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Documenting the importance of NK cell function as a biomarker for diseases and physiologic conditions including myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), will require assays amenable to clinical implementation and standardization. Research studies typically perform NK functional assays on the day of sample collection. This pilot study was conducted to compare assay formats and specimen processing to identify those that are most tolerant of conditions required for shipping and amenable to standardization as shown by inter-assay and inter-laboratory correlation of results. We compared performance within and between assays that measure NK cell function using direct cytotoxicity [chromium-51 release (CRCA) or fluorescence (Flow Cytometry Cytotoxicity Assay, FCCA)] or an indirect surrogate marker (CD107a surface expression)]. Additional variables for within/between assay comparisons included time of testing (same day as specimen collection or next day within 24 h), specimen types [whole blood or isolated peripheral blood mononuclear cells (PBMCs)], and processing method (fresh or cryopreserved). Statistical measures included number of samples tested in assay conditions (n), medians (x), interquartile range (IQR), Pearson correlation coefficient (R2), and correlation p-value (p). Samples came from 3 clinics and included 31 participants. Same day testing was only available for the subset of participants enrolled from the site of the laboratory performing CRCA. Results from same day CRCA testing of whole blood were considered the gold standard [n = 10, x=10.0%, IQR = 7.2%], and correlated well with PBMCs isolated next day [n = 26, x= 15.6%, IQR = 13.1%] [R2 = 0.59, p = 0.03]. Next day CRCA results were compromised using whole blood or frozen PBMCs. Next day FCCA cytotoxicity in PBMC [n = 30, x=34.1%, IQR = 15.5%] correlated with same day CRCA PMBC [R2 = 0.8, p = 0.001] and next day CRCA PMBC [R2 = 0.5, p

Understanding how immune cells respond to external stimuli such as pathogens or drugs is a key component of biomedical research. Critical to the immune response are the expression of cell-surface receptors and the secretion of cytokines, which are tightly regulated by gene expression and protein synthesis. Previously, cytokine mRNA expression levels have been measured from bulk analysis of heterogeneous or sorted cell populations, and the correlation between cytokine mRNA expression and protein levels using these techniques can be highly variable. Flow cytometry is used to monitor changes in cell-surface and intracellular proteins, but some proteins such as cytokines may be transient and difficult to measure. Thus, a flow cytometry method that can simultaneously measure cytokine mRNA and protein levels in single cells is a very powerful tool. We defined a flow cytometry method that combines the conventional measurement of T cell surface proteins (CD45, CD3, CD4, CD8) and intracellular cytokines (IL-2, INF-γ) with fluorescent in situ hybridization and branched DNA technology for amplification and detection of IL-2 and INF-γ mRNA transcripts in activated T cells. This method has been applied to frozen peripheral mononuclear blood cells (PBMCs) and frozen blood samples, making it applicable to clinical trial specimens that require shipment to the test site. In CD4+ cells from activated PBMCs, the concordance between mRNA and protein levels was 41% for IL-2 and 21% for and INF-γ. In CD8+ cells from activated PBMCs, the concordance was 15% for IL-2 and 32% for INF-γ. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen PBMCs Alternate Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen blood.

Treatment for acute ischemic stroke must be initiated within hours of stroke symptom onset, and the sooner it is administered, the better. In South Carolina, 76% of the population can access expert stroke care, and rural hospitals may provide specialized treatment using telemedicine, but many stroke sufferers seek care too late to achieve full benefit. Using a community-engaged approach in a southern rural community, we explored barriers and facilitators to early stroke care and implications for improvement. The Community-Engaged Assessment to facilitate Stroke Elimination (CEASE) study was guided by a community advisory group to ensure community centeredness and local relevance. In a qualitative descriptive study, eight focus groups were conducted including 52 individuals: recent stroke survivors, family members, emergency medical personnel, hospital emergency department staff, primary care providers, and community leaders. From analysis of focus group transcripts came six themes: lack of trust in healthcare system and providers; weak relationships fueled by poor communication; low health literacy; financial limitations related to health care; community-based education; and faith as a message of hope. A hierarchy model for improving early community-based stroke care was developed through consensus dialogue by community representatives and the research team. This model can be used to inform a community-partnered, stakeholder-informed intervention to improve stroke care in a rural southern community with the goal of improving stroke education, care, and outcome. © 2016 Wiley Periodicals, Inc.

