In vivo Bruton's tyrosine kinase inhibition attenuates alcohol-associated liver disease by regulating CD84-mediated granulopoiesis.

Severe alcohol-associated hepatitis (AH) is a life-threatening form of alcohol-associated liver disease. Liver neutrophil infiltration is a hallmark of AH, yet the effects of alcohol on neutrophil functions remain elusive. Identifying therapeutic targets to reduce neutrophil-mediated liver damage is essential. Bruton's tyrosine kinase (BTK) plays an important role in neutrophil development and function; however, the role of BTK in AH is unknown. Using RNA sequencing of circulating neutrophils, we found an increase in Btk expression (P = 0.05) and phosphorylated BTK (pBTK) in patients with AH compared with healthy controls. In vitro, physiologically relevant doses of alcohol resulted in a rapid, TLR4-mediated induction of pBTK in neutrophils. In a preclinical model of AH, administration of a small-molecule BTK inhibitor (evobrutinib) or myeloid-specific Btk knockout decreased proinflammatory cytokines and attenuated neutrophil-mediated liver damage. We found that pBTK was essential for alcohol-induced bone marrow granulopoiesis and liver neutrophil infiltration. In vivo, BTK inhibition or myeloid-specific Btk knockout reduced granulopoiesis, circulating neutrophils, liver neutrophil infiltration, and liver damage in a mouse model of AH. Mechanistically, using liquid chromatography-tandem mass spectrometry, we identified CD84 as a kinase target of BTK, which is involved in granulopoiesis. In vitro, CD84 promoted alcohol-induced interleukin-1ß and tumor necrosis factor-α in primary human neutrophils, which was inhibited by CD84-blocking antibody treatment. Our findings define the role of BTK and CD84 in regulating neutrophil inflammation and granulopoiesis, with potential therapeutic implications in AH.

Kmt2c restricts G-CSF-driven HSC mobilization and granulocyte production in a methyltransferase-independent manner.

Granulocyte colony-stimulating factor (G-CSF) is widely used to enhance myeloid recovery after chemotherapy and to mobilize hematopoietic stem cells (HSCs) for transplantation. Unfortunately, through the course of chemotherapy, cancer patients can acquire leukemogenic mutations that cause therapy-related myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). This raises the question of whether therapeutic G-CSF might potentiate therapy-related MDS/AML by disproportionately stimulating mutant HSCs and other myeloid progenitors. A common mutation in therapy-related MDS/AML involves chromosome 7 deletions that inactivate many tumor suppressor genes, including KMT2C. Here, we show that Kmt2c deletions hypersensitize murine HSCs and myeloid progenitors to G-CSF, as evidenced by increased HSC mobilization and enhanced granulocyte production from granulocyte-monocyte progenitors (GMPs). Furthermore, Kmt2c attenuates the G-CSF response independently from its SET methyltransferase function. Altogether, the data raise concerns that monosomy 7 can hypersensitize progenitors to G-CSF, such that clinical use of G-CSF may amplify the risk of therapy-related MDS/AML.

The Syk Inhibitor Entospletinib Abolishes Dermal-Epidermal Separation in a Fully Human Ex Vivo Model of Bullous Pemphigoid.

Bullous pemphigoid (BP) is an autoantibody-mediated blistering skin disease characterized by local inflammation and dermal-epidermal separation, with no approved targeted therapy. The Syk tyrosine kinase is critical for various functions of the immune response. Second-generation Syk inhibitors such as entospletinib are currently being tested for hematological malignancies. Our aim was to test the effect of entospletinib in a fully human model system of BP. Incubating BP serum-treated human frozen skin sections with normal human granulocytes and fresh plasma triggered dermal-epidermal separation that was dependent on complement, NADPH oxidase, and protease activity. Entospletinib dramatically reduced dermal-epidermal separation with a half-maximal inhibitory concentration of ≈16 nM. Entospletinib also reduced ROS production, granule release, and spreading of human granulocytes plated on immobilized immune complexes consisting either of a generic antigen-antibody pair or of recombinant collagen type XVII (BPAg2) and BP serum components (supposedly autoantibodies). However, entospletinib did not affect the chemotactic migration of human granulocytes or their responses to nonphysiological stimulation by phorbol esters. Entospletinib had no effect on the survival of granulocytes either. Taken together, entospletinib abrogates dermal-epidermal separation, likely through inhibition of granulocyte responsiveness to deposited immune complexes. Entospletinib or other Syk inhibitors may provide therapeutic benefits in BP.

