Themis2 lowers the threshold for B cell activation during positive selection.

The positive and negative selection of lymphocytes by antigen is central to adaptive immunity and self-tolerance, yet how this is determined by different antigens is not completely understood. We found that thymocyte-selection-associated family member 2 (Themis2) increased the positive selection of B1 cells and germinal center B cells by self and foreign antigens. Themis2 lowered the threshold for B-cell activation by low-avidity, but not high-avidity, antigens. Themis2 constitutively bound the adaptor protein Grb2, src-kinase Lyn and signal transducer phospholipase γ2 (PLC-γ2), and increased activation of PLC-γ2 and its downstream pathways following B cell receptor stimulation. Our findings identify a unique function for Themis2 in differential signaling and provide insight into how B cells discriminate between antigens of different quantity and quality.

Cancer control in the Caribbean island countries and territories: some progress but the journey continues.

Cancer causes a fifth of deaths in the Caribbean region and its incidence is increasing. Incidence and mortality patterns of cancer in the Caribbean reflect globally widespread epidemiological transitions, and show cancer profiles that are unique to the region. Providing comprehensive and locally responsive cancer care is particularly challenging in the Caribbean because of the geographical spread of the islands, the frequently under-resourced health-care systems, and the absence of a cohesive approach to cancer control. In many Caribbean countries and territories, cancer surveillance systems are poorly developed, advanced disease presentations are commonplace, and access to cancer screening, diagnostics, and treatment is often suboptimal, with many patients with cancer seeking treatment abroad. Capacity building across the cancer-control continuum in the region is urgently needed and can be accomplished through collaborative efforts and increased investment in health care and cancer control.

Development of a robust immuno-S-FISH protocol using imaging flow cytometry.

Fluorescence in situ hybridization (FISH) is a microscopy technique which uses a fluorescent probe to detect DNA sequences and is generally performed on metaphase spreads or interphase nuclei of intact cells on a slide. In a diagnostic laboratory, cells are hybridized with fluorescent probes and up to 200 cells counted for the number of cells with probe "spots." Recent modifications to standard FISH include immuno-FISH, where chromosomal abnormalities are detected only in cells by their phenotype, and S-FISH where probe hybridization is performed on whole cells in suspension. Here we describe the development of an immuno-S-FISH method that combines immunophenotyping and FISH analysis of cells in suspension followed by analysis on an imaging flow cytometer. This single platform technique couples microscopy with flow cytometry and "spot" detection of bound FISH probe. Automated immuno-S-FISH enables large numbers of analyzed cells to be identified by phenotype and assessed for specific chromosomal determinants by FISH. This novel robust method enables quantitative cell population analysis and "spot" counting for large numbers of cells. We report method optimization of this imaging immuno-S-FISH flow cytometry protocol which has capability for many clinical applications. © 2016 International Society for Advancement of Cytometry.

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 (T(FH)) in the genesis of autoimmunity. To determine whether quantitative or qualitative abnormalities in the circulating T(FH) 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 T(FH) 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 T(FH) 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 T(FH) cells is not altered in the blood of patients with RA, the T(FH) cells have a distinct phenotype. These differences associate T(FH) cells with the pathogenesis of RA and support the relevance of the CD200/CD200R signalling pathway as a potential therapeutic target.

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

BACKGROUND: 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, ELISpots 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. METHODS: 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. RESULTS: 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 uL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing. CONCLUSIONS: 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 are required before processing.

Studying vertebrate topoisomerase 2 function using a conditional knockdown system in DT40 cells.

DT40 is a B-cell lymphoma-derived avian cell line widely used to study cell autonomous gene function because of the high rates with which DNA constructs are homologously recombined into its genome. Here, we demonstrate that the power of the DT40 system can be extended yet further through the use of RNA interference as an alternative to gene targeting. We have generated and characterized stable DT40 transfectants in which both topo 2 genes have been in situ tagged using gene targeting, and from which the mRNA of both topoisomerase 2 isoforms can be conditionally depleted through the tetracycline-induced expression of short hairpin RNAs. The cell cycle phenotype of topo 2-depleted DT40 cells has been compared with that previously reported for other vertebrate cells depleted either of topo 2alpha through gene targeting, or depleted of both isoforms simultaneously by transient RNAi. In addition, the DT40 knockdown system has been used to explore whether excess catenation arising through topo 2 depletion is sufficient to trigger the G2 catenation (or decatenation) checkpoint, proposed to exist in differentiated vertebrate cells.

Teaching provision for old age psychiatry in medical schools in the UK and Ireland: a survey.

