Knockout of the Tnfa Gene Decreases Influenza Virus-Induced Histological Reactions in Laboratory Mice.

Tumor necrosis factor alpha (TNF-α) is a cytokine that is responsible for many processes associated with immune response and inflammation. It is involved in the development of an antiviral response to many virus infections. This factor was shown to be activated in influenza A virus infection, which enhances production of other cytokines. The overexpression of these cytokines can lead to a cytokine storm. To study the role of TNF-α in the development of pathologies associated with viral infection, we generated a Tnfa knockout mouse strain. We demonstrated that these mice were characterized by a significant increase in the number of viral genomes compared to that in the parental strain, but the amount of live virus did not differ. A histopathology of the lungs in the genetically modified animals was significantly lower in terms of interalveolar septal infiltration. The generated model may be used to further study pathological processes in viral infections.

The Molecular Aspects of Functional Activity of Macrophage-Activating Factor GcMAF.

Group-specific component macrophage-activating factor (GcMAF) is the vitamin D3-binding protein (DBP) deglycosylated at Thr420. The protein is believed to exhibit a wide range of therapeutic properties associated with the activation of macrophagal immunity. An original method for GcMAF production, DBP conversion to GcMAF, and the analysis of the activating potency of GcMAF was developed in this study. Data unveiling the molecular causes of macrophage activation were obtained. GcMAF was found to interact with three CLEC10A derivatives having molecular weights of 29 kDa, 63 kDa, and 65 kDa. GcMAF interacts with high-molecular-weight derivatives via Ca2+-dependent receptor engagement. Binding to the 65 kDa or 63 kDa derivative determines the pro- and anti-inflammatory direction of cytokine mRNA expression: 65 kDa-pro-inflammatory (TNF-α, IL-1ß) and 63 kDa-anti-inflammatory (TGF-ß, IL-10). No Ca2+ ions are required for the interaction with the canonical 29 kDa CLEC10A. Both forms, DBP protein and GcMAF, bind to the 29 kDa CLEC10A. This interaction is characterized by the stochastic mRNA synthesis of the analyzed cytokines. Ex vivo experiments have demonstrated that when there is an excess of GcMAF ligand, CLEC10A forms aggregate, and the mRNA synthesis of analyzed cytokines is inhibited. A schematic diagram of the presumable mechanism of interaction between the CLEC10A derivatives and GcMAF is provided. The principles and elements of standardizing the GcMAF preparation are elaborated.

Impact of Double-Stranded RNA Internalization on Hematopoietic Progenitors and Krebs-2 Cells and Mechanism.

It is well-established that double-stranded RNA (dsRNA) exhibits noticeable radioprotective and radiotherapeutic effects. The experiments conducted in this study directly demonstrated that dsRNA was delivered into the cell in its native form and that it induced hematopoietic progenitor proliferation. The 68 bp synthetic dsRNA labeled with 6-carboxyfluorescein (FAM) was internalized into mouse hematopoietic progenitors, c-Kit+ (a marker of long-term hematopoietic stem cells) cells and CD34+ (a marker of short-term hematopoietic stem cells and multipotent progenitors) cells. Treating bone marrow cells with dsRNA stimulated the growth of colonies, mainly cells of the granulocyte-macrophage lineage. A total of 0.8% of Krebs-2 cells internalized FAM-dsRNA and were simultaneously CD34+ cells. dsRNA in its native state was delivered into the cell, where it was present without any signs of processing. dsRNA binding to a cell was independent of cell charge. dsRNA internalization was related to the receptor-mediated process that requires energy from ATP. Synthetic dsRNA did not degrade in the bloodstream for at least 2 h. Hematopoietic precursors that had captured dsRNA reinfused into the bloodstream and populated the bone marrow and spleen. This study, for the first time, directly proved that synthetic dsRNA is internalized into a eukaryotic cell via a natural mechanism.

Human Adenovirus and Influenza A Virus Exacerbate SARS-CoV-2 Infection in Animal Models.