Molecular and functional changes in neutrophilic granulocytes induced by nicotine: a systematic review and critical evaluation.

Background: Over 1.1 billion people smoke worldwide. The alkaloid nicotine is a prominent and addictive component of tobacco. In addition to tumors and cardiovascular disorders, tobacco consumption is associated with a variety of chronic-inflammatory diseases. Although neutrophilic granulocytes (neutrophils) play a role in the pathogenesis of many of these diseases, the impact of nicotine on neutrophils has not been systematically reviewed so far. Objectives: The aim of this systematic review was to evaluate the direct influence of nicotine on human neutrophil functions, specifically on cell death/damage, apoptosis, chemotaxis, general motility, adhesion molecule expression, eicosanoid synthesis, cytokine/chemokine expression, formation of neutrophil extracellular traps (NETs), phagocytosis, generation of reactive oxygen species (ROS), net antimicrobial activity, and enzyme release. Material and methods: This review was conducted according to the PRISMA guidelines. A literature search was performed in the databases NCBI Pubmed® and Web of Science™ in February 2023. Inclusion criteria comprised English written research articles, showing in vitro studies on the direct impact of nicotine on specified human neutrophil functions. Results: Of the 532 originally identified articles, data from 34 articles were finally compiled after several evaluation steps. The considered studies highly varied in methodological aspects. While at high concentrations (>3 mmol/l) nicotine started to be cytotoxic to neutrophils, concentrations typically achieved in blood of smokers (in the nmol/l range) applied for long exposure times (24-72h) supported the survival of neutrophils. Smoking-relevant nicotine concentrations also increased the chemotaxis of neutrophils towards several chemoattractants, elevated their production of elastase, lipocalin-2, CXCL8, leukotriene B4 and prostaglandin E2, and reduced their integrin expression. Moreover, while nicotine impaired the neutrophil phagocytotic and anti-microbial activity, a range of studies demonstrated increased NET formation. However, conflicting effects were found on ROS generation, selectin expression and release of ß-glucuronidase and myeloperoxidase. Conclusion: Nicotine seems to support the presence in the tissue and the inflammatory and selected tissue-damaging activity of neutrophils and reduces their antimicrobial functions, suggesting a direct contribution of nicotine to the pathogenesis of chronic-inflammatory diseases via influencing the neutrophil biology.

Targeting CAMKK2 and SOC Channels as a Novel Therapeutic Approach for Sensitizing Acute Promyelocytic Leukemia Cells to All-Trans Retinoic Acid.

Calcium ions (Ca2+) play important and diverse roles in the regulation of autophagy, cell death and differentiation. Here, we investigated the impact of Ca2+ in regulating acute promyelocytic leukemia (APL) cell fate in response to the anti-cancer agent all-trans retinoic acid (ATRA). We observed that ATRA promotes calcium entry through store-operated calcium (SOC) channels into acute promyelocytic leukemia (APL) cells. This response is associated with changes in the expression profiles of ORAI1 and STIM1, two proteins involved in SOC channels activation, as well as with a significant upregulation of several key proteins associated to calcium signaling. Moreover, ATRA treatment of APL cells led to a significant activation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and its downstream effector AMP-activated protein kinase (AMPK), linking Ca2+ signaling to autophagy. Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell differentiation and death. Altogether, our results unravel an ATRA-elicited signaling pathway that involves SOC channels/CAMKK2 activation, induction of autophagy, inhibition of cellular differentiation and suppression of cell death. We suggest that SOC channels and CAMKK2 may constitute novel drug targets for potentiating the anti-cancer effect of ATRA in APL patients.

Selective Biological Effects of Selenium-Enriched Polysaccharide (Se-Le-30) Isolated from Lentinula edodes Mycelium on Human Immune Cells.