Aims and method This work builds on a survey first done in 1999 to understand how old age psychiatry teaching is embedded in undergraduate medical schools in the UK and Ireland and the influence of academic old age psychiatrists on teaching processes. We invited deans of 31 medical schools in the UK and Ireland in 2015 to complete an online survey to reassess the situation 16 years later. Results Response rate was 74%. As found in the original survey, there was variation across medical schools in how old age psychiatry is taught. Half of schools stated there was not enough space in the curriculum dedicated to old age psychiatry, and not all medical school curricula offered a clinical attachment. Medical schools that involved academic old age psychiatrists in teaching (59%) showed a greater diversity of teaching methods. Clinical implications There is a need to recognise the importance of old age psychiatry teaching, with the consensus of opinion continuing to be that more curriculum space needs to be given to old age psychiatry. To achieve this we advocate increasing the number of old age psychiatrists with teaching roles, as relying on academics to teach and lead on curriculum development is challenging given their greater research pressures.

Role of Internal Mammary Node Radiation as a Part of Modern Breast Cancer Radiation Therapy: A Systematic Review.

PURPOSE: Despite data from multiple randomized trials, the role of internal mammary lymph node irradiation as a part of regional nodal irradiation (IMLN RT-RNI) remains unanswered. Recent noteworthy data and modern RT techniques might identify a subset of patients who will benefit from IMLN RT-RNI, lending insight into the balance between improved outcomes and acceptable toxicity. We evaluated the current role of IMLN RT-RNI by analyzing randomized, prospective, and retrospective data. METHODS AND MATERIALS: In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a review of the published data was performed using PubMed to evaluate published studies from 1994 to 2015. The information evaluated included the number of patients, follow-up period, technical aspects of RT, and outcomes (clinical outcomes, complications/toxicity). RESULTS: We included 16 studies (4 randomized, 4 nonrandomized, 7 retrospective, and 1 meta-analysis). Although older randomized trials failed to show differences in clinical outcomes or toxicity with IMLN RT-RNI, recent randomized data suggest the potential for improved outcomes, including overall survival, with IMLN RT-RNI. Furthermore, nonrandomized data have suggested a potential benefit for central tumors with IMLN RT-RNI. Although recent data have suggested a potential increase in pulmonary complications with IMLN RT-RNI with the use of advanced radiation techniques, toxicity rates remain low with limited cardiac toxicity data available. CONCLUSIONS: Increasing data from recent randomized trials support the use of IMLN RT-RNI. IMLN RT can be considered based on the inclusion of IMLN RT as a part of RNI in recent trials and the inclusion criteria from IMLN RT-RNI trials and for patients with central or medial tumors and axillary disease.

Preclinical assessment of adjunctive tPA and DNase for peritoneal dialysis associated peritonitis.

A major complication of peritoneal dialysis is the development of peritonitis, which is associated with reduced technique and patient survival. The inflammatory response elicited by infection results in a fibrin and debris-rich environment within the peritoneal cavity, which may reduce the effectiveness of antimicrobial agents and predispose to recurrence or relapse of infection. Strategies to enhance responses to antimicrobial agents therefore have the potential to improve patient outcomes. This study presents pre-clinical data describing the compatibility of tPA and DNase in combination with antimicrobial agents used for the treatment of PD peritonitis. tPA and DNase were stable in standard dialysate solution and in the presence of antimicrobial agents, and were safe when given intraperitoneally in a mouse model with no evidence of local or systemic toxicity. Adjunctive tPA and DNase may have a role in the management of patients presenting with PD peritonitis.

Depression and dementia: cause, consequence or coincidence?

The relationship between depression and dementia is complex and still not well understood. A number of different views exist regarding how the two conditions are linked as well as the underlying neurobiological mechanisms at work. This narrative review examined longitudinal and cross sectional studies in the existing literature and determined the evidence supporting depression being a risk factor, a prodrome, a consequence, or an independent comorbidity in dementia. Overall there is convincing evidence to support both the notion that early life depression can act as a risk factor for later life dementia, and that later life depression can be seen as a prodrome to dementia. There is also evidence to support both conditions showing similar neurobiological changes, particularly white matter disease, either indicating shared risk factors or a shared pattern of neuronal damage. These findings highlight the need to examine if effective treatment of depressive episodes has any effect in reducing the prevalence of dementia, as well as clinicians being vigilant for late life depression indicating the incipient development of dementia, and therefore carefully following up these individuals for future cognitive impairment.

Mutations in TCF12, encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis.

Craniosynostosis, the premature fusion of the cranial sutures, is a heterogeneous disorder with a prevalence of ∼1 in 2,200 (refs. 1,2). A specific genetic etiology can be identified in ∼21% of cases, including mutations of TWIST1, which encodes a class II basic helix-loop-helix (bHLH) transcription factor, and causes Saethre-Chotzen syndrome, typically associated with coronal synostosis. Using exome sequencing, we identified 38 heterozygous TCF12 mutations in 347 samples from unrelated individuals with craniosynostosis. The mutations predominantly occurred in individuals with coronal synostosis and accounted for 32% and 10% of subjects with bilateral and unilateral pathology, respectively. TCF12 encodes one of three class I E proteins that heterodimerize with class II bHLH proteins such as TWIST1. We show that TCF12 and TWIST1 act synergistically in a transactivation assay and that mice doubly heterozygous for loss-of-function mutations in Tcf12 and Twist1 have severe coronal synostosis. Hence, the dosage of TCF12-TWIST1 heterodimers is critical for normal coronal suture development.

