Long-term type 1 diabetes influences haematopoietic stem cells by reducing vascular repair potential and increasing inflammatory monocyte generation in a murine model

Aims/hypothesis

We sought to determine the impact of long-standing type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells.

Methods

Wild-type, gp130 ^?/? and GFP chimeric mice were treated with streptozotocin to induce type 1 diabetes. Bone marrow (BM)-derived cells were used for colony-formation assay, quantification of side population (SP) cells, examination of gene expression, nitric oxide measurement and migration studies. Endothelial progenitor cells (EPCs), a population of vascular precursors derived from HSCs, were compared in diabetic and control mice. Cytokines were measured in BM supernatant fractions by ELISA and protein array. Flow cytometry was performed on enzymatically dissociated retina from gfp ⁺ chimeric mice and used to assess BM cell recruitment to the retina, kidney and blood.

Results

BM cells from the 12-month-diabetic mice showed reduced colony-forming ability, depletion of SP-HSCs with a proportional increase in SP-HSCs residing in hypoxic regions of BM, decreased EPC numbers, and reduced eNos (also known as Nos3) but increased iNos (also known as Nos2) and oxidative stress-related genes. BM supernatant fraction showed increased cytokines, GP130 ligands and monocyte/macrophage stimulating factor. Retina, kidney and peripheral blood showed increased numbers of CD11b⁺/CD45^hi/ CCR2⁺/Ly6C^hi inflammatory monocytes. Diabetic gp130 ^?/? mice were protected from development of diabetes-induced changes in their HSCs.

Conclusions/interpretation

The BM microenvironment of type 1 diabetic mice can lead to changes in haematopoiesis, with generation of more monocytes and fewer EPCs contributing to development of microvascular complications. Inhibition of GP130 activation may serve as a therapeutic strategy to improve the key aspects of this dysfunction.     

The antirheumatic drug leflunomide inhibits osteoclastogenesis by interfering with receptor activator of NF‐κB ligand–stimulated induction of nuclear factor of activated T cells c1

Objective

Suppression of bone destruction is required as part of an effective therapeutic strategy for autoimmune arthritis. Although numerous antirheumatic drugs are in clinical use, little is known about whether they inhibit bone destruction by acting on activated T cells or other cell types, such as bone‐resorbing osteoclasts. This study was undertaken to determine whether leflunomide has a direct action on the osteoclast lineage and to gain insights into the molecular basis for the bone‐protective effect of leflunomide.

Methods

The direct effect of leflunomide on osteoclast differentiation was investigated using an in vitro culture system of bone marrow monocyte/macrophages stimulated with receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor. The molecular mechanism of the inhibition was analyzed by genome‐wide screening. The T cell–independent effect of leflunomide was examined in rag‐2^−/− mice.

Results

Leflunomide blocked de novo pyrimidine synthesis and RANKL‐induced calcium signaling in osteoclast precursor cells in vitro; hence, the induction of nuclear factor of activated T cells c1 (NF‐ATc1) was strongly inhibited. The inhibition of this pathway is central to the action of leflunomide, since the inhibition was overcome by ectopic expression of NF‐ATc1 in the precursor cells. Leflunomide suppressed endotoxin‐induced inflammatory bone destruction even in rag‐2^−/− mice.

Conclusion

Leflunomide has a direct inhibitory effect on RANKL‐mediated osteoclast differentiation by inhibiting the induction of NF‐ATc1, the master switch regulator for osteoclast differentiation. Our study suggests that the direct inhibitory action of leflunomide on osteoclast differentiation constitutes an important aspect in the amelioration of bone destruction, and that the RANKL‐dependent NF‐ATc1 induction pathway is a promising target for pharmacologic intervention in arthritic bone destruction.

     

A High Level of TM4SF5 Is Associated with Human Esophageal Cancer Progression and Poor Patient Survival

Purpose

We investigated expression of TM4SF5 and its involvement in human esophageal cancer (HEC).

Methods

We analyzed TM4SF5 expression in normal esophageal epithelial cells (HEEC), in four HEC cell lines, and in 20 HEC clinical tissue samples and matched nontumor samples. The effect of TM4SF5 on HEC cell proliferation and metastasis and invasion was assessed, and the relationship between TM4SF5 and integrin β1 was determined. Finally, TM4SF5 and integrin β1 expression were further examined by use of immunohistochemistry (IHC) and tissue microarray analysis, and the prognostic use of TM4SF5 and integrin β1 in HEC was evaluated.