In this study, we investigated the features of the infectious process by simulating co-infection with SARS-CoV-2 and human adenovirus type 5 (HAdV-5) or influenza A virus (IAV) in vitro and in vivo. The determination of infectious activity of viruses and digital PCR demonstrated that during simultaneous and sequential HAdV-5 followed by SARS-CoV-2 infection in vitro and in vivo, the HAdV-5 infection does not interfere with replication of SARS-CoV-2. The hamsters co-infected and mono-infected with SARS-CoV-2 exhibited nearly identical viral titers and viral loads of SARS-CoV-2 in the lungs. The hamsters and ferrets co-infected by SARS-CoV-2- and IAV demonstrated more pronounced clinical manifestations than mono-infected animals. Additionally, the lung histological data illustrate that HAdV-5 or IAV and SARS-CoV-2 co-infection induces more severe pathological changes in the lungs than mono-infection. The expression of several genes specific to interferon and cytokine signaling pathways in the lungs of co-infected hamsters was more upregulated compared to single infected with SARS-CoV-2 animals. Thus, co-infection with HAdV-5 or IAV and SARS-CoV-2 leads to more severe pulmonary disease in animals.

The New General Biological Property of Stem-like Tumor Cells (Part II: Surface Molecules, Which Belongs to Distinctive Groups with Particular Functions, Form a Unique Pattern Characteristic of a Certain Type of Tumor Stem-like Cells).

An ability of poorly differentiated cells of different genesis, including tumor stem-like cells (TSCs), to internalize extracellular double-stranded DNA (dsDNA) fragments was revealed in our studies. Using the models of Krebs-2 murine ascites carcinoma and EBV-induced human B-cell lymphoma culture, we demonstrated that dsDNA internalization into the cell consists of several mechanistically distinct phases. The primary contact with cell membrane factors is determined by electrostatic interactions. Firm contacts with cell envelope proteins are then formed, followed by internalization into the cell of the complex formed between the factor and the dsDNA probe bound to it. The key binding sites were found to be the heparin-binding domains, which are constituents of various cell surface proteins of TSCs-either the C1q domain, the collagen-binding domain, or domains of positively charged amino acids. These results imply that the interaction between extracellular dsDNA fragments and the cell, as well as their internalization, took place with the involvement of glycocalyx components (proteoglycans/glycoproteins (PGs/GPs) and glycosylphosphatidylinositol-anchored proteins (GPI-APs)) and the system of scavenger receptors (SRs), which are characteristic of TSCs and form functional clusters of cell surface proteins in TSCs. The key provisions of the concept characterizing the principle of organization of the "group-specific" cell surface factors of TSCs of various geneses were formulated. These factors belong to three protein clusters: GPs/PGs, GIP-APs, and SRs. For TSCs of different tumors, these clusters were found to be represented by different members with homotypic functions corresponding to the general function of the cluster to which they belong.

The new general biological property of stem-like tumor cells Part I. Peculiarities of the process of the double-stranded DNA fragments internalization into stem-like tumor cells.

Stem-like tumor cells of ascites carcinoma Krebs-2 and Epstein-Barr virus-induced B-lymphoma were shown to possess the innate capability of binding and internalizing the TAMRA-labeled double-stranded DNA (dsDNA) probe. The process of binding and internalizing is rather complicated and composed of the following successive stages: 1) initiating electrostatic interaction and contact of a negatively charged dsDNA molecule with a positively charged molecule(s) on the surface of a stem-like tumor cell; 2) binding of the dsDNA probe to a tumor stem cell surface protein(s) via the formation of a strong chemical/molecular bond; and 3) the very internalization of dsDNA into the cell. Binding of DNA to cell surface proteins is determined by the presence of heparin/polyanion-binding sites within the protein structure, which can be competitively blocked by heparin and/or dextran sulfate, wherein heparin blocks only the binding, while dextran sulfate abrogates both binding and internalization. The abrogation of internalization by dextran sulfate implies the role of scavenger receptors in this process. Cells were shown to uptake DNA in amounts constituting ∼0.008% of the haploid genome. Inhibitors of caveolae-dependent internalization abrogate the DNA uptake in Krebs-2 cells, and inhibitors of the clathrin/caveolar mechanism block the internalization in B-lymphoma cells. In the present report, it is shown for the first time that in contrast to the majority of committed tumor cells, stem-like tumor cells of Krebs-2 and B-lymphoma carry a general positive charge on their surface.

