Differentiation Of Bone Marrow Cells Research Articles

Influence of estradiol treatment on bone marrow cell differentiation in collagenase-induced arthritis.

Estrogen is one of the important regulators of the balance between bone formation and bone resorption that can modulate the levels and activity of certain growth factors and cytokines. In this study, we investigated the effect of 17β-estradiol (ED) on bone marrow (BM) cell differentiation in vivo and ex vivo in a mouse model of collagenase-induced osteoarthritis (CIOA). ICR (CD-2) female mice were used in present experiments (total number = 75) and bone marrow cells were used for in vitro studies. Mice were orally fed under different schemes with 17β-estradiol at a dose of 2μg or 4μg for 30days. The effect of estradiol was estimated by histopathological, flow cytometry, and ELISA assays. Statistical differences were determined by one-way ANOVA. Estradiol treatment ameliorated cartilage destruction and osteophyte formation if started from day 0 of CIOA induction, attended with a decrease of uterine and ovarian weights. Long time treatment lowered the percentage of megakaryocyte/platelet (CD62P+) populations and osteoclast (RANK+) populations in BM. Cells obtained from estradiol-treated CIOA mice showed inhibited capacity to differentiate into RANK+ and mesenchymal cells under osteoclastogenic conditions in vitro. Estrogen decreased serum IL-6 levels. Results indicate a potential protective role for estrogen against the development of OA.

Hypofractionated Irradiation Suppressed the Off-Target Mouse Hepatocarcinoma Growth by Inhibiting Myeloid-Derived Suppressor Cell-Mediated Immune Suppression.

Background: Stereotactic radiotherapy treats hepatocellular carcinoma (HCC) at different stages effectively and safely. Besides its direct killing of cancer cells, radiotherapy stimulates host immunity against hepatoma. However, the role of myeloid-derived suppressor cells (MDSCs) in on-target and off-target anti-HCC effects induced by hypofractionated irradiation (IR) is unclear.Methods and Materials: Hepa1-6 and H22 allogeneic transplanted tumors on hind limbs of C57BL/6 and Institute of Cancer Research (ICR) mice, respectively, were irradiated with 0, 2.5, 4, 6, or 8 Gy/fraction until the total dose reached 40 Gy. The off-target effect induced by the IR was investigated by subsequently inoculating the same HCC cells subcutaneously on the abdomen. MDSCs in peripheral blood and tumor tissues were measured by flow cytometry or immunofluorescence microscopy analysis. IL-6, regulated on activation normal T cell expressed and secreted (RANTES), and granulocyte colony-stimulating factor (G-CSF) in irradiated mouse plasma and hepatoma cell cultures were measured with ELISA kits. Conditioned media (CM) from irradiated HCC cell cultures on bone marrow cell differentiation and MDSC proliferation were examined by co-culture and flow cytometry.Results: Our study showed that the IR of primarily inoculated HCC on hind limbs created an “in situ tumor vaccine” and triggered the antitumor immunity. The immunity was capable of suppressing the growth of the same type of HCC subcutaneously implanted on the abdomen, accompanied with reduced MDSCs in both blood and tumors. The decreased MDSCs were associated with low plasma levels of IL-6, RANTES, and G-CSF. The cytokines IL-6 and RANTES in the CM were lower in the high single IR dose group than in the control groups, but G-CSF was higher. The CM from high single-dose IR-Hepa1-6 cell culture reduced the differentiation of C57BL/6 mouse bone marrow cells into MDSCs, whereas CM from high single-dose IR-H22 cells reduced the proliferation of MDSCs, which might be due to the decreased p-STAT3 in bone marrow cells.Conclusions: The hypofractionated IR on transplanted tumors at the primary location exerted a strong antitumor effect on the same tumor at a different location (off target). This abscopal effect is most likely through the reduction of MDSCs and decrease of IL-6, RANTES, and G-CSF.

Prolongation of rat-to-mouse islets xenograft survival by co-transplantation of autologous IL-10 differentiated murine tolerogenic dendritic cells.

