Evidence for Innate and Adaptive Immune Responses in a Cohort of Intractable Pediatric Epilepsy Surgery Patients.

Brain-infiltrating lymphocytes (BILs) were isolated from resected brain tissue from 10 pediatric epilepsy patients who had undergone surgery for Hemimegalencephaly (HME) (n = 1), Tuberous sclerosis complex (TSC) (n = 2), Focal cortical dysplasia (FCD) (n = 4), and Rasmussen encephalitis (RE) (n = 3). Peripheral blood mononuclear cells (PBMCs) were also isolated from blood collected at the time of the surgery. Cells were immunostained with a panel of 20 antibody markers, and analyzed by mass cytometry. To identify and quantify the immune cell types in the samples, an unbiased clustering method was applied to the entire data set. More than 85 percent of the CD45+ cells isolated from resected RE brain tissue comprised T cells; by contrast NK cells and myeloid cells constituted 80-95 percent of the CD45+ cells isolated from the TSC and the FCD brain specimens. Three populations of myeloid cells made up >50 percent of all of the myeloid cells in all of the samples of which a population of HLA-DR+ CD11b+ CD4- cells comprised the vast majority of myeloid cells in the BIL fractions from the FCD and TSC cases. CD45RA+ HLA-DR- CD11b+ CD16+ NK cells constituted the major population of NK cells in the blood from all of the cases. This subset also comprised the majority of NK cells in BILs from the resected RE and HME brain tissue, whereas NK cells defined as CD45RA- HLA-DR+ CD11b- CD16- cells comprised 86-96 percent of the NK cells isolated from the FCD and TSC brain tissue. Thirteen different subsets of CD4 and CD8 αβ T cells and γδ T cells accounted for over 80% of the CD3+ T cells in all of the BIL and PBMC samples. At least 90 percent of the T cells in the RE BILs, 80 percent of the T cells in the HME BILs and 40-66 percent in the TSC and FCD BILs comprised activated antigen-experienced (CD45RO+ HLA-DR+ CD69+) T cells. We conclude that even in cases where there is no evidence for an infection or an immune disorder, activated peripheral immune cells may be present in epileptogenic areas of the brain, possibly in response to seizure-driven brain inflammation

Quantitative PET reporter gene imaging of CD8+ T cells specific for a melanoma-expressed self-antigen

Adoptive transfer (AT) T-cell therapy provides significant clinical benefits in patients with advanced melanoma. However, approaches to non-invasively visualize the persistence of transferred T cells are lacking. We examined whether positron emission tomography (PET) can monitor the distribution of self-antigen-specific T cells engineered to express an herpes simplex virus 1 thymidine kinase (sr39tk) PET reporter gene. Micro-PET imaging using the sr39tk-specific substrate 9-[4-[18F]fluoro-3-(hydroxymethyl)-butyl]guanine ([18F]FHBG) enabled the detection of transplanted T cells in secondary lymphoid organs of recipient mice over a 3-week period. Tumor responses could be predicted as early as 3 days following AT when a >25-fold increase of micro-PET signal in the spleen and 2-fold increase in lymph nodes (LNs) were observed in mice receiving combined immunotherapy versus control mice. The lower limit of detection was ∼7 × 105 T cells in the spleen and 1 × 104 T cells in LNs. Quantification of transplanted T cells in the tumor was hampered by the sr39tk-independent trapping of [18F]FHBG within the tumor architecture. These data support the feasibility of using PET to visualize the expansion, homing and persistence of transferred T cells. PET may have significant clinical utility by providing the means to quantify anti-tumor T cells throughout the body and provide early correlates for treatment efficacy

RELIABILITY AND ACCURACY OF A STANDARDIZED SHALLOW WATER RUNNING TEST TO DETERMINE CARDIORESPIRATORY FITNESS

A standardized fitness assessment is critical for the development of an individualized exercise prescription. Although the benefits of aquatic exercise have been well established, there remains the need for a standardized nonswimming protocol to accurately assess cardiorespiratory fitness (CRF) in shallow water. The present investigation was designed to assess (a) the reliability of a standardized shallow water run (SWR) test of CRF and (b) the accuracy of a standardized SWR compared with a land-based treadmill (LTM)test. Twenty-three healthy women (20 6 3 years), with body mass index (23.5 6 3 kg$m22), performed 2 shallow water peak oxygen consumption (V_ O2peak) running tests (SWRa and SWRb), and 1 V_ O2max LTM. Intraclass correlation coefficients indicated moderately strong reliability for V_ O2peak (ml$kg21$min21) (r = 0.73, p , 0.01), HR peak (b$min21) (r = 0.82; p , 0.01), and O2pulse (V_ O2 [ml$kg21$min21]$HR [b$min21]) (r = 0.77, p , 0.01). Using paired t-tests and Pearson’s correlations, SWR V_ O2peak and HR peak were significantly lower than during LTM (p # 0.05) and showed moderate correlations of 0.60 and 0.58 (p , 0.001) to LTM. O2pulse was similar (p . 0.05) for the SWR and LTM tests with a moderate correlation of 0.63. A standardized SWR test asa measure of CRF is a reliable, and to some degree, valid alternative to conventional protocols and may be used by strength and conditioning professionals to measure program outcomes and monitor training progress. Furthermore, this protocol provides a water-based option for CRF assessment among healthy women and offers insight toward the development of an effective protocol that can accommodate individuals with limited mobility, or those seeking less musculoskeletal impact from traditional land-based types of training

