Fine-Tuning of Optimal TCR Signaling in Tumor-Redirected CD8 T Cells by Distinct TCR Affinity-Mediated Mechanisms

Redirecting CD8 T cell immunity with self/tumor-specific affinity-matured T cell receptors (TCRs) is a promising approach for clinical adoptive T cell therapy, with the aim to improve treatment efficacy. Despite numerous functional-based studies, little is known about the characteristics of TCR signaling (i.e., intensity, duration, and amplification) and the regulatory mechanisms underlying optimal therapeutic T cell responses. Using a panel of human SUP-T1 and primary CD8 T cells engineered with incremental affinity TCRs against the cancer-testis antigen NY-ESO-1, we found that upon activation, T cells with optimal-affinity TCRs generated intense and sustained proximal (CD3 zeta, LCK) signals associated with distal (ERK1/2) amplification-gain and increased function. In contrast, in T cells with very high affinity TCRs, signal initiation was rapid and strong yet only transient, resulting in poor MAPK activation and low proliferation potential even at high antigen stimulation dose. Under resting conditions, the levels of surface TCR/CD3e, CD8 beta, and CD28 expression and of CD3. phosphorylation were significantly reduced in those hypo-responsive cells, suggesting the presence of TCR affinity-related activation thresholds. We also show that SHP phosphatases were involved along the TCR affinity gradient, but displayed spatially distinct regulatory roles. While PTPN6/SHP-1 phosphatase activity controlled TCR signaling initiation and subsequent amplification by counteracting CD3. and ERK1/2 phosphorylation, PTPN11/SHP-2 augmented MAPK activation without affecting proximal TCR signaling. Together, our findings indicate that optimal TCR signaling can be finely tuned by TCR affinity-dependent SHP-1 and SHP-2 activity, and this may readily be determined at the TCR/CD3 complex level. We propose that these TCR affinity-associated regulations represent potential protective mechanisms preventing high affinity TCR-mediated autoimmune diseases

MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) activities. Genome-wide analysis of the integration loci and junction sequences validated the prevalent use of the SD-MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD-MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384-396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc

Decongestion improving right heart function ameliorates prognosis after an acute heart failure episode.

The prognostic role of decongestion-related change of cardiac morphology and in particular right heart function has not been investigated comprehensively in AHF patients. This prospective observational single-centre study included consecutive patients hospitalized for treatment of AHF with reduced, mildly-reduced or preserved left ventricular ejection fraction (LVEF). Comprehensive transthoracic echocardiography at admission and discharge assessed decongestion-related change of cardiac function and morphology. The combined endpoint of 1 year all-cause mortality and cardiovascular rehospitalization explored the prognostic importance of decongestion-related change. The 176 study participants were 83 years old [74-87] and 54% were men. Fifty one (29%) had rLVEF, 65 (37%) mrLVEF, and 60 (34%) pLVEF. The proportion of de novo or worsening chronic HF was not different between LVEF groups. HF aetiology and cardiovascular risk factors were equally distributed across all groups except for a higher BMI in the pLVEF group. Decongestion equally reduced body weight, heart rate, systolic and diastolic blood pressure, tricuspid regurgitation gradient, and inferior vena cava diameter across all groups (P < 0.004 for all). Decongestion-related increase in TAPSE independent of the LVEF was associated with improvement of right-ventricular-pulmonary artery coupling and a lower incidence of the combined outcome in the Cox proportional hazard risk analysis (unadjusted HR 0.50 95% CI 0.33-0.78, P = 0.002; adjusted HR 0.46 95% CI: 0.33-0.78, P = 0.001). Decongestion-related increase in TAPSE and recovery of RV/pulmonary artery coupling was observed across all LVEF groups and associated with a risk reduction for the combined endpoint highlighting the important prognostic role of right heart recovery after an AHF episode

Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons

Background Rhinovirus infections are the dominant cause of asthma exacerbations, and deficient virus induction of IFN-α/β/λ in asthmatic patients is important in asthma exacerbation pathogenesis. Mechanisms causing this interferon deficiency in asthmatic patients are unknown. Objective We sought to investigate the expression of suppressor of cytokine signaling (SOCS) 1 in tissues from asthmatic patients and its possible role in impaired virus-induced interferon induction in these patients. Methods We assessed SOCS1 mRNA and protein levels in vitro, bronchial biopsy specimens, and mice. The role of SOCS1 was inferred by proof-of-concept studies using overexpression with reporter genes and SOCS1-deficient mice. A nuclear role of SOCS1 was shown by using bronchial biopsy staining, overexpression of mutant SOCS1 constructs, and confocal microscopy. SOCS1 levels were also correlated with asthma-related clinical outcomes. Results We report induction of SOCS1 in bronchial epithelial cells (BECs) by asthma exacerbation–related cytokines and by rhinovirus infection in vitro. We found that SOCS1 was increased in vivo in bronchial epithelium and related to asthma severity. SOCS1 expression was also increased in primary BECs from asthmatic patients ex vivo and was related to interferon deficiency and increased viral replication. In primary human epithelium, mouse lung macrophages, and SOCS1-deficient mice, SOCS1 suppressed rhinovirus induction of interferons. Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors. Nuclear SOCS1 levels were also increased in BECs from asthmatic patients. Conclusion We describe a novel mechanism explaining interferon deficiency in asthmatic patients through a novel nuclear function of SOCS1 and identify SOCS1 as an important therapeutic target for asthma exacerbations

Human bocavirus (HBoV) in children with respiratory tract infection by enzyme linked immunosorbent assay (ELISA) and qualitative polymerase chain reaction (PCR)

<p>Abstract</p> <p>Background</p> <p>Human bocavirus (HBoV) is a recently discovered parvovirus associated with mild to severe lower respiratory tract infections in children, the aim of the work was determination of human bocavirus in nasopharyngeal aspirate (NPA) of infants by qualitative PCR and determination of acute human bocavirus infection by estimation of immunoglobulin M (IgM) antibodies in serum by enzyme linked immunosorbent assay.</p> <p>Results</p> <p>Twenty two (22%) out of the 100 NPA specimens of the patients with respiratory manifestations were positive for HBoV by qualitative PCR, while ELISA revealed positive HBoV IgM antibodies in 18 (18%) patients who were also positive by PCR. Non of the controls were positive by both techniques. The correlation study between ELISA and PCR revealed high significant association, (p < 0.001, X²= 36 and agreement = 96%). Also PCR detected 4 (18.1%) NPA samples as HBoV positive cases among the patients that were not identified by ELISA. This could be due to high sensitivity and efficacy of PCR. ELISA being less sensitive than RT-PCR, sensitivity was (81.8% vs 100%), the efficacy was 97.7% in ELISA versus 99.7% for RT-PCR.</p> <p>Conclusion</p> <p>HBoV infections could be diagnosed in NPA of children by conventional PCR as a rapid and sensitive technique. While ELISA was a reliable serologic analysis for diagnosis of acute HBoV infection by estimation IgM antibodies in serum.</p

Detection of Human Bocavirus mRNA in Respiratory Secretions Correlates with High Viral Load and Concurrent Diarrhea

Human bocavirus (HBoV) is a parvovirus recently identified in association with acute respiratory infections (ARI). Despite its worldwide occurrence, little is known on the pathogenesis of HBoV infections. In addition, few systematic studies of HBoV in ARI have been conducted in Latin America. Therefore, in order to test whether active viral replication of human bocavirus is associated with respiratory diseases and to understand the clinical impact of this virus in patients with these diseases, we performed a 3-year retrospective hospital-based study of HBoV in outpatients and inpatients with symptoms of Acute Respiratory Infections (ARI) in Brazil. Nasopharyngeal aspirates (NPAs) from 1015 patients with respiratory symptoms were tested for HBoV DNA by PCR. All samples positive for HBoV were tested by PCR for all other respiratory viruses, had HBoV viral loads determined by quantitative real time PCR and, when possible, were tested by RT-PCR for HBoV VP1 mRNA, as evidence of active viral replication. HBoV was detected in 4.8% of patients, with annual rates of 10.0%, 3.0% and 3.0% in 2005, 2006 and 2007, respectively. The range of respiratory symptoms was similar between HBoV-positive and HBoV-negative ARI patients. However, a higher rate of diarrhea was observed in HBoV-positive patients. High HBoV viral loads (>108 copies/mL) and diarrhea were significantly more frequent in patients with exclusive infection by HBoV and in patients with detection of HBoV VP1 mRNA than in patients with viral co-infection, detected in 72.9% of patients with HBoV. In summary, our data demonstrated that active HBoV replication was detected in a small percentage of patients with ARI and was correlated with concurrent diarrhea and lack of other viral co-infections