Tolerogenic dendritic cells (DCs) represent a promising approach to promote transplantation tolerance. In this study, the potential of autologous bone marrow (BM)-derived murine DC to protect rat-to-mouse islets xenografts was analyzed. Tolerogenic DCs were generated by differentiating BM cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin 10 (IL-10, IL-10 DC). The phenotype of IL-10 DC was characterized in vitro by expression of costimulatory/inhibitory molecules (flow cytometry) and cytokines (Luminex and ELISA), their function by phagocytosis and T-cell stimulation assays. To study transplant tolerance in vivo, rat islets were transplanted alone or in combination with autologous murine IL-10 DC under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. Xenograft survival was evaluated by monitoring glycemia, cellular infiltration of xenografts by microscopy and flow cytometry 10days post-transplantation. Compared with control DC, IL-10 DC exhibited lower levels of major histocompatibility complex class II, costimulatory molecules (CD40, CD86, CD205), lower production of pro-inflammatory cytokines (IL-12p70, TNF, IL-6), and higher production of IL-10. Phagocytosis of xenogeneic rat splenocytes was not impaired in IL-10 DC, whereas stimulation of T-cell proliferation was reduced in the presence of IL-10 DC. Xenograft survival of rat islets in diabetic mice co-transplanted with autologous murine IL-10 DC was significantly prolonged from 12 to 21days, without additional immunosuppressive treatment. Overall, infiltration of xenografts by T cells and myeloid cells was not different in IL-10 DC recipient mice, but enriched for CD8+ T cells and myeloid cells with suppressor-associated phenotype. Autologous IL-10-differentiated DC with tolerogenic properties prolong rat-to-mouse islets xenograft survival, potentially by locally inducing immune regulatory cells, indicating their potential for regulatory immune cell therapy in xenotransplantation.

Tumor Microenvironment following Gemcitabine Treatment Favors Differentiation of Immunosuppressive Ly6Chigh Myeloid Cells.

Regulation of myeloid-derived suppressor cells (MDSC) by ongoing inflammation following repeated chemotherapy remain elusive. In this study, we show that a multidose clinical regimen of gemcitabine (GEM) treatment enhances the immunosuppressive function of monocytic MDSC (M-MDSC), although tumor development is delayed in E0771 tumor-bearing mice. Accordingly, effector IFN-γ-producing CD4 and CD8 T cells are significantly decreased in the tumor microenvironment (TME) of GEM-treated mice. The conditioned medium of GEM-treated tumor cells enhances differentiation of mouse bone marrow cells and human PBMC into immunosuppressive M-MDSC. Cytokine profiling of GEM-treated tumor cells identifies GM-CSF as one of the most differentially expressed cytokines. Blockade or knockdown of GM-CSF can partially reduce immunosuppression of Ly6Chigh cells induced by GEM-conditioned medium. Knockdown of GM-CSF in tumor cells also delays tumor progression with decreased accumulation of M-MDSC in the TME. Mechanistically, enhanced production of reactive oxygen species and activation of NF-κB are observed in GEM-treated tumor cells. Treatment with the mitochondrial-targeted antioxidant and inhibitor of NF-κB signaling can abrogate GEM-induced hyperexpression of GM-CSF in E0771 cells. In addition, the phagocytic clearance of apoptotic tumor cells (efferocytosis) enhances the immunosuppressive function of bone marrow Ly6Chigh myeloid cells. Further, GEM treatment results in metabolic changes in residual tumor cells, leading to the resistance to T cell-mediated killing. Together, our results define an undesired effect of repeated GEM treatment promoting immunosuppression in TME via upregulation of GM-CSF and efferocytosis as well as deregulation of lipid metabolism in residual tumor cells.

The Coumarin Derivative 5'-Hydroxy Auraptene Suppresses Osteoclast Differentiation via Inhibiting MAPK and c-Fos/NFATc1 Pathways.

