Functional, morphologic, and molecular characterization of cold storage injury

ObjectiveCold storage is used to preserve tissue for later transplantation. There is particular interest in prolonging cold storage time for transplantation purposes. To date, the mechanisms that contribute to vascular dysfunction in response to cold storage are poorly understood. The present study aims to characterize cold storage injury of blood vessels on functional and molecular levels.MethodsTo assess vessel function of mouse aorta, isometric force measurements were performed in a Mulvany myograph after cold storage at 4°C for various intervals. Morphologic changes were judged by histologic analysis of aortic cross-sections. To characterize cold storage-induced alterations on RNA levels, microarray analysis with subsequent polymerase chain reaction analysis was performed.ResultsCold storage for 2 days revealed significant impairment of vessel function with respect to potassium-induced vessel tone development and acetylcholine-induced vessel relaxation. Detailed analysis of acetylcholine-mediated vascular response using specific pharmacologic blockers revealed that calcium-activated potassium channels seem to be impaired after 2 days of cold storage. At this point, no severe histologic changes (eg, elastic fiber disruption) were visible. RNA expression of 24 genes was significantly changed due to cold storage even after 2 hours. These include genes associated with vessel tone development (prostaglandin E3 receptor), cardiovascular function (adiponectin), electron transport chain (uncoupling protein 1), or calcium signaling (protein kinase A regulatory subunit 2b).ConclusionsLong-term cold storage impairs vascular function, especially with respect to potassium signaling by calcium-dependent potassium channels. Microarray analysis confirmed impairment of pathways that are involved in calcium signaling and vascular function. Furthermore, various genes were significantly altered even after 2 hours, significantly before functional impairment was observed.Clinical RelevanceIn bypass surgery, vessels are stored ex situ for <2 hours and for vessel banking for up to 48 hours during decontamination before cryopreservation. We investigated the onset of storage-induced alterations. The experiments reveal various genes were altered significantly, even within the first 2 hours of cold storage. This is of major importance, because of the time delay between regulation of messenger RNA and protein level and functional consequences. The alterations described here on the molecular level occur before any alterations on morphologic or functional levels are obvious. These molecular alterations, however, may affect later graft function

'Emerge' : benchmarking of clinical performance and patients experiences with emergency care in Switzerland

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch)Objective: To assess the effects of uniform indicator measurement and group benchmarking followed by hospital-specific activities on clinical performance measures and patients’ experiences with emergency care in Switzerland. Design: Data were collected in a pre-post design in two measurement cycles, before and after implementation of improvement activities. Trained hospital staff recorded patient characteristics and clinical performance data. Patients completed a questionnaire after discharge/transfer from the emergency unit. Setting: Emergency departments of 12 community hospitals in Switzerland, participating in the ‘Emerge’ project. Subjects: Eligible patients were entered into the study (18 544 in total: 9174 and 9370 in the first and second cycles, respectively), and 2916 and 3370 patients returned the questionnaire in the first and second measurement cycles, respectively (response rates 32% and 36%, respectively). Main outcome measures: Clinical performance measures (concordance of prospective and retrospective assessment of urgency of care needs, and time intervals between sequences of events) and patients’ reports about care provision in emergency departments (EDs), measured by a 22-item, self-administered questionnaire. Results: Concordance of prospective and retrospective assignments to one of three urgency categories improved significantly by 1%, and both under- and over-prioritization, were reduced. The median duration between ED admission and documentation of post-ED disposition fell from 137 minutes in 2001 to 130 minutes in 2002 (P < 0.001). Significant improvements in the reports provided by patients were achieved in 10 items, and were mainly demonstrated in structures of care provision and perceived humanity. Conclusion: Undertaken in a real-world setting, small but significant improvements in performance measures and patients’ perceptions of emergency care could be achieved. Hospitals accomplished these improvements mainly by averting strong outliers, and were most successful in preventing series of negative events. Uniform outcomes measurement, group benchmarking, and data-driven hospital-specific strategies for change are suggested as valuable tools for continuous improvement. Several hospitals have already implemented the developed measures in their internal quality systems and subsequent measurements are projected