Evidence for Innate and Adaptive Immune Responses in a Cohort of Intractable Pediatric Epilepsy Surgery Patients

Brain-infiltrating lymphocytes (BILs) were isolated from resected brain tissue from 10 pediatric epilepsy patients who had undergone surgery for Hemimegalencephaly (HME) (n = 1), Tuberous sclerosis complex (TSC) (n = 2), Focal cortical dysplasia (FCD) (n = 4), and Rasmussen encephalitis (RE) (n = 3). Peripheral blood mononuclear cells (PBMCs) were also isolated from blood collected at the time of the surgery. Cells were immunostained with a panel of 20 antibody markers, and analyzed by mass cytometry. To identify and quantify the immune cell types in the samples, an unbiased clustering method was applied to the entire data set. More than 85 percent of the CD45+ cells isolated from resected RE brain tissue comprised T cells; by contrast NK cells and myeloid cells constituted 80–95 percent of the CD45+ cells isolated from the TSC and the FCD brain specimens. Three populations of myeloid cells made up >50 percent of all of the myeloid cells in all of the samples of which a population of HLA-DR+ CD11b+ CD4− cells comprised the vast majority of myeloid cells in the BIL fractions from the FCD and TSC cases. CD45RA+ HLA-DR− CD11b+ CD16+ NK cells constituted the major population of NK cells in the blood from all of the cases. This subset also comprised the majority of NK cells in BILs from the resected RE and HME brain tissue, whereas NK cells defined as CD45RA− HLA-DR+ CD11b− CD16− cells comprised 86–96 percent of the NK cells isolated from the FCD and TSC brain tissue. Thirteen different subsets of CD4 and CD8 αβ T cells and γδ T cells accounted for over 80% of the CD3+ T cells in all of the BIL and PBMC samples. At least 90 percent of the T cells in the RE BILs, 80 percent of the T cells in the HME BILs and 40–66 percent in the TSC and FCD BILs comprised activated antigen-experienced (CD45RO+ HLA-DR+ CD69+) T cells. We conclude that even in cases where there is no evidence for an infection or an immune disorder, activated peripheral immune cells may be present in epileptogenic areas of the brain, possibly in response to seizure-driven brain inflammation

EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates

Existing methods for detecting RNA intermediates resulting from exonuclease degradation are low-throughput and laborious. In addition, mapping the 3′ ends of RNA molecules to the genome after high-throughput sequencing is challenging, particularly if the 3′ ends contain post-transcriptional modifications. To address these problems, we developed EnD-Seq, a high-throughput sequencing protocol that preserves the 3′ end of RNA molecules, and AppEnD, a computational method for analyzing high-throughput sequencing data. Together these allow determination of the 3′ ends of RNA molecules, including nontemplated additions. Applying EnD-Seq and AppEnD to histone mRNAs revealed that a significant fraction of cytoplasmic histone mRNAs end in one or two uridines, which have replaced the 1–2 nt at the 3′ end of mature histone mRNA maintaining the length of the histone transcripts. Histone mRNAs in fly embryos and ovaries show the same pattern, but with different tail nucleotide compositions. We increase the sensitivity of EnD-Seq by using cDNA priming to specifically enrich low-abundance tails of known sequence composition allowing identification of degradation intermediates. In addition, we show the broad applicability of our computational approach by using AppEnD to gain insight into 3′ additions from diverse types of sequencing data, including data from small capped RNA sequencing and some alternative polyadenylation protocols

Decitabine immunosensitizes human gliomas to NY-ESO-1 specific T lymphocyte targeting through the Fas/Fas Ligand pathway

