Correlation of Plasma FL Expression with Bone Marrow Irradiation Dose

Purpose

Ablative bone marrow irradiation is an integral part of hematopoietic stem cell transplantation. These treatment regimens are based on classically held models of radiation dose and the bone marrow response. Flt-3 ligand (FL) has been suggested as a marker of hematopoiesis and bone marrow status but the kinetics of its response to bone marrow irradiation has yet to be fully characterized. In the current study, we examine plasma FL response to total body and partial body irradiation in mice and its relationship with irradiation dose, time of collection and pattern of bone marrow exposure.

Materials/Methods

C57BL6 mice received a single whole body or partial body irradiation dose of 1–8 Gy. Plasma was collected by mandibular or cardiac puncture at 24, 48 and 72 hr post-irradiation as well as 1–3 weeks post-irradiation. FL levels were determined via ELISA assay and used to generate two models: a linear regression model and a gated values model correlating plasma FL levels with radiation dose.

Results

At all doses between 1–8 Gy, plasma FL levels were greater than control and the level of FL increased proportionally to the total body irradiation dose. Differences in FL levels were statistically significant at each dose and at all time points. Partial body irradiation of the trunk areas, encompassing the bulk of the hematopoietically active bone marrow, resulted in significantly increased FL levels over control but irradiation of only the head or extremities did not. FL levels were used to generate a dose prediction model for total body irradiation. In a blinded study, the model differentiated mice into dose received cohorts of 1, 4 or 8 Gy based on plasma FL levels at 24 or 72 hrs post-irradiation.

Conclusion

Our findings indicate that plasma FL levels might be used as a marker of hematopoietically active bone marrow and radiation exposure in mice.     

Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow

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Expression and Biological Significance of Leptin,Leptin Receptor,VEGF, and CD34 in Colorectal Carcinoma

To investigate the expression and biological significance of Leptin, Leptin receptor, Vascular Endothelial Growth Factor (VEGF), and CD34 protein in colorectal carcinoma tissues. The expression of Leptin, Leptin receptor, VEGF, and CD34 was detected in 68 cases of colorectal carcinoma tissues, paired para-carcinoma tissues and normal colorectal tissues by Immunohistochemical SP Method. The results and related clinicopathological data were analyzed. The positive rate of Leptin, Leptin receptor, and VEGF was significantly higher in colorectal carcinoma tissues than that in paired para-carcinoma tissues and normal colorectal tissues. The expression of Leptin, Leptin receptor, and VEGF was correlated with grade of tumor differentiation, depth of bowel wall invasion, lymph node metastasis, Dukes stage, distant metastasis, and lympho/vascular tumor embolization. Microvessel density (MVD) value in colorectal carcinoma was significantly higher than that in para-carcinoma tissues and normal colorectal tissues, and the density in para-carcinoma tissues was higher than that in normal colorectal tissues. The expression of Leptin, Leptin receptor, VEGF, and MVD value in colorectal carcinoma was positively correlated. In conclusion, microvessel density value is an important index of the growth, invasion, and metastasis of colorectal carcinoma. The binding of Leptin and Leptin receptor promotes the proliferation of colorectal carcinoma cells. The synergy between Leptin and VEGF accelerates the angiogenesis in colorectal carcinoma and accelerates the invasion and metastasis of the tumor cells.     

Soluble Leptin Receptor and Soluble Receptor‐Bound Fraction of Leptin in the Metabolic Syndrome

