Papers by Alexander Kovalchuk
A Candidate Ac3-S-LPS Vaccine Against S. flexneri 1b, 2a, 3a, 6, and Y Activates Long-Lived Systemic and Mucosal Immune Responses in Healthy Volunteers: Results of an Open-Label, Randomized Phase 2 Clinical Trial
Vaccines
Objectives: Determination of reactogenicity and immunogenicity of a pentavalent candidate vaccine... more Objectives: Determination of reactogenicity and immunogenicity of a pentavalent candidate vaccine against S. flexneri 1b, 2a, 3a, 6, and Y (PLVF). Methods: The study involved 80 healthy adult volunteers aged 18–55 years. Groups were subcutaneously immunized twice at a 30-day interval with 62.5 μg/0.5 mL or 125 μg/0.5 mL of the vaccine. Results: During the entire 8-month period of post-vaccination observation, the vaccine was well tolerated, with no local or systemic reactions detected objectively. The results of laboratory studies demonstrated no effect on the main indicators of hemogram, biochemical blood test, or urinalysis. IgA, IgG, and IgM levels against LPS S. flexneri 1b, 2a, 3a, 6, and Y were examined before vaccination, a month after each vaccination, and 6 months after booster vaccination. One month after vaccination, IgA and IgG seroconversions were observed in 67.5–82.5% (depending on serotype) and 60–77.5% of volunteers, respectively. Booster immunization did not have a...
Proceedings of the National Academy of Sciences of the United States of America, Aug 6, 2012
Oncotarget, May 22, 2018
The inhibitory effect of p53 on homologous recombination (HR) is exerted through sequestration of... more The inhibitory effect of p53 on homologous recombination (HR) is exerted through sequestration of replication protein A (RPA). Release of the p53/RPA complex in response to replication stress is crucially dependent on the phosphorylation status of both proteins and is required for efficient DNA repair by HR. Phosphorylation of RPA within its RPA2 subunit by cyclin-dependent kinases (CDK) is an early event in the replication stress response. Here we investigated the role of transcriptional activation of the p53 downstream target, p21Waf1, on RPA2 phosphorylation, the stability of the p53/RPA complex and HR in cells undergoing replication arrest induced by camptothecin (CPT). We show that in CPT-treated cells, activation of p53 and p21Waf1 impedes RPA2 phosphorylation, while their depletion by siRNA stimulates it. The p53/RPA complex is more stable in wild-type cells than in cells depleted of p21Waf1. We used nocodazole-synchronized cells treated with CPT at the entrance to S phase to...
Proceedings of the National Academy of Sciences of the United States of America, Jun 18, 2012
Human Burkitt lymphomas are divided into two main clinical variants: the endemic form, affecting ... more Human Burkitt lymphomas are divided into two main clinical variants: the endemic form, affecting African children infected with malaria and the Epstein-Barr virus, and the sporadic form, distributed across the rest of the world. However, whereas sporadic translocations decapitate Myc from 5′ proximal regulatory elements, most endemic events occur hundreds of kilobases away from Myc. The origin of these rearrangements and how they deregulate oncogenes at such distances remain unclear. We here recapitulate endemic Burkitt lymphoma-like translocations in plasmacytomas from uracil N-glycosylase and activation-induced cytidine deaminase-deficient mice. Mapping of translocation breakpoints using an acetylated histone H3 lysine 9 chromatin immunoprecipitation sequencing approach reveals Igh fusions up to ∼350 kb upstream of Myc or the related oncogene Mycn. A comprehensive analysis of epigenetic marks, PolII recruitment, and transcription in tumor cells demonstrates that the 3′ Igh enhancer (Eα) vastly remodels ∼450 kb of chromatin into translocated sequences, leading to significant polymerase occupancy and constitutive oncogene expression. We show that this longrange epigenetic reprogramming is directly proportional to the physical interaction of Eα with translocated sites. Our studies thus uncover the extent of epigenetic remodeling by Ig 3′ enhancers and provide a rationale for the long-range deregulation of translocated oncogenes in endemic Burkitt lymphomas. The data also shed light on the origin of endemic-like chromosomal rearrangements. chromosome conformation capture | chromosome translocations | activation-induced cytidine deamination | epigenetics
An essential role of transcription factors PU.1 and IRF8 in follicular B cell development and the germinal center response
Journal of Immunology, May 1, 2018
The transcription factors PU.1 and IRF8 regulate an unknown number of gene programs for different... more The transcription factors PU.1 and IRF8 regulate an unknown number of gene programs for differentiation of many hematopoietic cells including B cells, dendritic cells and myeloid cells. Their roles in B cell development were previously studied using B cell-specific conditional deletion mouse models, such as PU.1flox/flox-CD19Cre, IRF8flox/flox-CD19Cre, IRF8−/−PU.1flox/flox-CD19Cre, or IRF8flox/flox-PU.1flox/flox-CD19Cre mice. While PU.1-deficient B cells are phenotypically normal, deletion of IRF8 in B cells caused a moderate expansion of marginal zone B cells. Double deletion of PU.1 and IRF8 caused moderate expansion of plasma cells (PCs) in vitro. In this report, we investigated IRF8 and PU.1 double deletion mice using Mb1-Cre - termed DKO mice. FACS analysis revealed normal levels of early stage B cells in the bone marrow and transitional B cells in the spleens of DKO mice. However, follicular B cells were markedly reduced and the MZ B cell compartment was modestly expanded in DKO mice. The peritoneal CD5+ B-1a cells, which are a major source of circulating IgM, were completely absent in DKO mice. Surprisingly, the serum levels of IgM were higher and the levels of IgG3 and IgA were slightly lower in DKO than control mice. While the levels of serum IgG1 were comparable between DKO and control mice, DKO mice had no serum IgG2b or IgG2c antibodies. More intriguingly, following immunization with NP-KLH/alum, although the DKO mice produced more IgM-secreting PCs early on, they failed to generate germinal centers and produced markedly reduced levels of NP-specific switched IgG antibodies. Taken together, these data revealed a critical role of IRF8 and PU.1 in differentiation of follicular and germinal center B cells.
Abstract 772: Rap2b, a novel p53 downstream target, promotes cell survival after DNA damage
The tumor suppressor p53 is a critical regulator of apoptosis and cell survival. It evokes both p... more The tumor suppressor p53 is a critical regulator of apoptosis and cell survival. It evokes both pro-survival and pro-apoptosis programs upon DNA damage. However, the mechanisms underlying this cell fate decision are largely unclear. To identify p53 targets in a global and unbiased manner, we performed gene expression microarray and ChIP-chip assays using mouse embryonic fibroblasts (MEFs) and mouse embryonic stem (mES) cells. I treated MEFs and mES cells with adriamycin, a DNA damage agent, for 8 hours. Comparing to the untreated cells (negative controls), I obtained common genes that were dramatically induced both in MEFs and mES cells. Subsequently, we further analyzed these genes and obtained a list of 90 common genes. Then, I compared these 90 genes with published p53 targets in human cell lines U2OS and HCT116, and found 10 genes (Alox5, Eda2r, Btg2, Mdm2, Adrb2, Cdkn1a, Tnfrsf10b, Rap2b, Ddit4, Bbc3) that are conserved p53 targets between mouse and human. Most of these genes have been well studied except for Rap2b. Both mouse and human Rap2b share the same amino acid residue sequence, which indicating Rap2b might have important conserved functions both in human and in mouse. Using conventional chromatin immunoprecipitation (ChIP) assay and luciferase reporter assay, I identified one p53 binding motif in Rap2b promoter region. After DNA damage, p53 binds to the promoter of Rap2b and activates its transcription. The reduction of Rap2b levels by small interference RNA increases the apoptosis of cells under the damaged condition, suggesting that Rap2b helps cell survive upon DNA damage. This pro-survival role of Rap2b is very similar to the function of another well-known p53 target, Mdm2. Bioinformatic analysis revealed that Rap2b is over-expressed in many types of tumors, consistent with its pro-survival function. Anchorage independent growth assay showed that Rap2b only has weak transformation activity, suggesting that it is not a typical oncogene. Importantly, Rap2b activates RalGDS-Ral survival pathway after DNA damage. Therefore, our results identified a novel player, Rap2b, in the pro-survival program conducted by p53 and revealed a connection between the p53 signaling and the RalGDS-Ral cell survival pathway. Future studies will focus on investigating whether the inhibition of Rap2b may increase the apoptosis of tumor cells. Citation Format: Yunlong He, Mangmang Li, Wendy Dubois, Alexander Kovalchuk, Xiaolin Wu, Jing Huang. Rap2b, a novel p53 downstream target, promotes cell survival after DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 772. doi:10.1158/1538-7445.AM2013-772
Genetic Recombinations Between c-myc and Ighμ as Precursors for Recombinations Between c-myc and Ighα in BALB/c Plasmacytomas
Springer eBooks, 1997
In this paper we present evidence that primary plasmacytomas harboring a balanced chromosomal tra... more In this paper we present evidence that primary plasmacytomas harboring a balanced chromosomal translocation t(12;15) may consist of a mosaic of neoplastic clones of tumor cells, in which some of the clones are derived from common progenitors by large additional deletions which alter or “remodel” the fine structure of the t(12;15) on the c-myc-deregulating chromosome. This finding is based on the molecular analysis of the t(12;15) in three plasmacytomas, TEPC 1194, PCT 4127 and PCT 4132, in which primary clones of tumor cells with recombinations between the Ig heavy-chain (Igh) μ locus (Ighμ) and c-myc were found to be related to secondary clones of tumor cells with recombinations between the Igh a locus (Igha) and c-myc. Clonal relatedness was based on unique junction fragments between Sμ and c-myc that were found to be identical in Ighμ/c-myc and Ighα/c-myc breakpoint regions. Sμ/c-myc breakpoints with adjoining sequences can thus be used as clonotypic markers, providing molecular fingerprints for the evolution of precursor cells with Ighμ/c-myc recombinations into progenitor cells with Igha/c-myc recombinations. We propose that aberrant isotype switch recombination or illegitimate rearrangement in switch regions converts Ighμ/c-myc rearrangements, which are found very rarely in established plasmacytomas, into Ighα/c-myc rearrangements, which are present in about 80% of plasmacytomas with t(12;15). Remodeling of the t(12;15) results in closer juxtaposition of c-myc to the 3’-Cα locus control region and probably in enhanced transcription of c-myc. We suggest that remodeling of c-myc-deregulating rearrangements constitutes a noveln progression mechanism in malignant B-cell development.
Cell, Dec 1, 2013
A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes m... more A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoterenhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.
Data from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
We have compared histologic features and gene expression profiles of newly identified plasmacytom... more We have compared histologic features and gene expression profiles of newly identified plasmacytomas from NFS.V+ congenic mice with plasmacytomas of IL6 transgenic, Fasl mutant, and SJL-β2M−/− mice. NFS.V+ tumors comprised an overlapping morphologic spectrum of high-grade/anaplastic, intermediate-grade/plasmablastic, and low-grade/plasmacytic cases with similarities to subsets of human multiple myeloma and plasmacytoma. Microarray and immunohistochemical analyses of genes expressed by the most prevalent tumors, plasmablastic plasmacytomas, showed them to be most closely related to immunoblastic lymphomas, less so to plasmacytomas of Fasl mutant and SJL mice, and least to plasmacytic plasmacytomas of IL6 transgenic mice. Plasmablastic tumors seemed to develop in an inflammatory environment associated with gene signatures of T cells, natural killer cells, and macrophages not seen with plasmacytic plasmacytomas. Plasmablastic plasmacytomas from NFS.V+ and SJL-β2M−/− mice did not have structural alterations in Myc or T(12;15) translocations and did not express Myc at high levels, regular features of transgenic and pristane-induced plasmacytomas. These findings imply that, as for human multiple myeloma, Myc-independent routes of transformation contribute to the pathogenesis of these tumors. These findings suggest that plasma cell neoplasms of mice and humans exhibit similar degrees of complexity. Mouse plasmacytomas, previously considered to be homogeneous, may thus be as diverse as their human counterparts with respect to oncogenic mechanisms of plasma cell transformation. Selecting specific types of mouse plasmacytomas that relate most closely to subtypes of human multiple myeloma may provide new opportunities for preclinical testing of drugs for treatment of the human disease. [Cancer Res 2007;67(6):2439–47]
Constitutive Lymphoid Expression of the Nuclear Form of RNase H1 Is Associated with Development of Immature B Cell Lymphomas
Blood, Nov 20, 2009
Abstract 1613 Poster Board I-639 Ribonuclease H1, which cleaves the RNA of RNA/DNA hybrids, plays... more Abstract 1613 Poster Board I-639 Ribonuclease H1, which cleaves the RNA of RNA/DNA hybrids, plays a vital role in the replication of mitochondrial DNA during mouse embryogenesis, but its contributions to later mammalian development and adult biology are not known. In higher eukaryotes two isoforms of RNase H1, a nuclear form and mitochondrial form, are produced from a single transcript. To investigate the effects of constitutive high level RNase H1 expression, we generated transgenic (TG) mice, strain M27F7, with lymphocyte-specific over-expression of nuclear RNase H1. Transcripts in B cells from TG mice were ∼100-fold higher than in wild type B cells. Although there was no obvious phenotype in young mice, some of the older mice, especially females, developed lymphadenopathy, splenomegaly and mediastinal masses and were diagnosed histologically as having lymphoblastic lymphomas. Cell lines established from the tumors were examined by FACS, gene expression profiling and SKY as well as by FISH for Igh and Myc. By FACS criteria, the tumors were B cell lineage and were arrested at the pre-B to immature stage of differentiation. Expression profiles showed the lines to have increased expression of anti-apoptotic genes and genes promoting cell cycle progression. Chromosomal studies revealed no signs of genomic instability or translocations, although there were trisomies of chromosomes 3 and 15. Myc was structurally intact in all lines. These results suggest that RNase H1 plays a previously unappreciated role in early B cell development and that constitutive overexpression contributes to lymphomagenesis. This work was supported by the Intramural Research Program of the National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development and National Institute of Allergy and Infectious Diseases. Disclosures No relevant conflicts of interest to declare.