<p>Abstract</p> <p>Background</p> <p>The lack of effective treatments for gliomas makes them a significant health problem and highlights the need for the development of novel and innovative treatment approaches. Immunotherapy is an appealing strategy because of the potential ability for immune cells to traffic to and destroy infiltrating tumor cells. However, the absence of well-characterized, highly immunogenic tumor-rejection antigens (TRA) in gliomas has limited the implementation of targeted immune-based therapies.</p> <p>Methods</p> <p>We hypothesized that treatment with the demethylating agent, decitabine, would upregulate the expression of TRA on tumor cells, thereby facilitating enhanced surveillance by TRA-specific T cells.</p> <p>Results and Discussion</p> <p>Treatment of human glioma cells with decitabine increased the expression of NY-ESO-1 and other well characterized cancer testes antigens. The upregulation of NY-ESO-1 made these tumors susceptible to NY-ESO-1-specific T-cell recognition and lysis. Interestingly, decitabine treatment of T98 glioma cells also sensitized them to Fas-dependent apoptosis with an agonistic antibody, while a Fas blocking antibody could largely prevent the enhanced functional recognition by NY-ESO-1 specific T cells. Thus, decitabine treatment transformed a non-immunogenic glioma cell into an immunogenic target that was efficiently recognized by NY-ESO-1--specific T cells.</p> <p>Conclusions</p> <p>Such data supports the hypothesis that agents which alter epigenetic cellular processes may "immunosensitize" tumor cells to tumor-specific T cell-mediated lysis.</p

Deep Sequencing Shows Multiple Oligouridylations Are Required for 3′ to 5′ Degradation of Histone mRNAs on Polyribosomes

Histone mRNAs are rapidly degraded when DNA replication is inhibited during S-phase with degradation initiating with oligouridylation of the stemloop at the 3′ end. We developed a customized RNA-Seq strategy to identify the 3′ termini of degradation intermediates of histone mRNAs. Using this strategy, we identified two types of oligouridylated degradation intermediates: RNAs ending at different sites of the 3′ side of the stemloop that resulted from initial degradation by 3′hExo and intermediates near the stop codon and within the coding region. Sequencing of polyribosomal histone mRNAs revealed that degradation initiates and proceeds 3′ to 5′ on translating mRNA and many intermediates are capped. Knockdown of the exosome-associated exonuclease Pml/Scl-100, but not the Dis3L2 exonuclease, slows histone mRNA degradation, consistent with 3′ to 5′ degradation by the exosome containing PM/Scl-100. Knockdown of No-go decay factors also slowed histone mRNA degradation, suggesting a role in removing ribosomes from partially degraded mRNAs

Selection at the Y Chromosome of the African Buffalo Driven by Rainfall

Selection coefficients at the mammalian Y chromosome typically do not deviate strongly from neutrality. Here we show that strong balancing selection, maintaining intermediate frequencies of DNA sequence variants, acts on the Y chromosome in two populations of African buffalo (Syncerus caffer). Significant correlations exist between sequence variant frequencies and annual rainfall in the years before conception, with five- to eightfold frequency changes over short time periods. Annual rainfall variation drives the balancing of sequence variant frequencies, probably by affecting parental condition. We conclude that sequence variants confer improved male reproductive success after either dry or wet years, making the population composition and dynamics very sensitive to climate change. The mammalian Y chromosome, interacting with ecological processes, may affect male reproductive success much more strongly than previously thought

Effects and moderators of psychosocial interventions on quality of life, and emotional and social function in patients with cancer : an individual patient data meta-analysis of 21 RCTs

Objective: This individual patient data (IPD) meta‐analysis aimed to evaluate the effects of psychosocial interventions (PSI) on quality of life (QoL), emotional function (EF), and social function (SF) in patients with cancer, and to study moderator effects of demographic, clinical, personal, and intervention‐related characteristics. Methods: Relevant studies were identified via literature searches in 4 databases. We pooled IPD from 22 (n = 4217) of 61 eligible randomized controlled trials. Linear mixed‐effect model analyses were used to study intervention effects on the post‐intervention values of QoL, EF, and SF (z‐scores), adjusting for baseline values, age, and cancer type. We studied moderator effects by testing interactions with the intervention for demographic, clinical, personal, and intervention‐related characteristics, and conducted subsequent stratified analyses for significant moderator variables.Results: PSI significantly improved QoL (β = 0.14,95%CI = 0.06;0.21), EF (β = 0.13,95%CI = 0.05;0.20), and SF (β = 0.10,95%CI = 0.03;0.18). Significant differences in effects of different types of PSI were found, with largest effects of psychotherapy. The effects of coping skills training were moderated by age, treatment type, and targeted interventions. Effects of psychotherapy on EF may be moderated by cancer type, but these analyses were based on 2 randomized controlled trials with small sample sizes of some cancer types. Conclusions: PSI significantly improved QoL, EF, and SF, with small overall effects. However, the effects differed by several demographic, clinical, personal, and intervention‐related characteristics. Our study highlights the beneficial effects of coping skills training in patients treated with chemotherapy, the importance of targeted interventions, and the need of developing interventions tailored to the specific needs of elderly patients