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U266 cross-presents PR1 after exposure to NE/P3 from PMN co-culture

PDF file - 706k, Figure S1. CG is ubiquitinated in AML. Figure S2. CG1 is a naturally processed epitope that is eluted from leukemia cell surface. Figure S3. CG expression by each of the AML samples used in the cytotoxicity assays. Figure S4. Gating strategy used to determine CG1-CTL frequency in AML patient blood. Supplementary Table S1. Characteristics of cathepsin G-derived peptides with highest binding affinity to HLA-A*0201 Supplementary Table S2. AML samples used in determining cathepsin G expression

Purpose: Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy.Experimental Design: We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients.Results: CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL–mediated cytotoxicity, further confirming HLA-A*0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation.Conclusion: CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development. Clin Cancer Res; 19(1); 247–57. ©2012 AACR.

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies.Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples.Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivo.Conclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386–96. ©2018 AACR.

Supplementary Tables S1-S4

The article introduces a methodological approach to teaching English as a second language (ESL) to schoolchildren with general speech underdevelopment and identifies the main interlingual influences. The research featured various means that optimize the English language acquisition by schoolchildren with general speech underdevelopment. The research objective was to identify and substantiate the optimal methodological tools that take into account interlanguage influences. The study revealed the causes of the poor native grammar skills in this group of schoolchildren. A longitudinal study of interlingual influences made it possible to define several methodological tools that are able to improve the process of ESL acquisition by schoolchildren with general speech underdevelopment. These tools take into account the native vocabulary skills in children of this category. The authors propose to create additional adapted ESL syllabuses for schoolchildren with general speech underdevelopment. Such programs should be based on the peculiarities of the native vocabulary and grammar development in this category of children.

It is not a secret that every student, sooner or later, gets a job. Some students start their professional activities from the first year to pay for their studies. Others get a job after an internship at a university, for the sake of professional experience or out of personal interest. Regardless of the reason for the student's employment during the educational process, we cannot deny that work takes time and quite often affects the academic performance and the amount of extracurricular activities of university students. This article presents the results of research on the popularity of the idea about working in a company or setting up ones own business. The research was carried out at Baikal State University by conducting a survey, which was completed by 1664 full-time students from the first to the fifth year. The data obtained as a result of the survey show that the majority of respondents have worked for a company at least once. The most popular reason for employment is the desire to earn money. A third of students want to become entrepreneurs in order to be able to earn more money. Most of the students are attracted to business in the service sector, where the organization of leisure activities comes first, then come marketing and, finally, financial services or legal services.

Background: Despite recent advances in the treatment of AML, most approaches are rarely curative and most patients (pts) succumb to relapsed disease. The effectiveness of stem cell transplant and associated graft vs. leukemia effect implies an important role for immune-based therapy in producing long lasting remissions. Traditional approaches using immunotherapy have failed to establish a suitable surface target or treatment paradigm that is effective in myeloid malignancies. Hu8F4 is a humanized T-cell receptor-like monoclonal antibody that binds to the conformational epitope of PR1 bound to HLA-A2, which is highly, differentially expressed on the surface of AML compared to normal progenitors. Methods: We conducted a first in human, phase I dose escalation trial of Hu8F4 in pts with myeloid malignances. Pts with R/R AML, MDS, CMML, and myeloid blast phase of CML with adequate organ function and PS ≤ 2 were eligible. Pts were treated on 7 escalating dose levels, ranging from 0.01 mg/kg to 10 mg/kg IV on D1 & 15. Initial dose levels required 1 pt per dose (0.01, 0.03, 0.1, 0.3, 1), followed by 3 pts per dose (3, 10). Results: 10 pts with R/R AML have been enrolled, with a median age of 65 years (range, 23-77), including 6 females (60%). Pts had received a median of 4 (1-4) prior therapies; 5 pts (50%) had a PS of 2. At enrollment, the median WBC was 1.9 (0.1 - 18.4), median BM blasts were 32% (8 - 76); 9 (90%) pts had complex karyotype and 3 (30%) had a TP53 mutation. All pts had > 98% surface expression of PR1 on the myeloid blasts. Hu8F4 Cmax ranged up to 160,000 ng/mL with t1/2 of 48 hours and clearance of 2.61 hr*ng/mL at the highest dose. Weak anti-drug antibodies were observed after week 4 in 2 of 3 pts treated at 3 mg/kg. With a median follow up of 3.5 months (1.1 - 9.9), pts have received a median of 1 (1-4) cycle of therapy. Two pts had decline in BM blasts and 4 had stable disease. Routine peripheral blood testing revealed sharp decline in peripheral blasts immediately after infusion of Hu8F4 on D1 and 15 with associated elevation in serum LDH in some pts and a rise in normal granulocytes, consistent with on-tumor effects. The pharmacokinetic parameters and transient blast reduction indicated a possible sink effect mediated by high levels of circulating blasts. SAEs documented on study were mostly disease-related and included infections, cytopenias, hemoptysis, pneumonia, and GI bleeding. Treatment related AEs were temporally related to the infusion included hypotension (Grade 2: N=2), rigors (Grade 2: N=2; Grade 1: N=1). All infusion reactions were observed at dose levels of 3 and 10 mg/kg, but were transient, and managed with steroids and antihistamines. All pts proceeded with their next dose without further issues. No cytokine release syndrome or neurologic toxicity was observed. Correlative studies support antibody dependent cellular cytotoxicity and phagocytosis as important mechanisms of anit-AML activity. Conclusion: Hu8F4 was well tolerated with no dose-limiting toxicities observed at the maximum planned dose. On-target peripheral blast reduction temporally related to infusion suggests biological activity. Real-time pharmacokinetic data on study indicate a possible sink effect that may be overcome by a more frequent dosing strategy. Citation Format: Tapan M. Kadia, Hagop Kantarjian, Gheath Alatrash, Anna Sergeeva, Hong He, Lisa St. John, Priya Koppikar, Celine Kerros, Abhishek Maiti, Courtney Dinardo, Elias Jabbour, Serge Vesrstovsek, Naveen Pemmaraju, Nitin Jain, Ghayas Issa, Guillermo Montalban-Bravo, Aditi Shastri, Daniel Couriel, Rhona Pinsoy, Sapna Parshottam, Richard Champlin, Jorge Cortes, Jeffrey Molldrem. Phase I study of the T-cell receptor-like antibody Hu8F4 in patients with advanced hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT101.

