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There is a paucity of age data for chondrichthyan fishes owing, in large part, to limitations in traditional age estimation methods. Fourier transform near-infrared (FT-NIR) spectroscopy has shown promise as an alternative, more efficient method for acquiring age data from chondrichthyans. However, studies are limited to sharks in the southern hemisphere. We explored FT-NIR spectroscopy to predict age for a batoid species in the northern hemisphere. The longnose skate (Raja rhina) is one of a small number of batoids for which annual band periodicity in vertebral centra has been validated, allowing for traditional age estimation and making it an ideal candidate for this study. We fit a multivariate partial least-square predictive model between FT-NIR spectra collected from vertebral centra and traditional age estimates, and tested model predictive skill by using external validation. Using FT-NIR spectroscopy, we were able to predict age for longnose skates between the ages of 1 and 14 years with precision and bias near equal to those of traditional methods in less than a quarter of the time. These results support potential for FT-NIR spectroscopy to increase the amount of age data available for assessments used to inform the conservation and management of this sensitive group of species.

Background Metastasis is most often the root cause of cancer-related death. Human short stature homeobox 2 (SHOX2), a homeodomain transcription factor, is a novel inducer of epithelial-to-mesenchymal transition in breast cancer cells, though its exact role and underlying mechanisms in metastasis are not well understood. Methods TCGA analysis was performed to identify the clinical relevance of SHOX2 in breast cancer. Gene depletion was achieved by short hairpin RNA and small interfering RNA. Molecular regulations and alterations were assessed by Western blotting, immunoprecipitation, immunohistochemistry, qRT-PCR, chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR), and ChIP/re-ChIP. The impact of SHOX2 signaling on tumor growth and metastasis was evaluated in orthotopic breast tumor mice. Results The expression level of SHOX2 is strongly associated with poor distant metastasis-free survival in breast cancer patients and inactivation of SHOX2 suppresses breast tumor growth and metastasis in mice. In breast cancer cells, SHOX2 directly activates Wiskott-Aldridge syndrome protein family member 3 (WASF3), a metastasis-promoting gene, at the transcriptional level, leading to a significant increase in metastatic potential. Mechanistically, SHOX2 activates signal transducer and activator of transcription 3 (STAT3) and recruits it to the WASF3 promoter, where STAT3 cooperates with SHOX2 to form a functional immunocomplex to promote WASF3 transcriptional activity in breast cancer cells. WASF3 knockdown abrogates SHOX2-induced metastasis, but not SHOX2-dependent tumorigenesis. Conclusions These findings provide a critical link between the SHOX2-STAT3-WASF3 signaling axis and metastasis and suggest that the targeting of this signaling node may represent a valuable alternative strategy for combating breast cancer metastasis.

Small modifications in greenhouse agroenvironments can have a big impact on the success of biological control programs. For instance, the application of supplemental foods during and after the release of natural enemies onto crop plants, could considerably improve their long-term reproductive and population growth prospects. As such, food supplementation represents a valuable biological control supportive strategy, helping to grow natural enemy populations before pest establishment, akin to creating a standing-army to defend crops against future pest invasions. In many places of the world, food supplementation represents a relatively new but growing component or biological control research, with increasingly better resources available to guide producers, IPM practitioners, or researchers wanting to apply or optimize such strategies to their local agents and environments. In this review, we summarize the current stage of knowledge associated with various supplemental food types, which work best to support specific beneficial arthropods as well as some tools and techniques for successfully applying this biological control-enhancing strategy. We also summarize some current challenges to the use of supplemental foods and discuss what future research is needed to adapt and optimize food supplementation for a diversity of natural enemy species.

Autologous stem cell transplant (ASCT) is the standard of care for patients with multiple myeloma (MM). The clinical significance of peripheral blood T lymphocyte (PBTL) immunologic changes associated with ASCT is poorly understood. Here we evaluated T cell transcriptional messenger RNA profiles and immunophenotypes to correlate immunologic senescence, exhaustion, and anergy with clinical endpoints in a cohort of patients with MM undergoing ASCT. ASCT induced global transcriptional T cell changes and altered molecular levels of markers of T cell subtypes, T cell activation, and exhaustion. These included reduced CD4/CD8 ratio, skewing toward the Th1 subset, reduced expression of costimulatory receptors CD27 and CD28, heightened T cell activation, and increased expression of immune modulatory molecules LAG3 and PD1. Multicolor flow cytometry experiments confirmed altered circulating CD4 and CD8 subsets and skewing toward differentiated effector cells. Moreover, ASCT promoted an exhausted immunophenotype in CD3+CD4+ subsets and a senescent immunophenotype in CD3+CD8+ subsets. Subset-specific altered expression was also seen for surface molecules with immunomodulatory function. ASCT affected soluble levels of molecules with immunomodulatory function by increasing plasma HVEM and TIM3. High molecular LAG3 level was associated with inferior event-free survival post-ASCT (hazard ratio=5.44; confidence interval, 1.92 to 15.46; P = .001; adjusted P [controlling for false discovery rate] = .038). Using a comprehensive evaluation of PBTLs on a molecular and phenotypic level, we have identified that ASCT induces global T cell alterations with CD4 and CD8 subset-specific changes. Moreover, LAG3 emerged as an early biomarker of adverse events post-ASCT. These findings will support the development of treatment strategies targeting immune defects in MM to augment or restore T cell responses.

The appropriate dose of antithymocyte globulin (ATG) to be used in reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT) is yet to be determined. We retrospectively analyzed the outcomes of patients who underwent unrelated or mismatch related RIC allo-HSCT for hematologic malignancies and received r-ATG (4.5 mg/kg, 141 patients) versus R-ATG (6 mg/kg, 216 patients). There was a higher incidence of cytomegalovirus (P.001) and Epstein-Barr virus viremia (P =.03) in the R-ATG group than in the r-ATG group. The cumulative incidences of acute graft-versus-host disease (aGVHD) grades II to IV at day 180 in the r-ATG and R-ATG groups were 59% and 44% (P = .006) and grades III to IV 20% and 12% (P = .029), respectively. In multivariable models adjusting for disease diagnosis, the risk of aGVHD grades III to IV did not reach statistical significance (P = .087). The respective cumulative incidences of chronic GVHD in the r-ATG and R-ATG groups were 26% and 15% (P = .10), respectively. There were no significant differences in relapse rate (P = .24), nonrelapse mortality (P = .96), progression-free survival (P = .24), overall survival (P = .70), and GVHD-free relapse-free survival (P = .24). In this retrospective analysis, aGVHD incidence was higher in those treated with r-ATG compared with R-ATG, but this did not translate into significant differences of clinical outcome. Given the increasing use of RIC allo-HSCT for treating malignant hematologic conditions, the correct dose and schedule of ATG administration should be defined by prospective randomized controlled trials.

Blockade of PD-L1 expression on tumor cells via anti–PD-L1 monoclonal antibody (mAb) has shown great promise for successful cancer treatment by overcoming T-cell exhaustion; however, the function of PD-L1 on natural killer (NK) cells and the effects of anti–PD-L1 mAb on PD-L1+ NK cells remain unknown. Moreover, patients with PD-L1− tumors can respond favorably to anti–PD-L1 mAb therapy for unclear reasons. Here, we show that some tumors can induce PD-L1 on NK cells via AKT signaling, resulting in enhanced NK-cell function and preventing cell exhaustion. Anti–PD-L1 mAb directly acts on PD-L1+ NK cells against PD-L1− tumors via a p38 pathway. Combination therapy with anti–PD-L1 mAb and NK cell–activating cytokines significantly improves the therapeutic efficacy of human NK cells against PD-L1− human leukemia when compared with monotherapy. Our discovery of a PD-1–independent mechanism of antitumor efficacy via the activation of PD-L1+ NK cells with anti–PD-L1 mAb offers new insights into NK-cell activation and provides a potential explanation as to why some patients lacking PD-L1 expression on tumor cells still respond to anti–PD-L1 mAb therapy. Significance: Targeting PD-L1 expressed on PD-L1+ tumors with anti–PD-L1 mAb successfully overcomes T-cell exhaustion to control cancer, yet patients with PD-L1− tumors can respond to anti–PD-L1 mAb. Here, we show that anti–PD-L1 mAb activates PD-L1+ NK cells to control growth of PD-L1− tumors in vivo, and does so independent of PD-1. This article is highlighted in the In This Issue feature, p. 1325

Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development program to regional limb perfusion, comparing two methods previously established for gene therapy, peripheral venous limb perfusion (VLP) and an intra-arterial push and dwell (IAPD) using rAAV1 and rAAV8 in a non-human primate (rhesus macaque) study. The rhesus AAT transgene was used with a c-myc tag to enable quantification of transgene expression. 5 cohorts of animals were treated with rAAV1-IM, rAAV1-VLP, rAAV1-IAPD, rAAV8-VLP, and rAAV8-IAPD (n = 2–3), with a dose of 6 × 1012 vg/kg. All methods were well tolerated clinically. Potency, as determined by serum levels of AAT, of rAAV1 by the VLP method was twice that observed with direct IM injection; 90 μg/mL with VLP versus 38 μg/mL with direct IM injection. There was an approximately 25-fold advantage in estimated vector genomes retained within the muscle tissue with VLP and a 5-fold improvement in the ratio of total vector genomes retained within muscle as compared with liver. The other methods were intermediate in the potency and retention of vector genomes. Examination of muscle enzyme (CK) levels indicated rAAV1-VLP to be equally safe as compared with IM injection, while the IAPD method showed significant CK elevation. Overall, rAAV1-VLP demonstrates higher potency per vector genome injected and a greater total vector retention within the muscle, as compared to IM injection, while enabling a much greater total dose to be delivered, with equivalent safety. These data provide the basis for continuation of the dose escalation of the rAAV1-AAT program in patients and bode well for rAAV-VLP as a platform for replacement of secreted proteins.

We investigated the use of Fourier transform near infrared spectroscopy (FT-NIRS), which is a method of measuring light absorbance signatures, to derive ages from eastern Bering Sea walleye pollock (Gadus chalcogrammus) otoliths. This approach is based on a predictive model between near infrared spectra in the otolith and fish age, which is calibrated and validated. The advantage of FT-NIRS over traditional methods is the speed and repeatability with which age estimates are generated. The application of FT-NIRS to walleye pollock otoliths yielded r2 values between 0.91 and 0.95 for the calibration models and good validation performance (between 0.82 and 0.93). This approach can be expected to predict fish age within ±1.0 year of age 67% of the time. When comparing approaches, the FT-NIRS had as good or slightly better precision (75% agreement) than the traditional ageing (66% agreement) and showed little or no bias at age before 12 years of age. Once the predictive FT-NIR model is calibrated and validated, age estimates using FT-NIRS can be done at 10 times the rate compared to traditional methods.

Eulachon (Thaleichthys pacificus) are forage fish that play an important role in the ecosystem as prey for many species. Given their continuing population decline, determining accurate ages is necessary for age-structured stock assessments. This study employed microchemistry analysis to identify group differences and help interpret growth zone patterns on otolith surfaces. Specimens were collected off the coast of Oregon, in the coastal areas of Southeast Alaska, and in the southeastern Bering Sea. Laser-ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) was used to measure elemental ratios in the otoliths along a continuous track from the core to the proximoventral margin. Ba:Ca, Sr:Ca, Zn:Ca, and Mg:Ca signatures suggested that eulachon specimens from three geographic regions are different based on their elemental profiles. For the Oregon specimens, fluctuations in Ba:Ca and Zn:Ca signatures appeared consistent with otolith growth zones and most likely were the result of seasonal coastal upwelling events. Variations in Ba:Ca and Zn:Ca were useful as annual markers for eulachon otoliths from the Bering Sea. For the Southeast Alaska specimens, analysis of the Ba:Ca and Zn:Ca oscillations was not straightforward. Further work is needed to understand the link between otolith microchemistry, fish physiology, and regional environmental factors.

The etiology of multiple myeloma (MM) remains incompletely understood; however, epidemiologic studies have suggested a possible link between exposure to environmental aromatic hydrocarbons-which serve as exogenous ligands for the aryl hydrocarbon receptor (AHR), which has been implicated in cancer biology-and development of monoclonal gammopathy of undetermined significance (MGUS) and MM. Herein, we demonstrate the functional expression of AHR in MM cell lines and primary human MM samples. AHR is expressed in putative MM 'stem cells' and advanced clinical stages of MM, and functionally contributes to MM tumor cell phenotype and proliferation. Antagonism of AHR directly impairs MM cell viability and increases MM cell susceptibility to immune-mediated clearance. Furthermore, our findings indicate that AHR antagonism may represent an effective means to enhance the function of other drugs, such as anti-CD38 antibodies, in future clinical studies. Taken together, these data identify AHR as a novel target for MM therapy.

The programmed death-1 (PD-1) and the PD ligand 1 (PD-L1) interaction represents a key immune checkpoint within the tumor microenvironment (TME), and PD-1 blockade has led to exciting therapeutic advances in clinical oncology. Although IFN-γ–dependent PD-L1 induction on tumor cells was initially thought to mediate the suppression on effector cells, recent studies have shown that PD-L1 is also expressed at high level on tumor-associated macrophages (TAMs) in certain types of tumors. However, the precise role of PD-L1 expression on TAMs in suppressing antitumor immunity within the TME remains to be defined. Using a myeloid-specific Pdl1-knockout mouse model, here we showed definitive evidence that PD-L1 expression on TAMs is critical for suppression of intratumor CD8+ T cell function. We further demonstrated that tumor-derived Sonic hedgehog (Shh) drives PD-L1 expression in TAMs to suppress tumor-infiltrating CD8+ T cell function, leading to tumor progression. Mechanistically, Shh-dependent upregulation of PD-L1 in TAMs is mediated by signal transducer and activator of transcription 3, a cascade that has not been previously reported to our knowledge. Last, single-cell RNA sequencing analysis of human hepatocellular carcinoma revealed that PD-L1 is mainly expressed on M2 TAMs, supporting the clinical relevance of our findings. Collectively, our data revealed an essential role for Shh-dependent PD-L1 upregulation in TAMs in suppressing antitumor immunity within the TME, which could lead to the development of new immunotherapeutic strategies for treating cancer.

Daratumumab, an FDA approved antibody drug, displays specific targeting ability to abnormal white blood cells overexpressing CD38 and provides efficacious therapy for multiple myeloma. Here, in order to achieve enhanced remission of multiple myeloma, we designed Dara-DM4, antibody drug conjugates (ADCs) by conjugating Daratumumab and DM4 via a disulfide linker. Dara-DM4 showed significantly higher cellular uptake and inhibitory efficacy on MM1S cells that overexpressing CD38 with an IC50 of 0.88 µg/mL post 72 hr treatment. These results support a promising ADCs strategy for multiple myeloma treatment.

Interleukin 15 (IL-15) is one of the most important cytokines that regulate the biology of natural killer (NK) cells1. Here we identified a signaling pathway-involving the serine-threonine kinase AKT and the transcription factor XBP1s, which regulates unfolded protein response genes2,3-that was activated in response to IL-15 in human NK cells. IL-15 induced the phosphorylation of AKT, which led to the deubiquitination, increased stability and nuclear accumulation of XBP1s protein. XBP1s bound to and recruited the transcription factor T-BET to the gene encoding granzyme B, leading to increased transcription. XBP1s positively regulated the cytolytic activity of NK cells against leukemia cells and was also required for IL-15-mediated NK cell survival through an anti-apoptotic mechanism. Thus, the newly identified IL-15-AKT-XBP1s signaling pathway contributes to enhanced effector functions and survival of human NK cells.