Results

TM4SF5 was more highly expressed in HEC cells and in HEC tissues than in HEEC and matched nontumor tissues. Down-regulation of TM4SF5 in KYSE150 cells reduced cell proliferation and metastasis and invasion whereas metastasis and invasion by KYSE510 increased after TM4SF5 cDNA transfection. In HEC cells, TM4SF5 formed a complex with integrin β1, and interference with integrin β1 in KYSE510-TM4SF5 cells markedly inhibited cell invasion on laminin 5. Our findings also showed that TM4SF5 and integrin β1 overexpression correlated with low differentiation and high stage (p < 0.05, respectively). Postoperative 5-year overall survival of patients with TM4SF5^low and/or integrin β1^low was higher than for patients with TM4SF5^high and/or integrin β1^high. Multivariate analysis demonstrated that TM4SF5 and integrin β1 co-overexpression was an independent prognostic marker for HEC.

Conclusion

TM4SF5 is positively associated with HEC invasiveness. The combination of TM4SF5 with integrin β1 could potentially serve as a novel marker for predicting HEC prognosis.     

Patients with chronic hepatitis C express a high percentage of CD4+CXCR5+ T follicular helper cells

Background

T follicular helper (T_FH) cells are a subpopulation of T-helper cells which regulate humoral immune responses. The role of T_FH cells in viral infection is unclear. This study examined the possible involvement of CD4⁺CXCR5⁺ T_FH cells in chronic hepatitis C (HCV) infection.

Methods

The percentages of peripheral blood CD4⁺CXCR5⁺ T_FH cells, inducible T-cell costimulator cells, and/or programmed death 1-positive CD4⁺CXCR5⁺ T_FH cells in 39 HCV-infected patients, 12 patients with spontaneously resolved HCV infection (SR-HCV), and 12 healthy controls were characterized by flow cytometry analysis. The subjects’ serum HCV RNA loads and alanine aminotransferase and aspartate aminotransferase levels were measured. The potential association of the percentage of peripheral CD4⁺CXCR5⁺ T_FH cells with clinical data was analyzed.

Results

Higher percentages of peripheral blood CD4⁺CXCR5⁺ T_FH cells were found in SR-HCV and HCV-infected patients as compared with healthy controls. Interestingly, a statistically significant negative correlation was found between the percentage of CD4⁺CXCR5⁺ T_FH cells and the HCV RNA load.

Conclusions

These data suggest that CD4⁺CXCR5⁺ T_FH cells may participate in HCV-related immune responses. Increased T_FH cells in peripheral blood may help to control HCV infection.     

Insulin-Like Growth Factor 1 Receptor Promotes the Growth and Chemoresistance of Pancreatic Cancer

Background

Insulin-like growth factor 1 receptor (IGF1R) plays important roles in the progression of pancreatic cancer. However, the underlying mechanism remains unclear.

Aims

The purpose of this study was to investigate the effects of IGF1R knockdown on the proliferation, apoptosis and chemosensitivity of pancreatic cancer cells, and explore the possible mechanisms.

Methods

Pancreatic cancer cells expressing IGF1R shRNA were established, and the cell proliferation, colony formation, and chemosensitivity to gemcitabine were examined in vitro. The activation of AKT and NF-κB was detected by Western blot analysis and luciferase assay, respectively. Xenograft mice models were established to evaluate the in vivo anti-tumor effects of IGF1R knockdown.

Results

IGF1R knockdown notably inhibited pancreatic cancer cell proliferation and colony formation, induced apoptosis, and inhibited xenograft tumor growth. Moreover, IGF1R knockdown significantly enhanced chemosensitivity to gemcitabine in pancreatic cancer cells, and this was correlated with the inhibition of PI3K/AKT and NF-κB pathways.

Conclusions

IGF1R knockdown suppresses tumor growth and enhances chemosensitivity in pancreatic cancer via the inhibition of PI3K/AKT and NF-κB pathways, and is a promising approach to overcome the chemoresistance of pancreatic cancer.     

Sodium taurocholate cotransporting polypeptide mediates dual actions of deoxycholic acid in human hepatocellular carcinoma cells: enhanced apoptosis versus growth stimulation

Purpose

The hydrophobic bile acid, deoxycholic acid (DC), can induce apoptosis in hepatocytes. The roles of DC and its transporter are not yet established in hepatocellular carcinoma (HCC) cells. We investigated DC-induced alterations in HCC cell growth, with a particular focus on the effect of the expression of bile acid (BA)-transporting Na⁺-dependent taurocholic cotransporting polypeptides (NTCPs).