The phytochemical substances, coumarin derivatives, have demonstrated antiresorptive bone effects by suppressing osteoclast differentiation in vitro and in vivo. Recently, we have identified 5′-hydroxy auraptene (5′-HA), a coumarin derivative isolated from Lotus lalambensis Schweinf, as a novel stimulator for osteoblast differentiation. In this study, we investigated the effect of 5′-HA on osteoclast differentiation of mouse bone marrow (BM) cells. The effect of 5′-HA on BM cell proliferation and osteoclast differentiation was determined by measuring cell viability and tartrate-resistant acid phosphatase (TRAP) enzyme activity, quantification of TRAP+ multinucleated cells (TRAP+MNCs), and quantitative real-time PCR (qPCR) of osteoclastic gene expression. Regulation of NF-κB, c-Fos/NFATc1, and MAPK signaling pathways by 5′-HA during osteoclastogenesis was measured by the NF-κB reporter assay and Western blot analysis. 5′-HA significantly suppresses the receptor activator of NF-κB ligand (RANKL) induced osteoclast differentiation of BM cells in a dose-dependent manner. Consistently, treatment of BM cells with 5′-HA significantly inhibited RANKL-induced activation of NF-κB and c-Fos/NFATc1 pathways in a dose-dependent manner. Furthermore, RANKL-induced phosphorylation of ERK1/2, p-38, and JNK was significantly inhibited by 5′-HA in BM cells. In conclusion, we identified 5′-HA as a novel coumarin derivative that suppresses RANKL-induced osteoclastogenesis via inhibiting c-Fos/NFATc1 and MAPK signaling pathways.

Clinical Evidence Against the Continuum of Low-Primed Uncommitted Hematopoietic and Progenitor Cells (CLOUD-HSPC) Concept for Hematopoiesis

Introduction: The classical model of blood stem cell differentiation is branch-like linear HSPC progression from progenitor multi/oligo potent stem cells to differentiated mature cell types. An alternative model, referred to as CLOUD-HSPC, proposes bone marrow (BM) HSPCs continuous and direct differentiation into unipotent cells of myeloid lineage (neutrophils (N), basophils (B), eosinophils (E), monocytes (M), mast cells, megakaryocytes (platelets) and erythrocytes without linear stem cell commitment (Velten Nat Cell Biol 2017). Pegfilgrastim (Peg), and the non-colony stimulating agent Plinabulin (Plin) prevent chemotherapy (chemo) induced neutropenia (CIN) in cancer patients (pts), and both exert BM effects leading to CD34+ cell mobilization (Blayney ASH 2017, 2018). In mice, Plin enhances LSK (Lin-Sca+cKit3+) cell differentiation in BM (Ghosh, AACR 2018). Here we clinically validated the CLOUD-HSPC concept by analyzing peripheral blood mononuclear cell (PBMC) counts derived from two bifurcating progenitor cells 1. Granulocyte-Monocyte Progenitor (GMP) producing N,M,E and B and 2. Common Lymphoid Progenitor (CLP) producing lymphocytes (L), after exposure to Peg or Plin in a CIN setting. With linear stem cell commitment, increase in the differentiated mature cell counts would positively inter-correlate, and with CLOUD-HSPC, they would not. Methods: In the Phase 3 portion of study BPI-2358-105 (NCT03102606), pts with NSCLC, BC or HRPC received pre-medication with dexamethasone (Dex) on day (D) -1,0, and 1 and docetaxel (Doc) on day (D) 1. Pts were randomized 1:1 to either Plin 40 mg (n=52), given 30 min after Doc infusion D1, or Peg 6 mg (n=53), D2. Central laboratory (Covance Laboratory) PBMC counts were obtained at screening, D 1,2, 6,7,8,9,10, 15 in Cycle 1, and correlated with each other at a pre-planned interim analysis. D1 and D2 data points were omitted due to confounding effects of Dex and demargination. Data of Plin and Peg were combined, since both drugs exert BM effects leading to increased N cell counts and showed similar correlation trends. Results: Maximum increases in % from screening value on D5-D15 of each N,M,E,B and L counts correlated with each other. Pearson correlation coefficients (r) after linear regression and corresponding p-value are summarized below. Conclusions: There is a linear and positive inter-correlation between differentiated mature cells derived from the GMP and CLP lineage, proving strong clinical evidence against CLOUD-HSPC, but in favor of the classical linear commitment model. Table Disclosures Mohanlal: BeyondSpring Pharmaceuticals: Employment. Blayney:BeyondSpring Pharmaceuticals: Research Funding. Huang:BeyondSpring Pharmaceuticals: Employment.