A common edible mushroom Lentinula edodes, is an important source of numerous biologically active substances, including polysaccharides, with immunomodulatory and antitumor properties. In the present work, the biological activity of the crude, homogenous (Se)-enriched fraction (named Se-Le-30), which has been isolated from L. edodes mycelium by a modified Chihara method towards human peripheral blood mononuclear cells (PBMCs) and peripheral granulocytes, was investigated. The Se-Le-30 fraction, an analog of lentinan, significantly inhibited the proliferation of human PBMCs stimulated with anti-CD3 antibodies or allostimulated, and down-regulated the production of tumor necrosis factor (TNF)-α by CD3+ T cells. Moreover, it was found that Se-Le-30 significantly reduced the cytotoxic activity of human natural killer (NK) cells. The results suggested the selective immunosuppressive activity of this fraction, which is non-typical for mushroom derived polysaccharides.

Immunosuppressive effects and mechanisms of three myeloid-derived suppressor cells subsets including monocytic-myeloid-derived suppressor cells, granulocytic-myeloid-derived suppressor cells, and immature-myeloid-derived suppressor cells.

CONTEXT: Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immune cells of myeloid lineage. Recent reports have suggested that human MDSC are divided into three subsets: monocytic MDSC (M-MDSC), granulocytic MDSC (G-MDSC), and immature MDSC (I-MDSC). However, the characteristics of each human MDSC subset still remain unclear. MATERIALS AND METHODS: To evaluate the immunosuppressive effects and mechanisms, we first performed a T-cell suppression assay using cells obtained from healthy donor peripheral blood samples. The levels of immune inhibitory molecules in the culture supernatant of each MDSC subset were measured to reveal the T-cell suppressive mechanisms. Then, we compared these results with the results from cells obtained from cancer patient blood samples. Finally, we investigated the difference in the frequency of each MDSC subset between the healthy donors and the cancer patients. RESULTS: Although M-MDSC and G-MDSC suppressed T-cell activation, I-MDSC had no T-cell suppressive effect. We found that the culture supernatant of M-MDSC and G-MDSC contained high levels of interleukin-1 receptor antagonist (IL-1RA) and arginase, respectively, in both healthy donors and cancer patients. No inhibitory molecules were detected in the culture supernatant of I-MDSC. The population of functional MDSC (M-MDSC and G-MDSC) in the total MDSC was significantly increased in cancer patients compared with that in healthy donors. CONCLUSIONS: Although M-MDSC and G-MDSC, which released IL-1RA and arginase, respectively, suppressed T-cell activation, I-MDSC did not have an immunosuppressive effect. The population of functional MDSC was increased in cancer patients compared with that in healthy donors.

Inhibition of the PI3K/mTOR Pathway in Breast Cancer to Enhance Response to Immune Checkpoint Inhibitors in Breast Cancer.

OBJECTIVES: Inhibition of the PI3K/mTOR pathway suppresses breast cancer (BC) growth, enhances anti-tumor immune responses, and works synergistically with immune checkpoint inhibitors (ICI). The objective here was to identify a subclass of PI3K inhibitors that, when combined with paclitaxel, is effective in enhancing response to ICI. METHODS: C57BL/6 mice were orthotopically implanted with syngeneic luminal/triple-negative-like PyMT cells exhibiting high endogenous PI3K activity. Tumor growth in response to treatment with anti-PD-1 + anti-CTLA-4 (ICI), paclitaxel (PTX), and either the PI3Kα-specific inhibitor alpelisib, the pan-PI3K inhibitor copanlisib, or the broad spectrum PI3K/mTOR inhibitor gedatolisib was evaluated in reference to monotherapy or combinations of these therapies. Effects of these therapeutics on intratumoral immune populations were determined by multicolor FACS. RESULTS: Treatment with alpelisib + PTX inhibited PyMT tumor growth and increased tumor-infiltrating granulocytes but did not significantly affect the number of tumor-infiltrating CD8+ T cells and did not synergize with ICI. Copanlisib + PTX + ICI significantly inhibited PyMT growth and increased activation of intratumoral CD8+ T cells as compared to ICI alone, yet did not inhibit tumor growth more than ICI alone. In contrast, gedatolisib + ICI resulted in significantly greater inhibition of tumor growth compared to ICI alone and induced durable dendritic-cell, CD8+ T-cell, and NK-cell responses. Adding PTX to this regimen yielded complete regression in 60% of tumors. CONCLUSION: PI3K/mTOR inhibition plus PTX heightens response to ICI and may provide a viable therapeutic approach for treatment of metastatic BC.