Methods

We determined NTCP expression in four human HCC cell lines: Huh-BAT, Huh-7, SNU-761, and SNU-475. NTCP expression and apoptotic signaling cascades were examined by immunoblot analyses. Cell viability was assessed using the 3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt assay. Wound healing and invasion assays were performed to evaluate cell migration and invasion abilities. Real-time polymerase chain reaction was performed to measure IL-8 expression levels. Nuclear factor kappa B (NF-κB) activity was evaluated by enzyme-linked immunosorbent assay.

Results

The HCC cell lines revealed varying NTCP expression levels, and DC treatment had dual effects, depending on NTCP expression. DC induced apoptosis in NTCP-positive HCC cells, especially under hypoxic conditions. In NTCP-negative HCC cells, simultaneous treatment with DC and cyclooxygenase inhibitor markedly decreased aggressive cellular behaviors via the inhibition of NF-κB/COX-2/IL-8 pathways.

Conclusion

Hydrophobic bile acid offers therapeutic potential for patients with advanced HCC via different mechanisms depending on NTCP expression levels within the tumor.     

Abnormal Expression of Toll-Like Receptor 4 Is Associated with Susceptibility to Ethanol-Induced Gastric Mucosal Injury in Mice

Background and Aims

Toll-like receptor 4 (TLR4) contributes to ethanol-induced gastric mucosal injury. This study aimed to determine its precise role in this pathogenic state and the related signaling pathway.

Methods

Ethanol-induced gastric mucosal injury models were generated in TLR4^?/? mice (C3H/HeJ: point mutation; C57BL/10ScNJ: gene deletion), their respective TLR4^+/+ wild-type counterparts, and heterozygous TLR4^+/? mice. Lipopolysaccharide (LPS) or pyrrolidine dithiocarbamate (PDTC) was injected intraperitoneally 1 h or 30 min before ethanol administration. At 1 h post-ethanol treatment, gastric or serum specimens were evaluated.

Results

Ethanol intra-gastric administration induced significant gastric mucosal injury in all mice, but the damaged area was larger in TLR4^?/? mice. LPS preconditioning and up-regulated TLR4 expression led to significantly larger areas of gastric mucosal damage. Upon ethanol administration, TLR4^+/+, and not TLR4^?/?, mice showed significant increases in TLR4, myeloid differentiation factor 88 (MyD88), cytoplasmic high mobility group box 1 (HMGB1), and nuclear factor-kappa B p65 (NF-κB p65). PDTC pretreatment significantly attenuated the ethanol-induced gastric mucosal damaged areas, inhibited nuclear NF-κB p65 expression, and suppressed HMGB1 translocation out of the nucleus. In addition, PDTC pretreatment reduced ethanol-stimulated expression of the inflammatory modulators, interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), in serum.

Conclusions

Both deficient and excessive expression of TLR4 promotes ethanol-induced gastric mucosal injury. The underlying mechanism involves the MyD88/NF-κB signaling pathway and the HMGB1, TLR4 activator ligand. The increased expression of HMGB1 may lead to increased secretion and binding to TLR4, further stimulating the TLR4/MyD88/NF-κB signaling pathway and aggravating the ethanol-induced gastric mucosal injury.     

Chimeric 5/35 adenovirus-mediated Dickkopf-1 overexpression suppressed tumorigenicity of CD44+ gastric cancer cells via attenuating Wnt signaling

Background

Gastric cancer stem cells (CSCs), which require activation of Wnt signaling to maintain their self-renewal and tumorigenicity, are proposed to be critical targets for effective therapy of gastric carcinomas. Gene therapies that are delivered by adenovirus of serotype 5 (Ad5) or chimeric 5/35(Ad5/35) adenovirus have shown promise for treating various cancers. Here we aimed to develop a gene therapy strategy that targeted gastric CSCs (CD44⁺ cells).

Methods

CD44⁺ cells were isolated by fluorescence activated cell sorting from both primary gastric cancer cells and cell lines. Expression of adenovirus receptors was examined in CD44⁺ and CD44^? cells. A potent Wnt antagonist Dickkopf-1 (DKK1) was delivered into CD44⁺ cells using Ad5/35 (Ad5/35-DKK1). The therapeutic outcomes were evaluated.