The article discusses one of the newest ways to finance business projects — crowdfunding. The opinions of scientists on the essence of this concept are generalized. Particular attention is paid to various types of crowdfunding. The objectives of crowdfunding participants are considered. The advantages and disadvantages of crowdfunding, as a method of financing projects, are highlighted, the main difficulties of the development of the crowdfunding market in Russia are identified. In particular, the necessity of improving the regulatory framework and legal regulation of crowdfunding is shown. The features of government regulation of the crowdfunding market in the USA and the UK are investigated. The rules for the effective development and functioning of crowdfunding domestic and foreign systems, as well as the rules of behavior of project initiators for the effective attraction of investments are generalized. A comparative analysis of the main Russian and foreign crowdfunding platforms is done. Statistics on the activities of the largest crowdfunding venues are presented. Conclusions are drawn about the prospects and trends of crowdfunding in Russia.

Цель исследования – выявить особенности организации страховой защиты туристов в России. Для их понимания представлены результаты анализа российского рынка страхования туристов. Показаны изменения, произошедшие на нем в связи с пандемией. Представлены основные аспекты и особенности страхования туристов в разрезе видов туризма (внутренний, внешний). Раскрыты наиболее востребованные варианты страхового покрытия. Представлены этапы выбора оптимального варианта страховой защиты. Доказано, что страхование туристов – это комплексное направление страховой защиты, основу которого составляет рисковое личное страхование; в настоящее время ее активно дополняют различными сервисными услугами и опционными составляющими, направленными на расширение страхового покрытия. Исследование строилось на применении традиционных методов познания (синтез, анализ, обобщение).

The perennial life style is a successful ecological strategy, and Arabis alpina is a recently developed model Brassicaceae species for studying it. One aspect, poorly investigated until today, concerns the differing patterns of allocation, storage, and metabolism of nutrients between perennials and annuals and the yet unknown signals that regulate this process. A. alpina has a complex lateral stem architecture with a proximal vegetative perennial (PZ) and a distal annual flowering zone (AZ) inside the same stems. Lipid bodies (LBs) with triacylglycerols (TAGs) accumulate in the PZ. To identify potential processes of lipid metabolism linked with the perennial lifestyle, we analyzed lipid species in the PZ versus AZ. Glycerolipid fractions, including neutral lipids with mainly TAGs, phospholipids, and glycolipids, were present at higher levels in the PZ as compared to AZ or roots. Concomitantly, contents of specific long‐chain and very long‐chain fatty acids increased during formation of the PZ. Corresponding gene expression data, gene ontology term enrichment, and correlation analysis with lipid species pinpoint glycerolipid‐related genes to be active during the development of the PZ. Possibilities that lipid metabolism genes may be targets of regulatory mechanisms specifying PZ differentiation in A. alpina are discussed.

