TCRß sequencing reveals spatial and temporal evolution of clonal CD4 T cell responses in a breach of tolerance model of inflammatory arthritis

Effective tolerogenic intervention in Rheumatoid Arthritis (RA) will rely upon understanding the evolution of articular antigen specific CD4 T cell responses. TCR clonality of endogenous CD4 T cell infiltrates in early inflammatory arthritis was assessed to monitor evolution of the TCR repertoire in the inflamed joint and associated lymph node (LN). Mouse models of antigen-induced breach of self-tolerance and chronic polyarthritis were used to recapitulate early and late phases of RA. The infiltrating endogenous, antigen experienced CD4 T cells in inflamed joints and LNs were analysed using flow cytometry and TCRβ sequencing. TCR repertoires from inflamed late phase LNs displayed increased clonality and diversity compared to early phase LNs, while inflamed joints remained similar with time. Repertoires from late phase LNs accumulated clones with a diverse range of TRBV genes, while inflamed joints at both phases contained clones expressing similar TRBV genes. Repertoires from LNs and joints at the late phase displayed reduced CDR3β sequence overlap compared to the early disease phase, however the most abundant clones in LNs accumulate in the joint at the later phase. The results indicate CD4 T cell repertoire clonality and diversity broadens with progression of inflammatory arthritis and is first reflected in LNs before mirroring in the joint. These observations imply that antigen specific tolerogenic therapies could be more effective if targeted at earlier phases of disease when CD4 T cell clonality is least diverse

Cost-effectiveness of magnetic resonance-guided focused ultrasound surgery for treatment of uterine fibroids

OBJECTIVE: To estimate the cost-effectiveness of a treatment strategy for symptomatic uterine fibroids, which starts with Magnetic Resonance-guided Focused Ultrasound Surgery (MRgFUS) as compared with current practice comprising uterine artery embolisation, myomectomy and hysterectomy. DESIGN: Cost-utility analysis based on a Markov model. SETTING: National Health Service (NHS) Trusts in England and Wales. POPULATION: Women for whom surgical treatment for uterine fibroids is being considered. METHODS: The parameters of the Markov model of the treatment of uterine fibroids are drawn from a series of clinical studies of MRgFUS, and from the clinical effectiveness literature. Health-related quality of life is measured using the 6D. Costs are estimated from the perspective of the NHS. The impact of uncertainty is examined using deterministic and probabilistic sensitivity analysis. MAIN OUTCOME MEASURES: Incremental cost-effectiveness measured by cost per quality-adjusted life-year (QALY) gained. RESULTS: The base-case results imply a cost saving and a small QALY gain per woman as a result of an MRgFUS treatment strategy. The cost per QALY gained is sensitive to cost of MRgFUS relative to other treatments, the age of the woman and the nonperfused volume relative to the total fibroids volume. CONCLUSIONS: A treatment strategy for symptomatic uterine fibroids starting with MRgFUS is likely to be cost-effective

Molecular interactions of the plasma membrane calcium ATPase 2 at pre- and post-synaptic sites in rat cerebellum.

The plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events [Strehler EE, Filoteo AG, Penniston JT, Caride AJ (2007) Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 35 (Pt 5):919-922], its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre- and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull-down approaches confirmed that PMCA2 interacts with the post-synaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its C-terminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well-known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull-down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95-PMCA2 and syntaxin-1A-PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre- and post-synaptic signalling complexes

Preclinical models of arthritis for studying immunotherapy and immune tolerance

Increasingly earlier identification of individuals at high risk of rheumatoid arthritis (RA) (eg, with autoantibodies and mild symptoms) improves the feasibility of preventing or curing disease. The use of antigen-specific immunotherapies to reinstate immunological self-tolerance represent a highly attractive strategy due to their potential to induce disease resolution, in contrast to existing approaches that require long-term treatment of underlying symptoms. Preclinical animal models have been used to understand disease mechanisms and to evaluate novel immunotherapeutic approaches. However, models are required to understand critical processes supporting disease development such as the breach of self-tolerance that triggers autoimmunity and the progression from asymptomatic autoimmunity to joint pain and bone loss. These models would also be useful in evaluating the response to treatment in the pre-RA period. This review proposes that focusing on immune processes contributing to initial disease induction rather than end-stage pathological consequences is essential to allow development and evaluation of novel immunotherapies for early intervention. We will describe and critique existing models in arthritis and the broader field of autoimmunity that may fulfil these criteria. We will also identify key gaps in our ability to study these processes in animal models, to highlight where further research should be targeted