Analysis of the Biological Properties of Blood Plasma Protein with GcMAF Functional Activity.

The main problem related to the studies focusing on group-specific component protein-derived macrophage-activating factor (GcMAF) is the lack of clarity about changes occurring in different types of macrophages and related changes in their properties under the effect of GcMAF in various clinical conditions. We analyzed the antitumor therapeutic properties of GcMAF in a Lewis carcinoma model in two clinical conditions: untreated tumor lesion and tumor resorption after exposure to Karanahan therapy. GcMAF is formed during site-specific deglycosylation of vitamin D3 binding protein (DBP). DBP was obtained from the blood of healthy donors using affinity chromatography on a column with covalently bound actin. GcMAF-related factor (GcMAF-RF) was converted in a mixture with induced lymphocytes through the cellular enzymatic pathway. The obtained GcMAF-RF activates murine peritoneal macrophages (p < 0.05), induces functional properties of dendritic cells (p < 0.05) and promotes in vitro polarization of human M0 macrophages to M1 macrophages (p < 0.01). Treatment of whole blood cells with GcMAF-RF results in active production of both pro- and anti-inflammatory cytokines. It is shown that macrophage activation by GcMAF-RF is inhibited by tumor-secreted factors. In order to identify the specific antitumor effect of GcMAF-RF-activated macrophages, an approach to primary reduction of humoral suppressor activity of the tumor using the Karanahan therapy followed by macrophage activation in the tumor-associated stroma (TAS) was proposed. A prominent additive effect of GcMAF-RF, which enhances the primary immune response activation by the Karanahan therapy, was shown in the model of murine Lewis carcinoma. Inhibition of the suppressive effect of TAS is the main condition required for the manifestation of the antitumor effect of GcMAF-RF. When properly applied in combination with any chemotherapy, significantly reducing the humoral immune response at the advanced tumor site, GcMAF-RF is a promising antitumor therapeutic agent that additively destroys the pro-tumor properties of macrophages of the tumor stroma.

Efficacy of the new therapeutic approach in curing malignant neoplasms on the model of human glioblastoma.

OBJECTIVE: Glioma is a highly invasive tumor, frequently disposed in essential areas of the brain, which makes its surgical excision extremely difficult; meanwhile adjuvant therapy remains quite ineffective. METHODS: In the current report, a new therapeutic approach in curing malignant neoplasms has been performed on the U87 human glioblastoma model. This approach, termed "Karanahan", is aimed at the eradication of cancer stem cells (CSCs), which were recently shown to be capable of internalizing fragments of extracellular double-stranded DNA. After being internalized, these fragments interfere in the process of repairing interstrand cross-links caused by exposure to appropriate cytostatics, and such an interference results either in elimination of CSCs or in the loss of their tumorigenic potency. Implementation of the approach requires a scheduled administration of cytostatic and complex composite double-stranded DNA preparation. RESULTS: U87 cells treated in vitro in accordance with the Karanahan approach completely lost their tumorigenicity and produced no grafts upon intracerebral transplantation into immunodeficient mice. In SCID mice with developed subcutaneous grafts, the treatment resulted in reliable slowing down of tumor growth rate (P < 0.05). In the experiment with intracerebral transplantation of U87 cells followed by surgical excision of the developed graft and subsequent therapeutic treatment, the Karanahan approach was shown to reliably slow down the tumor growth rate and increase the median survival of the mice twofold relative to the control. CONCLUSIONS: The effectiveness of the Karanahan approach has been demonstrated both in vitro and in vivo in treating developed subcutaneous grafts as well as orthotopic grafts after surgical excision of the tumor.

