Nuclear localization of BCR and cortactin indicates their potential role in regulation of actin branching in nucleus.

AIM: To study cellular localization of full-length breakpoint cluster region (BCR), Pleckstrin homology domain of BCR and cortactin and determine whether they can coexist in cell nucleus. MATERIALS AND METHODS: HEK293T cell line was transfected with pECFP-BCR, pEGFP-PH and pmTagRFP-N1-CTTN using polyethyleneimine. Live cells were imaged in cell culture dishes with glass coverslip attached to the bottom with Leica SP8 STED 3D confocal microscope in the environmental chamber. Obtained images were processed and analyzed with Fiji software. RESULTS: We identified colocalization of full-length BCR and cortactin in nucleus of cell undergoing terminal phase of cell division. We did not observe nuclear localization of cortactin in non-dividing cell. Both Pleckstrin homology domain and full-length BCR exhibited cytoplasmic as well as nuclear localization. CONCLUSIONS: Colocalization of BCR with cortactin in cell nucleus indicates their potential role in regulation of actin network allowing for the maintenance of nuclear architecture and DNA integrity.

Inhibition of USP1, a new partner of Bcr-Abl, results in decrease of Bcr-Abl level in K562 cells.

AIM: To analyze interaction of ubiquitin specific peptidase 1 (USP1) with Bcr-Abl and to assess the relation between USP1 functional activity and Bcr-Abl expression in K562 chronic myeloid leukemia cells. MATERIALS AND METHODS: The interaction between USP1 and Bcr-Abl in K562 cells was analyzed by co-immunoprecipitation, Western blot analysis, and confocal microscopy. RESULTS: A direct interaction between Bcr-Abl oncoprotein and USP1 protein in K562 cells was established by co-immunoprecipitation. Immunofluorescence analysis and confocal microscopy revealed that Bcr-Abl/USP1 protein complex is formed in the cell nucleus. The inhibition of USP1 protein activity by ML323 reduced the level of Bcr-Abl oncoprotein in K562 cells. CONCLUSIONS: USP1 protein has been identified as a new protein partner of Bcr-Abl oncoprotein in chronic myeloid leukemia. The relationship between the functional activity of USP1 protein and the level of Bcr-Abl oncoprotein has been demonstrated, suggesting that the targeted inhibition of USP1 activity could be a challenging approach for reducing Bcr-Abl expression.

Colocalization of USP1 and РН domain of Bcr­Abl oncoprotein in terms of cronic myeloid leukemia cell rearrangements.

The development of chronic myeloid leukemia (CML) is the result of a reciprocal translocation between chromosomes 9 and 22 due to the emergence of Philadelphia chromosome. The product of this mutation is a hybrid oncoprotein Bcr-Abl. According to the results of mass spectrometric analysis, USP1 protein was identified as a potential candidate for interaction with the PH domain Bcr-Abl oncoprotein. Due to the deubiquitination properties, USP1 protein can prevent proteasomal degradation of Bcr-Abl oncoprotein in a cell and, consequently, contribute to its accumulation, and the progression of the disease. In this work, creating the genetic constructs, we detected the USP1 protein localization in the cell. Also, a nuclear colocalization of USP1 protein with PH domain of Bcr-Abl oncoprotein in HEK293T cells was shown. The results are important for understanding the implications of the Philadelphia chromosome emergence, and the development of new methods for CML treatment, since the recent techniques are not always effective due to the emergence of numerous mutations that cause drug resistance and relapse of the disease.

[Antiherpetic effect of RNA-tilorone molecular complex in cell culture].

The authors studied the antiviral effect of an interferonogenic yeast RNA-tilorone molecular complex (MC) compared to the Virolex, videly used antiherpetic drug, and standard interferon (IFN) alpha/beta inducer poly(I)poly(C) in Vero cells culture infected with herpes simplex type I virus (HSV-1). The tilorone contained by MC has been shown to be twice less toxic and twice more active against HSV than its free molecules. The value of chemotherapeutic index (CI) of Virolex in experiments with Vero cells reaches 2500, CI poly(I)poly(C) and MC being 324; the last value meets the requirements for promising drugs.