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

The appropriateness of gatekeeping in the provision of reproductive health care for adolescents in Lithuania:the general practice perspective

BACKGROUND: Adolescents' consultation of primary health care services remains problematic despite their accessibility. The reproductive health service seeking behavior of adolescents is the object of much research but little is known about how this behavior is influenced by the gatekeeping system. This study aimed to explore general practitioners' perceptions of the appropriateness of gatekeeping in adolescent reproductive health care. METHODS: Twenty in-depth interviews regarding factors affecting adolescent reproductive health care were carried out on a diverse sample of general practitioners and analyzed using grounded theory. RESULTS: The analysis identified several factors that shaped GPs' negative attitude to gatekeeping in adolescent reproductive health care. Its appropriateness in this field was questionable due to a lack of willingness on the part of GPs to provide reproductive health services for teenagers, their insufficient training, inadequately equipped surgeries and low perceived support for reproductive health service provision. CONCLUSION: Since factors for improving adolescent reproductive health concern not only physicians but also the health system and policy levels, complex measures should be designed to overcome these barriers. Discussion of a flexible model of gatekeeping, encompassing both co-ordination of care provided by GPs and the possibility of patients' self-referral, should be included in the political agenda. Adolescents tend to under-use rather than over-use reproductive health services and every effort should be made to facilitate the accessibility of such services

CCL25/CCR9 Interactions Regulate Large Intestinal Inflammation in a Murine Model of Acute Colitis

CCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine.Acute inflammation and recovery in wild-type (WT) and CCR9(-/-) mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level.CCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9(-/-) mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9(-/-) colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9(-/-) animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNFα, IFNγ, IL2, IL-6 and IL-17A while down modulating IL-10 production.Our results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation

The Accuracy of Survival Time Prediction for Patients with Glioma Is Improved by Measuring Mitotic Spindle Checkpoint Gene Expression

Identification of gene expression changes that improve prediction of survival time across all glioma grades would be clinically useful. Four Affymetrix GeneChip datasets from the literature, containing data from 771 glioma samples representing all WHO grades and eight normal brain samples, were used in an ANOVA model to screen for transcript changes that correlated with grade. Observations were confirmed and extended using qPCR assays on RNA derived from 38 additional glioma samples and eight normal samples for which survival data were available. RNA levels of eight major mitotic spindle assembly checkpoint (SAC) genes (BUB1, BUB1B, BUB3, CENPE, MAD1L1, MAD2L1, CDC20, TTK) significantly correlated with glioma grade and six also significantly correlated with survival time. In particular, the level of BUB1B expression was highly correlated with survival time (p<0.0001), and significantly outperformed all other measured parameters, including two standards; WHO grade and MIB-1 (Ki-67) labeling index. Measurement of the expression levels of a small set of SAC genes may complement histological grade and other clinical parameters for predicting survival time

Prophylactic ciprofloxacin treatment prevented high mortality, and modified systemic and intestinal immune function in tumour-bearing rats receiving dose-intensive CPT-11 chemotherapy

Infectious complications are a major cause of morbidity and mortality from dose-intensive cancer chemotherapy. In spite of the importance of intestinal bacteria translocation in these infections, information about the effect of high-dose chemotherapy on gut mucosal immunity is minimal. We studied prophylactic ciprofloxacin (Cipro) treatment on irinotecan (CPT-11) toxicity and host immunity in rats bearing Ward colon tumour. Cipro abolished chemotherapy-related mortality, which was 45% in animals that were not treated with Cipro. Although Cipro reduced body weight loss and muscle wasting, it was unable to prevent severe late-onset diarrhoea. Seven days after CPT-11, splenocytes were unable to proliferate (stimulation index=0.10±0.02) and produce proliferative and inflammatory cytokines (i.e., Interleukin (IL)-2, interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) IL-1β, IL-6) on mitogen stimulation in vitro (P<0.05 vs controls), whereas mesenteric lymph node (MLN) cells showed a hyper-proliferative response and a hyper-production of pro-inflammatory cytokines on mitogen stimulation. This suggests compartmentalised effects by CPT-11 chemotherapy on systemic and intestinal immunity. Cipro normalised the hyper-responsiveness of MLN cells, and in the spleen, it partially restored the proliferative response and normalised depressed production of IL-1β and IL-6. Taken together, Cipro prevented infectious challenges associated with immune hypo-responsiveness in systemic immune compartments, and it may also alleviate excessive pro-inflammatory responses mediating local gut injury

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention