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Background The impact of transarterial radioembolization (TARE) of breast cancer liver metastasis (BCLM) on antitumor immunity is unknown, which hinders the optimal selection of candidates for TARE. Purpose To determine whether response to TARE at PET/CT in participants with BCLM is associated with specific immune markers (cytokines and immune cell populations). Materials and Methods This prospective pilot study enrolled 23 women with BCLM who planned to undergo TARE (June 2018 to February 2020). Peripheral blood and liver tumor biopsies were collected at baseline and 1-2 months after TARE. Monocyte, myeloid-derived suppressor cell (MDSC), interleukin (IL), and tumor-infiltrating lymphocyte (TIL) levels were assessed with use of gene expression studies and flow cytometry, and immune checkpoint and cell surface marker levels with immunohistochemistry. Modified PET Response Criteria in Solid Tumors was used to determine complete response (CR) in treated tissue. After log-transformation, immune marker levels before and after TARE were compared using paired

Figure S1. Blockade of IL-7 but not IL-2 reduces engraftment of IL-12 conditioned (Tc1) CD8+ T cells in lymphodepleted mice. Figure S2. Blockade of cytokine signaling does not reduce the functionality of donor CD8+ T cells. Figure S3. Activated CD8+ T cells do not require TCR engagement for engraftment in an irradiated host. Figure S4. IL-7 mediated proliferation of Tc1 is STAT5 and PI3K dependent. Figure S5. Tc1 cells generated from IL-7Ralpha+/- and wildtype mice exhibit a similar profile except for IL-7Ralpha expression. Figure S6. The level of TCR stimulation, costimulation, and IL-12 drive IL-7Ralpha expression after CD8+ T cell activation.

Adoptive cellular therapy, in which activated tumor-reactive T cells are transferred into lymphodepleted recipients, is a promising cancer treatment option. Activation of T cells decreases IL7 responsiveness; therefore, IL15 is generally considered the main driver of effector T-cell responses in this setting. However, we found in lymphodepleted mice that CD8+ T cells activated with IL12 showed enhanced engraftment that was initially dependent on host IL7, but not IL15. Mechanistically, enhanced IL7 responsiveness was conferred by elevated IL7Rα expression, which was critical for antitumor immunity. Elevated IL7Rα expression was achievable without IL12, as polyclonal CD8+ T cells activated with high T-cell receptor (TCR) stimulation depended on T-cell IL7Rα expression and host IL7 for maximal engraftment. Finally, IL12 conditioning during the activation of human CD8+ T cells, including TCR-modified T cells generated using a clinically relevant protocol, led to enhanced IL7Rα expression. Our results demonstrate the importance of the donor IL7Rα/host IL7 axis for effector CD8+ T-cell engraftment and suggest novel strategies to improve adoptive cellular therapy as a cancer treatment. Cancer Immunol Res; 3(12); 1364–74. ©2015 AACR.

Effector CD8+ T cells conditioned with IL12 during activation mediate enhanced antitumor efficacy after adoptive transfer into lymphodepleted hosts; this is due in part to improved IL7 responsiveness. Therefore, we hypothesized that increasing the intensity or type of lymphodepletion would deplete more IL7-consuming host cells and improve the persistence and antitumor activity of IL12-conditioned CD8+ T cells. Using cyclophosphamide, fludarabine, and total body irradiation (TBI, 6 Gy) either individually or in combination, we found that combined lymphodepletion best enhanced T-cell engraftment in mice. This improvement was strongly related to the extent of leukopenia, as posttransfer levels of donor T cells inversely correlated to host cell counts after lymphodepletion. Despite the improvement in engraftment seen with combination lymphodepletion, dual-agent lymphodepletion did not augment the antitumor efficacy of donor T cells compared with TBI alone. Similarly, IL7 supplementation after TBI and transfer of tumor-reactive T cells failed to improve persistence or antitumor immunity. However, IL15 or IL2 supplementation greatly augmented the persistence and antitumor efficacy of donor tumor-reactive T cells. Our results indicate that the amount of host IL7 induced after single agent lymphodepletion is sufficient to potentiate the expansion and antitumor activity of donor T cells, and that the efficacy of future regimens may be improved by providing posttransfer support with IL2 or IL15.Significance: The relationship between lymphodepletion and cytokine support plays a critical role in determining donor T-cell engraftment and antitumor efficacy. Cancer Res; 78(11); 3067–74. ©2018 AACR.

This file contains the following figures: Supplemental Figure 1. The return of host lymphocytes is affected by the type of donor CD8+ T cells. Supplemental Figure 2. Return of myeloid cells after CTX leads to more rapid host cell reconstitution than TBI-containing regimens. Supplemental Figure 3. CTX and TBI differentially affect host T cells and NK cells. Supplemental Figure 4. Donor CD8+ T cells transferred into a TBI-conditioned host maintain functional efficacy. Supplemental Figure 5. ALT-803 (IL-15/IL-15Ra complexes) augment the persistence of Tc1 cells in irradiated hosts.