Multiple myeloma (MM) is an incurable hematologic malignancy of plasma cells, with an estimated 30,000 new cases diagnosed each year in the United States, signifying the need for new therapeutic approaches. We hypothesized that targeting MM using a bispecific antibody (biAb) to simultaneously engage both innate and adaptive cytolytic immune cells could present potent antitumor activity. We engineered a biAb by fusing an anti-CS1 single-chain variable fragment (scFv) and an anti-NKG2D scFv (CS1-NKG2D biAb). Although NKG2D is a potent activation receptor ubiquitously expressed on mostly cytolytic immune cells including NK cells, CD8+ T cells, γδ T cells, and NKT cells, the CS1 tumor-associated antigen on MM represents a promising target. CS1-NKG2D biAb engaged human MM cell lines and NKG2D+ immune cells, forming immune synapses. In effector cells, CS1-NKG2D biAb triggered the phosphorylation of AKT, a downstream protein kinase of the activated NKG2D–DAP10 complex. The EC50 values of CS1-NKG2D biAb for CS1high and for CS1low MM cell lines with effector PBMCs were 10−12 and 10−9 mol/L, respectively. CS1-NKG2D biAb acted through multiple types of immune cells, and this induced cytotoxicity was both CS1- and NKG2D-specific. In vivo, survival was significantly prolonged using CS1-NKG2D biAb in a xenograft NOD-SCIDIL2γc−/− (NSG) mouse model engrafted with both human PBMCs and MM cell lines. Collectively, we demonstrated that the CS1-NKG2D biAb facilitated an enhanced immune synapse between CS1+ MM cells and NKG2D+ cytolytic innate and antigen-specific effector cells, which, in turn, activated these immune cells for improved clearance of MM. Cancer Immunol Res; 6(7); 776–87. ©2018 AACR.

Despite recent advances, there is still a lack of treatment options for patients with high-risk multiple myeloma (MM), warranting the need for novel therapeutic targets. Monopolar spindle 1 (Mps1), also known as TTK protein kinase, is localized at the kinetochores and centromeres essential for the mitotic spindle checkpoint and centrosome duplication. In cancer cells, high levels of Mps1 help to support aneuploidy, a common malignant trait. In fact, high MPS1 expression correlates to unfavorable prognosis in colon and breast cancers. Here, we report the importance of Mps1 in MM and perform functional characterization of a novel Mps1 inhibitor, OSU-13. To assess the relevance of Mps1 in MM prognosis, we analyzed the CoMMpass database and examined the association of MPS1 expression with clinical outcome and genetic alterations in MM patients (n=769). Elevated MPS1 expression correlates with decreased overall survival (p-value = 0.0001) and decreased event-free survival (p-value < 0.0001). In addition, MPS1 expression is higher in high-risk MM with specific genetic alterations, such as deletion of 17p (p-value = 0.0003), Myc translocation t(8;14) (p-value = 0.02), and gain of 1q21 (p-value = 0.0001). We therefore compared MPS1 expression in eight different MM cell lines and primary CD138 - and CD138 + cells isolated from bone marrow (BM) of four recently diagnosed, untreated MM patients. Gene expression levels assessed by quantitative qPCR were normalized to 18S mRNA internal control, and relative quantification was performed using the ΔΔCt method. MPS1 expression was higher in all MM cell lines compared to the average expression level in primary CD138 + BM plasma cells, with a minimum of 3.2-fold increase in L363 and a maximum of 16.2-fold increase in MM1S. In addition, MPS1 expression was 2-fold higher in CD138 + MM cells compared to CD138 - counterparts from the BM of the same patient, suggesting its selectivity as a target. Next, we characterized the effects of OSU-13, a novel Mps1 inhibitor. In an Mps1 target engagement assay (NanoBRET™) in HEK293 cells, we measured the relative levels of OSU-13-mediated inhibition of Mps1-NanoLuc binding to a fluorescent tracer in comparison to MM clinical candidates. OSU-13 showed EC 50=10 nM, almost 10-fold lower than other agents tested. Endpoint measurement by MTT (3-[4,5- dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate cytotoxicity of OSU-13. MM cell lines were treated with increasing doses of OSU-13 for 72h and cell viability was measured. OPM-2 was the most sensitive cell line (IC 50 = 610 nM), followed by H929 (IC 50 = 1440 nM), RPMI-8226 (IC 50 = 4196 nM), and U266 (IC 50 = 5071 nM). To investigate the kinetics and the mechanisms used by OSU-13 to cause cell death, we treated H929 and OPM-2 cell lines with 500 nM of OSU-13 for 24h, 48h, 72h, 96h, and 120h and assessed both Annexin V-FITC and Zombie-Aqua staining by flow cytometry. Treatment with OSU-13 caused an increase in Annexin V staining as early as 24h in H929 (2.1-fold) and OPM-2 (1.6-fold). Maximum increase of apoptotic cells (Annexin V single positive) was observed after 72h, with a 5-fold increase in OPM-2 and 8.6-fold increase in H929 cell line. Cell death peaked at 120h, with Zombie staining 74% of OPM-2 and 60% of H929 cells. The activity of caspases 3/7 was further assessed in H929 myeloma cells at five time points after treatment with different concentrations of OSU-13. Caspases 3/7 activation started after 48h with 500nM OSU-13, and peaked after 72h post-treatment with the same dose, in agreement with the Annexin staining kinetics results. Finally, we evaluated the in vivo effect of OSU-13 in tumor growth in a H929 MM subcutaneous xenograft model in CB.17 SCID mice. Mice were treated with 5 mg/kg or 10 mg/kg daily dose of OSU-13 per oral gavage for 21 days. Treatment with 10 mg/kg daily dose of OSU-13 produced a 22% tumor growth delay (p Collectively, we show here that elevated MPS1/TTK expression correlates with poorer prognosis in MM, and OSU-13, a novel Mps1/TTK inhibitor, induces apoptosis and cell death in MM cell lines, and decreases tumor growth in mice containing H929 cell xenografts. Together, our findings reveal that targeting the mitotic checkpoint using OSU-13 is a potential novel strategy, particularly for high risk MM. Disclosures No relevant conflicts of interest to declare.

BACKGROUND Transthyretin-mediated amyloid cardiomyopathy (ATTR-CM) is a fatal condition caused by misfolding and aggregation of transthyretin (TTR). Despite the treatment with a TTR stabilizer, tafamidis, disease progression still occurs. IONIS-TTR-LRx is an antisense oligonucleotide that inhibits TTR production by the liver. It has the same sequence as inotersen but is conjugated to a triantennary N-acetyl galactosamine (GalNAc) moiety, which allows the use of a lower dose to achieve identical pharmacodynamic results. METHODS AND RESULTS CARDIO-TTRansform (ClinicalTrials.gov NCT04136171) is a Phase 3 global, double-blind, randomized, placebo-controlled study assessing the efficacy and safety of IONIS-TTR-LRx in hereditary and wild-type ATTR-CM patients. Approximately 750 patients will be randomized 1:1 to receive either IONIS-TTR-LRx 45 mg or placebo administered by monthly SC injection. Key inclusion criteria include ATTR-CM diagnosis by biopsy or positive PYP/DPD/HMDP scan, interventricular septum thickness >12mm, NT-proBNP >600 pg/mL, NYHA class I-III and 6-minute walk distance (6MWD) ≥150m. Concomitant treatment with tafamidis is allowed. Primary efficacy endpoint is the composite of cardiovascular (CV) mortality and recurrent CV clinical events at the Week 120 study visit using the Andersen-Gill method. CONCLUSION The CARDIO-TTRansform trial is a Phase 3 trial designed to evaluate the clinical efficacy and safety of IONIS-TTR-LRx for the treatment of ATTR-CM.

Cystic fibrosis (CF) is an autosomal recessive disease that is potentially treatable by gene therapy. Since the identification of the gene encoding CF transmembrane conductance regulator, a number of preclinical and clinical trials have been conducted using the first generation of adeno-associated virus, AAV2. All these studies showed that AAV gene therapy for CF is safe, but clinical benefit was not clearly demonstrated. Thus, a new generation of AAV vectors based on other serotypes is needed to move the field forward. This study tested two AAV serotypes (AAV1 and AAV5) using a dual-luciferase reporter system with firefly and Renilla luciferase genes packaged into AAV1 or AAV5, respectively. Two male and two female Rhesus macaques were each instilled in their lungs with both serotypes using a Penn-Century microsprayer. Both AAV1 and AAV5 vector genomes were detected in all the lung samples when measured at the time of necropsy, 45 days after instillation. However, the vector genome number for AAV1 was at least 10-fold higher than for AAV5. Likewise, luciferase activity was also detected in the same samples at 45 days. AAV1-derived activity was not statistically greater than that derived from AAV5. These data suggest that gene transfer is greater for AAV1 than for AAV5 in macaque lungs. Serum neutralizing antibodies were increased dramatically against both serotypes but were less abundant with AAV1 than with AAV5. No adverse events were noted, again indicating that AAV gene therapy is safe. These results suggest that with more lung-tropic serotypes such as AAV1, new clinical studies of gene therapy using AAV are warranted.

Von Willebrand factor (VWF), an exceptionally large multimeric plasma glycoprotein, functions to initiate coagulation by agglutinating platelets in the blood stream to sites of vascular injury. This primary hemostatic function is perturbed in type 2 dysfunctional subtypes of von Willebrand disease (VWD) by mutations that alter the structure and function of the platelet GPIbα adhesive VWF A1 domains. The resulting amino acid substitutions cause local disorder and misfold the native structure of the isolated platelet GPIbα-adhesive A1 domain of VWF in both gain-of-function (type 2B) and loss-of-function (type 2M) phenotypes. These structural effects have not been explicitly observed in A1 domains of VWF multimers native to blood plasma. New mass spectrometry strategies are applied to resolve the structural effects of 2B and 2M mutations in VWF to verify the presence of A1 domain structural disorder in multimeric VWF harboring type 2 VWD mutations. Limited trypsinolysis mass spectrometry (LTMS) and hydrogen-deuterium exchange mass spectrometry (HXMS) are applied to wild-type and VWD variants of the single A1, A2, and A3 domains, an A1A2A3 tridomain fragment of VWF, plasmin-cleaved dimers of VWF, multimeric recombinant VWF, and normal VWF plasma concentrates. Comparatively, these methods show that mutations known to misfold the isolated A1 domain increase the rate of trypsinolysis and the extent of hydrogen-deuterium exchange in local secondary structures of A1 within multimeric VWF. VWD mutation effects are localized to the A1 domain without appreciably affecting the structure and dynamics of other VWF domains. The intrinsic dynamics of A1 observed in recombinant fragments of VWF are conserved in plasma-derived VWF. These studies reveal that structural disorder does occur in VWD variants of the A1 domain within multimeric VWF and provides strong support for VWF misfolding as a result of some, but not all, type 2 VWD variants.

Background A molecular basis for von Willebrand factor (VWF) self-inhibition has been proposed by which the N-terminal and C-terminal flanking sequences of the globular A1 domain disulfide loop bind to and suppress the conformational dynamics of A1. These flanking sequences are rich in O-linked glycosylation (OLG), which is known to suppress platelet adhesion to VWF, presumably by steric hindrance. The inhibitory mechanism remains unresolved as to whether inhibition is due to steric exclusion by OLGs or a direct self-association interaction that stabilizes the domain. Objectives The platelet adhesive function, thermodynamic stability, and conformational dynamics of the wild-type and type 2M G1324S A1 domain lacking glycosylation (Escherichia coli) are compared with the wild-type glycosylated A1 domain (HEK293 cell culture) to decipher the self-inhibitory mechanism. Methods Surface plasmon resonance and analytical rheology are utilized to assess Glycoprotein Ibα (GPIbα) binding at equilibrium and platelet adhesion under shear flow. The conformational stability is assessed through a combination of protein unfolding thermodynamics and hydrogen-deuterium exchange mass spectrometry (HXMS). Results A1 glycosylation inhibits both GPIbα binding and platelet adhesion. Glycosylation increases the hydrodynamic size of A1 and stabilizes the thermal unfolding of A1 without changing its equilibrium stability. Glycosylation does not alter the intrinsic conformational dynamics of the A1 domain. Conclusions These studies invalidate the proposed inhibition through conformational suppression since glycosylation within these flanking sequences does not alter the native state stability or the conformational dynamics of A1. Rather, they confirm a mechanism by which glycosylation sterically hinders platelet adhesion to the A1 domain at equilibrium and under rheological shear stress.

Four isolates of Trichoderma (Gliocladium) virens (G-45, G-65, G-85, and G-93) and two isolates of binucleate Rhizoctonia spp. (BNR621 and P9023) were evaluated for biocontrol of preemergence damping-off of Catharanthus roseus (vinca) caused by Pythium ultimum. Putative biocontrol agents were amended to a soilless mix 1, 3, or 6 days prior to seeding and pathogen infestation to determine if colonization of the mix before infestation was important for biocontrol efficacy. Biocontrol of preemergence damping-off of vinca with the four isolates of T. virens was variable. Only isolate G-93 gave control of preemergence damping-off (10 to 18% disease) regardless of the length of time the mix was amended prior to seeding and infestation compared to the infested control (43% disease). In contrast, preemergence damping-off was 10 to 15% with SoilGard (based on isolate GL-21 of T. virens). For isolate G-65, preemergence damping-off of vinca was 0% in lots of mix amended 1 day prior to seeding, but over 60% in lots of mix amended 6 days prior to seeding, compared to 43% in the infested control. With the exception of isolate G-65 in the lot amended 6 days before seeding, the isolates of T. virens were as effective as metalaxyl (19% damping-off) for control of P. ultimum in lots of mix amended 1 to 6 days before seeding. In contrast to T. virens, biocontrol efficacy of isolates BNR621 and P9023 of binucleate Rhizoctonia spp. in a Pesta formulation improved as lots of mix were amended up to 6 days before seeding and infestation. As length of initial amendment increased from 1 to 6 days, preemergence damping-off decreased from 37 to 16% for BNR621, and from 42 to 22% for P9023. Preemergence damping-off was observed in vinca in control treatments with only the putative biocontrol agents (BNR621, 14% disease and P9023, 19.6%); therefore, additional bedding plant species were evaluated for susceptibility to the BNR isolates. In the absence of P. ultimum, isolates BNR621 and P9023 in a Pesta formulation caused an average 82.5, 56.5, and 5.8% damping-off of snapdragon, petunia, and impatiens, respectively. Our results suggest that binucleate Rhizoctonia isolates, although effective for biocontrol of P. ultimum on vinca, should be evaluated for pathogenicity on a crop by crop basis before use on other crops.

Isolates BNR621 and P9023 of binucleate Rhizoctonia spp. (BNR) in Pesta and rice flour formulations were evaluated for control of preemergence damping-off of impatiens caused by R. solani. Amendment of a soilless potting mix with the formulations at 0.47% (vol:vol) 3 days prior to seeding and infesting did not improve control compared to amendment 1 day prior to seeding and infesting regardless of whether the moistened amended potting mix was stored in closed plastic bags or in plug trays under a mist system. BNR fungi were no more effective in biocontrol of R. solani in formulations amended at 0.9%. Control of damping-off was comparable but not consistent between formulations of BNR fungi and the fungicide thiophanatemethyl. Damping-off was controlled better with formulations of BNR fungi than with SoilGard based on Trichoderma virens. Shelf life of Pesta and rice flour formulations at 4°C was determined by assessing viability of BNR isolates over time. Viability of the BNR isolates, measured as CFU/g of formulation, declined to approximately 68 to 79% of the original propagule concentration after 6 months in Pesta and rice flour formulations, with the greatest decline in the first 2 months. Shelf life of BNR isolates in formulation significantly affected control of preemergence damping-off but was isolate dependent. Preemergence damping-off was only 5 to 7% with fresh formulations but increased to 30 to 50% with 4-month-old formulations. Controlled atmospheres, maintained with saturated salt solutions, were established to measure the effect of water activity on shelf life of formulations. Water activities (aw) of 0.12 and 0.33 aw enhanced BNR survival in formulations by approximately 2 to 3 months compared with aw of 0.53 and 0.75 aw. Storage of Pesta and rice flour formulations at 4°C significantly improved BNR survival by 4 to 5 months compared with storage at 25°C. These results suggest that improved shelf life of BNR isolates is needed before formulated products can be developed for biocontrol of preemergence damping-off.

Phytophthora root rot, primarily caused by the oomycete Phytophthora cinnamomi, is a large problem for the Christmas tree industry in North Carolina. Fraser fir (Abies fraseri) has no known innate resistance to this pathogen while some exotic fir species, such as Trojan (Abies equi-trojani) and Turkish (Abies bornmuelleriana) fir display varying amounts of resistance. A Phytophthora-resistance screening trial was completed with 1600 seedlings from 12 Turkish and Trojan fir families and Fraser and momi fir (A. firma). Seedlings from each family or species were inoculated with each of eight Phytophthora isolates, six P. cinnamomi and two Phytophthora cryptogea, in an effort to describe variability in isolate aggressiveness. Mortality was assessed as percent shoot necrosis bi-weekly for 16 weeks. Overall, rankings of resistance in fir species confirmed previous single-isolate-based results; momi fir was the most resistant, followed by Turkish, Trojan, and Fraser fir. P. cinnamomi isolates were genera...