Perennial plants maintain their life span through several growth seasons. Arabis alpina serves as model Brassicaceae species to study perennial traits. A. alpina lateral stems have a proximal vegetative zone with a dormant bud zone, and a distal senescing seed-producing inflorescence zone. We addressed the questions of how this zonation is distinguished at the anatomical level, whether it is related to nutrient storage, and which signals affect the zonation. We found that the vegetative zone exhibits secondary growth, which we termed the perennial growth zone (PZ). High-molecular weight carbon compounds accumulate there in cambium and cambium derivatives. Neither vernalization nor flowering were requirements for secondary growth and sequestration of storage compounds. The inflorescence zone with only primary growth, termed annual growth zone (AZ), or roots exhibited different storage characteristics. Following cytokinin application, cambium activity was enhanced and secondary phloem parenchyma was formed in the PZ and also in the AZ. In transcriptome analysis cytokinin-related genes represented enriched gene ontology terms and were expressed at higher level in PZ than AZ. Thus, A. alpina uses primarily the vegetative PZ for nutrient storage, coupled to cytokinin-promoted secondary growth. This finding lays a foundation for future studies addressing signals for perennial growth.HighlightArabis alpina stems have a perennial zone with secondary growth, where cambium and derivatives store high-molecular weight compounds independent of vernalization. Cytokinins are signals for the perennial secondary growth zone.

Financial planning is the process of developing a set of strategic and tactical planning documents that define the goals, objectives and means of their implementation in the field of investment, financial and operational policies. The initial planning stage should be an assessment of the situation in the selected planning area. When planning investments, this is an analysis of the financial condition of the organization, reflecting the conditions for investment. Planning your own investments in your business, as well as investing in other business is carried out in conditions of financial well-being. The organization will be investment attractive in the face of unfavorable financial condition, if the financial condition of the organization is not a significant factor for the investor when choosing the object of investment. To conclude on the financial well-being of the organization can be based on the definition of the financial state scoring of the organization according to the proposed methodology.

Change in structural and functional properties of red cell membrane proteins in patients with the essential arterial hypertension (EAH) can promote development of significant dysfunction of these cells and can complicate the course of a system hypoxia in this category of patients. The aim of our research was to determine the interrelation between red cell sphericity and the level of proteins of their membrane in patients with EAH complicated and non-complicated with metabolic syndrome (MS). 51 male patients with EAH I and II (average age - 42 ± 1.5 years) were examined and divided into 2 groups: group 1 - 29 patients with EAH complicated with MS; group 2 - 22 patients with EAH non-complicated with MS. Protein spectrum was assessed by 10 red cell membrane proteins. Results. Patients with EAH complicated with MS had decrease in spectrin level and loss of correlations between the levels of red cell membrane proteins. Number of patients with diameter-thickness ratio < 3.4 (indicates the existence of cells prone to spherocytosis) in the group 1 was twice more than in the group 2 (29.4 % vs 13.7 %). Conclusion. We determined structural and functional disorders in interrelations of such membrane proteins as a-spektrin, ATP and GAPDH (in patients with EAH complicated with MS), and ATP, GAPDH and actin (in patients with EAH non-complicated with MS) which promote development of acquired spherocytosis and further impairments in microcirculation and gaseous metabolism in tissues.

Neutrophil elastase (NE) can be rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer, which results in cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapy target in hematological and solid tumor malignancies. The mechanism of NE uptake, however, remains unknown. Using the mass spectrometry-based approach, we identify neuropilin-1 (NRP1) as a NE receptor that mediates uptake and PR1 cross-presentation in breast cancer cells. We demonstrated that soluble NE is a specific, high-affinity ligand for NRP1 with a calculated Kd of 38.7 nm. Furthermore, we showed that NRP1 binds to the RRXR motif in NE. Notably, NRP1 knockdown with interfering RNA or CRISPR-cas9 system and blocking using anti-NRP1 antibody decreased NE uptake and, subsequently, susceptibility to lysis by PR1-specific cytotoxic T cells. Expression of NRP1 in NRP1-deficient cells was sufficient to induce NE uptake. Altogether, because NRP1 is broadly expressed in tumors, our findings suggest a role for this receptor in immunotherapy strategies that target cross-presented antigens.

In the modern world the institution of family and marriage is undergoing various changes. The attitude of Russian people towards the prenuptial agreement shows a tendency for acceptance and approval. The stereotypical perception of the prenuptial agreement as unacceptable between close people is in the past. Based on that, the article is focused on the comparative analysis of the factors that define the attitude of Russian people towards the prenuptial agreement. The key point in the research is evaluation of specific features of respondents’ opinions, expressed in the years of 2007 and 2018, in accordance with the following socio-psychological factors: awareness of the prenuptial agreement, aims for signing one or reasons to refuse doing so, gender and age factors. Research methods include: a preliminary survey of couples in order to define sample groups, a questionnaire, observation, a mathematical method of data analysis (Fisher’s φ-criterion). The research was conducted in two stages: the first diagnostics in the year 2007 (n=40 couples), and the second – in 2018 (n=50 couples). The respondents were divided into groups by the social status of their relationships: officially married, planning to get married, co-habiting couples and partner (guest) relationships. The age of respondents was 18-47. The results of the research showed the following: increase in age leads to clearer understanding and a better expressed opinion about the prenuptial agreement, positive dynamics in favourable perception of prenuptial agreement among the respondents who are officially married or are planning to marry, increased importance of motives for signing a prenuptial agreement – “protection of one’s own interests” and “opportunity to put pressure on the spouse”, lack of connection between the attitude towards prenuptial agreement and awareness of the document.