Engineered proteins have emerged as novel diagnostics, therapeutics, and catalysts. Often, poor protein developability – quantified by expression, solubility, and stability – hinders utility. The ability to predict protein developability from amino acid sequence would reduce the experimental burden when selecting candidates. Recent advances in screening technologies enabled a high-throughput developability dataset for 105of 1020possible variants of protein ligand scaffold Gp2. In this work, we evaluate the ability of neural networks to learn a developability representation from a high-throughput dataset and transfer this knowledge to predict recombinant expression beyond observed sequences. The model convolves learned amino acid properties to predict expression levels 44% closer to the experimental variance compared to a non-embedded control. Analysis of learned amino acid embeddings highlights the uniqueness of cysteine, the importance of hydrophobicity and charge, and the unimportance of aromaticity, when aiming to improve the developability of small proteins. We identify clusters of similar sequences with increased developability through nonlinear dimensionality reduction and we explore the inferred developability landscape via nested sampling. The analysis enables the first direct visualization of the fitness landscape and highlights the existence of evolutionary bottlenecks in sequence space giving rise to competing subpopulations of sequences with different developability. The work advances applied protein engineering efforts by predicting and interpreting protein scaffold developability from a limited dataset. Furthermore, our statistical mechanical treatment of the problem advances foundational efforts to characterize the structure of the protein fitness landscape and the amino acid characteristics that influence protein developability.Significance statementProtein developability prediction and understanding constitutes a critical limiting step in biologic discovery and engineering due to limited experimental throughput. We demonstrate the ability of a machine learning model to learn sequence-developability relationships first through the use of high-throughput assay data, followed by the transfer of the learned developability representation to predict the true metric of interest, recombinant yield in bacterial production. Model performance is 44% better than a model not pre-trained using the high-throughput assays. Analysis of model behavior reveals the importance of cysteine, charge, and hydrophobicity to developability, as well as of an evolutionary bottleneck that greatly limited sequence diversity above 1.3 mg/L yield. Experimental characterization of model predicted candidates confirms the benefit of this transfer learning and in-silico evolution approach.

While much progress has been made in oncologic care, metastatic solid organ cancer still carries a poor prognosis. Immunotherapy has emerged as a promising approach, though in most patients, does not control disease when given as a monotherapy. Combining immunotherapy with locoregional therapy is one approach to boost the efficacy of treatments and potentially prolong survival. Most of the researches regarding combination therapies have involved external radiation. This review describes the immunologic effects of external radiation and radioembolization, and how these effects provide a rationale for combining hepatic radioembolization with immunotherapy.

Objectives Although mindfulness meditation is the familiar and researched form of mental training derived from Buddhism, it represents but one form of practice. Monastic debate is an interactive and dyadic analytical meditation practice that originates from the Tibetan Buddhist tradition where monastics seek to jointly deepen their understanding of complicated philosophical issues. To date, monastic debate and analytic meditation have yet to be examined in the context of scientific investigation. Methods In the current study, we examined the neural correlates of this analytical meditation practice by means of hyperscanning electroencephalography, a method well-suited for examining social interactions. Results Consistent with the idea that analytical meditation helps to train concentration, we observed that over the course of the debate, mid-frontal theta oscillations—a correlate of absorption—increased significantly. This increase was stronger for more experienced monks as compared with monks at the beginning of their education. In addition, we found evidence for increases in synchrony in frontal alpha oscillations between paired debaters during moments of agreement as compared with disagreement on a set of premises. Conclusions Together, these findings provide an initial understanding of Tibetan monastic debate and analytical meditation using neuroscientific methods.

The development of the T- and natural killer (NK) cell growth factor IL-2 has been a sentinel force ushering in the era of immunotherapy in cancer. With the advent of clinical grade recombinant IL-2 in the mid-1980s, oncologists could for the first time directly manipulate lymphocyte populations with systemic therapy. By itself, recombinant IL-2 can induce clinical responses in up to 15% of patients with metastatic cancer or renal cell carcinoma. When administered with adoptively transferred tumor-reactive lymphocytes, IL-2 promotes T cell engraftment and response rates of up to 50% in metastatic melanoma patients. Importantly, these IL-2-driven responses can yield complete and durable responses in a subset of patients. However, the use of IL-2 is limited by toxicity and concern of the expansion of T regulatory cells. To overcome these limitations and improve response rates, other T cell growth factors, including IL-15 and modified forms of IL-2, are in clinical development. Administering T cell growth factors in combination with other agents, such as immune checkpoint pathway inhibitors, may also improve efficacy. In this study, we review the development of T- and NK cell growth factors and highlight current combinatorial approaches based on these reagents.

BACKGROUND A minimum male hemoglobin (Hb) level of 13.0 g/dL becomes a Food and Drug Administration requirement effective May 2016. In addition, extending whole blood (WB) interdonation intervals (IDIs) beyond 8 weeks has been considered to reduce iron depletion in repeat blood donors. This study estimates the impact these changes might have on blood availability and donor iron status. STUDY DESIGN AND METHODS Six blood centers participating in Retrovirus Epidemiology Donor Study-II (REDS-II) collected information on all donation visits from 2006 to 2009. Simulations were developed from these data using a multistage approach that first sought to adequately reproduce the patterns of donor return, Hb and ferritin levels, and outcomes of a donor's visit (successful single– or double–red blood cell donation, deferral for low Hb) observed in REDS-II data sets. Modified simulations were used to predict the potential impact on the blood supply and donor iron status under different Hb cutoff and IDI qualification criteria. RESULTS More than 10% of WB donations might require replacement under many simulated scenarios. Longer IDIs would reduce the proportion of donors with iron depletion, but 80% of these donors may remain iron-depleted if minimal IDIs increased to 12 or 16 weeks. CONCLUSION Higher Hb cutoffs and longer IDIs are predicted to have a potentially large impact on collections but only a modest impact on donor iron depletion. Efforts to address iron depletion should be targeted to at-risk donors, such as iron supplementation programs for frequent donors, and policy makers should try to avoid broadly restrictive donation requirements that could substantially reduce blood availability.

Analytical meditation and monastic debate are contemplative practices engaged in by Tibetan Buddhist monastics that have up to now been largely unexplored in Western contemplative science. The highly physical form of contemplative debating plays an important role in the monastic curriculum. Based on discussions and recorded interviews Tibetan monastic teachers and senior students at Sera Jey Monastic University and preliminary experiments, we outline an initial theory that elucidates the psychological mechanisms underlying this practice. We then make predictions about the potential effects of this form of debating on cognition and emotion. On the basis of initial observations, we propose that successful debating requires skills that include reasoning and critical thinking, attentional focus, working memory, emotion regulation, confidence in your own reasoning skills, and social connectedness. It is therefore likely that the many cumulative hours of debate practice over 20+ years of monastic training helps to cultivate these very skills. Scientific research is needed to examine these hypotheses and determine the role that monastic debate may play in terms of both psychological wellbeing and educational achievement.

Analytical meditation and monastic debate are contemplative practices engaged in by Tibetan Buddhist monastics that have up to now been largely unexplored in Western contemplative science. The highly physical form of contemplative debating plays an important role in the monastic curriculum. Based on discussions and recorded interviews Tibetan monastic teachers and senior students at Sera Jey Monastic University and preliminary experiments, we outline an initial theory that elucidates the psychological mechanisms underlying this practice. We then make predictions about the potential effects of this form of debating on cognition and emotion. On the basis of initial observations, we propose that successful debating requires skills that include reasoning and critical thinking, attentional focus, working memory, emotion regulation, confidence in your own reasoning skills, and social connectedness. It is therefore likely that the many cumulative hours of debate practice over 20+ years of monastic training helps to cultivate these very skills. Scientific research is needed to examine these hypotheses and determine the role that monastic debate may play in terms of both psychological wellbeing and educational achievement.

Monoclonal antibodies (mAbs) that block the programmed death 1 (PD-1) or programmed death-ligand 1 (PD-L1) receptors are the most clinically advanced tumor immunotherapies. Given the broad antitumor efficacy and novel mechanism of action, numerous combinatorial approaches incorporating PD-1/PD-L1 blockade have been suggested; herein we present a comprehensive analysis of these clinical trials. We queried clinicaltrials.gov for all PD-1/PD-L1 mAbs administered for cancer therapy with an end date of 4/30/2017. A total of 1,218 clinical trials met our search criteria. These trials have a planned enrollment of 227,190 patients, and approximately half (493) were initiated in 2016 alone. Of these over 1,200 trials, 916 combine PD-1/PD-L1 blockade with at least one additional therapy, ranging from traditional treatment modalities like surgery and chemoradiation to newer therapies like small molecule inhibitors and other immunotherapies. The staggering proliferation of clinical trials combining PD-1/PD-L1 blockade with disparate treatments necessitates careful accounting to maximize efficiency and highlight areas of unmet needs. We believe our analysis provides this data and expect it will facilitate the design of future clinical trials in this burgeoning area of oncology research.

Mouse CD8(+) T cells conditioned with interleukin (IL)-12 ex vivo mediate the potent regression of established melanoma when transferred into lymphodepleted mice. However, the quantitative and qualitative changes induced by IL-12 in the responding mouse CD8(+) T cells have not been well defined. Moreover, the mechanisms by which IL-12-conditioning impacts human CD8(+) T cells, and how such cells might be expanded prior to infusion into patients is not known. We found that ex vivo IL-12-conditioning of mouse CD8(+) T cells led to a tenfold-100-fold increase in persistence and anti-tumor efficacy upon adoptive transfer into lymphodepleted mice. The enhancing effect of IL-12 was associated with maintenance of functional avidity. Importantly, in the context of ongoing ACT clinical trials, human CD8(+) T cells genetically modified with a tyrosinase-specific T cell receptor (TCR) exhibited significantly enhanced functional activity when conditioned with IL-12 as indicated by heightened granzyme B expression and elevated peptide-specific CD107a degranulation. This effect was sustainable despite the 20 days of in vitro cellular expansion required to expand cells over 1,000-fold allowing adequate cell numbers for administration to cancer patients. Overall, these findings support the efficacy and feasibility of ex vivo IL-12-conditioning of TCR-modified human CD8(+) T cells for adoptive transfer and cancer therapy.

Effector CD8+ T cells conditioned with IL12 during activation mediate enhanced antitumor efficacy after adoptive transfer into lymphodepleted hosts; this is due in part to improved IL7 responsiveness. Therefore, we hypothesized that increasing the intensity or type of lymphodepletion would deplete more IL7-consuming host cells and improve the persistence and antitumor activity of IL12-conditioned CD8+ T cells. Using cyclophosphamide, fludarabine, and total body irradiation (TBI, 6 Gy) either individually or in combination, we found that combined lymphodepletion best enhanced T-cell engraftment in mice. This improvement was strongly related to the extent of leukopenia, as posttransfer levels of donor T cells inversely correlated to host cell counts after lymphodepletion. Despite the improvement in engraftment seen with combination lymphodepletion, dual-agent lymphodepletion did not augment the antitumor efficacy of donor T cells compared with TBI alone. Similarly, IL7 supplementation after TBI and transfer of tumor-reactive T cells failed to improve persistence or antitumor immunity. However, IL15 or IL2 supplementation greatly augmented the persistence and antitumor efficacy of donor tumor-reactive T cells. Our results indicate that the amount of host IL7 induced after single agent lymphodepletion is sufficient to potentiate the expansion and antitumor activity of donor T cells, and that the efficacy of future regimens may be improved by providing posttransfer support with IL2 or IL15. Significance: The relationship between lymphodepletion and cytokine support plays a critical role in determining donor T-cell engraftment and antitumor efficacy. Cancer Res; 78(11); 3067–74. ©2018 AACR.

Background Clinical application of adoptive T cell therapy has been hindered by an inability to generate adequate numbers of nontolerized, functionally active, tumor-specific T cells, which can persist in vivo. In order to address this, we evaluated the impact of interleukin (IL)-12 signaling during tumor-specific CD8+ T cell priming in terms of persistence and antitumor efficacy using an established B16 melanoma tumor adoptive therapy model. Study Design B6 mice were injected subcutaneously with B16 melanoma tumor cells. On day 12 of tumor growth, mice were preconditioned with cyclophosphamide (4mg dose, intraperitoneally), and 1 day later were treated by adoptive transfer of tumor-specific pmel-1 CD8+ T cells primed ex vivo 3 days earlier with both IL-12 and antigen (hGP10025-33 peptide) or antigen only. Tumors were measured biweekly, and infused donor T cells were analyzed for persistence, localization to the tumor, phenotype, and effector function. Results Adoptive transfer of tumor-specific CD8+ T cells primed with IL-12 was significantly more effective in reducing tumor burden in mice preconditioned with cyclophosphamide compared with transfer of T cells primed without IL-12. This enhanced antitumor response was associated with increased frequencies of infused T cells in the periphery and tumor as well as elevated expression of effector molecules including granzyme B and interferon-γ (IFNγ). Conclusions Our findings demonstrate that ex vivo priming of tumor-specific CD8+ T cells with IL-12 dramatically improves their in vivo persistence and therapeutic ability on transfer to tumor-bearing mice. These findings can be directly applied as novel clinical trial strategies.