Phytophthora root rot (PRR) disease afflicts significant economic losses to the Fraser fir Christmas tree industry. In previous surveys conducted in 1972 and from 1997 to 1998 in North Carolina, the incidence of PRR was ∼9.5% with Phytophthora cinnamomi identified as the predominant causal species isolated from infected roots of Fraser fir. Due to increased use of out-of-state planting stock since 2000, we suspected increased diversity of Phytophthora species. During 2014, we surveyed Fraser fir Christmas tree plantations in the Southern Appalachians of North Carolina, Tennessee and Virginia to determine the occurrence of pathogenic root-rotting species of Phytophthora. A weighted sampling strategy based on Christmas tree acreage was deployed to collect symptomatic Fraser fir roots from 103 commercial production fields in 14 counties. Six species of Phytophthora were isolated from infected roots sampled from 82 sites in 13 counties. Phytophthora cinnamomi, P. cryptogea and P. pini represented 70.3...

Spring harvest timing is critical to ensure high quality forage for dairy cattle.Spring harvest timing for high quality depends on botanical composition.Local binary patterns in images accurately estimate botanical composition.Accurate estimate reduces harvest uncertainty and improves economic outcomes. Botanical composition in mixed stands of alfalfa and grass is a critical parameter in equations estimating harvest fiber concentration for dairy rations. Composition is difficult to estimate by visual observation. Digital image analysis in mixed stands could reduce botanical composition uncertainty and improve spring harvest management decisions. Mixed stands were sampled (n=168) in farmers' fields in Tompkins County, New York in May 2011. A digital image was taken of standing samples at 5-Megapixels resolution using a Canon PowerShot A3100IS, and alfalfa and grass height relationships were recorded. After clipping representative samples at 10-cm above ground level, samples were manually separated into alfalfa (Medicago sativa L.) and timothy grass (Phleum pratense L.), and dried to calculate fractions on a dry matter basis. Uniform rotation invariant local binary patterns (LBP) were extracted from whole images and 64×64pixel tiles, and were used to develop regression equations estimating grass fraction. Tiles were manually classified as alfalfa (0), grass (1) or unclassifiable. An iterative process selected most accurate local binary pattern operator settings. Grass fraction was estimated in three regression model development approaches: (1) using average tile LBP histogram bins from whole images and botanical height relationships, (2) developing a binary tile classification model from tile LBP histogram bins, and using tile model-predicted grass probability averaged for tiles in whole images (grass coverage estimate) and botanical height relationships as inputs in whole image models, and (3) using LBP histogram bins extracted directly from whole images (1024 by 1024pixel square) and height relationships. Predictive accuracy in whole image models using tile LBP histogram averages was highest for models generated from LBP tile histogram bin means (R2pred up to 0.847), followed closely by combined tile models and whole image models (R2pred up to 0.807), with pairwise correlations between tile model-generated grass coverage estimates and sample grass fraction up to 0.895. Local binary patterns are effective in differentiating alfalfa and grass under field conditions, because the method is robust to changes in color and illumination. Furthermore, key LBP histogram bins (e.g., symmetric edges) strongly differentiate alfalfa and grass in tiles. The LBP method is promising based on this study, but further evaluation under diverse field conditions, including different cameras and grass species, is necessary to assess usefulness.

Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells in the bone marrow (BM). The microenvironment plays a key role in MM cell survival and drug resistance through release of soluble factors, expression of adhesion molecules and release of extracellular vesicles (EVs). The aim of this manuscript is to use proteomic profiling of EVs as a tool to identify circulating tumor associated markers in MM patients. First, we characterized the EV protein content obtained from different MM cell lines. Then, we established differences in protein abundance among EVs isolated from MM patient serum and BM and the serum of healthy donors. These data show that the Major Histocompatibility Complex Class I is highly enriched in EVs of MM cell lines and MM patient's serum. Next, we show that CD44 is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R. Furthermore, CD44 was found to be differentially expressed in EVs isolated from newly diagnosed MM patients. Finally through ELISA analysis, we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as an easily accessible source for MM biomarkers. Biological significance Extracellular vesicles are becoming a research focus due to their roles in cancer cell biology such as immune evasion, therapeutic resistance, proliferation and metastases. While numerous studies of vesicle characterization and biology have been conducted in many cancer models, the role of EV in MM remains relatively unstudied. Here we found that EVs isolated from MM cells are enriched in MHC-1 antigen presenting complex and its binding protein β2-MG, this observation is compatible with the enhanced proteasome activity of MM cells compared to other cancers and the ability of functional MHC-1 to bind and present peptides, generated from protein degradation by the proteasome. Additionally, our experiments show that CD44 is particularly enriched in the EV fraction of corticosteroid resistant MM.1R cells and is differentially expressed in the EV fraction of MM patients. This is of high significance due to the established role of CD44 in adhesion of MM cells to BMSC and induction of IL-6, the primary cytokine for MM cell survival, secretion by the BMSC. Furthermore, ELISA assays for CD44 content from the serum of 254 newly diagnosed MM patients enrolled in a Phase 3 randomized trial show highly variable CD44 levels and those patients with > 280 ng/mL serum CD44 showing a reduced overall survival time. These results suggest the potential use of CD44 as a prognostic biomarker in MM.

HdeA is a small acid-stress chaperone protein with a unique activity profile. At physiological pH, it forms a folded, but inactive, dimer. Below pH 3.0, HdeA unfolds and dissociates into disordered monomers, utilizing exposed hydrophobic patches to bind other unfolded proteins and prevent their irreversible aggregation. In this way, HdeA has a key role in helping pathogenic bacteria survive our acidic stomach and colonize our intestines, facilitating the spread of dysentery. Despite numerous publications on the topic, there remain questions about the mechanism by which HdeA unfolding and activation are triggered. Previous studies usually assessed HdeA unfolding over pH increments that are too far apart to gain fine detail of the process of unfolding and dimer dissociation, and often employed techniques that prevented thorough evaluation of specific regions of the protein. We used a variety of heteronuclear NMR experiments to investigate changes to backbone and side chain structure and dynamics of HdeA at four pHs between 3.0 and 2.0. We found that the long loop in the dimer interface is an early site of initiation of dimer dissociation, and that a molecular “clasp” near the disulfide bond is broken open at low pH as part, or as a trigger, of unfolding; this process also results in the separation of C-terminal helices and exposure of key hydrophobic client binding sites. Our results highlight important regions of HdeA that may have previously been overlooked because they lie too close to the disulfide bond or are thought to be too dynamic in the folded state to influence unfolding processes.

Sustainable management of fishery resources is predicated on a foundational understanding of the biogeography of fish stocks and the delineation of stocks into appropriate management units. Despite notable fluctuations in the catch of commercially valuable Pacific cod (Gadus macrocephalus) in South Korea, relatively little is known regarding the stock structure and migratory trends of this species in the region. Here, otolith microchemistry was used to evaluate the stock structure and ontogenetic migratory trends of Pacific cod from five spawning grounds around the Korean Peninsula. Statistically significant between-region discrimination was evident and resolved Pacific cod around the Korean Peninsula into two distinct stocks. Specimens were classified to region of capture using quadratic discriminant analysis of age-0 and capture elemental signatures with overall accuracies of 71.12 and 79.1% respectively, lending support to the notion that Pacific cod demonstrate natal philopatry. Analysis of the elemental signatures over the first 2 years of life exhibited clear trends indicative of shifts in habitat use, suggesting that trace elements function well as indicators of ontogenetic migration. Our work provides information that is complementary to other direct and indirect methods of monitoring migratory trends and delineating stocks, which are integral components of effective species and ecosystem management plans.