Characterization of biological peculiarities of the radioprotective activity of double-stranded RNA isolated from Saccharomyces сerevisiae.

THE PURPOSE OF THE ARTICLE: Protection from ionizing radiation is the most important component in the curing malignant neoplasms, servicing atomic reactors, and resolving the situations associated with uncontrolled radioactive pollutions. In this regard, discovering new effective radioprotectors as well as novel principles of protecting living organisms from high-dose radiation is the most important factor, determining the new approaches in medical and technical usage of radiation. MATERIALS AND METHODS: Experimental animals were irradiated on the γ-emitter (Cs137) with a dose of 9.4 Gy. Radioprotective properties of several agents (total RNA, single-stranded RNA, double-stranded RNA and B-190) were estimated by the survival/death rates of experimental animals within 30-90 d. Pathomorphological examination of internal organs end electron microscope assay was done on days 9-12 after irradiation. Cloning and other molecular procedures were performed accordingly to commonly accepted protocols. For assessment of the internalization of labeled nucleic acid, bone marrow cells were incubated with double-stranded RNA labeled with 6-FAM fluorescent dye. Cells with internalized double-stranded RNA were assayed using Axio Imager M1 microscope. In the other experiment, bone marrow cells after incubation with double-stranded RNA were stained with Cy5-labeled anti-CD34 antibodies and assayed using Axioskop 2 microscope. RESULTS: In this study, several biological features of the radioprotective action of double-stranded RNA are characterized. It was shown that 160 µg of the double-stranded RNA per mouse protect experimental animals from the absolutely lethal dose of γ-radiation of 9.4 Gy. In different experiments, 80-100% of irradiated animals survive and live until their natural death. Radioprotective properties of double-stranded RNA were found to be independent on its sequence, but strictly dependent on its double-stranded form. Moreover, double-stranded RNA must have 'open' ends of the molecule to exert its radioprotective activity. CONCLUSIONS: Experiments indicate that radioprotective effect of double-stranded RNA is tightly bound to its internalization into hematopoietic stem cells, which further repopulate the spleen parenchyma of irradiated mice. Actively proliferating progenitors form the splenic colonies, which further serve as the basis for restoration of hematopoiesis and immune function and determine the survival of animals received the lethal dose of radiation.

Approbation of the Cancer Treatment Approach Based on the Eradication of TAMRA+ Cancer Stem Cells in a Model of Murine Cyclophosphamide Resistant Lymphosarcoma.

BACKGROUND/AIM: We previously have described the "3+1" tumors cure approach consisting of individual time schedule of cyclophosphamide and dsDNA preparation administrations. The aim of the study was to adapt the "3+1" approach based on eradication of cancer stem cells to the model of murine ascitic cyclophosphamide-resistant lymphosarcoma (RLS). MATERIALS AND METHODS: Adaptation of the "3+1" approach includes the identification of the timing to disrupt the tumorigenic potential of a certain tumor. RESULTS: The proposed therapeutic scheme allowed complete reduction of primary RLS ascites in experimental animals. However, reduction of primary ascites due to the complementary action of cyclophosphamide and dsDNA was inevitably followed by the development of a secondary one, most likely arising from a solid carcinomatous formation in the peritoneal wall. CONCLUSION: The "3+1" approach resulted in the elimination of cancer stem cells, and, as a consequence, in the complete reduction of RLS ascites.

Oncolytic Effect of Adenoviruses Serotypes 5 and 6 Against U87 Glioblastoma Cancer Stem Cells.