Hematopoietic stem progenitor cells lacking HLA differ from those lacking GPI-anchored proteins in the hierarchical stage and sensitivity to immune attack in patients with acquired aplastic anemia.

To characterize glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) and HLA-class I allele-lacking (HLA[-]) hematopoietic stem progenitor cells (HSPCs) in acquired aplastic anemia (AA), we studied the peripheral blood (PB) of 56 AA patients in remission who possessed both (n = 13, Group A) or either GPI(-) (n = 34, Group B) and HLA(-) (n = 9, Group C) cell populations. Seventy-seven percent (10/13) of Group A had HLA(-) cells in all lineages of PB cells, including platelets, while only 23% (3/13) had GPI(-) cells in all lineages, and the median percentage of HLA(-) granulocytes in the total granulocytes (21.2%) was significantly higher than that of GPI(-) granulocytes (0.28%, P < 0.05). The greater lineage diversity in HLA(-) cells than in GPI(-) cells was also seen when Group B and Group C were compared. Longitudinal studies of seven patients in Group A showed a gradual decrease in the percentage of HLA(-) granulocytes, with a reciprocal increase in the GPI(-) granulocytes in four patients responding to cyclosporine (CsA) and an increase in the HLA(-) granulocytes with a stable or declining GPI(-) granulocytes in three patients in sustained remission off CsA therapy. These findings suggest that HLA(-) HSPCs differ from GPI(-) HSPCs in the hierarchical stage and sensitivity to immune attack in AA.

Ze-Qi-Tang Formula Induces Granulocytic Myeloid-Derived Suppressor Cell Apoptosis via STAT3/S100A9/Bcl-2/Caspase-3 Signaling to Prolong the Survival of Mice with Orthotopic Lung Cancer.

Non-small-cell lung cancer (NSCLC) remains the most common malignancy with the highest morbidity and mortality worldwide. In our previous study, we found that a classic traditional Chinese medicine (TCM) formula Ze-Qi-Tang (ZQT), which has been used in the treatment of respiratory diseases for thousands of years, could directly inhibit the growth of human NSCLC cells via the p53 signaling pathway. In this study, we explored the immunomodulatory functions of ZQT. We found that ZQT significantly prolonged the survival of orthotopic lung cancer model mice by modulating the tumor microenvironment (TME). ZQT remarkably reduced the number of MDSCs (especially G-MDSCs) and inhibited their immunosuppressive activity by inducing apoptosis in these cells via the STAT3/S100A9/Bcl-2/caspase-3 signaling pathway. When G-MDSCs were depleted, the survival promotion effect of ZQT and its inhibitory effect on lung luminescence signal disappeared in tumor-bearing mice. This is the first study to illustrate the immunomodulatory effect of ZQT in NSCLC and the underlying molecular mechanism.

Calcium activity in response to nAChR ligands in murine bone marrow granulocytes with different Gr-1 expression.

Polymorphonuclear neutrophilic granulocytes (PMNs) are the largest proportion of leukocytes in adult human blood that perform numerous functions, including phagocytosis, degranulation, generation of reactive oxygen species, and NETosis. Excessive neutrophil activity associates with hyperinflammation and tissue damage during pathologies such as inflammatory bowel disease, diabetes mellitus, tuberculosis, and coronavirus disease 2019. Nicotinic acetylcholine receptors (nAChRs) can modulate immune cells, including neutrophils, functions, therefore, nAChR ligands are considered as the potent agents for therapy of inflammation. Earlier it was shown, that about 30% of PMNs from the acute inflammatory site responded to nicotine by calcium spikes. In this study, we studied the generation of calcium spikes in murine granulocytes with different maturity level (evaluated by Gr-1 expression) isolated from bone marrow in response to ligands of nAChRs in control and under chronic nicotine consumption. It was found that nearly 20%-25% cells in the granulocyte population responded to nicotine or selective antagonists of different type of nAChRs (α-cobratoxin, GIC, and Vc1.1). We demonstrated that in the control group Ca2+ -mobilizing activity was regulated through α7 and α9α10 nAChRs in immature granulocytes (Gr-1int ), whereas in mature granulocytes (Gr-1hi ) it was regulated through α7, α3ß2, and α9-contained nAChRs. Sensitivity of PMNs to nicotine depended on their maturity level after chronic nicotine consumption. Gr-1int cells responded to nicotine through α7 and α9-contained nAChRs, while Gr-1hi did not respond to nicotine. Thus, calcium response to nAChR ligands in bone marrow PMNs depends on their maturity level.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig-a mutation and loss of GPI anchors was further strengthened and the genesis of Pig-a mutation in PCZ-dosed rats was evaluated by analyzing bone-marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48-deficient mutant phenotype bone-marrow granulocytes (BMGs [CD11b+ ]) were isolated by flow-cytometric sorting. Sequencing data showed that the CD48-deficient mutant phenotype BMGs contained mutations in the Pig-a gene while wild-type BMGs did not. PCZ-induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ-induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig-a assay and mutation in the Pig-a gene. Also, PCZ mutation spectra were similar in bone-marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig-a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.