Objective: In obesity, plasma leptin is high and soluble leptin receptor (sOb‐R) levels are low, resulting in a low fraction of bound leptin. The aim of this study was to investigate the influence of insulin resistance (IR) and the metabolic syndrome (MS) on sOb‐R concentration and the bound‐free ratio of leptin. Research Methods and Procedures: sOb‐R, leptin levels, and homeostasis model assessment (HOMA) index for IR were determined in 76 middle‐aged obese or overweight men. Results: Concentration of sOb‐R and soluble receptor‐bound fraction of leptin were lowest in the highest tertile of HOMA‐IR. sOb‐R and the bound‐free ratio of leptin correlated with HOMA‐IR, leptin concentration, and waist‐to‐hip ratio independently of age, BMI, and fat mass. Leptin and waist‐to‐hip ratio were the sole independent determinants of sOb‐R concentration, and BMI, HOMA‐IR, and visceral adipose tissue were independent determinants of the bound fractin of leptin. sOb‐R concentration and the bound fraction of leptin decreased with increasing numbers of components of the MS, resulting in lower sOb‐R concentration and a lower fraction of bound leptin in men with the MS. Discussion: IR and abdominal obesity are associated with low sOb‐R concentration and low bound‐free ratio of leptin independent of fat mass. Low sOb‐R concentration and low bound‐free ratio of leptin segregate with components of the MS. We suggest that low sOb‐R levels and a low fraction of specifically bound leptin are markers of leptin resistance, which is independently associated with IR and abdominal obesity and may constitute an additional component of the MS.     

Evaluation of Toll-Like Receptor 2 and 4 RNA Expression and the Cytokine Profile in Postmenopausal Women with Metabolic Syndrome

Objective

To evaluate the gene expression of Toll-Like (TLR-2 and TLR-4) receptors and cytokine profile in postmenopausal women with or without metabolic syndrome (MetS).

Methods

In this cross-sectional study, 311 Brazilian women (age≥45 years and amenorrhea≥12 months) were included. Women showing three or more of the following diagnostic criteria were diagnosed as positive for MetS: waist circumference>88 cm, triglycerides≥150 mg/dL, HDL cholesterol<50 mg/dL, blood pressure≥130/85 mmHg, and fasting glucose≥100 mg/dL. The expression of TLR-2 and TLR-4 in peripheral blood was evaluated by RNA extraction and subsequent real time PCR analysis. The cytokine profile, tumor necrosis factor alpha (TNF-α) and interleukins 1β, 6, and 10, were measured by ELISA.

Results

The expression of TLR-2 RNA was demonstrated in 32.5% and TLR-4 in 20.6% of the subjects. There was no association between the expression of TLR-2 and TLR-4 and the presence or absence of MetS (P>0.05). A greater production of IL-6 was associated with TLR-2 and TLR-4 expressions and greater production of TNF-α was associated only with TLR-2 expression (P>0.05). Only the lower quartile of IL-10 was associated with the presence of the MetS (P>0.05).

Conclusions

TLR-2 and TLR-4 expressions were associated with increased pro-inflammatory cytokines, IL-6 and TNF-α, with no association with biomarkers of MetS. The low concentrations of IL-10 may suggest an anti-inflammatory modulation in postmenopausal women with MetS.     

矿化液和地塞米松增强大鼠原代骨髓基质细胞NF-kB受体活化剂配体的表达

NF-KB受体活化剂配体（receptor activator for NF-KB ligand,RANKL）是调节破骨细胞生成的重要因子,它可在骨髓基质细胞中表达。矿化液（含10书mol／L地塞米松、10mmol／LB．甘油磷酸钠、50ug／ml L-抗坏血酸）能够诱导骨髓基质细胞向成骨细胞分化,为探讨矿化液及其主要成分地塞米松对大鼠原代骨髓基质细胞表达RANKL的影响,采用矿化液培养原代大鼠骨髓基质细胞48h,通过免疫荧光染色观察RANKL的表达变化。结果显示矿化液和地塞米松在短期内均能增强鼠骨髓基质细胞RANKL的表达,提示地塞米松促进破骨细胞形成的分子机制可能与骨髓基质细胞RANKL表达的改变密切相关。     

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.     

