A whole blood monokine-based reporter assay provides a sensitive and robust measurement of the antigen-specific T cell response

<p>Abstract</p> <p>Background</p> <p>The ability to measure T-cell responses to antigens is proving critical in the field of vaccine development and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of technologies exist for this purpose IFNγ-ELISpot assays are widely used because of their sensitivity and simplicity. However, ELISpot assays cannot be performed on whole blood, and require relatively large volumes of blood to yield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay that measures antigen-specific T cell responses through changes in monokine gene transcription. The biological amplification of the IFNγ signal generated by this assay provides sensitivity comparable to ELISpot, but with the advantage that responses can be quantified using small volumes of whole blood.</p> <p>Methods</p> <p>Whole blood or peripheral blood mononuclear cells (PBMCs) from healthy controls and immunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and control antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in a TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9), IP-10 (CXCL10) and HPRT. The induction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those obtained by ELISpot.</p> <p>Results</p> <p>Antigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression in PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification generated by IFNγ-R signaling allows responses to be detected in as little as 25 μL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing.</p> <p>Conclusions</p> <p>A monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation. Assays can be performed on small volumes of whole blood and remain accurate despite delays in processing. This assay may be a useful tool for studying T cell responses, particularly when samples are limited in quantity or when storage or transportation is required before processing.</p

With a little help from my friends: Developing an assisted automated peritoneal dialysis program in Western Australia

Background: Nurse-assisted automated peritoneal dialysis (AAPD) offers a model of care that has been successfully used in frail dialysis populations internationally. AAPD offers cost savings over hospitalisation on peritoneal dialysis (PD) or in-centre haemodialysis (HD). Method: A pilot AAPD model of care was developed in Western Australia (WA). Patient evaluation was measured utilising a perceptions of dialysis survey, clinical events, hospitalisation and peritonitis rates, Charlson Comorbidity Index (CCI), KDQoL-SF 36 and a survey. Staff opinions and perceived competency were measured by an online survey. Economic analysis was undertaken. Results: A successful collaborative model was developed. 40 staff were trained and competency significantly improved during program delivery (p &lt; 0.0001). 15 patients with an average CCI score of 8.7 used the service for 18 periods of care over 18 months (mean 33 days SD 47). Two non-renal cause deaths and two episodes of peritonitis occurred. Patient opinions were extremely positive. Cost savings were estimated at $620,000. Conclusion: In WA, an AAPD pilot program has been successfully developed and delivered. A sustainable model has overcome initial hurdles. Staff have gained new skills and delivered effective care, demonstrated by high patient acceptance. The program was cost-effective compared to staying in hospital or transferring to HD

The phenotype of circulating follicular-helper T cells in patients with rheumatoid arthritis defines CD200 as a potential therapeutic target

Rheumatoid arthritis (RA) is a systemic autoimmune disease primarily affecting synovial joints in which the development of autoantibodies represents a failure of normal tolerance mechanisms, suggesting a role for follicular helper T cells (TFH) in the genesis of autoimmunity. To determine whether quantitative or qualitative abnormalities in the circulating TFH cell population exist, we analysed by flow cytometry the number and profile of these cells in 35 patients with RA and 15 matched controls. Results were correlated with patient characteristics, including the presence of autoantibodies, disease activity, and treatment with biologic agents. Circulating TFH cells from patients with RA show significantly increased expression of the immunoglobulin superfamily receptor CD200, with highest levels seen in seropositive patients (P=0.0045) and patients treated with anti-TNFα agents (P=0.0008). This occurs in the absence of any change in TFH numbers or overt bias towards Th1, Th2, or Th17 phenotypes. CD200 levels did not correlate with DAS28 scores (P=0.887). Although the number of circulating TFH cells is not altered in the blood of patients with RA, the TFH cells have a distinct phenotype. These differences associate TFH cells with the pathogenesis of RA and support the relevance of the CD200/CD200R signalling pathway as a potential therapeutic target