Results

Expression of Coxsakievirus adenovirus receptor for Ad5 was significantly reduced, while abundance of CD46, the receptor for Ad5/35, was slightly higher in CD44⁺ cells. Accordingly, CD44⁺ cells were sensitive to Ad5/35 infection, but not to Ad5. Ad5/35-DKK1 introduced DKK1 into CD44⁺ cells and deactivated endogenous Wnt/β-catenin signaling efficiently. Overexpression of DKK1 inhibited survival, anchorage-independent colony formation, and invasion of CD44⁺ cells, which were restored by a GSK-3 specific inhibitor BIO-acetoxime. More importantly, introduction of DKK1 abrogated the tumorigenicity of CD44⁺ cells in vivo. However, Ad5/35-DKK1 only showed minimal cytotoxicity to normal tissue-derived cells, L-02 and GES-1.

Conclusions

We developed, for the first time, a novel Ad5/35-DKK1-based approach to abrogate Wnt signaling in CSCs and demonstrated that gastric CSC-targeting gene therapy was effective in preclinical experiments.     

Deguelin, an Akt Inhibitor, Down-Regulates NF-κB Signaling and Induces Apoptosis in Colon Cancer Cells and Inhibits Tumor Growth in Mice

Background

Deguelin, a naturally occurring rotenoid, is known to be an Akt inhibitor and to have an anti-tumor effect on several cancers.

Aims

This study was performed to elucidate the effect of deguelin on apoptotic pathways related to NF-κB signaling in colon cancer cells and on the anti-tumor effect in colon cancer xenograft mice.

Methods

We studied COLO 205 and HCT116 cells in the presence or absence of deguelin. NF-κB signaling was examined by real-time RT-PCR for interleukin (IL)-8, by Western blotting for IκB phosphorylation/degradation, and by the electrophoretic mobility shift assay. Cell death was determined by the MTT assay, and apoptosis by Annexin V-FITC staining and caspase-3 activity. We also assessed the expression of antiapoptotic and proapoptotic factors by use of RT-PCR. In colon cancer xenograft mice, we evaluated the effect of deguelin on inoculated tumor growth, and apoptotic index was measured by the in vivo TUNEL assay.

Results

Deguelin significantly inhibited IL-8 gene expression, IκB phosphorylation/degradation, and DNA binding activity of NF-κB in colon cancer cells. Deguelin induced cell death and apoptosis in colon cancer cells in a dose and time-dependent manner. Deguelin down-regulated expression of NF-κB-mediated antiapoptotic factors such as cFLIP, Bcl-2, and Bcl-X_L. In the colon cancer xenograft model, the volume of the tumor treated with deguelin was significantly lower than that of the control, and the apoptotic index for deguelin-treated mice was much higher.

Conclusion

Deguelin might be a potential therapeutic agent for treatment of colorectal cancer.     

The Noncytotoxic Dose of Sorafenib Sensitizes Bel-7402/5-FU Cells to 5-FU by Down-Regulating 5-FU-Induced Nrf2 Expression

Background

Acquired resistance to 5-fluorouracil (5-FU) is a serious therapeutic obstacle in advanced hepatocellular carcinoma (HCC) patients.

Aim

To investigate whether nuclear factor erythroid 2-related factor 2 (Nrf2) was associated with drug resistance in 5-FU resistant Bel-7402 (Bel-7402/5-FU) cells, and if sorafenib, an oral multikinase inhibitor targeting the tumor and vasculature, could reverse drug resistance in Bel-7402/5-FU cells at the noncytotoxic dosage.

Methods

We used MTT to detect the resistance reversal activity of sorafenib, compared Nrf2 expression in various conditions by western blot and qRT-PCR, and analyzed subcellular localization of Nrf2 by immunofluorescence.

Results

The endogenous expression of Nrf2 in Bel-7402/5-FU cells was similar to that observed in Bel-7402 cells. However, Nrf2 expression levels were increased by 5-FU treatment in Bel-7402/5-FU cells higher than that in Bel-7402 cells, which is to highlight the Nrf2 contribution to the enhanced resistance of Bel-7402/5-FU cells to 5-FU. Moreover, intracellular Nrf2 protein level was significantly down-regulated by Nrf2-shRNA in Bel-7402/5-FU cells, resulting in partial reversal of 5-FU resistance. Sorafenib down-regulated the increased expression of Nrf2 induced by 5-FU treatment and partly reversed 5-FU resistance in Bel-7402/5-FU cells.