Pulmonary epithelial barrier and immunological functions at birth and in early life - key determinants of the development of asthma? A description of the protocol for the Breathing Together study

Acknowledgements The authors are indebted to the participants and parents who have already been recruited. We also acknowledge the enthusiasm and endeavour of the research nurse team which includes: Stephen Main, Margaret Connon, Catherine Beveridge, Julie Baggott, Kay Riding, Ellie McCamie, Maria Larsson, Lynda Melvin, Mumtaz Idris, Tara Murray, Nicky Tongue, Nicolene Plaatjies, Sheila Mortimer, Sally Spedding, Susy Grevatt, Victoria Welch, Morag Zelisko, Jillian Doherty, Jane Martin, Emma Macleod and Cilla Snape. We are also delighted to be working alongside the following colleagues in laboratories: Marie Craigon, Marie McWilliam, Maria Zarconi, Judit Barabas, Lindsay Broadbent, Ceyda Oksel and Sheerien Manzoor. Grant information The study is supported by the Wellcome Trust [108818]; and the PHA HSC R&D Division, Northern Ireland.Peer reviewedPublisher PD

TRAIP/RNF206 is required for recruitment of RAP80 to sites of DNA damage

RAP80 localizes to sites of DNA insults to enhance the DNA-damage responses. Here we identify TRAIP/RNF206 as a novel RAP80-interacting protein and find that TRAIP is necessary for translocation of RAP80 to DNA lesions. Depletion of TRAIP results in impaired accumulation of RAP80 and functional downstream partners, including BRCA1, at DNA lesions. Conversely, accumulation of TRAIP is normal in RAP80-depleted cells, implying that TRAIP acts upstream of RAP80 recruitment to DNA lesions. TRAIP localizes to sites of DNA damage and cells lacking TRAIP exhibit classical DNA-damage response-defect phenotypes. Biochemical analysis reveals that the N terminus of TRAIP is crucial for RAP80 interaction, while the C terminus of TRAIP is required for TRAIP localization to sites of DNA damage through a direct interaction with RNF20-RNF40. Taken together, our findings demonstrate that the novel RAP80-binding partner TRAIP regulates recruitment of the damage signalling machinery and promotes homologous recombinationopen

Detection of human bocavirus from children and adults with acute respiratory tract illness in Guangzhou, southern China

<p>Abstract</p> <p>Background</p> <p>Human bocavirus (HBoV) is a newly discovered parvovirus associated with acute respiratory tract illness (ARTI) and gastrointestinal illness. Our study is the first to analyze the characteristics of HBoV-positive samples from ARTI patients with a wide age distribution from Guangzhou, southern China.</p> <p>Methods</p> <p>Throat swabs (n=2811) were collected and analyzed from children and adults with ARTI over a 13-month period. The HBoV complete genome from a 60 year-old female patient isolate was also determined.</p> <p>Results</p> <p>HBoV DNA was detected in 65/2811 (2.3%) samples, of which 61/1797 were from children (<18 years old) and 4/1014 from adults (≥18 years old). Seasonal peaks of 4.8% and 7.7% were detected in May and June, respectively. 28 of 65 (43.1%) HBoV-positive samples were co-detected with 11/16 other potential pathogens. <it>Mycoplasma pneumoniae </it>had the highest frequency of 16.9% (11/65). Upper and lower respiratory tract illness were common symptoms, with 19/65 (29.2%) patients diagnosed with pneumonia by chest radiography. All four adult patients had systemic influenza-like symptoms. Phylogenetic analysis of the complete genome revealed a close relationship with other HBoVs, and a more distant relationship with HBoV2 and HBoV3.</p> <p>Conclusions</p> <p>HBoV was detected from children and adults with ARTI from Guangzhou, southern China. Elderly people were also susceptive to HBoV. A single lineage of HBoV was detected among a wide age distribution of patients with ARTI.</p