Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation

Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or intracellular cytokine production, neither of which measure the cytolytic potential of these cells. Here we present a novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide, concordant with production of intracellular IFNgamma. Finally, CD107-expressing CD8+ T cells are shown to mediate cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined with MHC-class I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8+T cells expressing cognate T cell receptors (TCR).     

Negative selection by endogenous antigen and superantigen occurs at multiple thymic sites

The site of negative selection in the thymus has been inferredfrom a range of different experiments. Analysis of thymic deletionof Vß5⁺, V_ß11⁺ or Vß17a⁺ cellsH-2E transgenic mice led to the theory that negative selectionoccurs predominantly in the medulla (specifically, through presentationby medullary dendritic cells). Other experiments investigatedwhether transgenic TCR are deleted at the double-positive (DP)or single-positive stage following encounter with peptide ligand:by flow cytometric analysis deletion is generally found to occurat the DP thymocyte stage and as these cells are found predominantlyin the cortex, it has been inferred that this is the key siteof negative selection. The visualization of apoptotic thymocytesin situ has recently been reported for specific examples ofnegative selection. Using a panel of TCR transgenic lines inwhich negative selection occurs at different stages of thymocytedevelopment, we have used TUNEL staining to analyse the anatomicalsites of thymocyte apoptosis. For the first time we have beenable to compare directly the sites of deletion induced by theendogenous cognate peptides or by endogenous superantigen. Weshow that generalization from the medullary deletion of V_ß5⁺,V_ß11⁺ or V_ß17a⁺ cells by the endogenoussuperantigens Mtv 8 and 9 and from limited examples of corticaldeletion by exogenous peptide administered to TCR transgenicmice is over-simplified. Apoptotic thymocytes in mice lackingMtv superantigens are indeed localized in the cortex. However,when deletion is induced by cognate self peptide, apoptosiscan occur in the cortex, the medulla or at the junction betweenthe two.     

DicomWorks: Software for Reviewing DICOM Studies and Promoting Low-cost Teleradiology

DicomWorks is freeware software for reading and working on medical images [digital imaging and communication in medicine (DICOM)]. It was jointly developed by two research laboratories, with the feedback of more than 35,000 registered users throughout the world who provided information to guide its development. We detail their occupations (50% radiologists, 20% engineers, 9% medical physicists, 7% cardiologists, 6% neurologists, and 8% others), geographic origins, and main interests in the software. The viewer’s interface is similar to that of a picture archiving and communication system viewing station. It provides basic but efficient tools for opening DICOM images and reviewing and exporting them to teaching files or digital presentations. E-mail, FTP, or DICOM protocols are supported for transmitting images through a local network or the Internet. Thanks to its wide compatibility, a localized (15 languages) and user-friendly interface, and its opened architecture, DicomWorks helps quick development of non proprietary, low-cost image review or teleradiology solutions in developed and emerging countries.     

Why should breast surgeons use ultrasound?

Portable ultrasound is now used in a variety of clinical settings by specialties outside of radiology. Despite increased accessibility to ultrasound, the overall performance of ultrasound by breast surgeons is consistently low. We discuss the reasons why this is unacceptable for future patient care and answer the question, ‘Why should breast surgeons use ultrasound?’ We reviewed the literature for evidence assessing the outcomes of breast surgeon-performed ultrasound both intra-operatively and in the outpatient department. Intra-operative ultrasound performed by surgeons reduces re-excision rates in breast-conserving surgery. Outpatient-based ultrasound performed by surgeons frees up the resources of radiology departments, allowing them to focus upon patients requiring more complex diagnostic and interventional procedures. For surgeons to competently perform intra-operative and outpatient-based ultrasound, a period of formal ultrasound training is necessary to acquire knowledge of ultrasound skills and techniques. This should be followed by a period of mentorship and supervised training with an experienced breast radiologist. Breast surgeon-performed ultrasound is beneficial to the multi-disciplinary care of breast cancer patients. To further improve multidisciplinary care, breast surgeons and radiologists should work more collaboratively to optimise imaging applications both in the operating theatre and outpatient department. Current advances in therapeutic percutaneous techniques are of interest to both surgeons and radiologists. In future, a hybrid specialisation should be considered to incorporate accreditation in both specialties for breast interventional procedures.     