Comprehensive analysis of molecular pathology requires a collection of reference samples representing normal tissues from healthy donors. For the available limited collections of normal tissues from postmortal donors, there is a problem of data incompatibility, as different datasets generated using different experimental platforms often cannot be merged in a single panel. Here, we constructed and deposited the gene expression database of normal human tissues based on uniformly screened original sequencing data. In total, 142 solid tissue samples representing 20 organs were taken from post-mortal human healthy donors of different age killed in road accidents no later than 36 hours after death. Blood samples were taken from 17 healthy volunteers. We then compared them with the 758 transcriptomic profiles taken from the other databases. We found that overall 463 biosamples showed tissue-specific rather than platform- or database-specific clustering and could be aggregated in a single database termed Oncobox Atlas of Normal Tissue Expression (ANTE). Our data will be useful to all those working with the analysis of human gene expression., Design Type(s)gene expression analysis objective • data integration objective • organism part comparison design • transcription profiling designMeasurement Type(s)transcription profiling assayTechnology Type(s)RNA sequencingFactor Type(s)sex • age • organism subdivisionSample Characteristic(s)Homo sapiens • kidney • Kidney • Colon • Liver • brain • Lung • endometrium • Ovary • prostate gland • Esophagus • Stomach • Mammary gland • Thyroid gland • Pancreas • Tonsil • skeletal muscle tissue • small intestine • Adrenal gland • urinary bladder • skin of body • uterus • uterine cervix • Small intestine • tonsil • blood • bone marrow • esophagus • liver Machine-accessible metadata file describing the reported data (ISA-Tab format)

Acute myeloid leukaemia (AML) is a lethal haematological malignancy characterized by an immunosuppressive milieu in the tumour microenvironment (TME) that fosters disease growth and therapeutic resistance. Hypomethylating agents (HMAs) demonstrate clinical efficacy in AML patients and exert immunomodulatory activities. In the present study, we show that guadecitabine augments both antigen processing and presentation, resulting in increased AML susceptibility to T cell-mediated killing. Exposure to HMA results in the activation of the endogenous retroviral pathway with concomitant downstream amplification of critical mediators of inflammation. In an immunocompetent murine leukaemia model, guadecitabine negatively regulates inhibitory accessory cells in the TME by decreasing PD-1 (also termed PDCD1) expressing T cells and reducing AML-mediated expansion of myeloid-derived suppressor cells. Therapy with guadecitabine results in enhanced leukaemia-specific immunity, as manifested by increased CD4 and CD8 cells targeting syngeneic leukaemia cells. We have previously reported that vaccination with AML/dendritic cell fusions elicits the expansion of leukaemia-specific T cells and protects against disease relapse. In the present study, we demonstrate that vaccination in conjunction with HMA therapy results in enhanced anti-leukaemia immunity and survival. The combination of a novel personalized dendritic cell/AML fusion vaccine and an HMA has therapeutic potential, and a clinical trial investigating this combination is planned.

Despite substantial advances in the treatment of acute myeloid leukemia (AML), only 30% of patients survive more than 5 years. Therefore, new therapeutics are much needed. Here, we present a novel therapeutic strategy targeting PR1, an HLA-A2 restricted myeloid leukemia antigen. Previously, we have developed and characterized a novel T-cell receptor-like monoclonal antibody (8F4) that targets PR1/HLA-A2 and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC). To improve the potency of 8F4, we adopted a strategy to link T-cell cytotoxicity with a bi-specific T-cell-engaging antibody that binds PR1/HLA-A2 on leukemia and CD3 on neighboring T-cells. The 8F4 bi-specific antibody maintained high affinity and specific binding to PR1/HLA-A2 comparable to parent 8F4 antibody, shown by flow cytometry and Bio-Layer Interferometry. In addition, 8F4 bi-specific antibody activated donor T-cells in the presence of HLA-A2+ primary AML blasts and cell lines in a dose dependent manner. Importantly, activated T-cells lysed HLA-A2+ primary AML blasts and cell lines after addition of 8F4 bi-specific antibody. In conclusion, our studies demonstrate the therapeutic potential of a novel bi-specific antibody targeting the PR1/HLA-A2 leukemia-associated antigen, justifying further clinical development of this strategy.

O-Phthalimide oximes with an N O N fragment were synthesized in high yields via the reaction of vinyl azides with phthalimide-N-oxyl radicals derived from N-hydroxyphthalimide under the action of cerium(IV) ammonium nitrate. The disclosed process is based on the radical transformation of vinyl azides with the elimination of nitrogen and the formation of iminyl N-radicals. The developed approach exploited the dual reactivity of imide-N-oxyl radicals. They act as O-components for oxidative C O coupling with vinyl azides and participate in subsequent formation of the N O bond via their recombination with intermediate iminyl radicals.

Cathepsin G is broadly expressed in acute myeloid leukemia and is an effective immunotherapeutic target

We studied the effect of different levels of membrane-bound hemoglobin on the level of red-cell membrane proteins and also their interrelation in patients with essential hypertension with and without metabolic syndrome. It was found that high membrane-bound hemoglobin is closely related to the low level of high-density lipoproteins and high level of low-density lipoproteins in patients with essential hypertension complicated with metabolic syndrome. In patients with essential hypertension not complicated with metabolic syndrome high membrane-bound hemoglobin is related to the increased prothrombin time and decreased blood urea nitrogen. In patients with essential hypertension com-plicated with metabolic syndrome high membrane-bound hemoglobin significantly influences the level of membrane contractile proteins (actin, tropomiosine). In patients with essential hypertension without metabolic syndrome high membrane-bound hemoglobin is accompanied by the decrease of structural and integral membrane proteins levels (anion-transport protein and protein 4.1). As the result of quantitative changes in these proteins and change in their interrelations in patients with ssential hypertension complicated with metabolic syndrome more intensive disorders of structural and functional organization of red-cell membrane can appear.

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies. Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples. Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivo. Conclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386–96. ©2018 AACR.

Human prostate cancer often metastasizes to bone, but the biological basis for such site-specific tropism remains largely unresolved. Recent work led us to hypothesize that this tropism may reflect pathogenic interactions between RAGE, a cell surface receptor expressed on malignant cells in advanced prostate cancer, and proteinase 3 (PR3), a serine protease present in inflammatory neutrophils and hematopoietic cells within the bone marrow microenvironment. In this study, we establish that RAGE–PR3 interaction mediates homing of prostate cancer cells to the bone marrow. PR3 bound to RAGE on the surface of prostate cancer cells in vitro, inducing tumor cell motility through a nonproteolytic signal transduction cascade involving activation and phosphorylation of ERK1/2 and JNK1. In preclinical models of experimental metastasis, ectopic expression of RAGE on human prostate cancer cells was sufficient to promote bone marrow homing within a short timeframe. Our findings demonstrate how RAGE–PR3 interactions between human prostate cancer cells and the bone marrow microenvironment mediate bone metastasis during prostate cancer progression, with potential implications for prognosis and therapeutic intervention. Cancer Res; 77(12); 3144–50. ©2017 AACR.

Crucial importance of a business entity’s market value for its investment attractiveness is proved. Factors determining market value of a business entity and its attractiveness for investments are summarized. The main points of concept of value-based management and its impact on investment attractiveness are highlighted. In particular, the nature and contents of entity re-structuring process as an integral phase of value delivery are briefly considered, restructuring goals of national business entities are outlined from the standpoint of larger national and foreign companies operating in this country. Approaches and techniques of business valuation, proposed by Jay Fishman , Shannon Pratt, Clifford Griffith, Keith Wilson, as well as terms for use of each. Special attention is given to value added as an economic measure and contributor to both the investment value dynamics and business value. It is concluded that value-based management has to include the development of remuneration mechanism closely tied to changes in the value and investment attractiveness of a business entity.

Background aims The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a T-cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells, efficiently killing them in vitro and eliminating them in preclinical models. Humanized 8F4 (h8F4) with high affinity for the PR1/HLA-A2 epitope was used to construct an h8F4- chimeric antigen receptor (CAR) that was transduced into T cells to mediate anti-leukemia activity. Methods Human T cells were transduced to express the PR1/HLA-A2-specific CAR (h8F4-CAR-T cells) containing the scFv of h8F4 fused to the intracellular signaling endo-domain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28. Results Adult human normal peripheral blood (PB) T cells were efficiently transduced with the h8F4-CAR construct and predominantly displayed an effector memory phenotype with a minor population (12%) of central memory cells in vitro . Umbilical cord blood (UCB) T cells could also be efficiently transduced with the h8F4-CAR. The PB and UCB-derived h8F4-CAR-T cells specifically recognized the PR1/HLA-A2 complex and were capable of killing leukemia cell lines and primary AML blasts in an HLA-A2-dependent manner. Conclusions Human adult PB and UCB-derived T cells expressing a CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro . Our data could lead to a new treatment paradigm for AML in which targeting leukemia stem cells could transfer long-term immunity to protect against relapse.

Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Because neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination, and proteasome processing for cross-presentation. Using anti-PR1/human leukocyte antigen-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, whereas DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classic major histocompatibility class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia.