Effect of Perfluorooctanesulfonic acid (PFOS) on immune cell development and function in mice.

Perfluoroctanesulfonate (PFOS) belongs to a larger family of compounds known as Per- and polyfluoroalkyl substances (PFAS). The strength of the carbon-fluorine bond makes PFOS extremely resistant to environmental degradation. Due to its persistent nature, research has been directed to elucidating possible health effects of PFOS on humans and laboratory animals. Here we have explored the effects of PFOS exposure on immune development and function in mice. We exposed adult mice to 3 and 1.5 µg/kg/day of PFOS for 2 and 4 weeks, respectively, and examined the effects of PFOS exposure on populations of T cells, B cells, and granulocytes. These doses of PFOS resulted in serum levels of approximately 100 ng/mL with no weight loss during exposure. We find that PFOS does not affect T-cell development during this time. However, while PFOS exposure reduced immune cell populations in some organs, it also led to an increase in the numbers of cells in others, suggesting possible relocalization of cells. We also examined the effect of PFOS on the response to influenza virus infection. We find that exposure to PFOS at 1.5 µg/kg/day of PFOS for 4 weeks does not affect weight loss or survival, nor is viral clearance affected. Analysis of antibody and T cell specific antiviral responses indicate that at this concentration, PFOS does not suppress the immune cell development or antigen specific immune response.

HDN-1 induces cell differentiation toward apoptosis in promyelocytic leukemia cells depending on its selective effect on client proteins of Hsp90.

Heat Shock Protein 90 (Hsp90) is frequently upregulated in many cancers, and its inhibition simultaneously blocks multiple signaling pathways, resulting in cell differentiation or apoptosis. However, the complexity of Hsp90 in differentiation and its relation with apoptosis have remained unsettled. In this study, we demonstrated that HDN-1, a C-terminal inhibitor of Hsp90, induced the differentiation of HL-60 cells toward apoptosis. HDN-1 induced the differentiation of cells containing mutant AML1-ETO into mature granulocytes, which was related to its selective effect on client proteins of Hsp90. HDN-1 destabilized AML1-ETO and preserved C/EBPß at the same time, thereby induced a total increase in C/EBPß levels because of AML1-ETO negative regulation to C/EBPß expression. Neither HDN-1 nor 17-AAG (an N-terminal inhibitor of Hsp90) led to the differentiation of NB4 cells because mutant PML-RARα was not affected as a client protein of Hsp90; thus, no additional expression of C/EBPß was induced. 17-AAG did not affect the differentiation of HL-60 cells due to decreased AML1-ETO and C/EBPß levels. These results indicate that HDN-1 drives cell differentiation toward apoptosis depending on its selective influence on client proteins of Hsp90, establishing the relationship between differentiation and apoptosis and uncovering the mechanism of HDN-1 in promyelocytic leukemia cell differentiation. Moreover, HDN-1 is very promising for the development of anticancer agents with the induction of differentiation.

Pre-clinical evaluation of an innovative oral nano-formulation of baicalein for modulation of radiation responses.