The Journal of Pathology, Jan 21, 2010
Anaplastic plasmacytomas (APCTs) from NFS.V + congenic mice and pristane-induced plasmacytic PCTs... more Anaplastic plasmacytomas (APCTs) from NFS.V + congenic mice and pristane-induced plasmacytic PCTs from BALB/c mice were previously shown to be histologically and molecularly distinct subsets of plasma cell neoplasms (PCNs). Here we extended these comparisons, contrasting primary APCTs and PCTs by gene expression profiling in relation to the expression profiles of normal naïve, germinal centre, and memory B cells and plasma cells. We also sequenced immunoglobulin genes from APCT and APCT-derived cell lines and defined surface phenotypes and chromosomal features of the cell lines by flow cytometry and by spectral karyotyping and fluorescence in situ hybridization. The results indicate that APCTs share many features with normal memory cells and the plasma cell-related neoplasms (PLs) of FASL-deficient mice, suggesting that APCTs and PLs are related and that both derive from memory B cells.
Рецепторсвязывающий домен S-белка SARS-CoV-2, слитый с негликозилированным кристаллизующимся фрагментом IgG1 человека: получение и оценка иммуногенности
Biotehnologiâ, 2022
Leukemia Research, Dec 1, 2013
The E3 ligase ARF-BP1 governs the balance of life and death decisions by directing the degradatio... more The E3 ligase ARF-BP1 governs the balance of life and death decisions by directing the degradation of p53 and enhancing the transcriptional activity of MYC. We find B cells selectively deficient in ARF-BP1 have many defects in developing and mature B cells associated with increased expression of p53 and reduced expression of Myc. Overexpression of Myc results in suppression of p53 and complete reversal of defects induced by ARF-BP1 deficiency. These
Immunologic Research, Jul 29, 2008
IRF8, a transcription factor restricted primarily to hematopoietic cells, is known to influence t... more IRF8, a transcription factor restricted primarily to hematopoietic cells, is known to influence the differentiation and function of dendritic cells (DC), macrophages, granulocytes and B cells. In human tonsil, IRF8 is expressed at high levels by intrafollicular macrophages and DC, but at much lower levels by tingible body macrophages in germinal centers (GCs) and little, if at all, by follicular DC. Spleens of IRF8-defficient mice had reduced numbers of white pulp follicles and GCs that were irregular in shape. The frequency of follicular B cells was significantly reduced while the population of marginal zone (MZ) B cells was increased. In addition, MZ macrophages were reduced in number and abnormally distributed, while metallophilic macrophages were normal. These findings demonstrate differential requirements for IRF8 among distinct subsets of B cells, DC, and macrophages.
Supplementary Figure 3 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
Supplementary Figure 3 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Rel... more Supplementary Figure 3 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
Supplementary Data 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
Supplementary Data 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relat... more Supplementary Data 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
Supplementary Figure 2 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
Supplementary Figure 2 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Rel... more Supplementary Figure 2 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
Supplementary Figure 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
Supplementary Figure 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Rel... more Supplementary Figure 1 from Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells
Obtaining and Assessing Immunogenicity of a Fusion Protein Which Consists of a Receptor-Binding Domain (RBD) of Coronavirus SARS-COV-2 Spike Protein and a Monomeric Non- Glycosylated FC-Fragment of Human IGG1
Materialy ... Moskovskogo meždunarodnogo kongressa "Biotehnologiâ: sostoânie i perspektivy razvitiâ", 2022
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Papers by Alexander Kovalchuk