Titania nanotube morphologies for osseointegration via models of in vitro osseointegrative potential and in vivo intramedullary fixation.

As total joint replacements increase annually, new strategies to attain solid bone-implant fixation are needed to increase implant survivorship. This study evaluated two morphologies of titania nanotubes (TiNT) in in vitro experiments and an in vivo rodent model of intramedullary fixation, to simulate joint arthroplasty conditions. TiNT surfaces were prepared via an electrochemical etching process, resulting in two different TiNT morphologies, an aligned structure with nanotubes in parallel and a trabecular bone-like structure. in vitro data showed bone marrow cell differentiation into osteoblasts as well as osteoblastic phenotypic behavior through 21 days. In vivo, both TiNT morphologies generated greater bone formation and bone-implant contact than control at 12 weeks, as indicated by μCT analyses and histology, respectively. TiNT groups also exhibited greater strength of fixation compared to controls, when subjected to wire pull-out testing. TiNT may be a promising surface modification for promoting osseointegration.

Kinesin family member KIF18A is a critical cellular factor that regulates the differentiation and activation of dendritic cells.

KIF18A is a kinesin family member that is involved in various cellular processes including cell division, cell transformation, and carcinogenesis. However, its possible role in the regulation of host immunity has not been examined. The aim of this study is to investigate the functional role of KIF18A in the differentiation and activation of dendritic cells (DCs) that are the most efficient antigen-presenting cells. A bioinformatic analysis of the KIF18A gene family was performed to understand its sequence variability and evolutionary history. To inhibit KIF18A activity, a highly specific small molecule inhibitor for KIF18A, BTB-1 was used. DCs were differentiated from mouse bone marrow (BM) cells from 6 to 7week old C57BL/6 mice with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of KIF18A was measured by Western blotting. The surface expression of differentiation and activation markers on DCs were analyzed by flow cytometry. The phylogenetic analysis revealed that the KIF18A gene family is remarkably conserved across vertebrates. Interestingly, the expression of KIF18A was increased as BM precursor cells differentiated into DCs. BTB-1 treatment strongly inhibited the differentiation of BM cells into DCs in a dose-dependent manner. Furthermore, treatment of immature DCs with BTB-1 significantly impaired the expression of activation markers on DCs including MHC class I, CD80, and CD86 upon TLR4 or TLR7 treatment. Our results reveal that KIF18A is a critical DC differentiation and activation regulator. Therefore, KIF18A could be a potential therapeutic target for immune-mediated disorders.

C5a/C5aR1 Pathway Is Critical for the Pathogenesis of Psoriasis.

Psoriasis is one of the most common chronic inflammatory skin diseases, affecting ~2% of the population. The lack of characterization of the pathogenesis of psoriasis has hindered efficient clinical treatment of the disease. In our study, we observed that expression of complement component 5a receptor 1(C5aR1) was significantly increased in skin lesions of both imiquimod (IMQ) and IL23-induced psoriatic mice and patients with psoriasis. C5aR1 deficiency or treatment with C5a receptor 1 antagonist (C5aR1a) in mice significantly attenuated psoriasis-like skin lesions and expression of inflammatory cytokines and chemokines. Moreover, C5aR1 deficiency significantly decreased IMQ-induced infiltration of plasmacytoid dendritic cells (pDCs), monocytes and neutrophils in psoriatic skin lesions and functions of pDCs, evidenced by the remarkable reduction in the IMQ-induced production of interferon-α (IFN-α) and tumor necrosis factor α (TNF-α), and FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent pDCs differentiation. Accordingly, in vitro treatment with recombinant C5a accelerated pDCs migration and the differentiation of bone marrow cells into pDCs. Furthermore, biopsies of psoriatic patients showed a dramatic increase of C5aR1+ pDCs infiltration in psoriatic skin lesions, compared to healthy subjects. Our results provide direct evidence that C5a/C5aR1 signaling plays a critical role in the pathogenesis of psoriasis. Inhibition of C5a/C5aR1 pathway is expected to be beneficial in the treatment of patients with psoriasis.