There is an unmet medical need for non-toxic and effective radiation countermeasures for prevention of radiation toxicity during planned exposures. We have earlier shown that intraperitoneal administration of baicalein (BCL) offers significant survival benefit in animal model. Safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of baicalein has been reported in pre-clinical model systems and also in healthy human volunteers. However, clinical translation of baicalein is hindered owing to poor bioavailability due to lipophilicity. In view of this, we fabricated and characterized in-situ solid lipid nanoparticles of baicalein (SLNB) with effective drug entrapment and release kinetics. SLNB offered significant protection to murine splenic lymphocytes against 4 Gy ionizing radiation (IR) induced apoptosis. Oral administration of SLNB exhibited ~70% protection to mice against whole body irradiation (WBI 7.5 Gy) induced mortality. Oral relative bioavailability of BCL was enhanced by over ~300% after entrapment in the SLNB as compared to BCL. Oral dosing of SLNB resulted in transient increase in neutrophil abundance in peripheral blood. Interestingly, we observed that treatment of human lung cancer cells (A549) with radioprotective dose of SLNB exhibited radio-sensitization as evinced by decrease in survival and clonogenic potential. Contrary to antioxidant nature of baicalein in normal cells, SLNB treatment induced significant increase in cellular ROS levels in A549 cells probably due to higher uptake and inhibition of TrxR. Thus, a pharmaceutically acceptable SLNB exhibited improved bioavailability, better radioprotection to normal cells and sensitized cancer cells to radiation induced killing as compared to BCL suggesting its possible utility as an adjuvant during cancer radiotherapy.

EFFICIENCY OF BONE MARROW PRECURSOR CELL COLONY-FORMING AS A PREDICTOR OF DISEASE COURSE IN PLASMA CELL MYELOMA PATIENTS WITH A HISTORY OF RADIATION EXPOSURE. / EFEKTYVNIST' KOLONIIeUTVORENNIa KLITYN-POPEREDNYKIV KISTKOVOGO MOZKU, IaK PROGNOSTYChNYI FAKTOR PEREBIGU PLAZMOKLITYNNOÏ MIIeLOMY U OSIB Z RADIATsIINYM ANAMNEZOM.

OBJECTIVE: Assessment of role of the bone marrow colony-forming efficiency in plasma cell myeloma patients at different stages of treatment as a prognostic criterion for the disease course. MATERIALS AND METHODS: The colony forming efficiency (CFE) was assayed in stage I-II plasma cell myeloma (PCM)patients (n = 37) aged 42-73, namely in patients survived after the Chornobyl NPP accident (n = 21) and persons notexposed to ionizing radiation (n = 16). There were 11 males exposed to ionizing radiation and having got stage I PCM,9 males and 3 females exposed and having got stage II PCM, 3 males and 3 females not exposed and having got stageI PCM, 6 males and 2 females not exposed and having got stage II PCM. Healthy persons (n = 20) were included in thecontrol group. RESULTS: Number of the bone marrow (BM) granulocyte-macrophage colony-forming units (CFU-GM) in both exposedand not exposed PCM patients depended on a disease stage. CFU-GM was (16.7 ± 1.2) in the stage I PCM patients vs.(11.1 ± 1.1) in the stage II PCM ones both being lower (p < 0.05) compared to control (64.5 ± 2.2). Changes in cluster formation were similar, i.e. (37.7 ± 1.6) and (19.4 ± 1.3) correspondingly in the stage I and stage II PCM patients.Respective values in control were (89.8 ± 3.6). The CFE in stage I and stage II PCM patients at the time of diagnosiswas lower (5.7 ± 1.5 and 2.4 ± 1.1 respectively) vs. control (39.5 ± 1.51, p < 0.05), but has increased in remission upto (29. 6 ± 1.8) and (13.8 ± 1.2) respectively. There was no difference at that between the irradiated and non-irradiated patients. Number of the fibroblast colony-forming units (CFU-F) in the stage I and stage II PCM patients duringdiagnosis, namely (43.9 ± 5.4) and (22.5 ± 3.7), was lower (p < 0.05) vs. control (110.5 ± 4.9). Upon reaching remission the CFU-F value increased significantly (p < 0.05), reaching (87.4 ± 4.2) and (55.6 ± 2.7) correspondingly in thestage I and stage II PCM patients. CONCLUSION: Dependence of the BM cell CFE on the stage of PCM and presence or absence of remission was established. Prognostic value of the CFE of BM CFU-GM in terms of life span of patients was shown (Ro Spearm = 0.39,p < 0.02), namely in case of CFE > 20 before the polychemotherapy administration the life span of PCM patients wassignificantly longer vs. cases of CFE < 20.