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Ambulatory Assessment in the Research on Aging

Older adults have surprisingly high levels of well-being, which has been referred to as a paradox in the past. Improved emotion regulation has been suggested to underlie these high levels of well-being. Later life is also a period with enhanced exposure to critical life events, and this comes with risks. During such times, and towards the end of life, emotional well-being may and eventually does decline. We suggest that ambulatory assessment (AA) is ideally suited for the investigation of the above phenomena and for intervention purposes. More precisely, AA can be used to thoroughly examine within-person processes of emotion regulation, including the multiple levels on which emotions occur (physiology, experience, behavior, context, and nonverbal expressions). It thereby provides a basis for understanding competent emotion regulation, the well-being paradox, and emotionally critical periods. Such insights can be utilized to detect person-specific critical periods and for designing immediate person-specific interventions. Although this is still a vision, the benefits of such an approach seem invaluable. The major part of this paper is organized around three general principles that we suggest to further tap the potential of AA in aging research, namely (1) identify within-subject processes and their relations to important life outcomes; (2) capitalize on the full scope of AA technology via multivariate assessments, and (3) combine real-time monitoring with real-time interventions.Peer Reviewe

Tetrahydrobiopteryna, kofaktor śródbłonkowej syntazy tlenku azotu, chroni odległe obszary miokardium przed apoptozą po wywołanym doświadczalnie zawale serca w warunkach in vivo