Activation of Bone Marrow–Derived Cells and Resident Aortic Cells During Aortic Injury

BackgroundThe process of aortic injury, repair, and remodeling during aortic aneurysm and dissection is poorly understood. We examined the activation of bone marrow (BM)-derived and resident aortic cells in response to aortic injury in a mouse model of sporadic aortic aneurysm and dissection. Materials and methodsWild-type C57BL/6 mice were transplanted with green fluorescent protein (GFP)+ BM cells. For 4 wk, these mice were either unchallenged with chow diet and saline infusion or challenged with high-fat diet and angiotensin II infusion. We then examined the aortic recruitment of GFP+ BM-derived cells, growth factor production, and the differentiation potential of GFP+ BM-derived and GFP− resident aortic cells. ResultsAortic challenge induced recruitment of GFP+ BM cells and activation of GFP− resident aortic cells, both of which produced growth factors. Although BM cells and resident aortic cells equally contributed to the fibroblast populations, we did not detect the differentiation of BM cells into smooth muscle cells. Interestingly, aortic macrophages were both of BM-derived (45%) and of non−BM-derived (55%) origin. We also observed a significant increase in stem cell antigen-1 (Sca-1)+ stem/progenitor cells and neural/glial antigen 2 (NG2+) cells in the aortic wall of challenged mice. Although some of the Sca-1+ cells and NG2+ cells were BM derived, most of these cells were resident aortic cells. Sca-1+ cells produced growth factors and differentiated into fibroblasts and NG2+ cells. ConclusionsBM-derived and resident aortic cells are activated in response to aortic injury and contribute to aortic inflammation, repair, and remodeling by producing growth factors and differentiating into fibroblasts and inflammatory cells.

Thermoresponsive Stiffness Softening of Hierarchically Porous Nanohybrid Membranes Promotes Niches for Mesenchymal Stem Cell Differentiation.

Despite the attention given to the development of novel responsive implants for regenerative medicine applications, the lack of integration with the surrounding tissues and the mismatch with the dynamic mechanobiological nature of native soft tissues remain in the current products. Hierarchical porous membranes based on a poly (urea-urethane) (PUU) nanohybrid have been fabricated by thermally induced phase separation (TIPS) of the polymer solution at different temperatures. Thermoresponsive stiffness softening of the membranes through phase transition from the semicrystalline phase to rubber phase and reverse self-assembly of the quasi-random nanophase structure is characterized at body temperature near the melting point of the crystalline domains of soft segments. The effects of the porous structure and stiffness softening on proliferation and differentiation of human bone-marrow mesenchymal stem cells (hBM-MSCs) are investigated. The results of immunohistochemistry, histological, ELISA, and qPCR demonstrate that hBM-MSCs maintain their lineage commitment during stiffness relaxation; chondrogenic differentiation is favored on the soft and porous scaffold, while osteogenic differentiation is more prominent on the initial stiff one. Stiffness relaxation stimulates more osteogenic activity than chondrogenesis, the latter being more influenced by the synergetic coupling effect of softness and porosity.

Gender difference of CCAAT/enhancer binding protein homologous protein deficiency in susceptibility to osteopenia.