This case uncovers the balance between school culture and academic success that school leaders must consider while leading their schools. New school leaders as well as experienced leaders transitioning into new buildings find that each school has its own distinctive culture. The existing culture and ethos of a school must be considered as leaders gauge the need for short-term and long-term improvements. This must be balanced with the experiences and expectations brought by the leader that have the ability to create positive change. This transition process should not be taken lightly, and administrators should carefully observe what traditions their new school communities value when considering what change needs to be incorporated. This case uncovers the need for new building principals to reflect and analyze background data, both qualitative and quantitative, prior to mandating change in their building. In that sense, it gives aspiring leaders an opportunity to reflect on leadership style and whether there is an opportunity to contextualize that style.

Frequent blood donors become iron deficient. HFE mutations are present in over 30% of donors. A 24-month study of 888 first time/reactivated donors and 1537 frequent donors measured haemoglobin and iron status to assess how HFE mutations impact the development of iron deficiency erythropoiesis. Donors with two HFE mutations had increased baseline haemoglobin and iron stores as did those with one mutation, albeit to a lesser extent. Over multiple donations haemoglobin and iron status of donors with HFE mutations paralleled those lacking mutations. The prevalence of HFE mutations was not increased in higher intensity donors. Thus, in general, HFE mutations do not temper donation-induced changes in haemoglobin and iron status. However, in Black donors there was an increase of H63D carriers at baseline, from 3·7% in first time/reactivated donors to 15·8% in frequent donors, suggesting that the relative effects of HFE mutations on iron absorption may vary between racial/ethnic groups. In secondary analyses, venous haemoglobin decreased more slowly in donors with ferritin ≥12μg/l; and haemoglobin recovery time was shorter in donors with reticulocyte haemoglobin (CHr) ≥32·6pg, indicating that these biochemical measures are better indicators of a donor's response to phlebotomy than their HFE mutation status.

Blood donor qualification requires demonstration that the donor’s hemoglobin (Hb) or hematocrit (Hct) meets requirements. In the United States donor Hb must be greater than or equal to 12.5 g/dL or Hct at least 38%. Nearly all blood donor qualification schemes depend on capillary sampling of the donor’s finger. Earstick sampling has been used in the past but has been shown to produce values that are higher than venous and fingerstick values.1 Fingerstick sampling has been shown to more closely approximate venous Hb values, although reports differ on the nature and degree of the difference.2 Previous studies comparing capillary versus venous samples in blood donors have been limited. First, studies in operational settings have often used copper sulfate screening of capillary samples, using quantitative methods only to retest donors who failed the copper sulfate screen. Second, the various quantitative methods used have varied in reliability, user training, and the precision of measurement, which has often been poor. Finally the venous samples have often been obtained after whole blood donations, along with routine testing samples. Postdonation values have been shown in one small study to be significantly lower than venous samples collected before donation.1 Further, donor positional effects (sitting, recumbent) at the time of the venous sample collection have not been well controlled. Most comparisons of the two methods have demonstrated that fingerstick sampling has higher variability than venous sampling and that a number of donors accepted on the basis of a fingerstick sample have venous Hb values below the acceptable minimum.2 Recently, a study in Ireland has demonstrated in routinely collected fingerstick Hb deferred donors that venous Hb is higher than the fingerstick value by 1.07 g/dL (males) and 0.67 g/dL (females).3 The authors used this result to validate a decision to change the acceptance level for fingerstick Hb from 12.5 g/dL to 12.0 g/dL for females and 13.5 g/dL to 13.0 g/dL for males. Although this was the primary study focus, they also measured simultaneous fingerstick and venous Hb values across a wider range of fingerstick Hb on 155 accepted first-time donors and demonstrated that the difference between fingerstick and venous values was dependent on Hb level. Below fingerstick Hb of 15.4 g/dL for men and 13.4 g/dL for women, venous Hb values were higher than fingerstick, while above this level venous Hb values were lower than fingerstick. However, this relationship was based on only a small number of donors. Also because only first-time donors were studied, the applicability of this phenomenon to repeat blood donors is not clear. The REDS-II (Retrovirus Epidemiology Donor Study-II) blood centers have completed a study of blood donor iron status (RISE), which provides a large multicenter assessment of fingerstick and venous sampling methods. At enrollment blood donors had quantitative measurements of fingerstick Hb or Hct and venous Hb, as well as measurement of plasma ferritin. Several different quantitative fingerstick methods were used at the centers, allowing indirect assessment of methodologic differences. Pre-and postdonation venous samples were both obtained from some donors, allowing development of an algorithm for conversion of one sample type to the other. Finally, a multivariable model incorporating sex and body iron stores was developed to predict predonation venous Hb from the fingerstick Hb or Hct values.