Conclusions

These results suggested that more sensitive cell defense mediated by Nrf2 was associated with drug resistance of Bel-7402/5-FU cells. Sorafenib reversed drug resistance, and its reversal mechanism might be due to the suppression of Nrf2 expression induced by 5-FU, indicating the feasibility of using Nrf2 inhibitors to increase efficacy of chemotherapeutic drugs in HCC patients.     

CD34+ Hematopoietic Stem Cells Mobilization, Paralleled with Multiple Cytokines Elevated in Patients with HBV-Related Acute-on-Chronic Liver Failure

Background

Recent studies indicate that bone marrow (BM)-derived stem cells contribute to liver regeneration. But limited information is available on the dynamic and mechanisms of mobilization of BM-derived hematopoietic stem cells (HSCs) after acute-on-chronic liver failure (ACLF).

Aims

The purpose of this study was to assess the mobilization of BM-derived CD34+ HSCs in ACLF patients, and elucidate the association of stress-induced cytokines in HSCs mobilization and/or liver repair in ACLF patients.

Methods

Thirty patients with HBV-related ACLF, 30 patients undergoing chronic hepatitis B, and 20 healthy controls were enrolled. The percentages of peripheral blood CD34+ cells were determined by two-color flow cytometry. The hepatic commitment of mobilized CD34+ cells was investigated by RT-PCR. The serum levels of stress-induced cytokines were determined by enzyme-linked immunosorbent assays.

Results

A significant increase of circulating CD34+ cells was observed in ACLF patients. RT-PCR analyses showed that the mobilized CD34+ cells expressed both CD34 mRNA and liver-specific markers including cytokeratin 19 and α-fetoprotein. In parallel with mobilization of BM-derived CD34+ cells, elevated serum levels of hepatocyte growth factor, interleukin-6, stem cell factor, granulocyte colony-stimulating factor and matrix metalloproteinase 9 were observed in ACLF patients.

Conclusion

We demonstrated that ACLF led to mobilization of CD34+ cells, which had a hepatic differentiation potential.     

Human gastric cancer organizes neighboring lymphatic vessels via recruitment of bone marrow-derived lymphatic endothelial progenitor cells

Background

Lymphatic metastasis is a critical determinant of prognosis in human gastrointestinal cancers. Studies suggest that lymphatic metastasis has been linked to lymphangiogenesis, the growth of lymphatic vessels, while the mechanisms of tumor lymphangiogenesis remain poorly characterized.

Methods

Human gastric cancer cells, MKN45, were implanted under the gastric submucosa of nude mice receiving green fluorescent protein-positive bone marrow (BM) transplants. In addition, MKN45 cells were subcutaneously injected into the back of each mouse as a model of human tumor xenografts. The tumor tissue was analyzed 3?weeks after implantation.

Results

The mice with MKN45 cells represent recruitment and incorporation of BM-derived lymphatic endothelial progenitor cells (LEPC) into gastric lymphatics. Moreover, in a xenograft model, MKN45 cells induced lymphangiogenesis as well as recruitment of BM-derived LEPC in tumor lymphatics in a xenograft model.

Conclusions

These findings of this study suggest that human gastric adenocarcinoma induces tumor lymphangiogenesis via recruitment of LEPC from BM.     

Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Cancer Cells Promotes Cell Survival and Proliferation in Hepatocellular Carcinoma

Background

Recent studies have shown that toll-like receptor 4 (TLR4) is involved in hepatocarcinogenesis. However, the significance of TLR4 signaling in cancer development and progression remains unclear.

Aim

The purpose of this study was to investigate the role of TLR4 in cancer cell survival and proliferation in hepatocellular carcinoma (HCC).

Methods

Fifty-three HCC and ten normal liver specimens were analyzed by immunohistochemistry, and three cell lines (HL-7702, PLC/PRF/5 and HepG2) were used for in vitro studies. Lipopolysaccharide (LPS), a specific ligand of TLR4, was used to activate TLR4 signaling. The effects of LPS-TLR4 signaling on cell survival, proliferation and invasion were examined. Specific inhibitors of NF-κB and MAPK (JNK, ERK and p38) signaling pathways were used to explore the role of each pathway in LPS-TLR4 signaling.