Xu, Z., Gleason, M. L., Mueller, D. S., Esker, P. D., Bradley, C. A., Buck, J. W., Benson, D. M., Dixon, P. M., and Monteiro, J. E. B. A. 2008. Overwintering of Sclerotium rolfsii and S. rolfsii var. delphinii in different latitudes of the United States. Plant Dis. 92:719-724. Previously known only from the southern United States, hosta petiole rot recently appeared in the northern United States. Sclerotium rolfsii var. delphinii is believed to be the predominant petiole rot pathogen in the northern United States, whereas S. rolfsii is most prevalent in the southern United States. In order to test the hypothesis that different tolerance to climate extremes affects the geographic distribution of these fungi, the survival of S. rolfsii and S. rolfsii var. delphinii in the northern and southeastern United States was investigated. At each of four locations, nylon screen bags containing sclerotia were placed on the surface of bare soil and at 20-cm depth. Sclerotia were recovered six times from November 2005 to July 2006 in North Dakota and Iowa, and from December 2005 to August 2006 in North Carolina and Georgia. Survival was estimated by quantifying percentage of sclerotium survival on carrot agar. Sclerotia of S. rolfsii var. delphinii survived until at least late July in all four states. In contrast, no S. rolfsii sclerotia survived until June in North Dakota or Iowa, whereas 18.5% survived until August in North Carolina and 10.3% survived in Georgia. The results suggest that inability to tolerate low temperature extremes limits the northern range of S. rolfsii. Additional keywords: Hosta spp., ornamental crops

Phytophthora root rot of Fraser fir, caused by several Phytophthora spp., is a severe problem in Christmas tree production. Since fungicides are not economically viable for disease management in field plantings and host resistance is not available, cultural control methods were investigated. Mulches, dairy compost, and soil pH adjustment were tested at five field sites in North Carolina. Treatments included wood chips, wood chips plus compost, or pine bark as raised beds, and compost or sulfur tilled into soil. Soil and mulch microbial populations were characterized by dilution plating and calculation of a log series diversity index, and by enzyme analyses at 5, 12, 17, and 24 months after planting. Bacterial and fungal counts, microbial activity, and cellulase activity were higher in mulch than in soil at all sites and times (P < 0.01), and generally did not differ among mulch types or among soils. Treatments significantly affected disease ratings and tree survival at three of five sites, with one or more mulch treatments yielding lower disease ratings and greater survival than controls. Tree mortality at each time point varied significantly with cellulase activity in the upper root zone (P = 0.005). Other biological variables did not show significant relationships with disease ratings or mortality.

Isolates of Phytophthora in floriculture crops were collected from North Carolina commercial greenhouse facilities in 2007 and 2008, identified, and characterized for mating type and mefenoxam sensitivity. In all, 163 isolates from 13 host species at 11 locations were identified primarily as Phytophthora nicotianae (59%), P. drechsleri (23%), P. cryptogea (9%), and P. tropicalis (4%). Multiple Phytophthora spp. were found at five locations. DNA sequencing was more reliable for differentiating P. cryptogea and P. drechsleri. Only the A1 mating type of P. drechsleri was collected; however, both mating types of P. nicotianae, P. cryptogea, and P. tropicalis were found. Overall, 66% of Phytophthora isolates were resistant or intermediate in resistance to mefenoxam at 1 μg a.i./ml. Three groups of P. drechsleri isolates had effective concentration of mefenoxam providing 50% growth inhibition (EC50) estimates over 700 μg a.i./ml. EC50 estimates for P. nicotianae ranged from 246 to 435 μg a.i./ml. Isolates of P. nicotianae exhibited phenotypic heterogeneity at several locations, suggesting multiple separate introductions to the facilities. In contrast, P. nicotianae and P. drechsleri isolates from two locations were phenotypically identical to a previous survey, suggesting that the facilities have Phytophthora spp. established onsite.

Phytophthora isolates associated with ornamental plants or recovered from irrigation water in six states in the southeastern United States (Georgia, North Carolina, South Carolina, Tennessee, Texas, and Virginia) were identified and screened for sensitivity to mefenoxam. Isolates from forest and suburban streams in Georgia and Virginia were included for comparison. A new in vitro assay, utilizing 48-well tissue culture plates, was used to screen for mefenoxam sensitivity; this assay allowed high throughput of isolates and used less material than the traditional petri plate assay. In total, 1,483 Phytophthora isolates were evaluated, and 27 species were identified with Phytophthora nicotianae, P. hydropathica, and P. gonapodyides, the most abundant species associated with plants, irrigation water, and streams, respectively. Only 6% of isolates associated with plants and 9% from irrigation water were insensitive to mefenoxam at 100 μg a.i./ml. Approximately 78% of insensitive isolates associated with plants were P. nicotianae, and most of these (67%) came from herbaceous annual plants. Most of the insensitive isolates recovered from irrigation water were P. gonapodyides, P. hydropathica, P. megasperma, and P. pini, and 83% of the insensitive isolates from streams were P. gonapodyides. Overall, this study suggests that mefenoxam should continue to be a valuable tool in the management of Phytophthora diseases affecting ornamental plants in the southeastern United States.

Three isolates of Phytophthora cryptogea and three isolates of P. drechsleri were evaluated for host specificity and variations in aggressiveness on eight floriculture crops commonly grown in North Carolina. Plants were monitored for the development of foliar symptoms stemming from root rot caused by Phytophthora. No isolates of P. cryptogea or P. drechsleri had the same host range, though all P. cryptogea isolates caused foliar symptoms on gerbera daisy and annual stock, and all P. drechsleri isolates incited disease on osteospermum. No African marigold plants developed foliar symptoms of Phytophthora root rot. All P. cryptogea isolates caused foliar symptoms on at least three tested plant species. Isolates of P. drechsleri varied from highly specific and only causing disease on a single host species to relatively nonspecific and causing foliar symptoms on the majority of host plants. This is the first report of host specificity of ornamental isolates of P. drechsleri, and several of the tested plants are not reported hosts of P. drechsleri. Differences in isolate aggressiveness were found and depended on plant species; overall, isolates of P. drechsleri were more aggressive than P. cryptogea isolates. Isolations from nonsymptomatic inoculated plants suggest that these Phytophthora spp. could be moved between growers or from growers to consumers on nonsymptomatic infected ornamentals.

Acute myeloid leukemia (AML) can evade the mouse and human innate immune system by suppressing natural killer (NK) cell development and NK cell function. This is driven in part by the overexpression of microRNA (miR)-29b in the NK cells of AML patients, but how this occurs is unknown. In the current study, we demonstrate that the transcription factor aryl hydrocarbon receptor (AHR) directly regulates miR-29b expression. We show that human AML blasts activate the AHR pathway and induce miR-29b expression in NK cells, thereby impairing NK cell maturation and NK cell function, which can be reversed by treating NK cells with an AHR antagonist. Finally, we show that inhibition of constitutive AHR activation in AML blasts lowers their threshold for apoptosis and decreases their resistance to NK cell cytotoxicity. Together, these results identify the AHR pathway as a molecular mechanism by which AML impairs NK cell development and function. The results lay the groundwork in establishing AHR antagonists as potential therapeutic agents for clinical development in the treatment of AML.

PHLPP2, a member of the PH-domain leucine-rich repeat protein phosphatase (PHLPP) family, which targets oncogenic kinases, has been actively investigated as a tumor suppressor in solid tumors. Little is known, however, regarding its regulation in hematological malignancies. We observed that PHLPP2 protein expression, but not its mRNA, was suppressed in late differentiation stage acute myeloid leukemia (AML) subtypes. MicroRNAs (miR or miRNAs) from the miR-17-92 cluster, oncomir-1, were shown to inhibit PHLPP2 expression and these miRNAs were highly expressed in AML cells that lacked PHLPP2 protein. Studies showed that miR-17-92 cluster regulation was, surprisingly, independent of transcription factors c-MYC and E2F in these cells; instead all-trans-retinoic acid (ATRA), a drug used for terminally differentiating AML subtypes, markedly suppressed miR-17-92 expression and increased PHLPP2 protein levels and phosphatase activity. Finally, we demonstrate that the effect of ATRA on miR-17-92 expression is mediated through its target, transcription factor C/EBPβ, which interacts with the intronic promoter of the miR-17-92 gene to inhibit transactivation of the cluster. These studies reveal a novel mechanism for upregulation of the phosphatase activity of PHLPP2 through C/EBPβ-mediated repression of the miR-17-92 cluster in terminally differentiating myeloid cells.