Radioactive seed localisation (RSL) in the treatment of non-palpable breast cancers: Systematic review and meta-analysis

IntroductionThis systematic review compares the outcomes of radioactive seed localisation (RSL) versus standard wire-guided localisation (WGL) in the management of non-palpable breast cancers.MethodsWe performed a literature search of PubMed, EMBASE and the Cochrane database to identify clinical studies using RSL. Included studies examined invasive breast cancer and reported objective pathological outcome measurements. Quantitative data analyses were performed.Results197 papers were identified with eight being clinically relevant to our study. From these eight studies there was only one identified as being a randomised controlled trial (RCT) and four as cohort studies having a control WGL group and included in the quantitative analysis. This provided an overall combined odds ratio (OR) 0.51 (95% CI, 0.36–0.72; z = 3.88; p = 0.0001) for involved surgical margin status; OR, 0.47 (95% CI, 0.33–0.69; z = 3.96; p < 0.0001) for re-operation rates and mean difference (MD) ?1.32 (95% CI, ?2.32, ?0.32; z = 2.58; p = 0.01) for operative time favouring RSL over WGL. In the case of volume of specimens excised, MD 1.46; (95% CI, ?22.35, 25.26; z = 0.12; p = 0.90) showing no statistical significance for volume of tissue excised in specimens between the two groups.ConclusionsThe results of this systematic review demonstrate a statistically significant benefit of RSL over the gold standard of WGL in terms of involved margin status, re-operation rates and reduced operative time but no statistically significant difference with WGL in terms of volume of tissue excised in the treatment of non-palpable breast cancers. Adequately powered, multicentre RCTs are needed to validate these results.     

Effect of HIV on thymic function before and after antiretroviral therapy in children

Studies were undertaken to investigate the role of the thymus in T cell reconstitution in human immunodeficiency virus (HIV)-infected children treated with antiretroviral therapy. Nine pediatric patients who acquired HIV perinatally were treated with multidrug combinations of antiretroviral agents. Plasma virus load and CD4+ and CD8+ T cell subsets were measured, and thymus function was measured by quantifying T cell receptor rearrangement excision circles in peripheral blood. Patients with virus loads remaining >400 RNA copies/mL plasma were classified as virologic nonresponders. Thymus function was initially decreased in all subjects. After antiretrovirus therapy, peripheral CD4+ T cells increased in all subjects. Thymus function was restored in 4 of 5 virologic responders but in only 1 of 4 virologic nonresponders. This suggests that HIV has an adverse effect upon thymic function in pediatric HIV infection. Potent antiretroviral therapy restores thymic function but is affected by the degree to which virus suppression is achieved.     

Immune activation and HIV persistence: implications for curative approaches to HIV infection

Despite complete or near-complete suppression of human immunodeficiency virus (HIV) replication with combination antiretroviral therapy, both HIV and chronic inflammation/immune dysfunction persist indefinitely. Untangling the association between the virus and the host immune environment during therapy might lead to novel interventions aimed at either curing the infection or preventing the development of inflammation-associated end-organ disease. Chronic inflammation and immune dysfunction might lead to HIV persistence by causing virus production, generating new target cells, enabling infecting of activated and resting target cells, altering the migration patterns of susceptible target cells, increasing the proliferation of infected cells, and preventing normal HIV-specific clearance mechanisms from function. Chronic HIV production or replication might contribute to persistent inflammation and immune dysfunction. The rapidly evolving data on these issues strongly suggest that a vicious cycle might exist in which HIV persistence causes inflammation that in turn contributes to HIV persistence.     