Ciprofloxacin Modulates Cytokine/Chemokine Profile in Serum,Improves Bone Marrow Repopulation,and Limits Apoptosis and Autophagy in Ileum after Whole Body Ionizing Irradiation Combined with Skin-Wound Trauma

Radiation combined injury (CI) is a radiation injury (RI) combined with other types of injury, which generally leads to greater mortality than RI alone. A spectrum of specific, time-dependent pathophysiological changes is associated with CI. Of these changes, the massive release of pro-inflammatory cytokines, severe hematopoietic and gastrointestinal losses and bacterial sepsis are important treatment targets to improve survival. Ciprofloxacin (CIP) is known to have immunomodulatory effect besides the antimicrobial activity. The present study reports that CIP ameliorated pathophysiological changes unique to CI that later led to major mortality. B6D2F1/J mice received CI on day 0, by RI followed by wound trauma, and were treated with CIP (90 mg/kg p.o., q.d. within 2 h after CI through day 10). At day 10, CIP treatment not only significantly reduced pro-inflammatory cytokine and chemokine concentrations, including interleukin-6 (IL-6) and KC (i.e., IL-8 in human), but it also enhanced IL-3 production compared to vehicle-treated controls. Mice treated with CIP displayed a greater repopulation of bone marrow cells. CIP also limited CI-induced apoptosis and autophagy in ileal villi, systemic bacterial infection, and IgA production. CIP treatment led to LD_0/10 compared to LD_20/10 for vehicle-treated group after CI. Given the multiple beneficial activities of CIP shown in our experiments, CIP may prove to be a useful therapeutic drug for CI.     

西藏小型猪Leptin及其受体胞外区片段的克隆及原核表达

目的克隆及原核表达西藏小型猪瘦素（Leptin）成熟肽及瘦素受体胞外区片段。方法根据西藏小型猪瘦素序列（GenBank号：GQ240885.1）和猪瘦素受体基因胞外域序列（GenBank号：AF167719.1）分别设计并合成两对引物扩增瘦素、瘦素受体基因胞外域编码区1654-2319位片段,以西藏小型猪组织总RNA为模板,经反转录-聚合酶链反应（RT-PCR）方法获得了特异性片段。再以该两个特异性片段为模板,另外设计两对带有BanHⅠ和HidⅢ酶切位点的套式引物分别扩增瘦素64-504位（成熟肽编码区）和瘦素受体基因胞外域编码区1655-2314位的cDNA片段,将该两片段克隆入pMD18-T载体并转化感受态细菌E.coli DH5α测序并永久保存。此两片段经酶切后克隆到表达载体pRSET A的BamHⅠ和HindⅢ两酶切位点之间,构建重组质粒pR-OB和pR-OBR-a并在大肠杆菌E.coli BL21（DE3）中表达,SDS-PAGE电泳鉴定表达产物。结果在IPTG诱导下促使重组菌pR-OB表达了相对分子质量约18×103左右的融合蛋白;重组菌pR-OBR-a表达了相对分子质量约27×103左右的融合蛋白。结论说明重组质粒pR-OB、pR-OBR-a在大肠杆菌BL21（DE3）中分别可表达西藏小型猪瘦素成熟肽、瘦素受体片段蛋白,为进一步研究瘦素、瘦素受体功能和应用提供了基础。     

Induction of Unresponsiveness to Canine Renal Allografts by Total Body Irradiation and Bone Marrow Transplantation

RECENT progress in the definition of the DL-A antigens within a closely bred colony of beagles maintained at the Mary Imogene Basset Hospital (Cooperstown colony) has shown that these antigens are components of the principal system of histocompatibility (DL-A) in the dog^1–3. Dausset and associates⁴ have described the segregation of DL-A antigens in 679 offspring of 151 consecutive matings within the Coopers-town colony. Through methods of backcross analysis, 23 DL-A haplotypes (that is, specificities determined by the DL-A region of one single chromosome) have been identified and the DL-A genotypes of 1,302 offspring of 517 matings in this colony have been defined⁴. This combined genetic and serological information has provided an opportunity to reassess the reported survival rates of allogeneic bone marrow transplantation in serologically matched littermate dogs⁵. We describe here the long term survival of bone marrow transplants and renal allografts obtained from the same donors in nine littermate and three non-littermate recipients selected on the basis of genetic and serological criteria of DL-A compatibility in the Cooperstown colony. The results are contrasted with the survival of twenty-two renal allografts performed in untreated recipients selected on the basis of similar criteria.     