BACKGROUND/AIM: Oncolytic adenoviruses are promising therapeutic agents against both the bulk of tumor cells and cancer stem cells. The present study intended to test the oncolytic capability of adenovirus serotype 6 (Ad6), which has a lower seroprevalence and hepatotoxicity relatively to adenovirus 5 (Ad5), against the glioblastoma and its cancer stem cells. MATERIALS AND METHODS: Oncolytic efficacy of Ad6 was compared to widespread Ad5 both in vitro and in vivo, using the U87 and U251 human glioblastoma cell lines and subcutaneously transplanted U87 cells in SCID mice, respectively. RESULTS: Ad6 had a dose-dependent cytotoxicity toward glioblastoma cells in vitro and its intratumoral injections lead to a significant (p<0.05) decrease in volume of U87 xenografts, similarly to Ad5. Based on the innate capability of glioblastoma cancer stem cells to internalize a fluorescent-labeled double-stranded DNA probe, the spatial localization of these cells was estimated and it was shown that the number of cancer stem cells tended to decrease under adenovirus therapy as compared to the control group. CONCLUSION: Ad6 was shown to be a promising agent for treating glioblastomas.

Features of monocyte-derived dendritic cells encompassing a rare subpopulation of cells that are capable of natural internalization of extracellular dsDNA.

The present study demonstrates that monocyte-derived dendritic cells (moDCs) produced in vitro using a GM-CSF and IFN-α differentiation protocol encompass a rare (∼5%) subpopulation of cells showing classical dendritic cell morphology and capable of natural internalization of extracellular self-DNA. We established that DEFB, HMGB1, LL-37 and RAGE antigens, which mediate the process of DNA internalization, are expressed on the surface of moDCs similar to plasmacytoid dendritic cells. However, in constrast to the latter subpopulation, these cells do not produce interleukin (IL)-37. Nonetheless, the process of DNA internalization was not in direct relation to the presence of the above antigens on the surface of these cells. Dendritic cells were sorted into total and non-DNA-internalizing populations and cytokine production was analyzed at 24-48 hours post-DNA treatment. We show that massive secretion of cytokines by dendritic cells is associated with the dsDNA-internalizing subpopulation. A total pool of IFN-moDCs secrete pro-inflammatory "first-wave" cytokines (IL-2, IL-6, IL-8, TNF-α) at both 24 and 48 hours time points. The anti-inflammatory cytokines IL-4 and IL-10 were found to be modestly induced, whereas GM-CSF, G-CSF, and IFN-γ production was strongly induced. Treatment of moDCs with dsDNA results in the up-regulated transcription of IFN-α, IFN-ß, IFN-γ, IL-8, IL-10, and VEGF by 6 hours. Combined dsDNA + chloroquine treatment has a synergistic effect on transcription of only one of the genes tested, with the pro-inflammatory cytokine IFN-ß displaying the strongest fold induction by 24 hours.

Identification of the xenograft and its ascendant sphere-forming cell line as belonging to EBV-induced lymphoma, and characterization of the status of sphere-forming cells.

BACKGROUND: We have characterized the human cell line arised from the Epstein-Barr virus (EBV) positive multiple myeloma aspirate subjected to the long-term cultivation. This cell line has acquired the ability to form free-floating spheres and to produce a xenograft upon transplantation into NOD/SCID mice. METHODS: Cells from both in vitro culture and developed xenografts were investigated with a number of analytical approaches, including pathomorphological analysis, FISH analysis, and analysis of the surface antigens and of the VDJ locus rearrangement. RESULTS: The obtained results, as well as the confirmed presence of EBV, testify that both biological systems are derived from B-cells, which, in turn, is a progeny of the EBV-transformed B-cellular clone that supplanted the primordial multiple myeloma cells. Next we assessed whether cells that (i) were constantly present in vitro in the investigated cell line, (ii) were among the sphere-forming cells, and (iii) were capable of internalizing a fluorescent TAMRA-labeled DNA probe (TAMRA+ cells) belonged to one of the three types of undifferentiated bone marrow cells of a multiple myeloma patient: CD34+ hematopoietic stem cells, CD90+ mesenchymal stem cells, and clonotypic multiple myeloma cell. CONCLUSION: TAMRA+ cells were shown to constitute the fourth independent subpopulation of undifferentiated bone marrow cells of the multiple myeloma patient. We have demonstrated the formation of ectopic contacts between TAMRA+ cells and cells of other types in culture, in particular with CD90+ mesenchymal stem cells, followed by the transfer of some TAMRA+ cell material into the contacted cell.