[Effect of interferonogenic molecular complex of yeast RNA--tilorone on DNA, RNA and protein synthesis in vitro]. / Vplyv interferonohennoho molekuliarnoho kompleksu drizhdzhova RNK--tyloron na syntez DNK, RNK ta bilka in vitro.

In the experiments in vitro using the primary mononuclear cells (MNC) culture of the human peripheral blood the influence of interferonogenic yeast RNA-tilorone molecular complex on the DNA, RNA and protein synthesis was studied. The complex was shown to inhibit the insertion of 3H-thymidine, 3H-uridine and 3H-leucine into DNA, RNA and protein of MNC total pool (by 13, 1 and 40% respectively); that was practically conformed with this synthesis inhibition upon to a natural origin polynucleotide interferon inducers--lariphan (9, 0 and 57% respectively) and ridostin (9, 0 and 56% respectively) action, and at the same time rather less than poly(I)-poly(C) (14, 5 and 62% respectively). In the case of preliminary cell stimulation by the mitogen PHA the complex revealed comitogenic action at a concentration 25 micrograms/ml, that corresponded to optimal for interferonogenesis; the increase of the doses till 100-1000 micrograms/ml lead to in the reversal effect. To proceed from mutual relation between interferonogen preparations influence on the mentioned synthesis and their cytotoxicity the conclusion was about made the complex promising usage as an interferon inducer both in vitro and in vivo conditions.

Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.

Toll-like receptors (TLRs) are ancient microbial pattern recognition receptors highly conserved from Drosophila to humans. To investigate if subsets of human dendritic cell precursors (pre-DC), including monocytes (pre-DC1), plasmacytoid DC precursors (pre-DC2), and CD11c(+) immature DCs (imDCs) are developed to recognize different microbes or microbial antigens, we studied their TLR expression and responses to microbial antigens. We demonstrate that whereas monocytes preferentially express TLR 1, 2, 4, 5, and 8, plasmacytoid pre-DC strongly express TLR 7 and 9. In accordance with these TLR expression profiles, monocytes respond to the known microbial ligands for TLR2 (peptidoglycan [PGN], lipoteichoic acid) and TLR4 (lipopolysaccharide), by producing tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. In contrast, plasmacytoid pre-DCs only respond to the microbial TLR9-ligand, CpG-ODNs (oligodeoxynucleotides [ODNs] containing unmethylated CpG motifs), by producing IFN-alpha. CD11c(+) imDCs preferentially express TLR 1, 2, and 3 and respond to TLR 2-ligand PGN by producing large amounts of TNF-alpha, and to viral double-stranded RNA-like molecule poly I:C, by producing IFN-alpha and IL-12. The expression of distinct sets of TLRs and the corresponding difference in reactivity to microbial molecules among subsets of pre-DCs and imDCs support the concept that they have developed through distinct evolutionary pathways to recognize different microbial antigens.

Distinct cytokine profiles of neonatal natural killer T cells after expansion with subsets of dendritic cells.

Natural killer T (NKT) cells are a highly conserved subset of T cells that have been shown to play a critical role in suppressing T helper cell type 1-mediated autoimmune diseases and graft versus host disease in an interleukin (IL)-4-dependent manner. Thus, it is important to understand how the development of IL-4- versus interferon (IFN)-gamma-producing NKT cells is regulated. Here, we show that NKT cells from adult blood and those from cord blood undergo massive expansion in cell numbers (500-70,000-fold) during a 4-wk culture with IL-2, IL-7, phytohemagglutinin, anti-CD3, and anti-CD28 mAbs. Unlike adult NKT cells that preferentially produce both IL-4 and IFN-gamma, neonatal NKT cells preferentially produce IL-4 after polyclonal activation. Addition of type 2 dendritic cells (DC2) enhances the development of neonatal NKT cells into IL-4(+)IFN-gamma(-) NKT2 cells, whereas addition of type 1 dendritic cells (DC1) induces polarization towards IL-4(-)IFN-gamma(+) NKT1 cells. Adult NKT cells display limited plasticity for polarization induced by DC1 or DC2. Thus, newly generated NKT cells may possess the potent ability to develop into IL-4(+)IFN-gamma(-) NKT2 cells in response to appropriate stimuli and may thereafter acquire the tendency to produce both IL-4 and IFN-gamma.