Expression of CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is induced during endoplasmic reticulum (ER) stress, which is related to apoptosis in several cell types. CHOP null mice have been exhibited to decrease bone formation. However, a study of transgenic mice overexpressing CHOP in the bone microenvironment showed that CHOP overexpression impairs the osteoblastic function leading to osteopenia. The regulatory role of CHOP in bone formation is controversial and still remains to be clarified. Here, we investigated the alterations in bone microstructure of CHOP knockout (Chop-/- ) mice and tested the gender difference of CHOP deficiency in susceptibility to osteopenia. Adult female and male mice (WT) and Chop-/- mice were used. The microcomputed tomography (µCT) analysis in trabecular bone and cortical bone of tibia was determined. Trabecular bone volume fraction (BV/TV), trabecular number, and bone mineral density (BMD) in tibia are markedly decreased in both male and female Chop-/- mice compared to the control WT mice. Unexpectedly, the BMD and BV/TV in trabecular bone of tibia in female Chop-/- mice were significantly lower than in male Chop-/- mice. The similar results could also be observed in the cortical bone of tibia in Chop-/- mice. This gender difference was also observed in the decreased capacity of osteoblast differentiation of bone marrow cells isolated from Chop-/- mice. These results indicated that ER stress-related CHOP signaling might play an important role in the bone formation in a mouse model, especially in females. There is the gender difference of CHOP deficiency in susceptibility to osteopenia. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

The Effect of Dissociated Soft Tissue on Osteogenesis: A Preliminary In Vitro Study.

The purpose of this in vitro study was to examine the effect of dissociated soft tissue on bone marrow cell proliferation and differentiation under osteogenic conditions. Rat bone marrow cells were cultured to assess the stimulation of cell proliferation and differentiation. Harvested palatal mucosa was dissociated using a device (Rigenera; Human Brain Wave) and the dissociated soft tissue was cultured with rat bone marrow cells. Cell proliferation, differentiation, and mineralized nodule formation were assessed after 2 or 5 days of culturing. Bone marrow cell proliferation was assessed by quantifying the absorbance of a water-soluble tetrazolium salt using a cell proliferation assay kit. Bone marrow cell differentiation was assessed by alkaline phosphatase staining and real-time polymerase chain reaction. Mineralized nodule formation was assessed by Alizarin red staining. At day 2, cell proliferation, osteoblast specific gene expression, and mineralized nodule formation were significantly higher in the experimental group than in the control group. Alkaline phosphatase staining was also higher in the experimental group on day 2. Mineralized nodule formation area and osteoblast specific gene expression were also statistically higher in the experimental group on day 5. This study demonstrates that dissociated soft tissue elevates bone marrow cell proliferation and differentiation under osteogenic conditions.

Osseointegration of Alkali-Modified NANOZR Implants: An In Vivo Study.

Ingredients and surface modification methods are being continually developed to improve osseointegration of dental implants and reduce healing times. In this study, we demonstrate in vitro that, by applying concentrated alkali treatment to NANOZR with strong bending strength and fracture toughness, a significant improvement in the bone differentiation of rat bone marrow cells can be achieved. We investigated the influence of materials modified with this treatment in vivo, on implanted surrounding tissues using polychrome sequential fluorescent labeling and micro-computer tomography scanning. NANOZR implant screws in the alkali-treated group and the untreated group were evaluated after implantation in the femur of Sprague–Dawley male rats, indicating that the amount of new bone in the alkali-modified NANOZR was higher than that of unmodified NANOZR. Alkali-modified NANOZR implants proved to be useful for the creation of new implant materials.

Heterogeneity of Stem Cells in theHuman Vocal Fold Mucosa.

1. There is growing evidence to suggest that the cells in the maculae flavae are tissue stem cells of the human vocal fold and maculae flavae are a candidate for a stem cell niche. 2. The latest research shows that the cells in the human maculae flavae are involved in the metabolism of extracellular matrices that are essential for viscoelasticity in the human vocal fold mucosa as a vibrating tissue and are considered to be important cells in the growth, development, and aging of the human vocal fold mucosa. 3. Recent evidence has indicated that the cells including vocal fold stellate cells in the maculae flavae of the human vocal fold mucosa are a functionally heterogenous population. 4. The cells in the human maculae flavae possess proteins of all three germ layers, indicating that they are undifferentiated and have the ability of multipotency. 5. The cell division in the human adult maculae flavae is reflective of asymmetric self-renewal, and cultured cells form a colony-forming unit. Therefore, the phenomenon gives rise to the strong possibility that the cells in the human maculae flavae are putative stem cells. 6. Recent research has suggested that the cells in the human maculae flavae arise from the differentiation of bone marrow cells via peripheral circulation. 7. Cultured cell populations in the human maculae flavae are roughly divided into three groups by morphological features: cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells. However, at the present state of our investigation, it is difficult to clarify the stem cell system and hierarchy of stem cells in the human maculae flavae. 8. Subpopulations of cells in the maculae flavae proliferate extremely slowly and retain stem cell properties. 9. Tension caused by phonation seems to regulate the behavior and heterogeneity of the cells (mechanical regulation) in the maculae flavae of the human vocal fold. 10. The putative stem cells in the maculae flavae appear to differentiate into other kind of cells in the surrounding tissue.

