Role of Immune Microenvironmental Factors for Improving the IPI-related Risk Stratification of Aggressive B Cell Lymphoma.

OBJECTIVE: To investigate the risk stratification of aggressive B cell lymphoma using the immune microenvironment and clinical factors. METHODS: A total of 127 patients with aggressive B cell lymphoma between 2014 and 2015 were enrolled in this study. CD4, Foxp3, CD8, CD68, CD163, PD-1, and PD-L1 expression levels were evaluated in paraffin-embedded lymphoma tissues to identify their roles in the risk stratification. Eleven factors were identified for further evaluation using analysis of variance, chi-square, and multinomial logistic regression analysis. RESULTS: Significant differences in 11 factors (age, Ann Arbor stage, B symptom, ECOG performance status, infiltrating CD8+ T cells, PD-L1 expression, absolute blood monocyte count, serum lactate dehydrogenase, serum iron, serum albumin, and serum ß2-microglobulin) were observed among patient groups stratified by at least two risk stratification methods [International Prognostic Index (IPI), revised IPI, and NCCN-IPI models] (P < 0.05). Concordance rates were high (81.4%-100.0%) when these factors were used for the risk stratification. No difference in the risk stratification results was observed with or without the Ann Arbor stage data. CONCLUSION: We developed a convenient and inexpensive tool for use in risk stratification of aggressive B cell lymphomas, although further studies on the role of immune microenvironmental factors are needed.

Valproic Acid Enhances the Anti-tumor Effect of (-)-gossypol to Burkitt Lymphoma Namalwa Cells.

Burkitt lymphoma is a highly aggressive B-cell neoplasm. New therapeutic methods are needed to overcome the adverse effect of intensive chemotherapy regimens. Valproic acid and (-)-gossypol are two kinds of chemical compounds used as new anti-tumor drugs in recent years. To investigate the anti-tumor effect of valproic acid and (-)-gossypol, Burkitt lymphoma Namalwa cells were cultured and treated with valproic acid and (-)-gossypol at different concentrations. The proliferation of Namalwa cells was dramatically suppressed after the combination treatment with 2 mmol/L valproic acid and 5 µmol/L (-)-gossypol. The combined treatment also enhanced intrinsic apoptosis by down-regulating anti-apoptotic protein Mcl-1. Moreover, the autophagy flux significantly increased in Namalwa cells after combined treatment. However, the enhanced autophagy showed little effect on cell survival with present regimen. The results confirmed that combination of valproic acid and (-)-gossypol had synergistic anti-tumor effect to Burkitt lymphoma Namalwa cells. The related mechanisms might include the down-regulation of anti-apoptotic protein Mcl-1 and avianized pro-survival role of autophagy.

Different roles of TGF-ß in the multi-lineage differentiation of stem cells.

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells. Transforming growth factor ß (TGF-ß) family is a superfamily of growth factors, including TGF-ß1, TGF-ß2 and TGF-ß3, bone morphogenetic proteins, activin/inhibin, and some other cytokines such as nodal, which plays very important roles in regulating a wide variety of biological processes, such as cell growth, differentiation, cell death. TGF-ß, a pleiotropic cytokine, has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells, through the Smad pathway, non-Smad pathways including mitogen-activated protein kinase pathways, phosphatidylinositol-3-kinase/AKT pathways and Rho-like GTPase signaling pathways, and their cross-talks. For instance, it is generally known that TGF-ß promotes the differentiation of stem cells into smooth muscle cells, immature cardiomyocytes, chondrocytes, neurocytes, hepatic stellate cells, Th17 cells, and dendritic cells. However, TGF-ß inhibits the differentiation of stem cells into myotubes, adipocytes, endothelial cells, and natural killer cells. Additionally, TGF-ß can provide competence for early stages of osteoblastic differentiation, but at late stages TGF-ß acts as an inhibitor. The three mammalian isoforms (TGF-ß1, 2 and 3) have distinct but overlapping effects on hematopoiesis. Understanding the mechanisms underlying the regulatory effect of TGF-ß in the stem cell multi-lineage differentiation is of importance in stem cell biology, and will facilitate both basic research and clinical applications of stem cells. In this article, we discuss the current status and progress in our understanding of different mechanisms by which TGF-ß controls multi-lineage differentiation of stem cells.