Design of novel granulopoietic proteins by topological rescaffolding.

Computational protein design is rapidly becoming more powerful, and improving the accuracy of computational methods would greatly streamline protein engineering by eliminating the need for empirical optimization in the laboratory. In this work, we set out to design novel granulopoietic agents using a rescaffolding strategy with the goal of achieving simpler and more stable proteins. All of the 4 experimentally tested designs were folded, monomeric, and stable, while the 2 determined structures agreed with the design models within less than 2.5 Å. Despite the lack of significant topological or sequence similarity to their natural granulopoietic counterpart, 2 designs bound to the granulocyte colony-stimulating factor (G-CSF) receptor and exhibited potent, but delayed, in vitro proliferative activity in a G-CSF-dependent cell line. Interestingly, the designs also induced proliferation and differentiation of primary human hematopoietic stem cells into mature granulocytes, highlighting the utility of our approach to develop highly active therapeutic leads purely based on computational design.

Intratumoral Activation of 41BB Costimulatory Signals Enhances CD8 T Cell Expansion and Modulates Tumor-Infiltrating Myeloid Cells.

The activation of 41BB costimulatory signals by agonistic Abs enhances the expansion and function of tumor-infiltrating lymphocytes (TILs) for treating cancer patients with adoptive cell therapy. However, the impact of 41BB agonism is not limited to enhancing the activity of T cells, and the mechanism by which additional activation of this costimulatory axis in tumor-associated myeloid cells is poorly understood. In this study, we describe that the intratumoral administration of 41BB agonistic Abs led to increases in CD8 T cell infiltration followed by tumor regression in murine models. We found that granulocytes and monocytes rapidly replaced macrophages and dendritic cells in tumors following administration of anti-41BB Abs. Overall, myeloid cells from anti-41BB-treated tumors had an improved capacity to stimulate T cells in comparison with control-treated tumors. In human coculture systems, we demonstrated that the agonism of the 41BB-41BBL axis enhanced costimulatory signals and effector functions among APC and autologous TILs. Overall, these findings suggest that the effect of 41BB agonistic Abs are supported by additional costimulatory signals from tumor-associated myeloid cells,v leading to enhanced TIL expansion and function.

Involvement of Signaling Cascades in Granulocytopoiesis Regulation under Conditions of Cytostatic Treatment.

Suppression of the production of granulocytic CSF under the effect of 5-fluorouracyl is related to disorders in the NF-κB-, cAMP-dependent signaling pathways and MAPK cascade. These secondary messengers are involved in the regulation of functional activity of nonadherent myelokaryocytes starting from day 10 of the experiment (initial period of the hemopoietic granulocytic stem regeneration after antimetabolite challenge). Granulocytic CSF does not play essential role in the formation of colony-stimulating activity of cells of the adherent and nonadherent fractions of the bone marrow. Only cAMP-dependent pathway is involved in the regulation of the realization of the granulocytic precursor growth potential in response to the challenge.

Hemostimulating Effects of c-Jun N-Terminal Kinase (JNK) Inhibitor during Cytostatic Myelosuppression and Mechanisms of Their Development.

The hemostimulating effects of c-Jun N-terminal kinase (JNK) inhibitor were examined on the mouse model of myelosuppression provoked by 5-fluorouracil. Blockade of JNK during postcytostatic period accelerated recovery of granulomonocytopoiesis and erythropoiesis. It also increased the content of neutrophilic granulocytes and erythroid cells in the hematopoietic tissue and elevated the counts of neutrophils and reticulocytes in the peripheral blood. The development of these phenomena resulted from elevated content and up-regulated functional activity of bone marrow hematopoietic progenitors associated with the direct action of JNK inhibitor on these progenitors and enhanced secretion of hemopoietins by stromal elements of the hematopoiesis-inducing microenvironment.