Differentiation of bone marrow cells in arthritic mice with decreased complement activity

Differentiation of bone marrow cells in arthritic mice with decreased complement activity

Expression of interferon regulatory factors (IRF-1 and IRF-2) during radiation-induced damage and regeneration of bone marrow by transplantation in mouse.

Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors of IRF-family that regulate expression of genes for cytokines, chemokines and growth factors in mammalian cells. IRF-1 and IRF-2 play crucial roles in the differentiation of bone marrow cells for immune response. Bone marrow (BM) is the soft lymphoid organ that contains many types of stem cells and produces different types of cells of the blood and immune system. Genetic alterations and damage of the bone marrow cells can lead to different types of blood and immune system-related diseases including anemia and cancer. We have studied the expression of IRF-1 and IRF-2 during radiation-induced damage and regeneration of bone marrow cells after transplantation of freshly isolated bone marrow cells in the mouse. Cell cycle analysis, colony forming unit-fibroblast (CFU-F) assay and bone marrow histology showed that after radiation-induced damage, the bone marrow transplantation resulted in regeneration of the bone marrow up to 24-35% recovery. Real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) for the mRNA expression showed that IRF-1 and IRF-2 were expressed at higher levels in the bone marrow cells of the irradiated (4.34× fold for IRF-1, and 3.87× fold for IRF-2) compared to control and transplanted (1.13× fold for IRF-1, and 1.12× fold IRF-2) mice and immuno-fluorescence analysis for the protein expression showed that IRF-1 and IRF-2 were expressed at higher levels in the bone marrow cells of the irradiated (2.12× fold for IRF-1 and 1.71× fold for IRF-2) compared to control and transplanted (1.73× fold for IRF-1 and 1.21× fold for IRF-2) mice. Thus, IRF-1 and IRF-2 are sensitive and responsive to radiation-induced damage in the bone marrow cells and may also be involved in the bone marrow regeneration process.

Downregulation of Blimp1 inhibits the maturation of bone marrow-derived dendritic cells.

Modulation of differentiation of dendritic cells (DCs), which are derived from bone marrow cells, may influence their maturation and consequently regulate their ability to present antigens to alloreactive T lymphocytes. B lymphocyte‑induced maturation protein‑1 (Blimp1) is a master regulator of immunocyte differentiation, which has been investigated for its effect on DCs. In the present study, a lentivirus was used as a vector to transduce Blimp1‑short hairpin (sh)RNA into primary bone marrow cells during their differentiation to DCs. Lentiviral‑mediated Blimp1‑shRNA (lenti‑shRNA‑Blimp1) had a transduction efficiency of >60% in DC precursors. Lenti‑shRNA‑Blimp1 significantly downregulated the expression levels of Blimp1 and modulated the expression of its target proteins, including class II major histocompatibility complex (MHC) transactivator, c‑myc and interleukin‑6. Although lenti‑shRNA‑Blimp1 did not interfere with the differentiation of bone marrow cells to DCs, it inhibited DC maturation by decreasing the expression of surface MHC‑II molecules, but not the expression of MHC‑I molecules and co‑stimulatory molecules [cluster of differentiation (CD)80/CD86]. Subsequently, alloreactive T cell proliferation was alleviated and regulatory T cells were expanded in response to lenti‑shRNA‑Blimp1. A toxicity assay indicated that the morphology and proliferation of cultured DCs were mildly influenced by the lentiviral vector, indicating that the use of alternative vectors with minimal or no toxicity could be investigated in future studies. In conclusion, transduction with lenti‑shRNA‑Blimp1 modulated the maturation of DCs via MHC‑II molecule suppression and inhibited alloreactive T cell activation. The present findings supported the application of Blimp1‑based intervention as a novel approach to induce immature DCs for further immunological research.