As the US population ages, and those over 65, who represent 53% of the red blood cell (RBC) transfusion recipients, grows from 40 to 72 million by 2030, it is assumed that there will be an increase in the transfusion needs of the US population. 1,2 As outlined by Riley and coworkers,3 approximately 37% of the US population is eligible to donate blood but only 5% actually participate in blood donor programs. This raises concern as to the viability of the blood donor pool, because the majority of current donors are older and may make fewer donations and become recipients as they age. Several studies have investigated the reasons first-time donors do not return for a subsequent donation. These studies have documented the results of age,4 sex,5,6 education,5,7 race/ethnicity,8–10 convenience,8,9,11 donor intention to return,4,8 and donor return patterns.7,8 In 2009, Notari and colleagues12 reported on donor age and its impact on return behavior in first-time donors over a 13-month period. Sixteen- and 17-year-old donors had the highest return rate with 62 and 52% returning, respectively, whereas donors 60 years and older had the next highest return rate at 48%. The frequencies of donations in the 13-month follow-up period were 2.61, 2.46, and 3.09 donations for 16-, 17-, and 60+-year-old donors, respectively, compared to 2.40 to 2.95 for donors aged 18 to 59 years. Others have summarized the issues related to the donation experience that, if addressed, may change blood donation behavior. These issues include: 1) understanding the motivations of first-time versus long-term donors and their differing responses to incentives;10 2) contacting first- and second-time donors in a timely fashion to schedule a repeat donation;5,7 and 3) deferral, reactions, pain, or anxiety during the first and second donation, all of which lead to lower return rates.4 Since donation frequency in the first year is correlated with an increased likelihood of returning during the next 6 years, encouraging first-time donors to make additional donations in the first year may produce more dedicated lifetime donors.7 This could be facilitated by providing these donors information on convenient donation locations for future donations, thereby potentially reducing blood donation barriers, especially among those who are interested and willing to become a regular donor.7 Convenience is important for donors. Bringing the donation site closer to the donor by conducting more mobile blood drives is one way to increase convenience, while at the same time contributing to blood center goals of increased numbers of donors and increased donation frequency. Of course, in this scenario, the number of donations is tied to the frequency the mobile site presents itself to the donor. With this in mind, the characteristics of donations at fixed versus mobile locations should be examined. Specifically, more information is needed about donors who visit fixed and mobile sites to better understand donation patterns and behavior for movement within and between these donation locations. This study investigates the demographic characteristics of donors by donation type (whole blood [WB], plateletpheresis [PP], and double RBC [R2]), the median number of donations (frequency), median interdonation interval, and the median return time (controlling for mandatory wait time).

BACKGROUND It is thought that HLA antibodies might contribute to the pathogenesis of transfusion-related acute lung injury (TRALI). Many methods exist to detect HLA antibodies, including ELISA, flow cytometry, and multiplex bead-based assays, as well as the older lymphocytotoxicity assay. The sensitivity of each of these testing platforms varies, and it is not obvious how to compare HLA antibody results obtained on different platforms. This issue has become increasingly important as some blood centers have begun HLA antibody apheresis donor screening as a TRALI risk-reduction measure.

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca(2+)-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O(2)(-)) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O(2)(-) from the IMS and sensitizing to the Ca(2+)-induced MPT. Treatment of isolated rat liver mitochondria with 5microM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8+/-1.4min within a range of 100-250microM Ca(2+). G3139-mediated acceleration of the MPT was reversed by 20microM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O(2)(-) in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O(2)(-) accumulation. Mathematical modeling of generation and turnover of O(2)(-) within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O(2)(-). In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O(2)(-), which causes an internal oxidative stress and sensitizes mitochondria toward the Ca(2+)-induced MPT.

IL-7 and IL-15 are critical for supporting T cells transferred into a lymphopenic environment. As activated CD8(+) T cells downregulate IL-7Rα, it is thought IL-15 is more important. However, we find that CD8(+) T cells activated with IL-12 have elevated IL-7Rα and rely on IL-7 for persistence and antitumor immunity.

Interleukin-2 (IL-2) is a lymphocyte growth factor that is an important component of many immune-based cancer therapies. The efficacy of IL-2 is thought to be limited by the expansion of T regulatory cells, which express the high-affinity IL-2 receptor subunit IL-2Rα. IL-15 is under investigation as an alternative to IL-2. Although both cytokines signal through IL-2Rβγ, IL-15 does not bind IL-2Rα and therefore induces less T regulatory cell expansion. However, we found that transferred effector CD8(+) T cells induced curative responses in lymphoreplete mice only with IL-2-based therapy. Although conventional in vitro assays showed similar effector T cell responsiveness to IL-2 and IL-15, upon removal of free cytokine, IL-2 mediated sustained signaling dependent on IL-2Rα. Mechanistically, IL-2Rα sustained signaling by promoting a cell surface IL-2 reservoir and recycling of IL-2 back to the cell surface. Our results demonstrate that IL-2Rα endows T cells with the ability to compete temporally for limited IL-2 via mechanisms beyond ligand affinity. These results suggest that strategies to enhance IL-2Rα expression on tumor-reactive lymphocytes may facilitate the development of more effective IL-2-based therapies.

Background: CPR duty cycle is the portion of time spent in compression relative to the total time of the compression-decompression cycle. Guidelines recommend a 50% duty cycle based largely on animal investigation. Little is known about duty cycle in human resuscitation and whether duty cycle correlates with other CPR measures. Methods: We calculated the duty cycle, compression depth, and compression rate during EMS resuscitation of 164 patients with out-of-hospital ventricular fibrillation cardiac arrest. We captured force recordings from a chest accelerometer to measure ten-second CPR epochs that immediately preceded each scheduled rhythm analysis. Duty cycle was calculated using two methods. The effective compression time (ECT) is the time from beginning to end of compression divided by total period for that compression-decompression cycle. The area duty cycle (ADC) is the ratio of area under the force curve divided by total area of one compression-decompression cycle. We evaluated the compression depth and compression rate according to duty cycle quartiles. Results: There were 369 ten-second epochs among 164 patients. The median duty cycle was 38.8% (SD=5.5%) using ECT and 32.2% (SD=4.3%) using ADC. A relatively shorter compression phase (lower duty cycle) was associated with greater compression depth (test for trend < 0.05 for ECT and ADC) and slower compression rate (test for trend < 0.05 for ADC) (Table). Sixty-one patients (37%) survived to hospital discharge. Conclusions: In this system with high survival, duty cycle was well below the 50% recommended guideline, and was associated with compression depth and rate. These findings provide rationale to incorporate duty cycle into future research to evaluate how CPR influences resuscitation.