Bone marrow mesenchymal stem cells can be mobilized into peripheral blood by G-CSF in vivo and integrate into traumatically injured cerebral tissue.

The efficacy of granulocyte colony-stimulating factor (G-CSF) in mobilizing mesenchymal stem cells (MSCs) into peripheral blood (PB) and the ability of PB-MSCs incorporated into injured brain were tested. Colony forming, cell phenotype and differentiation potential of mouse MSCs mobilized by G-CSF (40 µg/kg) were evaluated. Mortality and pathological changes in mice with serious craniocerebral trauma plus G-CSF treatment (40 µg/kg) were investigated. Bone marrow (BM) cells derived from GFP mice were fractionated into MSCs, hematopoietic stem cells (HSCs), and non-MSC/HSCs using magnetic beads and adherent culture. The resultant cell populations were transplanted into injured mice. The in vivo integration and differentiation of the transplanted cells were detected immunocytochemically. The expression of SDF-1 in injured area of brain was tested by Western blot. G-CSF was able to mobilize MSCs into PB (fourfold increase). PB-MSCs possessed similar characteristics as BM-MSCs in terms of colony formation, the expression pattern of CD73, 44, 90, 106, 31 and 45, and multipotential of differentiation. Accumulative total mice mortality was lower in TG group (5/14) than that in T group (7/14). It was MSCs, not HSCs or non-MSC/HSC cells integrated into the damaged cerebral tissue and differentiated into cells expressing neural markers. Increased SDF-1 expression in injured area of brain was confirmed, which could facilitate the homing of MSCs to brain. G-CSF can mobilize MSCs into PB and MSCs in PB can integrate into injured cerebral tissue and transdifferentiated into neural cells and may benefit the repair of trauma.

Human umbilical cord blood-derived stromal cells prevent graft-versus-host disease in mice following haplo-identical stem cell transplantation.

BACKGROUND AIMS: Human umbilical cord blood-derived stromal cells (hUCBDSC) comprise a novel population of CD34(+) cells that has been isolated in our laboratory. They have been shown previously not only to be non-immunogenic but also to exert immunosuppressive effects on xenogenic T cells in vitro. This study investigated the role of hUCBDSC in immunomodulation in an acute graft-versus-host disease (GvHD) mouse model after haplo-identical stem cell transplantation. METHODS: Acute GvHD was induced in recipient (B6 × BALB/c)F(1) mice by irradiation (750 cGy) followed by infusion of bone marrow cells and splenocytes from donor C57BL/6 mice. hUCBDSC were co-transplanted in the experimental group. The survival time, body weight and clinical and histopathologic scores were recorded after transplantation. The expression of surface markers [major histocompatibility complex (MHC) I, MHC II, CD80 and CD86] on CD11c(+) dendritic cells (DC), and the percentage of CD4(+) regulatory T cells (Treg), in the spleens of recipient mice were examined by flow cytometry. RESULTS: The survival time was significantly prolonged, and the clinical and histopathologic scores were reduced in mice co-transplanted with hUCBDSC. The expression levels of the surface markers on DC were significantly lower in mice transplanted with hUCBDSC compared with those without. The proportion of CD4(+) Treg in the spleen was also increased in mice transplanted with hUCBDSC. CONCLUSIONS: These results from a GvHD mouse model are in agreement with previous in vitro findings, suggesting that hUCBDSC possess immunosuppressive properties and may act via influencing DC and CD4(+) Treg.

Human bone marrow mesenchymal stem cells expressing SDF-1 promote hematopoietic stem cell function of human mobilised peripheral blood CD34+ cells in vivo and in vitro.