Chemokine (C-X-C motif) ligand 1 and CXCL2 produced by tumor promote the generation of monocytic myeloid-derived suppressor cells.

Accumulation of myeloid‐derived suppressor cells (MDSC) in tumor‐bearing hosts is a hallmark of tumor‐associated inflammation, which is thought to be a barrier to immunosurveillance. Multiple factors secreted by tumor cells and tumor stromal cells are reported to be involved in promoting the expansion of MDSC. Herein, we showed that s.c. inoculation of tumor cells and i.v. injection of tumor‐conditioned medium increased the number of MDSC. Subsequent investigation elucidated that chemokine (C‐X‐C motif) ligand 1 (CXCL1) and CXCL2, which were originally characterized as the chemokines of neutrophils, specifically promoted the expansion of monocytic MDSC (mo‐MDSC), a subtype of MDSC, in the presence of granulocyte‐macrophage colony‐stimulating factor. Depletion of CXCL1 or CXCL2 in B16F10 cells or in B16F10‐bearing mice noticeably decreased the generation of mo‐MDSC in bone marrow. Moreover, we found that, in addition to the tumor cells, tumor‐infiltrated CD11b+ myeloid cells also expressed CXCL1 and CXCL2. Furthermore, CXCL1‐ and CXCL2‐induced increase of mo‐MDSC was not correlated with chemotaxis, proliferation or apoptosis of mo‐MDSC. These findings show a novel role of CXCL1 and CXCL2 in promoting mo‐MDSC generation by favoring the differentiation of bone marrow cells in tumor‐bearing conditions, which suggests that inhibition of CXCL1 and CXCL2 could decrease mo‐MDSC generation and improve host immunosurveillance.

NOVAL COMBINED IMMUNODEFICIENCY, HYPOGAMMAGLOBINEMIA, BONE MARROW FAILURE AND RADIOSENSITIVITY DUE TO PATHOGENIC MUTATION IN MYSM1

Introduction Using radio-sensitivity testing plays a critical role in diagnosis and management of primary immunodeficiency. Case Description We describe a clinical presentation of a premature newborn male who was diagnosed with a rare radio-sensitivity disorder after a positive newborn screen for SCID. Pregnancy was complicated by maternal Type I DM and IUGR. Physical examination remarkable for underweight. The immunological workups confirmed the diagnosis of combined immunodeficiency with B cell aplasia, low T cell and normal NK cell and mildly impaired thymic function. T cell proliferation to mitogen was normal. Negative STR for maternal T cell engraftment. IVIG was started for hypogammaglobinemia. SCID gene sequencing panel was negative. Chromosomal microarray analysis showed region of homozygositiy involving the entire chromosome1. Bone marrow biopsy was done for severe anemia and worsening leukopenia that showed a normal immunophenotyping without abnormal blasts. Radio-sensitivity testing on peripheral blood mononuclear cells demonstrated increased cell death compared to controls following irradiation. Additionally, the patient's unirradiated B-cells radio-sensitivity testing showed spontaneous increased DNA damage signaling. WES identified a homozygous splicing variant in MYSM1 due to paternal uniparental disomy of chromosome1. Discussion MYSM1 is intracellulare enzyme that plays a major role on hematopoietic stem cell maintenance and differentiation in lymphoid cells and bone marrow. In the literature, there were 5 cases reported for pathogenic mutations in MYSM1 that were detected in patients with hypogammaglobulinemia, B-cell deficiency and idiopathic bone marrow failure. 2 patients were shown to have increased radio-sensitivity. We report the first patient with a novel MYSM1 variant who presented with abnormal newborn screen.