CD47 is an anti-phagocytic (i.e. "don't eat me") signal and macrophage checkpoint that cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with SIRPα, its cognate inhibitory receptor.5F9 is undergoing investigation in several clinical trials and preliminary analyses have revealed encouraging therapeutic potential. We previously established that administration of 5F9 clears a subset of aged RBCs, resulting in a compensatory reticulocytosis. This occurs transiently, predominantly after the first dose, and does not worsen with subsequent doses. These findings, aligned with the observations of Oldenborg et al., which established that removal of aged RBCs, is dependent on the balance between the accumulating pro-phagocytic signals and loss of inhibitory anti-phagocytic signals. We therefore hypothesized that an initial lower 5F9 "priming dose", sufficient to trigger clearance of aged RBCs, would yield an overall younger pool of RBCs that are less vulnerable to subsequent higher 5F9 therapeutic "maintenance doses". Indeed, this priming and maintenance dosing strategy has substantially mitigated the on-target anemia and has allowed for 5F9 treatment in multiple clinical trials and indications. To further probe this observed selective RBC clearance, we employed a receptor occupancy assay to quantitate the CD47 expression on the cell surface of RBCs, leukocytes (WBCs), and acute myeloid leukemia (AML) blasts. We discovered that the priming dose of 5F9 not only triggered clearance of a subset of RBCs, but also resulted in a near complete loss of CD47 - a phenomenon we term CD47 pruning. This effect was RBC-specific, as WBCs and AML bone marrow (BM) blasts did not exhibit this same CD47 pruning (Fig 1). To explore the functional consequence of these unexpected clinical findings, we investigated whether we could recapitulate them in a preclinical mouse model. Similar to the clinical setting, anti-mouse CD47 blocking Ab treatments exhibited a similar pruning effect and transient anemia. Using this model, we asked whether, in the context of anti-CD47 treatment, these pruned RBCs were protected compared to unpruned RBCs. We transfused fluorescently labeled RBCs from CD47 Ab-untreated and CD47-treated mice into animals receiving continuous anti-CD47 treatment. We observed that CD47-pruned RBCs exhibited a significantly longer half-life compared to unpruned RBCs, suggesting that CD47 pruning is protective for RBCs. To distinguish whether this protection is cell-intrinsic or cell-extrinsic, we transfused fluorescently labeled RBCs into CD47 Ab-untreated and -treated mice and observed that CD47-pruned RBCs are rapidly cleared in untreated recipients, suggesting that this treatment-induced protection of RBCs is cell extrinsic and a host-based alteration. Through depletion of distinct immune subsets by chemical depletion, antibody depletion, splenectomy, and partial hepatectomies, we demonstrated that host immune effector cells (e.g. red-pulp macrophages, neutrophils, T cells etc.) were not necessary for this pruning phenomenon. Critically, we also found that this CD47 pruning and resulting tolerance was Fc-independent. In order to understand whether these findings were generalizable to other anti-human CD47 blocking agents, we conducted similar studies in double humanized huCD47 and huSIRPa micewith additional agents and observed this same CD47 pruning and tolerance phenomenon. Thereby suggesting this represents a class-effect of this emerging potential therapy class. Notably, these clinical and pre-clinical findings have been RBC-specific and have not precluded anti-tumor efficacy. The loss of CD47 on RBCs after the priming dose also suggests that the potential risk of CD47 Ab-mediated RBC agglutination following subsequent maintenance dosing is substantially reduced. Unlike prior RBC antigen modulation reports, to our knowledge, these findings represent a novel RBC antigen depletion phenomenon that is independent of the known RBC regulatory compartments, including the spleen, liver, major immune effectors cells, complement, and is critically Fc-independent. These findings provide fundamental insight into the mechanism underlying how anti-CD47 Abs are tolerated without impairing therapeutic efficacy. Disclosures Chen: Forty Seven Inc: Consultancy, Equity Ownership. McKenna:Forty Seven Inc.: Equity Ownership. Choi:Forty Seven Inc: Employment, Equity Ownership. Duan:Forty Seven Inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Schrier:Forty Seven Inc.: Consultancy. Weissman:Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Elrod:Forty Seven Inc.: Employment, Equity Ownership. Chao:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties.