Ultrastructural analysis of the Krebs-2 ascites cancer cells treated with extracellular double-stranded DNA preparation.

Electron-microscopic analysis of the ultrastructure of the Krebs-2 carcinoma ascites cells in the first 90 min immediately after their exposure to fragmented double-stranded DNA has been performed. Morphological attributes of the treated cancer cells indicate the induction in these cells of destructive processes of presumably apoptotic type. The predominance of dystrophic-destructive changes in cells after the addition of DNA is supposed to be a consequence of the disturbance in metabolic processes caused by the experimental action.

Cancer Stem Cells: Emergent Nature of Tumor Emergency.

A functional analysis of 167 genes overexpressed in Krebs-2 tumor initiating cells was performed. In the first part of the study, the genes were analyzed for their belonging to one or more of the three groups, which represent the three major phenotypic manifestation of malignancy of cancer cells, namely (1) proliferative self-sufficiency, (2) invasive growth and metastasis, and (3) multiple drug resistance. 96 genes out of 167 were identified as possible contributors to at least one of these fundamental properties. It was also found that substantial part of these genes are also known as genes responsible for formation and/or maintenance of the stemness of normal pluri-/multipotent stem cells. These results suggest that the malignancy is simply the ability to maintain the stem cell specific genes expression profile, and, as a consequence, the stemness itself regardless of the controlling effect of stem niches. In the second part of the study, three stress factors combined into the single concept of "generalized cellular stress," which are assumed to activate the expression of these genes, were defined. In addition, possible mechanisms for such activation were identified. The data obtained suggest the existence of a mechanism for the de novo formation of a pluripotent/stem phenotype in the subpopulation of "committed" tumor cells.

Efficacy of a new cancer treatment strategy based on eradication of tumor-initiating stem cells in a mouse model of Krebs-2 solid adenocarcinoma.

Krebs-2 solid carcinoma was cured using a new "3+1" strategy for eradication of Krebs-2 tumor-initiating stem cells. This strategy was based on synchronization of these cells in a treatment-sensitive phase of the cell cycle. The synchronization mechanism, subsequent destruction of Krebs-2 tumor-initiating stem cells, and cure of mice from a solid graft were found to depend on the temporal profile of the interstrand cross-link repair cycle. Also, the temporal profile of the Krebs-2 interstrand repair cycle was found to have a pronounced seasonal cyclicity at the place of experiments (Novosibirsk, Russia). As a result, the therapeutic effect that is based on application of the described strategy, originally developed for the "winter repair cycle" (November-April), is completely eliminated in the summer period (June-September). We conclude that оne of the possible and the likeliest reasons for our failure to observe the therapeutic effects was the seasonal cyclicity in the duration of the interstrand repair cycle, the parameter that is central to our strategy.

Gene expression profiling of tumor-initiating stem cells from mouse Krebs-2 carcinoma using a novel marker of poorly differentiated cells.

Using the ability of poorly differentiated cells to natively internalize fragments of extracellular double-stranded DNA as a marker, we isolated a tumorigenic subpopulation present in Krebs-2 ascites that demonstrated the features of tumor-inducing cancer stem cells. Having combined TAMRA-labeled DNA probe and the power of RNA-seq technology, we identified a set of 168 genes specifically expressed in TAMRA-positive cells (tumor-initiating stem cells), these genes remaining silent in TAMRA-negative cancer cells. TAMRA+ cells displayed gene expression signatures characteristic of both stem cells and cancer cells. The observed expression differences between TAMRA+ and TAMRA- cells were validated by Real Time PCR. The results obtained corroborated the biological data that TAMRA+ murine Krebs-2 tumor cells are tumor-initiating stem cells. The approach developed can be applied to profile any poorly differentiated cell types that are capable of immanent internalization of double-stranded DNA.

A strategy to eradicate well-developed Krebs-2 ascites in mice.