Thymus transplantation restores the repertoires of forkhead box protein 3 (FoxP3)+ and FoxP3− T cells in complete DiGeorge anomaly

The development of T cells with a regulatory phenotype after thymus transplantation has not been examined previously in complete DiGeorge anomaly (cDGA). Seven athymic infants with cDGA and non-maternal pretransplantation T cell clones were assessed. Pretransplantation forkhead box protein 3 (Foxp3)⁺ T cells were detected in five of the subjects. Two subjects were studied in greater depth. T cell receptor variable β chain (TCR-Vβ) expression was assessed by flow cytometry. In both subjects, pretransplantation FoxP3⁺ and total CD4⁺ T cells showed restricted TCR-Vβ expression. The development of naive T cells and diverse CD4⁺ TCR-Vβ repertoires following thymic transplantation indicated successful thymopoiesis from the thymic tissue grafts. Infants with atypical cDGA develop rashes and autoimmune phenomena before transplantation, requiring treatment with immunosuppression, which was discontinued successfully subsequent to the observed thymopoiesis. Post-transplantation, diverse TCR-Vβ family expression was also observed in FoxP3⁺ CD4⁺ T cells. Interestingly, the percentages of each of the TCR-Vβ families expressed on FoxP3⁺ and total CD4⁺ T cells differed significantly between these T lymphocyte subpopulations before transplantation. By 16 months post-transplantation, however, the percentages of expression of each TCR-Vβ family became significantly similar between FoxP3⁺ and total CD4⁺ T cells. Sequencing of TCRBV DNA confirmed the presence of clonally amplified pretransplantation FoxP3⁺ and FoxP3⁻ T cells. After thymus transplantation, increased polyclonality was observed for both FoxP3⁺ and FoxP3⁻ cells, and pretransplantation FoxP3⁺ and FoxP3⁻ clonotypes essentially disappeared. Thus, post-transplantation thymic function was associated with the development of a diverse repertoire of FoxP3⁺ T cells in cDGA, corresponding with immunological and clinical recovery.     

Pre-operative sentinel lymph node localization in breast cancer with superparamagnetic iron oxide MRI: the SentiMAG Multicentre Trial imaging subprotocol

Objective:

Sentinel lymph node biopsy (SLNB) with a superparamagnetic iron oxide (SPIO) tracer was shown to be non-inferior to the standard combined technique in the SentiMAG Multicentre Trial. The MRI subprotocol of this trial aimed to develop a magnetic alternative for pre-operative lymphoscintigraphy (LS). We evaluated the feasibility of using MRI following the administration of magnetic tracer for pre-operative localization of sentinel lymph nodes (SLNs) and its potential for non-invasive identification of lymph node (LN) metastases.

Methods:

Patients with breast cancer scheduled to undergo SLNB were recruited for pre-operative LS, single photon emission CT (SPECT)-CT and SPIO MRI. T₁ weighted turbo spin echo and T₂ weighted gradient echo sequences were used before and after interstitial injection of magnetic tracer into the breast. SLNs on MRI were defined as LNs with signal drop and direct lymphatic drainage from the injection site. LNs showing inhomogeneous SPIO uptake were classified as metastatic. During surgery, a handheld magnetometer was used for SLNB. Blue or radioactive nodes were also excised. The number of SLNs and MR assessment of metastatic involvement were compared with surgical and histological outcomes.

Results:

11 patients were recruited. SPIO MRI successfully identified SLNs in 10 of 11 patients vs 11 of 11 patients with LS/SPECT-CT. One patient had metastatic involvement of four LNs, and this was identified in one node on pre-operative MRI.

Conclusion:

SPIO MRI is a feasible technique for pre-operative localization of SLNs and, in combination with intraoperative use of a handheld magnetometer, provides an entirely radioisotope-free technique for SLNB. Further research is needed for the evaluation of MRI characterization of LN involvement using subcutaneous injection of magnetic tracer.

Advances in knowledge:

This study is the first to demonstrate that an interstitially administered magnetic tracer can be used both for pre-operative imaging and intraoperative SLNB, with equal performance to imaging and localization with radioisotopes.