The objective of this manuscript is to present an approach for evaluating specimen stability for flow cytometric methods used during drug development. While this approach specifically addresses stability assessment for assays to be used in clinical trials with centralized testing facilities, the concepts can be applied to any stability assessment for flow cytometric methods. The proposed approach is implemented during assay development and optimization, and includes suggestions for designing a stability assessment plan, data evaluation and acceptance criteria. Given that no single solution will be applicable in all scenarios, this manuscript offers the reader a roadmap for stability assessment and is intended to guide the investigator during both the method development phase and in the experimental design of the validation plan.

Many rural diabetes educators feel isolated professionally. Access to resources, skilled peers and opportunities for ongoing education may be lacking or not known. Rural diabetes educators often have multiple roles and may be confused about their responsibilities in specific areas. Geographic and professional isolation and lack of role definition may prevent appropriate diabetes care. Some sole educators in metropolitan hospitals also experience lack of support and interaction with their peers, A survey was designed to identify and address the issues facing isolated educators, determine the proportion of diabetes educators who consider themselves isolated, identify the reasons for isolation and the difficulties which result from isolation, and elicit suggestions for addressing the problems identified. Reasons given for isolation included professional isolation, distance, lack of resources and lack of network/support. The most frequently suggested methods of decreasing isolation were networking, regional meetings, teleconferences, distance education and provision of a practice manual. Professionals working in isolated areas of Australia need organised continuing medical education, quality assurance programs, systems for networking and access to expert advice. Modem information technology, distance education programs and models for effective networking offer the potential to meet these needs.

In 1967, the American Board of Pediatrics and the American Board of Internal Medicine uniformly recognized the 4-year combined medicine and pediatrics training program. Since that time, the number of combined internal medicine and pediatrics programs has increased. Today, there are more than 78 combined residency programs with more than 354 first-year residency positions throughout the United States. Medicine/pediatrics residency programs give the resident the opportunity to rotate 24 months in each specialty. Graduates of combined medicine/pediatrics residency programs have several career options available to them. In 2007, a total of 55% of medicine/pediatrics residents went into primary care practice, 18% went into subspecialty training, 17% went into hospital medicine, and 10% chose other careers.

Introduction Evaluation of right ventricular (RV) function is an important prognostic factor in many cardiac and/or pulmonary conditions. Pulmonary arterial hypertension (PAH) is a major cause of RV dysfunction and failure, which in turn affects left ventricular performance due to ventricular interdependence. Cardiac MRI is a reproducible three dimensional tomographic tool providing accurate measurements of global right ventricular function. For the RV, measuring regional strain using MRI (e.g., using tagging) is limited by thin wall. Strain encoded MRI (SENC), however, enables the quantification of RV strain (1) in the longitudinal direction (Ell), which is the main contributor to RV contractile function. On the other hand, fast strain encoded MRI (fSENC) allows for single heartbeat, non breath hold, acquisitions; thus providing an alternative to patients experiencing difficulty in breath holding (2).