Distinct CpG DNA and polyinosinic-polycytidylic acid double-stranded RNA, respectively, stimulate CD11c- type 2 dendritic cell precursors and CD11c+ dendritic cells to produce type I IFN.

Two classes of nucleic acids, bacterial DNA containing unmethylated CpG motifs and dsRNA in viruses, induce the production of type I IFN that contributes to the immunostimulatory effects of these microbial molecules. Thus, it is important to determine which cells produce type I IFN in response to CpG DNA and dsRNA. CD4(+)CD11c(-) type 2 dendritic cell precursors (pre-DC2) were identified as the main producers of type I IFN in human blood in response to viruses. Here we asked whether pre-DC2 also produce type I IFN in response to CpG DNA and dsRNA. Oligodeoxynucleotides containing particular palindromic CpG motifs induced pre-DC2, but not CD11c(+) blood DC or monocytes, to produce IFN-alpha. In contrast, a synthetic dsRNA, polyinosinic polycytidylic-acid, induced CD11c(+) DC, but not pre-DC2 or monocytes, to produce IFN-alphabeta. These data indicate that CpG DNA and polyinosinic-polycytidylic acid stimulate different types of cells to produce type I IFN and that it is important to select oligodeoxynucleotides containing particular CpG motifs to induce pre-DC2 to produce type I IFN, which may play a key role in the strong adjuvant effects of CpG DNA.

Virus-inhibitory effect of a yeast RNA-tilorone molecular complex in cell cultures.

The virus-inhibitory activity of a molecular complex (MC) of tilorone and yeast RNA was studied in vitro on three virus-cell systems: vesicular stomatitis virus (VSV) - murine fibroblast L929 cells, Venezuelan equine encephalittis virus (VEEV) - swine embryo kidney (SEK) cells and encephalomyocarditis virus (EMCV) - established piglet testicular (EPT) cells. In all these systems the MC exerted an antiviral effect similar to that of polynucleotide interferon (IFN) inducers such as poly(I)-poly(C), larifan and ridostin. The antiviral effect of the MC was similar when the compound was applied before or after virus adsorption to cells. The MC may be regarded as a perspective antiviral agent of common use.

Generation of interferon alpha-producing predendritic cell (Pre-DC)2 from human CD34(+) hematopoietic stem cells.

Upon viral stimulation, the natural interferon (IFN)-alpha/beta-producing cells (IPCs; also known as pre-dendritic cells (DCs 2) in human blood and peripheral lymphoid tissues rapidly produce huge amounts of IFN-alpha/beta. After performing this innate antiviral immune response, IPCs can differentiate into DCs and strongly stimulate T cell-mediated adaptive immune responses. Using four-color immunofluorescence flow cytometry, we have mapped the developmental pathway of pre-DC2/IPCs from CD34(+) hematopoietic stem cells in human fetal liver, bone marrow, and cord blood. At least four developmental stages were identified, including CD34(++)CD45RA(-) early progenitor cells, CD34(++)CD45RA(+) late progenitor cells, CD34(+)CD45RA(++)CD4(+)interleukin (IL)-3Ralpha(++) pro-DC2, and CD34(-)CD45RA(++) CD4(+)IL-3Ralpha(++) pre-DC2/IPCs. Pro-DC2s have already acquired the capacity to produce large amounts of IFN-alpha/beta upon viral stimulation and to differentiate into DCs in culture with IL-3 and CD40 ligand. CD34(++)CD45RA(-) early progenitor cells did not have the capacity to produce large amounts of IFN-alpha/beta in response to viral stimulation; however, they can be induced to undergo proliferation and differentiation into IPCs/pre-DC2 in culture with FLT3 ligand.