Purpose: Natural killer (NK) cells may play an important role in the immune response to multiple myeloma; however, multiple myeloma cells express killer immunoglobulin-like receptor (KIR) ligands to prevent NK cell cytotoxicity. Lenalidomide can expand and activate NK cells in parallel with its direct effects against multiple myeloma; however, dexamethasone may impair these favorable immunomodulatory properties. IPH2101, a first-in-class antiinhibitory KIR antibody, has acceptable safety and tolerability in multiple myeloma as a single agent. The present work sought to characterize lenalidomide and IPH2101 as a novel, steroid-sparing, dual immune therapy for multiple myeloma. Experimental Design: A phase I trial enrolled 15 patients in three cohorts. Lenalidomide was administered per os at 10 mg on cohort 1 and 25 mg on cohorts 2 and 3 days 1 to 21 on a 28-day cycle with IPH2101 given intravenously on day 1 of each cycle at 0.2 mg/kg in cohort 1, 1 mg/kg in cohort 2, and 2 mg/kg in cohort 3. No corticosteroids were utilized. The primary endpoint was safety, and secondary endpoints included clinical activity, pharmacokinetics (PK), and pharmacodynamics (PD). Results: The biologic endpoint of full KIR occupancy was achieved across the IPH2101 dosing interval. PD and PK of IPH2101 with lenalidomide were similar to data from a prior single-agent IPH2101 trial. Five serious adverse events (SAE) were reported. Five objective responses occurred. No autoimmunity was seen. Conclusions: These findings suggest that lenalidomide in combination with antiinhibitory KIR therapy warrants further investigation in multiple myeloma as a steroid-sparing, dual immune therapy. This trial was registered at www.clinicaltrials.gov (reference: NCT01217203). Clin Cancer Res; 21(18); 4055–61. ©2015 AACR.

A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon-optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e., rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early- and late-onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be readministered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies that may reduce the effectiveness of the vector, especially if readministration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa(-/-) mice, an animal model of Pompe disease. This article describes the IND-enabling preclinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for readministration of rAAV9-DES-hGAA injected intramuscularly into the tibialis anterior muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with late-onset Pompe disease will be enrolled. The goal of the immune modulation strategy is to ablate B-cells before the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of the active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the evidence generated from gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the three months following each dosing will assess the safety, biochemical, and functional impact of the vector.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

In rockfish (Family Scorpaenidae), age determination is difficult and the annual nature of otolith growth zones must be validated independently. We applied routine age determination to four species of Gulf of Alaska rockfish: two shallower-water species, namely harlequin rockfish (Sebastes variegatus) and redstripe rockfish (Sebastes proriger), and two deep-water species, namely shortspine thornyhead (Sebastolobus alascanus) and shortraker rockfish (Sebastes borealis). The estimated ages (counts of presumed annual growth zones in the otoliths) were then evaluated with bomb-produced radiocarbon (14C) and Bayesian modelling with Markov chain Monte Carlo simulations. This study successfully demonstrated the level of accuracy in estimated ages of redstripe rockfish (a 35% probability of underageing, and ~5% probability of overageing) and harlequin rockfish (a 100% probability that they were underaged by ~3 or 4 years). Measured Δ14C in shortspine thornyhead and shortraker rockfish otoliths was lower and increased later than expected. Hence, incorrect age determination could not be evaluated. This is likely caused by dissimilar environmental and biological availability of 14C between these two species and the Pacific halibut (Hippoglossus stenolepis) reference chronology, or underageing of these two species.

The appropriate dose of antithymocyte globulin (ATG) to be utilized in reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplant (alloHSCT) is as yet unknown. We retrospectively compared patients who received 7.5 mg/kg (R-ATG, 39 patients) and 6 mg/kg (r-ATG, 97 patients). The cumulative incidences of acute graft-versus-host disease (aGVHD) grade II-IV at 180 days were 46% and 41% and of aGVHD grade III-IV were 11% and 18% in r-ATG and R-ATG, respectively (p > 0.30). The respective estimated cumulative incidences at 24 months of cGVHD were 42% and 44% (p > 0.30). There was no significant difference in non-relapse mortality (p = 0.22), cumulative incidence of relapse (p = 0.53), progression-free survival (p = 0.69) or overall survival (p = 0.95). In conclusion, a decreased ATG dose of 6 mg/kg was associated with a similar proportion of GVHD to 7.5 mg/kg ATG. Given the increasing number of RIC HSCTs performed worldwide, the correct dose and preparation of ATG should be defined by prospective randomized trials.

Phytophthora spp. are waterborne plant pathogens that are commonly found in streams, rivers, and reclaimed irrigation water. Rhododendron and Pieris trap plants at two commercial nurseries were irrigated with water naturally infested with Phytophthora spp. during the 2011 and 2012 growing seasons to assess the frequency of disease. Phytophthora spp. were consistently recovered from water samples at every collection time but detected on only 2 of the 384 trap plants during the two growing seasons. Pathogenicity assays proved that Phytophthora hydropathica and Phytophthora taxon PgChlamydo, commonly recovered taxa in irrigation water at the nurseries, were foliar pathogens of Rhododendron and Pieris; however, neither species was able to cause root rot on these same hosts. Overall, Phytophthora spp.-infested irrigation water did not act as a primary source of infection on Rhododendron and Pieris, even though foliar pathogenic species of Phytophthora were present in the water.

Accumulating evidence indicates that human natural killer (NK) cells develop in secondary lymphoid tissue (SLT) through a so-called "stage 3" developmental intermediate minimally characterized by a CD34(-)CD117(+)CD94(-) immunophenotype that lacks mature NK cell function. This stage 3 population is heterogeneous, potentially composed of functionally distinct innate lymphoid cell (ILC) types that include interleukin-1 receptor (IL-1R1)-positive, IL-22-producing ILC3s. Whether human ILC3s are developmentally related to NK cells is a subject of ongoing investigation. Here, we show that antagonism of the aryl hydrocarbon receptor (AHR) or silencing of AHR gene expression promotes the differentiation of tonsillar IL-22-producing IL-1R1(hi) human ILC3s to CD56(bright)CD94(+) interferon (IFN)-γ-producing cytolytic mature NK cells expressing eomesodermin (EOMES) and T-Box Protein 21 (TBX21 or TBET). Hence, we demonstrate the lineage plasticity of human ILCs by identifying AHR as a transcription factor that prevents IL-1R1(hi) ILC3s from differentiating into NK cells.

Idelalisib, an oral inhibitor of phosphatidylinositol-3-kinase δ (PI3Kδ), was evaluated in a 48-week phase 1 study (50-350 mg daily or twice daily) enrolling 40 patients with relapsed or refractory mantle cell lymphoma (MCL). Primary outcome was safety and dose-limiting toxicity (DLT). Secondary outcomes were pharmacokinetic parameters, pharmacodynamic effects, overall response rate (ORR), progression-free survival (PFS), and duration of response (DOR). Patients without DLT and no evidence of disease progression after 48 weeks enrolled in the extension study. Patients had median age of 69 years (range, 52-83) and received median of 4 prior therapies (1-14); 17 of 40 patients (43%) were refractory to their most recent treatment. Median duration of idelalisib treatment was 3.5 months (range, 0.7-30.7), with 6 (15%) continuing extension treatment. Common grade ≥3 adverse events (AEs) included (total%/grade ≥3%) diarrhea (40/18), nausea (33/5), pyrexia (28/0), fatigue (25/3), rash (23/3), decreased appetite (20/15), upper respiratory infection (20/0), pneumonia (13/10), and alanine transaminase or aspartate transaminase elevations (60/20). ORR was 16 of 40 patients (40%), with CR in 2 of 40 patients (5%). Median DOR was 2.7 months, median PFS was 3.7 months, and 1-year PFS was 22%. These data provide proof of concept that targeting PI3Kδ is a viable strategy and worthy of additional study in MCL. This trial was registered at www.clinicaltrials.gov as #NCT00710528.