We describe the strategy, which allows curing experimental mice engrafted with Krebs-2 ascites. The strategy is based on the facts that i) Krebs-2 tumor-initiating stem cells (TISCs) are naturally capable of internalizing fragments of extracellular double-stranded DNA (dsDNA); ii) upon delivery into TISCs, these dsDNA fragments interfere with the on-going DNA repair process so that TISCs either die or lose their tumorigenic potential. The following 3-step regimen of therapeutic procedures leading to eradication of Krebs-2 ascites is considered. Firstly, three timed injections of cyclophosphamide (CP) exactly matching the interstrand cross-link (ICL) repair phases that lead to synchronization of ascites cells in late S/G2/M. Secondly, additional treatment of ascites 18 hours post each CP injection (at NER/HR transition timepoint) with a composite dsDNA-based preparation interfering with the NER and HR repair pathways, so that tumorigenic properties of ascites cells are compromised. Thirdly, final treatment of mice with a combination of CP and dsDNA injections as ascites cells undergo apoptotic destruction, and the surviving TAMRA+ TISCs arrested in late S/G2/M phases massively enter into G1/S, when they regain sensitivity to CP+dsDNA treatment. Thus, this regimen assures that no viable cells, particularly Krebs-2 TISCs, remain.

Comparative analysis of pathologic processes developing in mice housed in SPF vs non-SPF conditions and treated with cyclophosphamide and dsDNA preparation.

In our earlier studies, we observed that when mice are treated with cyclophosphamide and fragmented exogenous dsDNA (18-30 h post cytostatic treatment), they develop a very characteristic set of symptoms and 80-90% of such animals succumb within 6-25 days. This was called "delayed death" phenomenon, and the gap between cyclophosphamide and DNA injections required for such phenotype to develop was termed "death window". We established that mice succumbed to multi-organ failure, which was caused by systemic inflammation and sepsis. These processes unfolded along with accidental involution of lymphoid organs, which resulted from the failure of CD34(+) hematopoietic stem cells to differentiate into lymphoid lineage progenitors. Here we compare SPF and non-SPF animals, and demonstrate that the major cause of systemic inflammation and sepsis observed upon such treatments is activation of an opportunistic infection. Mice of the same strain (CBA) housed under SPF conditions do not develop the characteristic symptoms, nor do they become moribund. Yet, regardless of the breeding conditions, upon synergistic action of cyclophosphamide and dsDNA, CD34(+) hematopoietic stem cells consistently fail to give rise to lymphoid lineage progenitors. We demonstrate that this differentiation defect is reversible and that population of lymphoid progenitors is restored by day 29 after cyclophosphamide injection.

Combination of cyclophosphamide and double-stranded DNA demonstrates synergistic toxicity against established xenografts.

BACKGROUND: Extracellular double-stranded DNA participates in various processes in an organism. Here we report the suppressive effects of fragmented human double-stranded DNA along or in combination with cyclophosphamide on solid and ascites grafts of mouse Krebs-2 tumor cells and DNA preparation on human breast adenocarcinoma cell line MCF-7. METHODS: Apoptosis and necrosis were assayed by electrophoretic analysis (DNA nucleosomal fragmentation) and by measurements of LDH levels in ascitic fluid, respectively. DNA internalization into MCF-7 was analyzed by flow cytometry and fluorescence microscopy. RESULTS: Direct cytotoxic activity of double-stranded DNA (along or in combination with cyclophosphamide) on a solid transplant was demonstrated. This resulted in delayed solid tumor proliferation and partial tumor lysis due to necrosis of the tumor and adjacent tissues. In the case of ascites form of tumor, extensive apoptosis and secondary necrosis were observed. Similarly, MCF-7 cells showed induction of massive apoptosis (up to 45%) as a result of treatments with double-stranded DNA preparation. CONCLUSIONS: Double-stranded DNA (along or in combination with cyclophosphamide) induces massive apoptosis of Krebs-2 ascite cells and MCF-7 cell line (DNA only). In treated mice it reduces the integrity of gut wall cells and contributes to the development of systemic inflammatory reaction.