Controversies on cosmetic outcomes in black women after breast conservation therapy: hyperperception or hyperpigmentation?

Multiple studies have reported inferior cosmetic outcomes after breast conservation surgery and adjuvant radiation therapy in black women. However, cosmetic analysis scales contemporarily utilized in the field of radiation oncology rely largely on subjective visual and tactile perception. These methods are undeniably fraught with intraobserver and interobserver variability. Herein, we uncover how and why these methods may unwittingly and disparately misjudge cosmetic outcomes in black women, and the clinical ramifications thereof. In addition, we highlight more objective cosmetic outcomes assessment programs that promise to yield more reproducible and unbiased results.

Detection of polyomavirus BK reactivation after renal transplantation using an intensive decoy cell surveillance program is cost-effective.

BACKGROUND: Reactivation of polyomavirus BK (BKV) after renal transplantation can lead to allograft dysfunction or loss with early detection improving outcomes. Current guidelines recommend quantitative polymerase chain reaction for surveillance; however, urinary decoy cell detection is a potentially cost-effective alternative. We present the outcomes from an early intensive BKV surveillance program using decoy cell detection for initial screening starting 2 weeks after transplantation. METHODS: Records for all recipients of kidney (n=211) or simultaneous kidney and pancreas (n=102) transplants performed over 2 years in a single center were reviewed. Follow-up was for a minimum of 1 year. Urine cytology screening was performed fortnightly from 0 to 3 months after transplantation, monthly from 3 to 6 months then every 2 months from 6 to 12 months. RESULTS: Decoy cell positivity occurred in 56 of 313 patients (17.9%) with sustained decoy cell positivity (≥2 positive urine samples >2 weeks apart) present in 32 patients (10.2%). Twenty-four patients (7.6%) became viremic and three patients (1%) developed polyoma virus nephropathy. The median time after transplantation until decoy cell positivity was 78 days, decreasing to 67 days for patients with sustained positivity and 57 days for patients who developed polyoma virus nephropathy. No grafts were lost due to BKV during the study period. Decoy cell screening resulted in savings of approximately £135,000 over 2 years, when compared with routine surveillance by quantitative polymerase chain reaction. CONCLUSIONS: Clinically significant BKV reactivation occurs early after transplantation and can be reliably detected by decoy cell screening. A surveillance strategy for detecting BKV reactivation based on urine cytology is cost-effective.

Optimization of Adjuvant Radiation in Breast Conservation Therapy: Can We Minimize without Compromise?

Adjuvant breast radiation therapy after breast conservation surgery is recommended as it yields significant reduction in the risk of local recurrence, and confers a potential overall survival benefit. Although the standard breast radiation regimen has historically been delivered over 5-7 weeks; more novel, shorter courses of breast radiation are currently being employed, offering the advantage of more convenience and less time-commitment. Herein, we review the recent literature substantiating these abbreviated radiation treatment approaches and the methods of delivery thereof. In addition, we discuss imaged guided techniques currently being utilized to further refine the delivery of adjuvant breast radiation therapy.

Patterns of utilization of adjuvant radiotherapy and outcomes in black women after breast conservation at a large multidisciplinary cancer center.

PURPOSE: Population-based studies have reported that as many of 35% of black women do not undergo radiotherapy (RT) after breast conservation surgery (BCS). The objective of the present study was to determine whether this trend persisted at a large multidisciplinary cancer center, and to identify the factors that predict for noncompliance with RT and determine the outcomes for this subset of patients. METHODS AND MATERIALS: Between January 2002 and December 2007, 83 black women underwent BCS at Memorial Sloan-Kettering Cancer Center and were therefore eligible for the present study. Of the 83 women, 38 (46%) had Stage I, 38 (46%) Stage II, and 7 (8%) Stage III disease. Of the study cohort, 31 (37%) had triple hormone receptor-negative tumors. RT was recommended for 81 (98%) of the 83 patients (median dose, 60 Gy). RESULTS: Of the 81 women, 12 (15%) did not receive the recommended adjuvant breast RT. Nonreceipt of chemotherapy (p = .003) and older age (p = .009) were associated with nonreceipt of RT. With a median follow-up of 70 months, the 3-year local control, locoregional control, recurrence-free survival, disease-free survival, and overall survival rate was 99% (actuarial 5-year rate, 97%), 96% (actuarial 5-year rate, 93%), 95% (actuarial 5-year rate, 92%), 92% (actuarial 5-year rate, 89%), and 95% (actuarial 5-year rate, 91%), respectively. CONCLUSION: We found a greater rate of utilization adjuvant breast RT (85%) among black women after BCS than has been reported in recent studies, indicating that excellent outcomes are attainable for black women after BCS when care is administered in a multidisciplinary cancer center.

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)-γ 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-γ 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 immunosuppression on these effector cells. This information may be helpful in guiding the titration of immunosuppression in the clinical setting.