Results

TLR4 was overexpressed in HCC cell lines and in human HCC tissues, where it correlated with Ki-67 expression. LPS-induced activation of TLR4 signaling promoted cancer cell survival and proliferation. LPS-TLR4 signaling was associated with regulation on the activation of NF-κB and MAPK signaling pathways. LPS-TLR4-induced activation of ERK and JNK signaling promotes cell proliferation through regulating Bax translocation to mitochondria. Activation of NF-κB and p38 mediates cytotoxicity of LPS, and inhibition on these two pathways promotes cell proliferation in HCC cells.

Conclusion

Our results indicate that TLR4 signaling in cancer cells promotes cell survival and proliferation in HCC.     

Sulforaphane protects H9c2 cardiomyocytes from angiotensin II-induced hypertrophy

Background

Cardiac hypertrophy is an adaptive process of the heart in response to various stimuli, but sustained cardiac hypertrophy will finally lead to heart failure. Sulforaphane—extracted from cruciferous vegetables of the genus Brassica such as broccoli, brussels sprouts, and cabbage—has been evaluated for its anticarcinogenic and antioxidant effects.

Aims

To investigate the effect of sulforaphane on angiotensin II (Ang II)-induced cardiac hypertrophy in vitro.

Methods

Embryonic rat heart-derived H9c2 cells were co-incubated with sulforaphane and Ang II. The cell surface area and mRNA levels of hypertrophic markers were measured to clarify the effect of sulforaphane on cardiac hypertrophy. The underlying mechanism was further investigated by detecting the activation of Akt and NF-κB signaling pathways.

Results

We found that H9c2 cells pretreated with sulforaphane were protected from Ang II-induced hypertrophy. The increasing mRNA levels of ANP, BNP, and β-MHC in Ang II-stimulated cells were also down-regulated after sulforaphane treatment. Moreover, sulforaphane repressed the Ang II-induced phosphorylation of Akt, GSK3β, mTOR, eIF4e, as well as of IκBα and NF-κB.

Conclusion

Based on our results, sulforaphane attenuates Ang II-induced hypertrophy of H9c2 cardiomyocytes mediated by the inhibition of intracellular signaling pathways including Akt and NF-κB.     

An unbalanced monocyte polarisation in peripheral blood and bone marrow of patients with type 2 diabetes has an impact on microangiopathy

Aim/hypothesis

Monocytes/macrophages play important roles in adipose and vascular tissues and can be polarised as inflammatory M1 or anti-inflammatory M2. We sought to analyse monocyte polarisation status in type 2 diabetes, which is characterised by chronic inflammation.

Methods

We enrolled 60 individuals without diabetes and 53 patients with type 2 diabetes. We quantified standard monocyte subsets defined by cluster of differentiation (CD)14 and CD16. In addition, based on the phenotype of polarised macrophages in vitro, we characterised and quantified more definite M1 (CD68⁺CCR2⁺) and M2 (CX3CR1⁺CD206⁺/CD163⁺) monocytes. We also analysed bone marrow (BM) samples and the effects of granulocyte-colony stimulating factor (G-CSF) stimulation in diabetic and control individuals.

Results

We found no alterations in standard monocyte subsets (classical, intermediate and non-classical) when comparing groups. For validation of M1 and M2 phenotypes, we observed that M2 were enriched in non-classical monocytes and had lower TNF-α content, higher LDL scavenging and lower transendothelial migratory capacity than M1. Diabetic patients displayed an imbalanced M1/M2 ratio compared with the control group, attributable to a reduction in M2. The M1/M2 ratio was directly correlated with waist circumference and HbA_1c and, among diabetic patients, M2 reduction and M1/M2 increase were associated with microangiopathy. A decrease in M2 was also found in the BM from diabetic patients, with a relative M2 excess compared with the bloodstream. BM stimulation with G-CSF mobilised M2 macrophages in diabetic but not in healthy individuals.

Conclusions/interpretation

We show that type 2 diabetes markedly reduces anti-inflammatory M2 monocytes through a dysregulation in bone-marrow function. This defect may have a negative impact on microangiopathy.     

Transformation potential of bone marrow stromal cells into undifferentiated high-grade pleomorphic sarcoma

Purpose

Bone marrow adherent cells contain conventional bone marrow stromal cells and mesenchymal stem cells and these cells constitute the hematopoietic microenvironment. Mesenchymal stem cells have the capacity to give rise to multiple mesenchymal lineage cells and even ectodermal lineage cells. In the present study, we investigated what types of tumor cells are inducible from BM adherent cells by chemical carcinogens.