Natural interferon alpha/beta-producing cells link innate and adaptive immunity.

Innate immune responses to pathogens critically impact the development of adaptive immune responses. However, it is not completely understood how innate immunity controls the initiation of adaptive immunities or how it determines which type of adaptive immunity will be induced to eliminate a given pathogen. Here we show that viral stimulation not only triggers natural interferon (IFN)-alpha/beta-producing cells (IPCs) to produce vast amounts of antiviral IFN-alpha/beta but also induces these cells to differentiate into dendritic cells (DCs). IFN-alpha/beta and tumor necrosis factor alpha produced by virus-activated IPCs act as autocrine survival and DC differentiation factors, respectively. The virus-induced DCs stimulate naive CD4(+) T cells to produce IFN-gamma and interleukin (IL)-10, in contrast to IL-3-induced DCs, which stimulate naive CD4(+) T cells to produce T helper type 2 cytokines IL-4, IL-5, and IL-10. Thus, IPCs may play two master roles in antiviral immune responses: directly inhibiting viral replication by producing large amounts of IFN-alpha/beta, and subsequently triggering adaptive T cell-mediated immunity by differentiating into DCs. IPCs constitute a critical link between innate and adaptive immunity.

[Comparative cytotoxic characteristics of the interferon-inducing yeast RNA-tilorone complex]. / Sravnitel'naia kharakteristika tsitotoksichnosti interferon-indutsiruiushchego kompleksa drozhzhevaia RNK-tiloron.

Cytotoxicity of the yeast RNA-tilorona molecular complex (MC) with interferonogenic properties and its influence on the DNA replicative synthesis were studied in experiments with human lymphocytes and 3 cell lines. It was shown that the MC doses of 25, 100 and 250 micrograms/ml were absolutely nontoxic for all the cell lines. The main parameters of the MC toxicity based on the cell viability were calculated. The parameters were found to correlate in the order of their magnitude with those relating to interferonogens of the polynucleotide nature. Within the dose ranges of 10 to 100 micrograms/ml the MC had a stimulating effect on replicative processes in the cells. It was concluded that the use of the MC as an inductor in large-scale manufacture of human and animal interferons of type 1 was promising.

IL-10 transgenic mice present a defect in T cell development reminiscent of SCID patients.

To analyze the effect of IL-10 overexpressed by APCs as observed in some SCID patients, we have expressed the human IL-10 cDNA under the control of the murine MHC class II promoter in transgenic mice. Similar to SCID patients, these mice presented a defect in T cell maturation characterized by a rapid thymic aplasia that started after birth. The blockage in T cell maturation was strictly restricted to TCR-alpha beta T cells as the absolute number of thymic dendritic, TCR-gamma delta and NK1.1 T cells were equivalent to control littermates. Crossing IL-10 transgenic mice with TCR transgenic mice or treatment with staphylococcal enterotoxin B showed that the defect was not related to the impairment of positive or negative selection. However, repopulating of IL-10 transgenic mouse-fetal thymic organ culture with different stages of triple negative T cells isolated from control mice showed that the blockage occurred specifically at the pre-T cell stage and was reverted by treatment with blocking anti-IL-10 mAbs. These results demonstrate that IL-10 regulates T cell maturation and that dysregulation of IL-10 expression can lead to severe T cell immunodeficiency.

The nature of the principal type 1 interferon-producing cells in human blood.

Interferons (IFNs) are the most important cytokines in antiviral immune responses. "Natural IFN-producing cells" (IPCs) in human blood express CD4 and major histocompatibility complex class II proteins, but have not been isolated and further characterized because of their rarity, rapid apoptosis, and lack of lineage markers. Purified IPCs are here shown to be the CD4(+)CD11c- type 2 dendritic cell precursors (pDC2s), which produce 200 to 1000 times more IFN than other blood cells after microbial challenge. pDC2s are thus an effector cell type of the immune system, critical for antiviral and antitumor immune responses.