The results of numerical and laboratory simulation of ensembles of quasi-random unidirectional intense surface gravity waves in deep water have been summarized. The role of nonlinear self-modulation of the waves applied to the problem of ocean rogue waves, as well as the appearance, dynamics, and manifestation of non-linear wave packets in stochastic wave fields, is discussed.

The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a high-affinity T-cell receptor-like murine monoclonal antibody, 8F4, that binds to the PR1/HLA-A2 complex, mediates lysis of AML and inhibits leukemia colony formation. Here, we explored whether 8F4 was active in vivo against chemotherapy-resistant AML, including secondary AML. In a screening model, coincubation of AML with 8F4 ex vivo prevented engraftment of all tested AML subtypes in immunodeficient NSG (NOD scid IL-2 receptor γ-chain knockout) mice. In a treatment model of established human AML, administration of 8F4 significantly reduced or eliminated AML xenografts and extended survival compared with isotype antibody-treated mice. Moreover, in secondary transfer experiments, mice inoculated with bone marrow from 8F4-treated mice showed no evidence of AML engraftment, supporting the possible activity of 8F4 against the subset of AML with self-renewing potential. Our data provide evidence that 8F4 antibody is highly active in AML, including chemotherapy-resistant disease, supporting its potential use as a therapeutic agent in patients with AML.

In the Drosophila germline, retrotransposons are silenced by the PIWI-interacting RNA (piRNA) pathway. Telomeric retroelements HeT-A, TART and TAHRE, which are involved in telomere maintenance in Drosophila, are also the targets of piRNA-mediated silencing. We have demonstrated that expression of reporter genes driven by the HeT-A promoter is under the control of the piRNA silencing pathway independent of the transgene location. In order to test directly whether piRNAs affect the transcriptional state of retrotransposons we performed a nuclear run-on (NRO) assay and revealed increased density of the active RNA polymerase complexes at the sequences of endogenous HeT-A and TART telomeric retroelements as well as HeT-A-containing constructs in the ovaries of spn-E mutants and in flies with piwi knockdown. This strongly correlates with enrichment of two histone H3 modifications (dimethylation of lysine 79 and dimethylation of lysine 4), which mark transcriptionally active chromatin, on the same sequences in the piRNA pathway mutants. spn-E mutation and piwi knockdown results in transcriptional activation of some other non-telomeric retrotransposons in the ovaries, such as I-element and HMS Beagle. Therefore piRNA-mediated transcriptional mode of silencing is involved in the control of retrotransposon expression in the Drosophila germline.

The rewarding influence of drugs of abuse varies with time of day and appears to involve interactions between the circadian and the mesocorticolimbic dopamine systems. The circadian system is also intimately involved in measuring daylength. Thus, the present study examined the impact of changing daylength (photoperiod) on cocaine-seeking behaviors. Male Sprague-Dawley rats were trained and tested on a 12L:12D light:dark schedule for cocaine-induced reinstatement of conditioned place preference (CPP) at three times of day (Zeitgeber time (ZT): 4, 12, and 20) to determine a preference score. Rats were then shifted to either shorter (6L:18D) or longer (18L:6D) photoperiods and then to constant conditions, re-tested for cocaine-induced reinstatement under each different condition, and then returned to their original photoperiod (12L:12D) and tested once more. Rats exhibited a circadian profile of preference score in constant darkness with a peak at 12 h after lights-off. At both ZT4 and ZT20, but not at ZT12, shorter photoperiods profoundly suppressed cocaine reinstatement, which did not recover even after switching back to 12L:12D. In contrast, longer photoperiods did not alter reinstatement. Separate studies showed that the suppression of cocaine reinstatement was not due to repeated testing. In an additional experiment, we examined the photoperiodic regulation of tyrosine hydroxylase (TH) and dopamine transporter (DAT) proteins in drug-naive rats. These results revealed photoperiodic modulation of proteins in the prefrontal cortex and dorsal striatum, but not in the nucleus accumbens or ventral tegmental area. Together, these findings add further support to the circadian genesis of cocaine-seeking behaviors and demonstrate that drug-induced reinstatement is modulated by photoperiod. Furthermore, the results suggest that photoperiod partly contributes to the seasonal expression of certain drug-related behaviors in humans living at different latitudes and thus our findings may have implications for novel targeting of circadian rhythms in the treatment of addiction.