Paracrine Effects of Bone Marrow Soup Restore Organ Function,Regeneration, and Repair in Salivary Glands Damaged by Irradiation

Background

There are reports that bone marrow cell (BM) transplants repaired irradiated salivary glands (SGs) and re-established saliva secretion. However, the mechanisms of action behind these reports have not been elucidated.

Methods

To test if a paracrine mechanism was the main effect behind this reported improvement in salivary organ function, whole BM cells were lysed and its soluble intracellular contents (termed as “BM Soup”) injected into mice with irradiation-injured SGs. The hypothesis was that BM Soup would protect salivary cells, increase tissue neovascularization, function, and regeneration. Two minor aims were also tested a) comparing two routes of delivering BM Soup, intravenous (I.V.) versus intra-glandular injections, and b) comparing the age of the BM Soup’s donors. The treatment-comparison group consisted of irradiated mice receiving injections of living whole BM cells. Control mice received irradiation and injections of saline or sham-irradiation. All mice were followed for 8 weeks post-irradiation.

Results

BM Soup restored salivary flow rates to normal levels, protected salivary acinar, ductal, myoepithelial, and progenitor cells, increased cell proliferation and blood vessels, and up-regulated expression of tissue remodeling/repair/regenerative genes (MMP2, CyclinD1, BMP7, EGF, NGF). BM Soup was as an efficient therapeutic agent as injections of live BM cells. Both intra-glandular or I.V. injections of BM Soup, and from both young and older mouse donors were as effective in repairing irradiated SGs. The intra-glandular route reduced injection frequency/dosage by four-fold.

Conclusion

BM Soup, which contains only the cell by-products, can be advantageously used to repair irradiation-damaged SGs rather than transplanting whole live BM cells which carry the risk of differentiating into unwanted/tumorigenic cell types in SGs.     

小鼠骨髓间充质干细胞的培养、鉴定和Survivin的表达研究

为培养及鉴定小鼠来源骨髓间充质干细胞,并测定细胞中Survivin的表达情况,采用全骨髓培养法获取骨髓间充质干细胞,绘制生长曲线,流式细胞仪检测细胞表面标志物,行成骨、成脂检测,RT-PCR测定Survivin表达情况.结果表明培养出的细胞呈长梭状成纤维细胞样,经流式细胞仪检测细胞表面高表达CD29、CD34、CD44、SCA-1,低表达CD117;细胞曲线显示传代细胞培养1～3d生长缓慢,第4d生长加快并于第7d达到高峰;成骨诱导20d经茜素红染色呈红色结节,成脂诱导14d油红O染色显示有大量脂质沉淀;RT-PCR结果显示Survivin mRNA阳性表达.经全骨髓培养法可以培养出大量骨髓间充质干细胞,同时Survivin在小鼠骨髓间充质干细胞中正常表达,提示可能参与骨髓间充质干细胞抗凋亡过程.     

Generation of Mature Murine Monocytes from Heterogeneous Bone Marrow and Description of Their Properties

Monocytes are involved in a wide range of physiological and pathological processes, many of which are studied in mouse models. Current protocols to isolate murine monocytes are few and result in unsatisfactory cell yield and purity. Here, we describe a novel approach to efficiently differentiate large numbers of mature inflammatory monocytes from heterogeneous bone marrow cell suspensions. Bone marrow cell suspensions were isolated by flushing femurs and tibias from Balb/c and C57Bl/6 mice, supplemented with macrophage colony–stimulating factor (M-CSF), and were cultured on ultra-low attachment surfaces to inhibit adherence-mediated maturation. Cells were harvested at indicated time points, underwent time-line analysis of the differentiation processes, and were subsequently extensively phenotyped to verify their monocytotic properties. In order to confirm downstream compatibility, we tested for typical monocyte behavior. Our protocol yielded 24 ± 6 × 10⁶ differentiated cells per donor mouse, 10-fold higher than yields obtained using previously described peripheral blood isolation methods. Differentiated cells consisted of approximately 47% ± 12% monocytes, the rest being mature macrophages. We increased monocyte purity to 86% ± 6% by depleting adherent macrophages. Our findings indicate that bone marrow–derived monocytes (BMDMs) are an attractive tool to study, for example, the innate and adaptive immune system, atherosclerosis, and cellular migration during infection. Moreover, BMDM transplantation could be used to test novel, therapeutic in vivo approaches in mice disease models.     

