Chunmin Liang

Local expression of secondary lymphoid tissue chemokine delivered by adeno-associated virus within the tumor bed stimulates strong anti-liver tumor immunity

ABSTRACT Development of an effective antitumor immune response depends on the appropriate interaction of effector and target cells. Thus, the expression of chemokines within the tumor may induce a more potent antitumor immune response. Secondary lymphoid tissue chemokine (SLC) is known to play a critical role in establishing a functional microenvironment in secondary lymphoid tissues. Its capacity to attract dendritic cells (DCs) and colocalize them with T cells makes it a good therapeutic candidate against cancer. In this study, we used SLC as a treatment for tumors established from a murine hepatocellular carcinoma model. SLC was encoded by recombinant adeno-associated virus (rAAV), a system chosen for the low host immunity and high efficiency of transduction, enabling long-term expression of the gene of interest. As a result, rAAV-SLC induced a significant delay of tumor progression, which was paralleled by a profound infiltration of DCs and activated CD4+ T cells and CD8+ T cells (CD3+ CD69+ cells) into the tumor site. In addition, rAAV-SLC treatment was also found to reduce tumor growth in nude mice, most likely due to inhibition of neoangiogenesis. In conclusion, local expression of SLC by rAAV represents a promising approach to induce immune-mediated regression of malignant tumors.

Detection of Aβ Plaques by a Novel Specific MRI Probe Precursor CR-BSA-(Gd-DTPA)n in APP/PS1 Transgenic Mice

Amyloid plaques are one of the hallmarks of Alzheimers disease (AD). The lack of specific probes that can detect individual senile plaques in AD has prompted the development of magnetic resonance imaging (MRI) probes. In this study, based on DTPA‐gadolinium (III) and congo red (CR), a novel specific MRI probe precursor CR‐BSA‐(Gd‐DTPA)n was successfully synthesized. Its ability to bind to amyloid plaques was evaluated by brain sections from APP/PS1 transgenic mice. Its specificity for Aβ plaques was further demonstrated by immunohistochemistry (IHC) staining with the monoclonal antibody to the Aβ protein. Meanwhile, the amyloid deposits detected by the CR‐BSA‐(Gd‐DTPA)n were matched to the amyloid deposits detected by Aβ specific antibody. We also found that a few amyloid‐like deposits which was not detected by IHC. The findings indicated that the probe perhaps could detect the neurofibrillary tangles (NFT) similar to the effect of CR itself, and this will be verified in future experiments. The works suggested that the Aβ protein‐specific magnetic resonance contrast agent precursor CR‐BSA‐(Gd‐DTPA)n can be used as a potential fluorescence and MR multi‐modal imaging probe precursor to display individual senile plaques in AD. Anat Rec, 2010.

Overexpression of suppressor of cytokine signaling 1 in islet grafts results in anti-apoptotic effects and prolongs graft survival

AIMS A significant portion of islet grafts are destroyed by apoptosis and fail to become functional after transplantation. Strategies that enhance islet resistance to apoptosis may prevent graft loss. The aim of this study was to investigate whether overexpression of suppressor of cytokine signaling 1 (SOCS1) in islet grafts could achieve an anti-apoptotic effect and prolong graft survival. MAIN METHODS We used a chimeric adenovirus vector (Ad5F35) to enhance SOCS1 expression in isolated rat islets, and assessed its protective action against TNF-alpha-induced apoptosis. After transplanting SOCS1-overexpressing islets into allogeneic recipients with streptozotocin-induced diabetes, graft survival and in situ apoptosis were analyzed using immunohistochemistry. KEY FINDINGS The isolated rat islets infected with Ad5F35-SOCS1 showed significantly higher SOCS1 expression than Ad5F35-EGFP and mock infected islets. The Ad5F35 transfection and SOCS1 overexpression on islets did not affect their insulin secretory function. After treatment with rat TNF-alpha and cycloheximide in vitro, Ad5F35-SOCS1 infected islets exhibited a lower apoptotic ratio than controls (Ad5F35-EGFP and mock infected islets). The diabetic recipients transplanted with Ad5F35-SOCS1 infected islets displayed longer time of normoglycemia than recipients transplanted with mock infected islets. Furthermore, histological analysis indicated that the infected grafts with local overexpression of SOCS1 showed decreased apoptosis in the early post-transplant period. SIGNIFICANCE These results demonstrate that overexpression of SOCS1 in islet grafts prior to transplantation can significantly protect them from apoptotic loss and prolong their survival. This approach might find a clinical counterpart.