PURPOSE: There is mounting evidence demonstrating that stromal cell derived factor-1 (SDF-1) plays an important role in homing of hematopoietic progenitor cells to bone marrow. This study was aimed to assess whether bone marrow mesenchymal stem cells overexpressing exogenous SDF-1 could synergistically promote the homing of CD34(+) (Cluster of Differentiation [CD]) cells to bone marrow of lethally irradiated severe combined immunodeficiency (SCID) mice. METHODS: Human SDF-1 complementary Deoxyribonucleic acid (cDNA) was transfected into bone marrow-derived mesenchymal stem cells with recombinant lentiviral vector. The expression of SDF-1 was detected by real-time Polymerase Chain Reaction (PCR) and Enzyme-Linked Immunosorbent Assay (ELISA), and the ex vivo chemotaxis function on CD34(+) cells was measured by coculture system and Transwell system. SDF-1 gene-modified mesenchymal stem cell (MSC) and CD34(+) cells were infused into lethally irradiated SCID mice and the hematopoietic reconstitution in the recipient mice was examined. RESULTS: Messenger ribonucleic acid (mRNA) and protein of SDF-1 in infected MSC were significantly higher than that of the non-infected control MSC (p < 0.05). The infected MSC have significant chemotaxis effect on CD34(+) cells in vitro and promote hematopoietic reconstitution after CD34(+) cell transplantation in vivo. CONCLUSION: MSC with high-level expression of SDF-1 can synergistically promote hematopoietic reconstitution after CD34(+) cell transplantation in lethally irradiated SCID mice.

Human umbilical cord blood-derived stromal cells suppress xenogeneic immune cell response in vitro.

AIM: To explore immunological properties of human umbilical cord blood-derived stromal cells (hUCBDSC) and their effect on xenogeneic immune cells in vitro. METHODS: Immunological phenotype of freshly isolated and cryopreserved hUCBDSCs was evaluated by flow cytometry. Xenogeneic splenic T-cells were stimulated by phytohemaglutinin A (PHA) or dendritic cells in the absence or presence of hUCBDSCs. T-cell proliferation was measured by cell counting kit-8 after 7-day incubation. The proportion of apoptotic cells and CD4+CD25+Foxp3+ regulatory T-cells (Tregs) was determined in T-cells activated by PHA in the absence or presence of hUCBDSCs by flow cytometry. Phenotype of dendritic cells, cultured alone or with hUCBDSCs, was analyzed by flow cytometry. RESULTS: Levels of immune molecule expression on freshly isolated hUCBDSCs were as follows: human leukocyte antigen-I (HLA-I) (84.1+/-2.9%), HLA-II (1.6+/-0.3%), CD80 (0.8+/-0.1%), CD86 (0.8+/-0.1%), CD40 (0.6+/-0.1%), and CD40L (0.5+/-0.1%), which was not influenced by cryopreservation. T-cell proliferation in the presence of hUCBDSCs was significantly lower than that of positive control. The coculture led to a 10-fold increase (from 1.2+/-0.3% to 12.1+/-1.4%, P<0.001) in the proportion of CD4+CD25+Foxp3+ regulatory T-cells (Tregs) and a reversion of mature dendritic cells, as indicated by the down-regulation of major histocompatibility complex (MHC)-II molecule (49.3% vs 25.9%, P=0.001), CD80 (47.2% vs 23.3%, P=0.001), and CD86 (40.6% vs 25.1%, P=0.002). When subjected to annexin V binding and propidium iodide uptake assay, the hUCBDSCs did not show the ability to induce apoptosis of xenogeneic T-cells. CONCLUSION: These results demonstrate low immunogenicity and immunomodulation effect of the hUCBDSCs. Reversion of mature dendritic cells and increase in Treg proportion, but not cell apoptosis, can possibly contribute to the suppression of xenogeneic T-cell proliferation by the hUCBDSCs.

[Exogenous gene expression in vitro and in vivo in bone marrow mesenchymal stem cells modified by hPDGF-A and hBD(2)].