Ex vivo IL-12 priming of tumor-reactive CD8+ T-cells enhances their persistence and subsequent anti-tumor efficacy upon adoptive cell transfer (ACT) into lymphodepleted mice. Since IL-7 and IL-15 are considered critical for the persistence of adoptively transferred T-cells, we investigated the responsiveness of IL-12 primed CD8+ T-cells to these homeostatic cytokines to help elucidate the mechanisms behind their superior anti-tumor abilities. Using pmel-1 T-cell receptor transgenic mice, we found that pmel CD8+ T cells activated in the presence of IL-12 (pmelIL-12) showed much greater expansion in an irradiated (6 Gy) host compared to cells activated without IL-12 (pmelsham). This expansion was dependent on host IL-7, but not IL-15. Compared to pmelsham, pmelIL-12 demonstrated greatly enhanced in vitro IL-7 responsiveness, as measured by proliferation and phosphorylation of signaling molecules STAT5, S6 and AKT. These striking differences were not seen with IL-15. Despite not playing a major role in pmelIL-12 effector phase expansion, IL-15 was critical for maximum anti-tumor efficacy of pmelIL12, as irradiated mice devoid of IL-15, like mice receiving IL-7 neutralizing antibody (clone M25), showed reduced survival compared to irradiated mice receiving pmelIL-12 only. This decreased tumor control likely occurred because IL-15 was needed for long-term persistence of pmelIL-12. Together, these findings suggest that IL-12 priming of CD8 + T-cells augments the efficacy of ACT protocols in part by conferring an enhanced ability to respond to IL-7, thereby enabling transferred cells to persist in the IL-7 rich post-lymphodepletion environment.

Dietary iron absorption is regulated by hepcidin, an iron regulatory protein produced by the liver. Hepcidin production is regulated by iron stores, erythropoiesis and inflammation, but its physiology when repeated blood loss occurs has not been characterized. Hepcidin was assayed in plasma samples obtained from 114 first-time/reactivated (no blood donations in preceding 2 years) female donors and 34 frequent (≥3 red blood cell donations in preceding 12 months) male donors as they were phlebotomized ≥4 times over 18-24 months. Hepcidin levels were compared to ferritin and hemoglobin levels using multivariable repeated measures regression models. Hepcidin, ferritin and hemoglobin levels declined with increasing frequency of donation in the first-time/reactivated females. Hepcidin and ferritin levels correlated well with each other (Spearman's correlation of 0.74), but on average hepcidin varied more between donations for a given donor relative to ferritin. In a multivariable repeated measures regression model the predicted inter-donation decline in hemoglobin varied as a function of hepcidin and ferritin; hemoglobin was 0.51 g/dL lower for subjects with low (>45.7 ng/mL) or decreasing hepcidin and low ferritin (>26 ng/mL), and was essentially zero for other subjects including those with high (>45.7 ng/mL) or increasing hepcidin and low ferritin (>26 ng/mL) levels (P

ALT-803, a complex of an interleukin (IL)-15 superagonist mutant and a dimeric IL-15 receptor αSu/Fc fusion protein, was found to exhibit significantly stronger in vivo biologic activity on NK and T cells than IL-15. In this study, we show that a single dose of ALT-803, but not IL-15 alone, eliminated well-established 5T33P and MOPC-315P myeloma cells in the bone marrow of tumor-bearing mice. ALT-803 treatment also significantly prolonged survival of myeloma-bearing mice and provided resistance to rechallenge with the same tumor cells through a CD8+ T-cell–dependent mechanism. ALT-803 treatment stimulated CD8+ T cells to secrete large amounts of IFN-γ and promoted rapid expansion of CD8+CD44high memory T cells in vivo. These memory CD8+ T cells exhibited ALT-803–mediated upregulation of NKG2D (KLRK1) but not PD-1 (PDCD1) or CD25 (IL2RA) on their cell surfaces. ALT-803–activated CD8+ memory T cells also exhibited nonspecific cytotoxicity against myeloma and other tumor cells in vitro, whereas IFN-γ had no direct effect on myeloma cell growth. ALT-803 lost its antimyeloma activity in tumor-bearing IFN-γ knockout mice but retained the ability to promote CD8+CD44high memory T-cell proliferation, indicating that ALT-803–mediated stimulation of CD8+CD44high memory T cells is IFN-γ–independent. Thus, besides well-known IL-15 biologic functions in host immunity, this study shows that IL-15–based ALT-803 could activate CD8+CD44high memory T cells to acquire a unique innate-like phenotype and secrete IFN-γ for nonspecific tumor cell killing. This unique immunomodulatory property of ALT-803 strongly supports its clinical development as a novel immunotherapeutic agent against cancer and viral infections. Cancer Res; 73(10); 3075–86. ©2013 AACR.

e21013Background: Stage IV melanoma patients treated with ipilimumab (Ipi) have a median survival of 40 to 44 weeks (wks). Recent studies have demonstrated the combination of RT and Ipi can improve...

Background and Objectives LDL apheresis is used to treat patients with familial hypercholesterolaemia, and low-volume plasmapheresis for plasma donation may similarly lower cholesterol levels in some donors. This study was designed to assess the effect of plasmapheresis on total, LDL and HDL cholesterol levels in a plasma donor population. Materials and Methods This was a prospective, unblinded longitudinal cohort study in which a blood sample was obtained for analysis before each donation. Data from 663 donors were analysed using a multivariable repeated measures regression model with a general estimating equations approach with changes in cholesterol as the primary outcome measure. Results The model predicted a significant decrease in total and LDL cholesterol for both genders and all baseline cholesterol levels (P