The Evolving Role of Diagnostic Genomics in Kidney Transplantation

Monogenic forms of heritable kidney disease account for a significant proportion of chronic kidney disease (CKD) across both pediatric and adult patient populations and up to 11% of patients under 40 years reaching end-stage kidney failure (KF) and awaiting kidney transplant. Diagnostic genomics in the field of nephrology is ever evolving and now plays an important role in assessment and management of kidney transplant recipients and their related donor pairs. Genomic testing can help identify the cause of KF in kidney transplant recipients and assist in prognostication around graft survival and rate of recurrence of primary kidney disease. If a gene variant has been identified in the recipient, at-risk related donors can be assessed for the same and excluded if affected. This paper aims to address the indications for genomic testing in the context for kidney transplantation, the technologies available for testing, the conditions and groups in which testing should be most often considered, and the role for the renal genetics multidisci-plinary team in this process

Renal Transplant Immunosuppression Impairs Natural Killer Cell Function In Vitro and In Vivo

Background: Despite an increasing awareness of the importance of innate immunity, the roles of natural killer (NK) cells in transplant rejection and antiviral and cancer immunity during immunosuppression have not been clearly defined. Methods: To address this issue we have developed a quantitative assay of NK cell function that can be used on clinical samples and have studied the influence of immunosuppression on NK cell function. NK cell degranulation and intracellular interferon (IFN)-c production were determined by flow cytometry of peripheral blood samples. Results: Overnight ex vivo treatment of peripheral blood cells from healthy controls with ciclosporin or tacrolimus inhibited NK cell degranulation and IFN-c production in a dose-dependent manner. A similar impairment of function was seen in NK cells from patients treated in vivo with calcineurin inhibitors. In the early post-transplant period, there was a variable reduction of NK cell counts after treatment with alemtuzumab and basiliximab. Conclusions: The functional inhibition of NK cells in early transplant patients coincides with the period of maximum susceptibility to viral infections. The ability to assay NK cell function in clinical samples allows assessment of the impact of immunosuppressio

Analysis of early mesothelial cell responses to Staphylococcus epidermidis isolated from patients with peritoneal dialysis-associated peritonitis

The major complication of peritoneal dialysis (PD) is the development of peritonitis, an infection within the abdominal cavity, primarily caused by bacteria. PD peritonitis is associated with significant morbidity, mortality and health care costs. Staphylococcus epidermidis is the most frequently isolated cause of PD-associated peritonitis. Mesothelial cells are integral to the host response to peritonitis, and subsequent clinical outcomes, yet the effects of infection on mesothelial cells are not well characterised. We systematically investigated the early mesothelial cell response to clinical and reference isolates of S. epidermidis using primary mesothelial cells and the mesothelial cell line Met-5A. Using an unbiased whole genome microarray, followed by a targeted panel of genes known to be involved in the human antibacterial response, we identified 38 differentially regulated genes (adj. p-value &lt; 0.05) representing 35 canonical pathways after 1 hour exposure to S. epidermidis. The top 3 canonical pathways were TNFR2 signaling, IL-17A signaling, and TNFR1 signaling (adj. pvalues of 0.0012, 0.0012 and 0.0019, respectively). Subsequent qPCR validation confirmed significant differences in gene expression in a number of genes not previously described in mesothelial cell responses to infection, with heterogeneity observed between clinical isolates of S. epidermidis, and between Met-5A and primary mesothelial cells. Heterogeneity between different S. epidermidis isolates suggests that specific virulence factors may play critical roles in influencing outcomes from peritonitis. This study provides new insights into early mesothelial cell responses to infection with S. epidermidis, and confirms the importance of validating findings in primary mesothelial cells

Renal infarction caused by medium vessel vasculitis

A 44-year-old Italian man presented to the emergency department on three occasions over 4 days with severe left flank pain. Initial investigations including a renal tract ultrasound were normal and he was discharged with analgaesia. On his third presentation, a CT angiogram was performed due to persisting pain, which demonstrated infarction of his left kidney as well as thickening of the anterior branch of left renal artery and complete occlusion with focal intimal dissection of the coeliac artery. His antineutrophil cytoplasmic antibody was negative. A medium vessel vasculitis was suspected and confirmed on positron emission tomography-CT, which revealed increased metabolic activity involving the right internal mammary artery and coeliac artery. Treatment with pulse methylprednisolone was started followed by a tapering prednisolone regimen, with a rapid reduction in his inflammatory indices. Twenty-four months later his renal function remains normal off all immunosuppression