responses in tissues may therefore be modulated by the relative abundance of substrates in the inflamed site. In turn immune cell activity can feed back and affect metabolic behaviour of the tissues, as most clearly demonstrated in cachexia - the loss of cellular mass driven by tumour necrosis factor-alpha (TNF-α) a key mediator of the inflammatory response. Here we discuss the potential for metabolomic analysis to clarify the interactions between inflammation and metabolic changes underlying many diseases. We suggest that an increased understanding of the interaction between inflammation and cellular metabolism, energy substrate use, tissue breakdown markers, the microbiome and drug metabolites, may provide novel insight into the regulation of inflammatory diseases.

A Transendocytosis Model of CTLA-4 Function Predicts Its Suppressive Behavior on Regulatory T Cells

Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Although it is clear that CTLA-4 is a critical regulator of T cell responses, the immunological contexts in which CTLA-4 controls immune responses are not well defined. In this study, we show that whereas CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression, in this context CTLA-4 blocking Abs had no impact on the response. In contrast, in settings where CTLA-4+ cells were present as “regulators,” inhibition of resting T cell responses was dependent on CTLA-4 expression and specifically related to the number of APC. At low numbers of APC or low levels of ligand, CTLA-4–dependent suppression was highly effective whereas at higher APC numbers or high levels of ligand, inhibition was lost. Accordingly, the degree of suppression correlated with the level of CD86 expression remaining on the APC. These data reveal clear rules for the inhibitory function of CTLA-4 on regulatory T cells, which are predicted by its ability to remove ligands from APC.

Candidate T cell epitopes of the human La/SSB autoantigen†

OBJECTIVE To identify T cell epitopes of the human La autoantigen involved in the generation of anti-Ro/La autoantibodies. METHODS Molecular techniques were used for HLA typing of 219 white patients with systemic lupus erythematosus and 125 white patients with primary Sjögrens syndrome. Anti-Ro/La antibody levels were measured by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cell responses to an overlapping series of synthetic 15-mer peptides spanning the entire La sequence were examined in pools or individually in conventional 7-day proliferation assays. RESULTS HLA typing confirmed that the HLA-DR3/DQ2 haplotype is closely associated with the occurrence of anti-Ro/La antibodies, and that the frequency of HLA-DR1 and DR4 haplotypes is reduced among antibody-positive patients. We identified 3 regions of the La sequence likely to contain T cell epitopes and 1 peptide, La 49-63, that generated a low-level but clear-cut T cell proliferative response. The HLA restrictions of these responses mirrored the HLA association data from the cohort study. Among individuals who were HLA-DR3 positive, there was no difference between patients and controls in the proliferative response to the La 49-63 peptide. CONCLUSION Our data suggest that these are naive T cell responses, and that the identification of T cell epitopes involved in the generation of anti-Ro/La autoantibodies should focus on alternative candidate antigens.