Background: The Stroke Prevention Trial in Sickle Cell Anemia (STOP) and Optimizing Primary Stroke Prevention in Sickle Cell Anemia (STOP 2) established routine transcranial Doppler ultrasound (TCD) screening with indefinite chronic red cell transfusions (CRCT) for children with abnormal TCD as standard of care. To identify children at high-risk of stroke, annual TCD screening is recommended from ages 2 to 16 years, with more frequent monitoring if the result is not normal. A reduction in stroke incidence in children with SCD has been reported in several clinical series and analyses utilizing large hospital databases when comparing rates before and after the publication of the STOP study in 1998. We sought to determine the rate of first ischemic stroke in a multicenter cohort of children who had previously participated in the STOP and/or STOP 2 trials and to determine whether these strokes were screening or treatment failures. Subjects and Methods: Between 1995 and 2005, STOP and STOP 2 (STOP/2) were conducted at 26 sites in the US and Canada. These studies included 3,835 children, ages 2 to 16 y with SCD type SS or S-beta-0-thalassemia. Participation in STOP/2 ranged from a single screening TCD to randomization. STOP 2 also had an observational arm for children on CRCT for abnormal TCD whose TCD had not reverted to normal. The Post-STOP study was designed to follow-up the outcomes of children who participated in one or both trials. 19 of the 26 original study sites participated in Post-STOP, contributing a total of 3,539 (92%) of the STOP/2 subjects. After exit from STOP/2, these children received TCD screening and treatment according to local practices. Data abstractors visited each clinical site and obtained retrospective data from STOP/2 study exit to 2012-2014 (depending on site) including follow-up TCD and brain imaging results, clinical information, and laboratory results. Two vascular neurologists, blinded to STOP/2 status and prior TCD and neuroimaging results, reviewed source records to confirm all ischemic strokes, defined as a symptomatic cerebral infarction; discordant opinions were resolved through discussion. For the first Post-STOP ischemic stroke, prior TCD result and treatment history subsequently were analyzed. Results: Of the 3,539 subjects, follow-up data were available for 2,850 (81%). Twelve children who had a stroke during STOP or STOP2 were excluded from these analyses resulting in data on 2,838 subjects. The mean age at the start of Post-STOP was 10.5 y and mean duration of follow-up after exiting STOP/2 was 9.1 y. A total of 69 first ischemic strokes occurred in the Post-STOP observation period (incidence 0.27 per 100 pt years). The mean age at time of stroke was 14.4±6.2 (median 13.8, range 3.5-28.9) y. Twenty-five of the 69 patients (36%) had documented abnormal TCD (STOP/2 or Post-STOP) prior to the stroke; 15 (60%) were receiving CRCT and 9 (36%) were not (treatment data not available for 1 subject). Among the 44 subjects without documented abnormal TCD, 29 (66%) had not had TCD re-screen in the Post-STOP period prior to the event; 7 of these 29 (24%) were 16 y or older at the start of Post-STOP, which is beyond the recommended screening age. Four of the 44 (9%) patients had inadequate TCD in Post-STOP (1 to 10.7 y prior to event). Six (14%) had normal TCD more than a year before the event (1.2 - 4 y); all but one of these children were younger than 16 y at the time of that TCD. Only 5 (11%) had a documented normal TCD less than 1 year prior to the event. Conclusions: In the Post-STOP era, the rate of first ischemic stroke was substantially lower than that reported in the Cooperative Study of Sickle Cell Disease, prior to implementation of TCD screening. Many (39%) of the Post-STOP ischemic strokes were associated with a failure to re-screen according to current guidelines, while only 11% occurred in children who had had recent low-risk TCD. Among those known to be at high risk prior to stroke, treatment refusal or inadequate treatment may have contributed. While TCD screening and treatment are effective at reducing ischemic stroke in clinical practice, significant gaps in screening and treatment, even at sites experienced in the STOP protocol, remain to be addressed. Closing these gaps should provide yet further reduction of ischemic stroke in SCD. Disclosures No relevant conflicts of interest to declare.