Background: Following myocardial infarction (MI), apoptosis occurs early in the remote myocardium and contributes to the processes of myocardial remodelling. Increased nitrosative stress is a well-known and potent inductor of myocardial apopto¬sis. Excess activation of endothelial nitric oxide synthase (eNOS) increases its uncoupling potential and results in nitrosative stress via formation of peroxynitrite. However, the pathophysiological role of eNOS signalling in the remote myocardium after MI is as yet undefined. Aim: The impact of eNOS activation on pro- and anti-apoptotic signalling in the remote myocardium and the influence of pretreatment with the eNOS cofactor tetrahydrobiopterin (BH4) on eNOS activation, nitrosative stress level, and apoptosis induction and execution were studied in a rat MI model in vivo. Results: Twenty-four hours after anterior MI, eNOS activity in animals treated with left anterior descending coronary artery ligation (LIG) significantly increased in the posterior left ventricular (LV) myocardium as did protein nitrosylation when com¬pared to sham treatment. This was paralleled by induction of apoptosis via the extrinsic and intrinsic pathways. Moreover, anti-apoptotic signalling via protein kinase B/Akt and glycogen synthase-kinase 3 beta was suppressed. Notably, pretreatment with the eNOS cofactor BH4 reduced eNOS activation, prevented excess protein nitrosylation, blunted apoptosis induction, facilitated anti-apoptotic signalling, and eventually prevented apoptosis execution. Conclusions: Here we showed that 24 h after experimental MI in rats in vivo, apoptosis was induced in the posterior non-in¬farcted LV wall. Evidence is presented that pretreatment with the eNOS cofactor BH4 resulted in less nitrosative stress and weakened apoptotic processes, although the stabilisers contained did participate in this phenomenon. Because apoptosis is a crucial component of myocardial remodelling, influencing eNOS signalling might be an interesting pharmacological target for the development of novel anti-remodelling therapies.Wstęp: Apoptoza w odległych rejonach miokardium występuje wcześnie po zawale serca (MI) i przyczynia się do remodelingu mięśnia sercowego. Zwiększenie stresu nitrozacyjnego jest znanym silnym czynnikiem wywołującym apoptozę miokardium. Nadmierna aktywacja śródbłonkowej syntazy tlenku azotu (eNOS) zwiększa jej rozprzęganie i powoduje stres nitrozacyjny w wyniku syntezy peroksynitrytu. Jednak nie poznano dotychczas patofizjologicznej roli eNOS w przesyłaniu sygnałów do odległych rejonów miokardium po MI. Cel: Celem niniejszej pracy było przeanalizowanie wpływu aktywacji eNOS na przekazywanie sygnałów pro- i anty-apop¬tycznych w odległych rejonach miokardium oraz wpływu wstępnego leczenia kofaktorem, tetrahydrobiopteryną (BH4) na aktywację NOS, poziom stresu nitrozacyjnego oraz indukowanie i przebieg apoptozy w szczurzym modelu in vivo zawału serca. Wyniki: Dwadzieścia cztery godziny po zawale ściany przedniej stwierdzono istotne zwiększenie w porównaniu z wartościami obserwowanymi w grupie kontrolnej w zakresie aktywności eNOS w miokardium tylnej ściany lewej komory u zwierząt, u których podwiązano gałąź międzykomorową przednią lewej tętnicy wieńcowej, podobnie jak w przypadku nitrozylacji białek. Jednocześnie zaobserwowano indukcję apoptozy zarówno w mechanizmie zewnątrz-, jak i wewnątrzpochodnym. Ponadto zahamowane zostało przekazywanie sygnałów anty-apoptotycznych za pośrednictwem ścieżki sygnałowej kinazy proteinowej B/Akt i kinazy syntazy glikogenu 3 beta. Warto zaznaczyć, że podanie wcześniej kofaktora eNOS, BH4, zmniejszyło aktywację eNOS, zapobiegło nadmiernej nitrozylacji białek, stłumiło indukcję apoptozy, ułatwiło przekazywanie sygnałów anty-apoptotycznych i zatrzymało proces apoptozy. Wnioski: Wykazano, że 24 godziny po wywołanym eksperymentalnie MI u szczurów in vivo nastąpiła indukcja apoptozy w tylnej ścianie lewej komory nieobjętej zawałem. Przedstawiono dowody naukowe potwierdzające, że wcześniejsze podanie kofaktora eNOS, BH4, spowodowało zmniejszenie stresu nitrozacyjnego i osłabienie procesów apoptozy, chociaż zawarte w nim stabilizatory miały w tym swój udział. Ze względu na to, że apoptoza jest ważnym elementem remodelingu miokar¬dium, oddziaływanie na ścieżkę sygnałową eNOS może być interesującym celem leczenia w opracowywaniu nowych terapii przeciwdziałających remodelingowi

High Stroma T-Cell Infiltration is Associated with Better Survival in Stage pT1 Bladder Cancer

Stage pT1 bladder cancer (BC) shows highly diverse outcomes. Predictive markers are required to stratify patients for personalized treatment. The present study aimed to validate immune response quantification as a prognostic marker. Patients with pT1 BC (n = 167) treated by transurethral resection of the bladder (TURB) were enrolled. Formaldehyde-fixed paraffin-embedded material was stained for CD3 and CD8. Corresponding T cells were counted in three regions with the highest immune response. Numbers of tertiary lymphoid structures (TLS) and lymphocyte aggregates (LA) were quantified. High CD3(+) stroma T-cell infiltration was associated with improved survival (p = 0.045), especially in the G3 subgroup (p = 0.01). Cluster with higher immune response showed less recurrence (p = 0.034) and favorable overall survival (OS) (p = 0.019). In contrast, higher CD3(+) and CD8(+) tumor T-cell infiltration seemed to have a negative impact on prognosis. TLS and LA were more frequently observed in G3 tumors, indicating an increased anti-tumoral immune response. We proved the role of immune cell infiltration and showed that higher infiltration numbers of CD3(+) (not CD8(+)) lymphocytes in the stroma are associated with favorable outcome. Immune cell quantification could be used as a marker to help stratify patients' risk and therefore, to optimize patients' management and follow-up examination as well as possible therapies