The glycine cleavage system (GCS), the major pathway of glycine catabolism in liver, is found only in the mitochondria matrix and is regulated by the oxidized nicotinamide adenine dinucleotide (NAD(+) )/reduced nicotinamide adenine dinucleotide (NADH) ratio. In conjunction with serine hydroxymethyltransferase, glycine forms the 1 and 2 positions of serine, while the 3 position is formed exclusively by GCS. Therefore, we sought to exploit this pathway to show that quantitative measurements of serine isotopomers in liver can be used to monitor the NAD(+) /NADH ratio using (13) C NMR spectroscopy. Rat hepatocytes were treated with modulators of GCS activity followed by addition of 2-(13) C-glycine, and the changes in the proportions of newly synthesized serine isotopomers were compared to controls. Cysteamine, a competitive inhibitor of GCS, prevented formation of mitochondrial 3-(13) C-serine and 2,3-(13) C-serine isotopomers while reducing 2-(13) C-serine by 55%, demonstrating that ca. 20% of glycine-derived serine is produced in the cytosol. Glucagon, which activates GCS activity, and the mitochondrial uncoupler carbonyl cyanide-3-chlorophenylhydrazone both increased serine isotopomers, whereas rotenone, an inhibitor of complex I, had the opposite effect. These results demonstrate that (13) C magnetic resonance spectroscopy monitoring of the formation of serine isotopomers in isolated rat hepatocytes given 2-(13) C-glycine reflects the changes of mitochondrial redox status.

Fingerstick blood samples are used to estimate donor venous hemoglobin (Hb).Fingerstick Hb or hematocrit (Hct) was determined routinely for 2425 selected donors at six blood centers, along with venous Hb. Using sex and measures of iron status including absent iron stores (AIS; ferritin 12 ng/mL), linear regression models were developed to predict venous Hb from fingerstick.Across all subjects, fingerstick Hb was higher than venous Hb in the higher part of the clinical range, but lower in the lower part of the range. The relationship varied by sex and iron status. Across centers, a female donor had on average a venous Hb result 0.5 to 0.8 g/dL lower than a male donor with the same fingerstick Hb and iron status. Similarly, a donor with AIS had on average a venous Hb result 0.3 to 1.1 g/dL lower than an iron-replete donor with the same fingerstick value and sex. An iron-replete male donor with a fingerstick result at the cutoff (Hb 12.5 g/dL) had an acceptable expected venous Hb (12.8 to 13.8 g/dL). A female donor with AIS with a fingerstick result at the cutoff had an expected venous Hb below 12.5 g/dL (11.7 to 12.4 g/dL). Of females with AIS, 40.2% donated blood when their venous Hb was less than 12.5 g/dL.Fingerstick is considered a useful estimator of venous Hb. However, in some donor groups, particularly female donors with AIS, fingerstick overestimates venous Hb at the donation cutoff. This significant limitation should be considered in setting donor fingerstick Hb or Hct requirements.

Anemia is an important public health concern. Data from population-based surveys such as the National Health and Nutrition Examination Survey (NHANES) are the gold standard, but are obtained infrequently and include only small samples from certain minority groups. We assessed whether readily available databases of blood donor hemoglobin values could be used as a surrogate for population hemoglobin values from NHANES. Blood donor venous and fingerstick hemoglobin values were compared to 10,254 NHANES 2005-2008 venous hemoglobin values using demographically stratified analyses and ANOVA. Fingerstick hemoglobins or hematocrits were converted to venous hemoglobin estimates using regression analysis. Venous hemoglobin values from 1,609 first time donors correlated extremely well with NHANES data across different ages, genders, and demographic groups. Cigarette smoking increased hemoglobin by 0.26-0.59 g/dL depending on the intensity. Converted fingerstick hemoglobin from 36,793 first time donors agreed well with NHANES hemoglobin (weighted mean hemoglobin of 15.53 g/dL for donors and 15.73 g/dL for NHANES) with similar variation in mean hemoglobin by age. However, compared to NHANES, the larger donor data set showed reduced differences in mean hemoglobin between Blacks and other races/ethnicities. Overall, first-time donor fingerstick hemoglobins approximate US population data and represent a readily available public health resource for ongoing anemia surveillance.

The glycine cleavage system (GCS), the major pathway of glycine catabolism in liver, is found only in the mitochondria matrix and is regulated by the oxidized nicotinamide adenine dinucleotide (NAD(+) )/reduced nicotinamide adenine dinucleotide (NADH) ratio. In conjunction with serine hydroxymethyltransferase, glycine forms the 1 and 2 positions of serine, while the 3 position is formed exclusively by GCS. Therefore, we sought to exploit this pathway to show that quantitative measurements of serine isotopomers in liver can be used to monitor the NAD(+) /NADH ratio using (13) C NMR spectroscopy. Rat hepatocytes were treated with modulators of GCS activity followed by addition of 2-(13) C-glycine, and the changes in the proportions of newly synthesized serine isotopomers were compared to controls. Cysteamine, a competitive inhibitor of GCS, prevented formation of mitochondrial 3-(13) C-serine and 2,3-(13) C-serine isotopomers while reducing 2-(13) C-serine by 55%, demonstrating that ca. 20% of glycine-derived serine is produced in the cytosol. Glucagon, which activates GCS activity, and the mitochondrial uncoupler carbonyl cyanide-3-chlorophenylhydrazone both increased serine isotopomers, whereas rotenone, an inhibitor of complex I, had the opposite effect. These results demonstrate that (13) C magnetic resonance spectroscopy monitoring of the formation of serine isotopomers in isolated rat hepatocytes given 2-(13) C-glycine reflects the changes of mitochondrial redox status.

The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5-(13)C]-thiazolidine-4-carboxylate, (13)C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by (13)C magnetic resonance spectroscopy. Although uptake and clearance of (13)C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of (13)C-OTZ over 20 h, however, resulted in (13)C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.