对虾白斑综合征病毒的细胞因子受体基因的分析与表达

在对虾白斑综合征病毒(White spot syndrome virus,WSSV)的基因组中发现一个具有细胞因子受体特征的开放阅读框,该阅读框全长2022个核苷酸,编码674个氨基酸,蛋白质理论分子量为76kDa.该基因含有真核生物细胞因子gpl30受体特征序列.为了研究该基因的功能,采用PCR方法从病毒基因组中扩增出基因片段,克隆到pGEM-T Easy载体中,经BamH I和Sal I双酶切后插入pET28b表达载体中.重组质粒转化到大肠杆菌BL21中,IPTG诱导后,经SDS-PAGE电泳表明在76kDa处有目的蛋白表达.用冰浴超声波对诱导后的菌液进行处理以获得初步纯化的蛋白,作为抗原人工免疫实验兔子以获得含特异性抗体的抗血清.该基因的表达成功,为其功能的进一步深入研究奠定了基础.     

对虾白斑综合征病毒的细胞因子受体基因的分析与表达

在对虾白斑综合征病毒(White spot syndrome virus,WSSV)的基因组中发现一个具有细胞因子受体特征的开放阅读框,该阅读框全长2022个核苷酸,编码674个氨基酸,蛋白质理论分子量为76kDa。该基因含有真核生物细胞因子gp130受体特征序列。为了研究该基因的功能,采用PCR方法从病毒基因组中扩增出基因片段,克隆到pGEM-T Easy载体中,经BamH I和Sal I双酶切后插入pET28b表达载体中。重组质粒转化到大肠杆菌BL21中,IPTG诱导后,经SDS-PAGE电泳表明在。76kDa处有目的蛋白表达。用冰浴超声波对诱导后的菌液进行处理以获得初步纯化的蛋白,作为抗原人工免疫实验兔子以获得含特异性抗体的抗血清。该基因的表达成功,为其功能的进一步深入研究奠定了基础。     

PGK1 as Predictor of CXCR4 Expression,Bone Marrow Metastases and Survival in Neuroblastoma

Background and Aim

A close relationship between phosphoglycerate kinase 1 (PGK1) and the CXCR4/SDF1 axis (chemokine receptor 4/stromal cell derived factor 1) has been shown for several cancers. However, the role of PGK1 has not been investigated for neuroblastoma, and PGK1 might be a therapeutic target for this tumor entity. The aim of the current study was to evaluate the role of PGK1 expression in neuroblastoma patients, to determine the impact of PGK1 expression levels on survival, and to correlate PGK1 expression with CXCR4 expression and bone marrow dissemination.

Materials and Methods

Samples from 22 patients with neuroblastoma that were surgically treated at the University Medical Center Hamburg-Eppendorf were evaluated for expression of PGK1 and CXCR4 using immunohistochemistry. Results were correlated with clinical parameters, metastases and outcome of patients. Immunocytochemistry, proliferation and expression analysis of CXCR4 and PGK1 were performed in neuroblastoma cell lines.

Results

PGK1 is expressed in neuroblastoma cells. PGK1 expression is significantly positively correlated with CXCR4 expression and tumor dissemination to the bone marrow. Moreover the expression of PGK1 is significantly associated with a negative impact on survival in patients with neuroblastoma. PGK1 is downregulated by inhibition of CXCR4 in neuroblastoma cells.

Conclusion

PGK1 appears to play an important role for neuroblastoma, predicting survival and tumor dissemination. Further in vivo studies outstanding, it is a candidate target for novel therapeutic strategies.