Methods

Bone marrow cells from neonatal C3H/HeN mice were collected within 24 h after birth and then cultured. Four days later, bone marrow adherent cells were obtained and the cells were treated with 3-methylcholanthrene.

Results

By this treatment, some transformed clones consisting of large spindle cells were obtained. The transformed cells were highly positive for CD44 and were positive for Sca-1, CD49d and CD106, whereas the cells were negative for hematolymphoid markers. The cell clones had the ability to support hematopoiesis in vitro. These results indicate that the transformed cell lines have the characteristics of BM stromal cells/mesenchymal stem cells. Moreover, during culture of the transformed cells, spontaneous bone nodule formation was observed. When the transformed cells were inoculated into immunodeficient mice subcutaneously, the neoplasms grew in the subcutaneous tissue of the mice. Microscopically and ultrastructurally, the neoplasms showed the typical morphology of undifferentiated high-grade pleomorphic sarcoma (UHGPS). Bone-related genes have been found to be expressed in both transformed cells and UHGPSs.

Conclusion

The present study suggests that UHGPSs are derived from BM stromal cells, probably mesenchymal stem cells.     

Guggulsterone enhances antitumor activity of gemcitabine in gallbladder cancer cells through suppression of NF-κB

Purpose

Patients with gallbladder cancer usually have a poor prognosis, and effective standard chemotherapeutic regimens have not been established. The anticancer activities of guggulsterone have been demonstrated in various cancer cells. The aims of the study were to determine the effect of guggulsterone on gallbladder cancer cells and to investigate whether treatment with guggulsterone influences the antitumor activities of gemcitabine.

Methods

The Dojindo Cell Counting Kit-8 assay was used to determine the inhibition of proliferation by drugs in TGBC1 and TGBC2 cells. Cell migration and invasion were examined using 24-well inserts and Matrigel?-coated invasion chambers. The activities of NF-κB p65, VEGF-C, and MMP-2 were measured by ELISA.

Results

Guggulsterone inhibited the proliferation and suppressed migration and invasion of gallbladder cancer cells in a dose-dependent manner. Guggulsterone significantly decreased NF-κB p65, VEGF-C, and MMP-2 activities in the gallbladder cancer cells examined. Gallbladder cancer cells treated with a combination of guggulsterone and gemcitabine demonstrated significant inhibition of cell proliferation and invasion when compared to treatment with gemcitabine alone. In addition, NF-κB p65 activation decreased significantly in cells treated with a combination of guggulsterone and gemcitabine when compared to treatment with gemcitabine alone.

Conclusions

Guggulsterone exhibits anticancer activities and enhances the antitumor activities of gemcitabine through the suppression of NF-κB activation in gallbladder cancer cells. These results suggest that guggulsterone could be a potential therapeutic option for patients with gallbladder cancer.     

Increased bone density and resistance to ovariectomy‐induced bone loss in FoxP3‐transgenic mice based on impaired osteoclast differentiation

Objective

Immune activation triggers bone loss. Activated T cells are the cellular link between immune activation and bone destruction. The aim of this study was to determine whether immune regulatory mechanisms, such as naturally occurring Treg cells, also extend their protective effects to bone homeostasis in vivo.

Methods

Bone parameters in FoxP3‐transgenic (Tg) mice were compared with those in their wild‐type (WT) littermate controls. Ovariectomy was performed in FoxP3‐Tg mice as a model of postmenopausal osteoporosis, and the bone parameters were analyzed. The bones of RAG‐1^–/– mice were analyzed following the adoptive transfer of isolated CD4+CD25+ T cells. CD4+CD25+ T cells and CD4+ T cells isolated from FoxP3‐Tg mice and WT mice were cocultured with monocytes to determine their ability to suppress osteoclastogenesis in vitro.

Results

FoxP3‐Tg mice developed higher bone mass and were protected from ovariectomy‐induced bone loss. The increase in bone mass was found to be the result of impaired osteoclast differentiation and bone resorption in vivo. Bone formation was not affected. Adoptive transfer of CD4+CD25+ T cells into T cell–deficient RAG‐1^–/– mice also increased the bone mass, indicating that Treg cells directly affect bone homeostasis without the need to engage other T cell lineages.

Conclusion

These data demonstrate that Treg cells can control bone resorption in vivo and can preserve bone mass during physiologic and pathologic bone remodeling.