The objective of this study was to investigate the expression of exogenous hPDGF-A and hBD(2) in gene-modified bone marrow mesenchymal stem cells (BM-MSCs) in vitro and in vivo. Recombinant adenovirus vector expressing hPDGF-A/hBD(2) genes was constructed and packaged into virion. Primary isolated and cultured BM-MSCs were transfected by using hPDGF-A hBD(2), then the expressions of exogenous hPDGF-A/hBD(2) were detected by immunocytochemical staining in vitro. The conditioned medium (serum-free cultured supernatant of BM-MSCs transfected with recombinant adenovirus) collected from gene-modified BM-MSCs was applied to scratch wound on monolayer cells of multipotential cell line 10T1/2 in order to confirm the stimulative effect of hPDGF-A on cell migration. Gene-modified BM-MSCs were topically transplanted on wound of rats with radiation and skin excision combined injury. The distribution of BM-MSCs and expression of hPDGF-A/hBD(2) on the wound was observed by fluorescent microscopy and immunohistochemical staining respectively. The results indicated that the rat BM-MSCs transfected with recombinant adenovirus could express the EGFP in vitro. The immunofluorescent cytochemistry assay showed that the gene-modified BM-MSCs expressed the hPDGF-A and hBD(2). The scratch test confirmed that the percentage of healing area of wound in cultured supernatant group of gene-modified BM-MSCs was significant higher than that in control group on 8, 12, 24 and 48 hours (p < 0.05). The fluorescence microscopy of exogenous gene-modified BM-MSCs transplanted on wound revealed that the gene-modified BM-MSCs could higher express exogenous genes of EGFP at least within 2 weeks. The immunohistochemistry staining of wound indicated that the expression of exogenous genes began from day 3, reached to peak on day 7, and still visible on day 21 even though the expression became weak because of the possible dilution of the exogenous genes during cell division. It is concluded that efficient expression of exogenous hPDGF-A/hBD(2) in gene-modified BM-MSCs are demonstrated both in vitro and in vivo, which suggests that the molecular mechanism underlying chronic wound-healing accelerated by the strategy combining cell therapy with gene therapy.

[The effects of burn serum on the erythropoiesis and granulopoiesis in bone marrow in mice].

OBJECTIVE: To observe the effects of burn serum on the erythropoiesis and granulopoiesis in bone marrow in mice, and to explore the possible underlying mechanism. METHODS: Murine bone marrow cell (BMC) strain was prepared routinely and was employed in the establishment of the culture system of colony forming units of erythrocytes, or granulocytes and monocytes. To both sets of culture system normal murine serum (N group) and burn serum, which was collected from the mice with 15% full thickness burn at 12 postburn hours (PBH) and 1, 3, 5, 7 and 10 postburn days (PBD), (burn serum group) was added. In addition, positive control and blank control groups were set accordingly. The stimulating activity of all kinds of sera on the BMCs in the two sets of culture system was determined. The changes in the burn serum concentrations of EPO and GM-CSF were detected by radioimmunoassay, and the data were analyzed by logarithmic linear fitting correlation with the former influence of burn sera on the erythrocytes and granulocytes. RESULTS: (1) Burn sera exhibited obvious stimulation promoting activity on the erythropoiesis and granulopoiesis in BMC, and the activity peaked (384 +/- 60 and 127 +/- 16 CFU) on 1 PBD and decreased thereafter to approach the values found in normal sera group (125 +/- 14 and 34 +/- 20 CFU) on 7 PBD. (2) The EPO content in burn serum was evidently higher than the normal value (P < 0.01) during 12 PBH to 7PBD period. The GM-CSF concentration was obviously higher than the normal value (P < 0.05) at 12 PBH and on 1 PBD. (3) The EPO concentration in burn serum was significantly and logarithmically correlated with the stimulation promoting activity of burn serum on erythropoiesis (r = 0.8570, P = 0.0137). But the GM-CSF concentration in culture with burn serum was not correlated with the stimulation promoting activity of burn serum on granulopoiesis (r = 0.7049, P > 0.05). CONCLUSION: The sera harvested from burned mice during early postburn stage exhibited strong stimulation promoting activity on the erythropoiesis and granulopoiesis in bone marrow. The increased EPO level in burn serum might be the important factor contributing strong stimulation action on erythropoiesis, while increased GM-CSF level was not.