Natural killer (NK) cells, large granular lymphocytes comprising a key cellular subset of innate immunity, were originally named for their capacity to elicit potent cytotoxicity against tumor cells independent of prior sensitization or gene rearrangement. This process is facilitated through the expression of activating and inhibitory receptors that provide for NK cell “education” and a subsequent ability to survey, recognize, and lyse infected or transformed cells, especially those lacking or possessing mutated MHC class I expression. Since these original observations were made, how NK cells recognize candidate target cells continues to be the topic of ongoing investigation. It is now appreciated that NK cells express a diverse repertoire of activating and inhibitory receptors of which killer immunoglobulin-like receptors (KIR) appear to play a critical role in mediating self-tolerance as well as facilitating cytotoxicity against infected or transformed cells. In addition, in the presence of an activating signal, the absence or mismatch of MHC class I molecules on such targets (which serve as inhibitory KIR ligands) promotes NK cell–mediated lysis. An increasing understanding of the complexities of KIR biology has provided recent opportunities to leverage the NK cell versus tumor effect as a novel avenue of immunotherapy for cancer. The present review summarizes the current understanding of KIR expression and function and highlights ongoing efforts to translate these discoveries into novel NK cell–mediated immunotherapies for cancer. Cancer Immunol Res; 2(2); 99–104. ©2014 AACR.

The incidence of oesophagogastric junctional (OGJ) adenocarcinoma is rising rapidly in western countries, in contrast to the declining frequency of distal gastric carcinoma. Treatment options for adenocarcinomas involving the oesophagogastric junction are limited and the overall prognosis is extremely poor. To determine the genomic landscape of OGJ adenocarcinoma, exomes of eight tumours and matched germline DNA were subjected to massively parallel DNA sequencing. Microsatellite instability was observed in three tumours which coincided with an elevated number of somatic mutations. In total, 117 genes were identified that had predicted coding alterations in more than one tumour. Potentially actionable coding mutations were identified in 67 of these genes, including those in CR2, HGF , FGFR4, and ESRRB. Twenty-nine genes harbouring somatic coding mutations and copy number changes in the MSS OGJ dataset are also known to be altered with similar predicted functional consequence in other tumour types. Compared with the published mutational profile of gastric cancers, 49% (57/117) of recurrently mutated genes were unique to OGJ tumours. TP53, SYNE1, and ARID1A were amongst the most frequently mutated genes in a larger OGJ cohort. Our study provides an insight into the mutational landscape of OGJ adenocarcinomas and confirms that this is a highly mutated and heterogeneous disease. Furthermore, we have uncovered somatic mutations in therapeutically relevant genes which may represent candidate drug targets.

Multiple myeloma (MM) is an incurable hematological malignancy. Chimeric antigen receptor (CAR)-expressing T cells have been demonstrated successfully in the clinic to treat B-lymphoid malignancies. However, the potential utility of antigen-specific CAR-engineered natural-killer (NK) cells to treat MM has not been explored. In this study, we determined whether CS1, a surface protein that is highly expressed on MM cells, can be targeted by CAR NK cells to treat MM. We successfully generated a viral construct of a CS1-specific CAR and expressed it in human NK cells. In vitro, CS1-CAR NK cells displayed enhanced MM cytolysis and interferon-γ (IFN-γ) production, and showed a specific CS1-dependent recognition of MM cells. Ex vivo, CS1-CAR NK cells also showed similarly enhanced activities when responding to primary MM tumor cells. More importantly, in an aggressive orthotopic MM xenograft mouse model, adoptive transfer of NK-92 cells expressing CS1-CAR efficiently suppressed the growth of human IM9 MM cells and also significantly prolonged mouse survival. Thus, CS1 represents a viable target for CAR-expressing immune cells, and autologous or allogeneic transplantation of CS1-specific CAR NK cells may be a promising strategy to treat MM.

Patients with HER-2/neu–expressing breast cancer remain at risk for relapse following standard therapy. Vaccines targeting HER-2/neu to prevent relapse are in various phases of clinical testing. Many vaccines incorporate the HER-2/neu HLA-A2–binding peptide p369–377 (KIFGSLAFL), because it has been shown that CTLs specific for this epitope can directly kill HER-2/neu–overexpressing breast cancer cells. Thus, understanding how tumors process this epitope may be important for identifying those patients who would benefit from immunization. Proteasome preparations were used to determine if p369–377 was processed from larger HER-2/neu–derived fragments. HPLC, mass spectrometry, cytotoxicity assays, IFN-γ ELISPOT, and human breast cancer cell lines were used to assess the proteolytic fragments. Processing of p369–377 was not detected by purified 20S proteasome and immunoproteasome, indicating that tumor cells may not be capable of processing this Ag from the HER-2/neu protein and presenting it in the context of HLA class I. Instead, we show that other extracellular domain HER-2/neu peptide sequences are consistently processed by the proteasomes. One of these sequences, p373–382 (SLAFLPESFD), bound HLA-A2 stronger than did p369–377. CTLs specific for p373–382 recognized both p373–382 and p369–377 complexed with HLA-A2. CTLs specific for p373–382 also killed human breast cancer cell lines at higher levels than did CTLs specific for p369–377. Conversely, CTLs specific for p369–377 recognized p373–382. Peptide p373–382 is a candidate epitope for breast cancer vaccines, as it is processed by proteasomes and binds HLA-A2.

Genetic models for studying localized cell suicide that halt the spread of pathogen infection and immune response activation in plants include Arabidopsis accelerated-cell-death 11 mutant (acd11). In this mutant, sphingolipid homeostasis is disrupted via depletion of ACD11, a lipid transfer protein that is specific for ceramide 1-phosphate (C1P) and phyto-C1P. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we investigated the functional regulation of ACD11 and CPTP by anionic phosphoglycerides and found that 1-palmitoyl-2-oleoyl-phosphatidic acid or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (≤15 mol %) in C1P source vesicles depressed C1P intermembrane transfer. By contrast, replacement with 1-palmitoyl-2-oleoyl-phosphatidylserine stimulated C1P transfer by ACD11 and CPTP. Notably, "soluble" phosphatidylserine (dihexanoyl-phosphatidylserine) failed to stimulate C1P transfer. Also, none of the anionic phosphoglycerides affected transfer action by human glycolipid lipid transfer protein (GLTP), which is glycolipid-specific and has few cationic residues near its glycolipid binding site. These findings provide the first evidence for a potential phosphoglyceride headgroup-specific regulatory interaction site(s) existing on the surface of any GLTP-fold and delineate new differences between GLTP superfamily members that are specific for C1P versus glycolipid.

Historically, attempts at cancer immunotherapy have emphasized strategies designed to stimulate or augment the immune system into action. In the past decade, a complementary approach has developed, that of releasing immune cells from inhibitory restraint. Discoveries in the fundamental biology of how immunity is regulated, how the immune system interfaces with malignancy, and how cancer cells may exploit these processes to evade detection have all been translated into the rapidly growing field of therapeutic immune checkpoint inhibition for cancer. Myeloma is a malignancy associated with significant immune dysfunction imparted both by the disease itself as well as by many of the immunosuppressive therapies that have been used in the past. The growing body of preclinical data regarding immunoregulatory mechanisms that appear active in myeloma has begun to be translated to clinical trials targeting these signaling axes. This review will attempt to summarize the current understanding of the basic biology of several immune checkpoint pathways that may be important in myeloma and provide an up-to-date overview of recent and ongoing clinical trials of immune checkpoint inhibitors in myeloma. Finally, several current challenges and possible future directions of immune checkpoint blockade in myeloma will be reviewed.

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid lineage blasts. Due to its heterogeneity and to the high rate of acquired drug resistance and relapse, new treatment strategies are needed. Here, we demonstrate that IFNγ promotes AML blasts to act as effector cells within the context of antibody therapy. Treatment with IFNγ drove AML blasts toward a more differentiated state, wherein they showed increased expression of the M1-related markers HLA-DR and CD86, as well as of FcγRI, which mediates effector responses to therapeutic antibodies. Importantly, IFNγ was able to up-regulate CD38, the target of the therapeutic antibody daratumumab. Because the antigen (CD38) and effector receptor (FcγRI) were both simultaneously up-regulated on the AML blasts, we tested whether IFNγ treatment of the AML cell lines THP-1 and MV4-11 could stimulate them to target one another after the addition of daratumumab. Results showed that IFNγ significantly increased daratumumab-mediated cytotoxicity, as measured both by 51Cr release and lactate dehydrogenase release assays. We also found that the combination of IFNγ and activation of FcγR led to the release of granzyme B by AML cells. Finally, using a murine NSG model of subcutaneous AML, we found that treatment with IFNγ plus daratumumab significantly attenuated tumor growth. Taken together, these studies show a novel mechanism of daratumumab-mediated killing and a possible new therapeutic strategy for AML.