A transgenic model to analyze the immunoregulatory role of IL-10 secreted by antigen-presenting cells.

IL-10 is a cytokine secreted by a wide variety of cells type that has pleiotropic stimulatory and suppressive activities on both lymphoid and myeloid cells in vitro. To analyze the consequences of high IL-10 secretion by APCs in immune responses, we produced transgenic mice expressing human IL-10 directed by the MHC class II Ea promoter. Despite alterations in the development of T and B cells, no gross abnormalities were detected in peripheral lymphocyte populations or serum Ig levels. However, when immunized using conditions that give either a Th2-type or a Th1-type response, IL-10 transgenic mice failed to mount a significant T or B cell immune response to OVA. IL-10 transgenic mice were also highly susceptible to infection with intracellular pathogens like Listeria monocytogenes or Leishmania major, in contrast to IL-10 transgenic mice, where the transgene was express in T cells. Finally, the recently described stimulatory effect of IL-10 on CD8+ T cells was confirmed by the ability of IL-10 transgenic mice to limit the growth of immunogenic tumors by a CTL-mediated mechanism. These results demonstrate, that, depending on the type of immune response, IL-10 can mediate immunosuppressive or immunostimulatory activities in vivo.

A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis.

Induction and maintenance of peripheral tolerance are important mechanisms to maintain the balance of the immune system. In addition to the deletion of T cells and their failure to respond in certain circumstances, active suppression mediated by T cells or T-cell factors has been proposed as a mechanism for maintaining peripheral tolerance. However, the inability to isolate and clone regulatory T cells involved in antigen-specific inhibition of immune responses has made it difficult to understand the mechanisms underlying such active suppression. Here we show that chronic activation of both human and murine CD4+ T cells in the presence of interleukin (IL)-10 gives rise to CD4+ T-cell clones with low proliferative capacity, producing high levels of IL-10, low levels of IL-2 and no IL-4. These antigen-specific T-cell clones suppress the proliferation of CD4+ T cells in response to antigen, and prevent colitis induced in SCID mice by pathogenic CD4+CD45RB(high) splenic T cells. Thus IL-10 drives the generation of a CD4+ T-cell subset, designated T regulatory cells 1 (Tr1), which suppresses antigen-specific immune responses and actively downregulates a pathological immune response in vivo.

[Study of anti-HIV activity of the yeast RNA-tilorone molecular complex]. / Izuchenie anti-VICh-aktivnosti molekuliarnogo kompleksa drozhzhevaia RNK-tiloron.

Anti-HIV activity of the molecular complex forming during interaction between yeast RNA and tilorone was studied in vitro on the models of acute and chronic infection. Addition of this agent to the cells infected with HIV-1/IIIB and HIV-1/BRU decreased the virus reproduction controlled by assessing the viability of cells, syncytium production, and accumulation of p24 antigen in culture medium. The authors hypothesize that the detected anti-HIV effect is due to its capacity to produce type I interferon and direct antiviral action.

[The depression of the reproduction of the human immunodeficiency virus by the use of Bogomolets antireticular cytotoxic serum]. / Pryhnichennia reproduktsiï virusu imunodefitsytu liudyny za dopomohoiu antyretykuliarnoï tsytotoksychnoï syrovatky Bohomol'tsia.

The action of the Bogomolets' antireticular cytotoxic serum (ACS) as a HIV-1 reproduction inhibitor was studied on the model of human lymphoblastoid cell line MT-4/BRU (acute infection). The HIV-1 reproduction inhibition was estimated according to cell protection level for virus cytopathic action and p24 antigen level in the cultural media. The both tests data on ACS usage in dilutions 1:160-1:640 prove that the virus inhibition effect which may be compared with anti-HIV action of the well-known agent, azidothymidine, was observed. At this dilutions range ACS is devoid of the cell toxicity. The conclusion about perspectivity of ACS usage in the AIDS complex therapy was drawn.