Background: Primary hemorrhagic stroke is a rare complication of sickle cell disease (SCD) that usually occurs in adults. The Stroke Prevention Trial in Sickle Cell Anemia (STOP) and Optimizing Primary Stroke Prevention in Sickle Cell Anemia (STOP 2) established routine transcranial Doppler ultrasound (TCD) screening with indefinite chronic red cell transfusions (CRCT) for patients with abnormal TCD as standard of care. Despite a notable improvement in the incidence of infarctive stroke in children with SCD after the introduction of TCD screening protocols, it is unclear how this protocol will affect the rate of hemorrhagic stroke. Presumably, early TCD screening and subsequent initiation of CRCT in high risk patients will prevent the progression of cerebral vasculopathy, which should decrease the risk of hemorrhagic stroke; however this has not been proven. Using the large multicenter cohort of children who participated in STOP and/or STOP 2 trials, we sought to assess whether the rate of hemorrhagic stroke was impacted by the use of TCD screening and/or CRCT. Subjects and Methods: Between 1995 and 2005, STOP and STOP 2 (STOP/2) were conducted at 26 sites in the US and Canada. These studies included 3835 children, ages 2 to 16 y with SCD type SS or S-beta-0-thalassemia. Participation in STOP/2 required at least a single screening TCD for randomization. Patients on STOP 2 also had an observational arm for children started on CRCT who had an abnormal TCD. The Post-STOP study was designed to follow-up the outcomes of children who participated in one or both of trials. For all participants the date of their last encounter in STOP/2 was defined as the start of their Post-STOP period. 19 of the 26 original study sites participated in Post-STOP, contributing a total of 3539 (92%) of the STOP/2 subjects. After exit from STOP/2, these children received TCD screening and treatment according to local practices. Data abstractors visited each clinical site and obtained retrospective data from STOP/2 study exit to 2012-2014 including follow-up TCD and brain imaging results, clinical information, and laboratory results. Two separate neurologists, blinded to STOP/2 status and prior TCD and neuroimaging results adjudicated all suspected strokes. Results: Follow-up data were available for 2850 of the 3539 subjects (81%). Twelve children who had a stroke during the STOP study period were further excluded from this analysis resulting 2838 subjects. The mean age at the start of Post-STOP was 10.5 years and mean duration of follow-up after exiting STOP/2 until time of last medical encounter was 9.1 years. A total of 31 patients had a primary hemorrhagic stroke during the Post-STOP observation period (incidence 0.12 per 100 pt years). The mean age at time of stroke was 16.2+5.6 (median 15.3 range (4.8-30.2) years of age. Of those 31 patients, only 52% had a TCD during Post-STOP prior to the event. Seven of those children who underwent screening had documentation of an abnormal TCD prior to the event (5 during STOP era and/or 3 Post-STOP). However, only 1/7 patients (14%) were documented on CRCT at the time of the stroke (4 patients were receiving HU and 2 patients had unknown treatment). Discussion: Although less common than infarctive stroke, patients with SCD are at increased risk for hemorrhagic stroke. There is an increased risk of mortality for patients who suffer from hemorrhagic stroke (up to 26% in some reports in the 2 weeks after the event). It is unclear if TCD screening and subsequent initiation of CRCT will impact the rate of hemorrhagic stroke in the long term. In our results, a similar incidence of primary hemorrhagic stroke was noted although the patients were overall younger than previously reported (16.5+/- 5.5 years versus 20-29 years in Cooperative Study of Sickle Cell Disease). Many patients who had a hemorrhagic stroke (48%) had not undergone TCD screening during the Post-Stop period. Additionally, although a safe stopping point for CRCT has not been established in patients who have had an abnormal TCD, only 1 patient was documented on CRCT at the time of the event (14%). Thus, it is unclear at this time whether TCD screening and subsequent, lifelong continuation of CRCT could have prevented these other events. Clearly, these results demonstrate that improved implementation of STOP protocol is needed as well as further evaluation of the impact of this protocol on the incidence of hemorrhagic stroke. Disclosures No relevant conflicts of interest to declare.