Acute myeloid leukemia (AML) is associated with an immunosuppressive tumor microenvironment (TME) that prevents immune recognition and fosters disease growth. In an attempt to reverse this immune suppression, we developed a personalized vaccine in which patient-derived AML cells are fused with autologous dendritic cells (DCs) such that a broad array of leukemic antigens (Ags) is presented in the context of DC-mediated costimulation. We have examined strategies to enhance vaccine efficacy. In this regard, we previously demonstrated that the hypomethylating agent (HMA), SGI-110, augments leukemic Ag processing and increases AML cell susceptibility to T cell-mediated killing. In the present study, we investigated if SGI-110 enhances leukemic Ag presentation by measuring the binding capacity of a unique T-cell receptor-like (TCRL) antibody (Ab) to AML cells in vitro. The TCRL Ab is a TCR mimic that recognizes the PR1-HLA-A2-MHC complex on the surface of leukemic cells where PR-1 is a known leukemic Ag. Following exposure of SGI-110 to the AML cell line, THP-1 which endogenously expresses HLA-A2, we noted enhanced tumor Ag presentation and HLA-A2 upregulation via FACS analysis. Aside from facilitating Ag presentation, we investigated if SGI-110 affects myeloid-derived suppressor cell (MDSC) burden in the TME where MDSCs have immunosuppressive capabilities. In an in vitro assay where healthy donor-derived peripheral blood mononuclear cells were cocultured with 2 AML cell lines, SGI-110 treatment led to a significant decrease in MDSC expansion compared to control. Based on these in vitro data, we investigated the ability of SGI-110 to reverse critical aspects of the immunosuppressive milieu in AML and enhance vaccine efficacy in vivo. C57BL/6J mice were retro-orbitally inoculated with 1x105 syngeneic TIB-49 AML cells and treated with low-dose SGI-110 (1 mg/kg) to assess immunologic effects while not preventing engraftment. AML engraftment in the spleen and bone marrow (BM) was similarly observed in treated and control animals. However, SGI-110-treated animals showed decreased PD-1 expression on CD4 T cell splenocytes (mean 3.1% vs. 6.3%; n=4; p=0.01). Similarly, SGI-110 therapy was associated with a decrease in MDSC burden in the BM (n=5; p=0.01) and spleen (n=4; p=0.03) compared to control mice. Consistent with these findings, SGI-110-treated mice demonstrated increased AML-specific immunity as manifested by increased T cells isolated from the BM or spleen that expressed intracellular IFNγ following ex vivo exposure to TIB-49 lysate (mean 3% vs. 0.5%; n=5; p=0.02). We subsequently examined the potential synergy of SGI-110 plus the vaccine in this model. Mice treated with SGI-110 + vaccine showed a significant increase in BM and spleen CD4 (n=5; p=0.05) and CD8 (n=5; p=0.02) T cell IFNγ in response to tumor lysate as compared to vaccine or SGI-110 alone. Given these effects, we sought to examine whether treatment with the vaccine and SGI-110 would enhance survival in this model. Mice were treated with vehicle, SGI-110 X 5 days, vaccine, or both agents. Monotherapy with either agent led to a statistically significant survival advantage as compared to untreated mice; yet the combination arm further prolonged survival as compared to either monotherapy arm. In conclusion, SGI-110 enhances Ag presentation and modulates the AML-associated immunosuppressive milieu by decreasing both T-cell PD-1 expression and MDSC burden with a notable increase in AML-specific immunity. Moreover, SGI-110 increases the immunogenicity of the DC/AML vaccine through an enhanced AML-specific response with a survival benefit in a murine model. This combination strategy has promising translational potential and a phase 1 clinical trial with this synergistic approach is planned. Citation Format: Myrna Rita Nahas, Dina Stroopinksy, Anna Sergeeva, Athalia Pyzer, Abigail Washington, Leandra Cole, Salvia Jain, Malgorzata McMasters, James D. Levine, Rebecca Karp Leaf, Shira Orr, Matthew Weinstock, Jeffrey J. Molldrem, David Avigan, Jacalyn Rosenblatt. Hypomethylating agent, SGI-110, alters the immunosuppressive milieu in acute myeloid leukemia (AML) and enhances the immunogenicity of a dendritic cell/AML fusion vaccine [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 19.

Antibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF) can be induced when GM-CSF is used as an adjuvant to solid tumor vaccination. Neutralizing anti-GM-CSF IgG has been associated with pulmonary alveolar proteinosis (PAP), and secondary PAP has been linked to myeloid leukemia. We studied 69 patients with acute myeloid leukemia, chronic myeloid leukemia and myelodysplastic syndrome, including 19 patients who received GM-CSF with peptide antigen and incomplete Freund's adjuvant in a vaccine trial for the presence or induction of anti-GM-CSF antibodies. Anti-GM-CSF IgG were present in 36 (52%) patients with myeloid leukemia compared to only 1 of 33 (3%) healthy subjects (P=0.008) and in none of 6 patients with lymphoid leukemia (P=0.0001). Antibody titers were unaffected by vaccination. Anti-GM-CSF IgA and IgM were found in 33 and 20% of patients, respectively; IgA from two patients neutralized GM-CSF. Strikingly, while anti-GM-CSF IgG titers were higher in patients with active disease (n=52) versus those in complete remission (n=14, P=0.0009), GM-CSF expression was not increased in either group. These data are first to show that anti-GM-CSF antibodies of multiple isotypes are present in patients with active myeloid leukemia without PAP and may be useful markers of disease activity.