The synergistic activity between nitric oxide (NO) released from diazeniumdiolate-modified proline (PROLI/NO) and silver(I) sulfadiazine (AgSD) was evaluated against Escherichia coli, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis using a modified broth microdilution technique and a checkerboard-type assay. The combination of NO and AgSD was defined as synergistic when the fractional bactericidal concentration (FBC) was calculated to be

The Eastern oyster (Crassostrea virginica) is a useful, robust model marine organism for tissue metabolism studies. Its relatively few organs are easily delineated and there is sufficient understanding of their functions based on classical assays to support interpretation of advanced spectroscopic approaches. Here we apply high-resolution proton nuclear magnetic resonance (1H NMR)-based metabolomic analysis to C. virginica to investigate the differences in the metabolic profile of different organ groups, and magnetic resonance imaging (MRI) to non-invasively identify the well separated organs. Metabolites were identified in perchloric acid extracts of three portions of the oyster containing: (1) adductor muscle, (2) stomach and digestive gland, and (3) mantle and gills. Osmolytes dominated the metabolome in all three organ blocks with decreasing concentration as follows: betaine > taurine > proline > glycine > ß-alanine > hypotaurine. Mitochondrial metabolism appeared most pronounced in the adductor muscle with elevated levels of carnitine facilitating ß-oxidation, and ATP, and phosphoarginine synthesis, while glycogen was elevated in the mantle/gills and stomach/digestive gland. A biochemical schematic is presented that relates metabolites to biochemical pathways correlated with physiological organ functions. This study identifies metabolites and corresponding 1H NMR peak assignments for future NMR-based metabolomic studies in oysters.

We report the therapeutic potential of S-nitroso-N-acetylpenicillamine-derivatized generation-4 polyamidoamine dendrimers (G4-SNAP) for reducing ischemia/reperfusion (I/R) injury in an isolated, perfused rat heart. The use of this dendrimer scaffold to deliver the nitrosothiol therapeutic did not inhibit NO donor activity as the required dose of G4-SNAP to minimize I/R injury (31nM corresponding to 2microM SNAP) was consistent with the optimum concentration of small molecule SNAP alone. An exploration of G4-SNAP NO release kinetics in the presence of physiologically relevant concentrations of glutathione (GSH) indicated enhanced NO release (t[NO]=1.28microM NO/mg) at 500microM GSH. Reperfusion experiments conducted with 500microM GSH further lowered the optimal therapeutic G4-SNAP dose to 230pM (i.e., 15nM SNAP). The unique combination of G4-SNAP dendrimer and glutathione trigger represents a novel strategy with possible clinical relevance toward salvaging ischemic tissue.

The utility of nitric oxide (NO)-releasing silica nanoparticles as novel antibacterial agents is demonstrated against Pseudomonas aeruginosa. Nitric oxide-releasing nanoparticles were prepared via co-condensation of tetraalkoxysilane with aminoalkoxysilane modified with diazeniumdiolate NO donors, allowing for the storage of large NO payloads. Comparison of the bactericidal efficacy of the NO-releasing nanoparticles to 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO), a small molecule NO donor, demonstrated enhanced bactericidal efficacy of nanoparticle-derived NO and reduced cytotoxicity to healthy cells (mammalian fibroblasts). Confocal microscopy revealed that fluorescently labeled NO-releasing nanoparticles associated with the bacterial cells, providing rationale for the enhanced bactericidal efficacy of the nanoparticles. Intracellular NO concentrations were measurable when the NO was delivered from nanoparticles as opposed to PROLI/NO. Collectively, these results demonstrate the advantage of delivering NO via nanoparticles for antimicrobial applications.

The cytotoxicity and time-dependent membrane disruption by polypropylenimine dendrimer conjugates on cultured human umbilical vein endothelial cells (HUVEC) is reported. Fluorescently labeled derivatives of generation 5 polypropylenimine dendrimers were prepared via conversion of amines to acetamides or through the covalent attachment of high molecular weight poly(ethylene glycol) (PEG) chains. Direct interactions between the fluorescent dendrimer conjugates and HUVEC were monitored using confocal fluorescence microscopy to track dendrimer movement across the plasma membrane and the fluorescent staining of cell nuclei. Propidium iodide and lactate dehydrogenase cytotoxicity assays confirmed that chemical modification of the surface amines of the parental dendrimer to neutral acetamide or PEG functionalities eliminated their acute cytotoxicity. Cationic primary-amine-containing dendrimers demonstrated drastic time-dependent changes in the plasma membrane permeability and prominent cytotoxicity. However, complete removal of the primary amines or masking of the cationic surface via PEGylation decreased dendrimer cytotoxicity. Thus, preventing electrostatic interactions of dendrimers with cellular membranes apparently is a necessary step toward minimizing the toxicity of delivery vehicles to the endothelium.

The adoptive transfer of tumor-reactive T cells has shown great promise in treating patients with metastatic cancer. However, effective T cell responses are limited by the availability of T cell growth factors such as IL-2 and tumor-induced suppressive pathways. As tumor-induced suppression may hamper cytokine responsiveness, we hypothesized that combinatorial therapy providing exogenous cytokine with blockade of inhibitory pathways would lead to synergistic anti-tumor responses. We evaluated this hypothesis by treating mice with palpable B16 melanoma tumors with lymphodepletion and transfer of activated, tumor-reactive CD8 + T cells (pmel-1 TCR transgenic). The persistence of the adoptively transferred tumor-reactive CD8 + T cells was dramatically augmented in the recipient mice with injections of an IL-15 superagonist (ALT-803) which, compared with IL-2, has greater biological activity and does not expand T regulatory cells. The ALT-803-treated mice also survived significantly longer than the untreated mice. B16 melanoma tumor cells were found to express PD-L1 and activated CD8 + T cells have PD-1 on their surface. Thus, we also gave mice anti-PD-L1 mAb treatment to block this PD-1/PD-L-1 inhibitory pathway. Our preliminary data suggest that combinatorial therapy with anti-PD-L1 mAb led to synergistic improvement in anti-tumor efficacy. We are now determining the optimal timing and dosing of ALT-803 and anti-PD-L1 mAb therapy to confirm these results. Currently, ALT-803 is in clinical trials for treating patients with various solid and hematologic tumors. Our findings suggest combinatorial therapy relieving T cell dysfunction using checkpoint inhibitors and providing ALT-803 cytokine therapy may lead to substantially improved outcomes over currently available therapies for patients with metastatic cancer.