Background: The Stroke Prevention Trial in Sickle Cell Anemia (STOP) and Optimizing Primary Stroke Prevention in Sickle Cell Anemia (STOP 2) study established routine transcranial Doppler ultrasound (TCD) screening with indefinite transfusions for children with abnormal TCD as standard of care. Children with normal TCD studies have the lowest risk of stroke of ~0.5-1% per year (y). Annual TCD screening is usually recommended for these children to detect possible subsequent conversion to high risk. We sought to determine the frequency of TCD screening utilized in “real world” clinical practice and the TCD outcomes for children with prior normal TCD. Subjects and Methods: During STOP and STOP2 (STOP/2), 3,837 children, ages 2 to 16 y with sickle cell disease type SS or S-Beta-0-thalassemia underwent screening TCD. The Post-STOP study was designed to follow-up the outcomes of children who were screened for or participated in one or both of these randomized trials. 19 of the 26 original study sites participated in Post-STOP, contributing a total of 3,541 (92%) of the STOP/2 subjects. After exit from STOP/2, these children received TCD screening and treatment according to local practices. Data abstractors visited each clinical site and obtained retrospective data from STOP/2 study exit to 2012-2014 (depending on site) on follow-up TCD results and clinical information using standard data collection forms. The rates of TCD re-screening and the proportion of children who converted to abnormal TCD were calculated. Factors associated with conversion to abnormal TCD were assessed. Results: Of the 3,541 subjects, follow-up data were available for 2,838 (80%). The mean age at the last TCD study obtained in STOP/2 was 9.5 y and the mean age at last follow-up in Post-STOP was 19.6 y. The mean duration of follow-up after exiting STOP/2 was 9.2 y. Subjects were classified by their worst TCD in STOP/2: the TCD was normal in 1,814 (64%), conditional in 479 (17%), abnormal in 357 (13%) and inadequate 188 (7%). Among the 1,814 children with only normal studies in STOP/2, follow-up TCD screening was obtained in the Post-STOP era on 842 (46%) at a median rate of 0.28 TCD studies/y (range, 0.05-3.04/y). Among these children, 26 (3.1%) developed an abnormal TCD at a median of 11.5 y (2.2-18.2 y) from the last STOP/2 study, while 77.5% still had normal TCD at a median of 10.7 y (0.7-18.3 y) from last STOP/2 study. The worst follow-up TCD classification for this group with prior normal TCD was conditional in 9.7% and inadequate in 9.6%. Among those that converted from prior normal to abnormal TCD, 12 had an interval conditional study (at median 2.8 y, 0.98-9.2 y) while 14 children converted from normal to abnormal at a median of 4.2 y (1.4-12.7 y) without documented interval conditional study. Children who developed abnormal TCD were younger at STOP/2 study exit (4.9 vs. 7.8 y, p Conclusions: In clinical practice, follow-up TCD for children with prior normal TCD was performed less frequently than the generally recommended annual basis. Among children re-screened, the risk of conversion to abnormal TCD was relatively low, but re-screening with TCD identified a subset of at-risk children who could benefit from transfusions to prevent a potentially devastating outcome. Predictors of conversion to abnormal TCD included younger age and prior TCD velocity in the high normal range. Disclosures Adams: Novartis: Consultancy.

High‐quality semiconductor material is essential to the production of high performance, affordable semiconductor components. With infrared (IR) components, the maturity and quality of the material has an increased significance, affecting not only component characteristics, but also system performance and cost. The criteria for selection of the focal plane material involves consideration of the system cooling requirements, spectral band, nonuniformity correction, and even system reliability. Each of these system parameters has a direct relationship to the IR material. The system requirements place demands on the quality and cost of the starting material and help to define the appropriate material growth technique, wafer size, the requirements for on‐chip signal processing, and relevant material figures‐of‐merit. A concurrent engineering approach to IR material development is necessary, taking into account both the material limitations and the demands of the infrared imaging system for the proposed applicat...