This paper discusses the possibility to study propagation, shoaling and run-up of these waves over a slope in a 300-meter long large wave flume (GWK), Hannover. For this purpose long bell-shaped solitary waves (elongated solitons) of different amplitude and the same period of 30 s are generated. Experimental data of long wave propagation in the flume are compared with numerical simulations performed within the fully nonlinear potential flow theory and KdV equations. Shoaling and run-up of waves on different mild slopes is studied hypothetically using nonlinear shallow water theory. Conclusions about the feasibility of using large scale experimental facility (GWK) to study tsunami wave propagation and run-up are made. Alexander von Humboldt foundation RFBR/14-02-00983 RFBR/14-05-00092

Previously we developed a T cell receptor (TCR) -like murine IgG2a monoclonal antibody, m8F4, which binds to the PR1 peptide/HLA-A2 complex, and targets both HLA-A2+ leukemia cell lines and primary HLA-A2+ patient blasts of various AML subtypes. We reported that m8F4 mediated complement dependent cytotoxicity (CDC) of AML in vitro and actively depleted AML in vivoin AML patient-derived xenograft (PDX) models. Due to the therapeutic potential of 8F4, we humanized 8F4 for clinical development. Here we report the specificity of this humanized IgG1κ 8F4 (h8F4) to PR1/HLA-A2 and document the activity of h8F4 against AML. Both ELISA and surface plasmon resonance (SPR) were used to study h8F4 specificity to recombinant monomer, consisting of PR1 peptide, HLA-A2 and β2 microglobulin. Like mouse 8F4, human 8F4 bound PR1/HLA-A2 monomer with high affinity (KD= 6.5 nM), while no binding was detected to control monomers pp65/HLA-A2 and WT1/HLA-A2. Moreover, FACS of peptide-pulsed T2 cells showed that h8F4 bound PR1- pulsed T2, but did not bind T2 cells loaded with other HLA-A2-restricted peptides, including MART1, pp65, WT1 and gp100, thus confirming h8F4 specificity to PR1/HLA-A2. To test whether h8F4 retains the in vivoanti-leukemia activity seen with m8F4, we used an NSG mouse model. Similar to m8F4, h8F4 treatment reduced leukemia growth in NSG mice with established disease in 3 out of 4 AML PDX models tested. To determine the mechanism of action (MOA) of humanized 8F4, we first tested h8F4 activity in the presence of complement because we had previously demonstrated the primary MOA of m8F4 to be CDC. However, unlike m8F4, even when tested at high concentrations up to 20 µg/ml, h8F4 did not mediate CDC of HLA-A2 transduced U937, HL60, or PR1-loaded T2 cells. To further examine h8F4 MOA, we tested antibody dependent cellular cytotoxicity (ADCC) of h8F4 using an lactase dehydrogenase (LDH) assay with pooled human PBMC as effectors. H8F4 induced ADCC of U937-A2 at E:T ratio 50 (EC50=0.95 µg/ml), thus indicating improved ADCC anti-AML activity of h8F4. Introduction of alanine substitutions in positions 234-235 (AAh8F4), which disrupted the binding of h8F4 to Fc receptor (FcR), abolished ADCC activity against U937-A2 in vitro. In contrast, AA8F4 still retained partial in vivo activity against the same target cells in the NSG mouse model, suggesting that ADCC is important, but not the exclusive mechanism of h8F4 in vivo. To investigate whether h8F4 had direct activity against AML, as possible alternative MOA, we measured apoptosis of target cells by h8F4 with and without crosslinking anti-Fc antibody fragments (CLA). Indeed, we found, by using annexin V and propidium iodide staining, that a 24 hours incubation with h8F4 (1µg/ml) in the presence of CLA induced apoptosis of 55.5 ± 3.5% of U937-A2 cells, as compared to 23.5 ± 2.1% in CLA-only control (p < 0.05). Moreover, caspase 3 activation was observed as early as 10 minutes after adding h8F4+CLA, and was time- and h8F4 concentration- dependent. Our data demonstrate that specificity of humanized anti-PR1/HLA-A2 TCR-like antibody, h8F4, is preserved and that h8F4 has a similar affinity compared with m8F4. In addition to inducing apoptosis, h8F4 mediates greater ADCC compared with m8F4, but lacks CDC activity in vitro. Together, our data is consistent with multiple mechanisms of action in vivo, including ADCC. Our results support further testing of h8F4 for patients with AML. Disclosures Sergeeva: Astellas Pharma: Patents & Royalties. Molldrem:Astellas Pharma: Patents & Royalties.