Inflammatory microenvironment contributes to epithelial-mesenchymal transition in gastric cancer

Gastric cancer (GC) is the fifth most common malignancy in the world. The major cause of GC is chronic infection with Helicobacter pylori (H. pylori). Infection with H. pylori leads to an active inflammatory microenvironment that is maintained by immune cells such as T cells, macrophages, natural killer cells, among other cells. Immune cell dysfunction allows the initiation and accumulation of mutations in GC cells, inducing aberrant proliferation and protection from apoptosis. Meanwhile, immune cells can secrete certain signals, including cytokines, and chemokines, to alter intracellular signaling pathways in GC cells. Thus, GC cells obtain the ability to metastasize to lymph nodes by undergoing the epithelial-mesenchymal transition (EMT), whereby epithelial cells lose their epithelial attributes and acquire a mesenchymal cell phenotype. Metastasis is a leading cause of death for GC patients, and the involved mechanisms are still under investigation. In this review, we summarize the current research on how the inflammatory environment affects GC initiation and metastasis via EMT.

Special role of Foxp3 for the specifically altered microRNAs in Regulatory T cells of HCC patients

BackgroundRegulatory T cells (Tregs) exhibit functional abnormalities in the context of hepatocellular carcinoma (HCC). The microRNAs (miRNAs) are identified as the key modulators in Tregs. This study was to explore whether the expression profiles of miRNAs of Tregs were different in HCC-activated Tregs and whether Foxp3 had special effects on them.MethodsWe isolated HCC-activated Tregs from mice bearing HCC and compared the expression profiles of miRNAs between HCC-activated Tregs and control Tregs by microarray. RNA interference against Foxp3 was also performed through transfection of synthetic siRNAs to Tregs for analyzing the effect of Foxp3 on the expression of miRNAs. Tregs isolated from HCC patients (n = 12) and healthy controls (n = 7) were used for validation of the differentially expressed miRNAs. Finally, bioinformatic analysis was applied to infer their possible roles.ResultsWe found nine specifically altered miRNAs in HCC-activated Tregs from the murine model. After transfection with siRNAs against Foxp3, control Tregs showed obvious reduction of Foxp3 and five miRNAs were significantly changed; HCC-activated Tregs exhibited a slight reduction of Foxp3 with three miRNAs significantly changed. Tregs from HCC patients and healthy controls finally confirmed the up-regulation of four miRNAs (hsa-miR-182-5p, hsa-miR-214-3p, hsa-miR-129-5p and hsa-miR-30b-5p). Following bioinformatic analysis suggested these altered miRNAs would target eight important signaling pathways that could affect the functions of Tregs.ConclusionsOur studies provided the first evidence that Tregs in HCC had the specifically altered expression of miRNAs, which was affected by Foxp3. These results are useful both in finding new biomarkers and in further exploring the functions of Tregs in HCC patients.

Combination of SLC administration and Tregs depletion is an attractive strategy for targeting hepatocellular carcinoma

BackgroundSecondary lymphoid tissue chemokine (SLC) is a key CC chemokine for chemotaxis of immune cells and has been an attractive candidate for anti-tumor treatments. However, among the immune cells recruited by SLC to tumors, the CD25+ Foxp3+ regulatory T cells (Tregs) compromise the anti-tumor effects. In this study, we proposed the combination therapy of intratumoral co-administration of SLC and anti-CD25 monoclonal antibodies (mAbs). We hypothesized that the intratumoral injections of SLC and depletion of Tregs would have stronger inhibition effects on the progression of hepatocellular carcinoma (HCC) in mice.MethodsC57BL/6 mice were inoculated subcutaneously with the murine HCC cell line, and mice with visible tumors were treated intratumorally with SLC, SLC plus anti-CD25 mAbs or the control antibodies. The percentages of Tregs, effector CD8+ T cells and CD4+ T cells were checked in the tumors, lymph nodes, spleen and liver at regular intervals. The levels of intratumoral IL-12, IFN-γ, IL-10 and TGF-β1 were evaluated. The final anti-tumor effects were measured by the tumor volume and weight as well as the intratumoral activity of MMP2 and MMP9. Bone-marrow-derived dendritic cells were used to explore the mechanisms of maturation induced by SLC in vitro.ResultsOur experiments showed the combination therapy significantly decreased the frequency of Tregs, and increased CD8+ T cells and CD4+ T cells at tumor sites. These alterations were accompanied by an increased level of IL-12 and IFN-γ, and decreased level of IL-10 and TGF-β1. Unexpectedly, we observed a significantly decreased percentage of Tregs, and increased CD8+ T cells and CD4+ T cells in the lymph nodes, spleen and liver after the combination therapy. The growth and invasiveness of HCC was also maximally inhibited in the combination therapy compared with the SLC alone. Furthermore, we confirmed SLC induced the maturation of DCs via NF-κB p65 and this maturation would benefit the combination therapy.ConclusionsOur data demonstrated that intratumoral co-administration of SLC and anti-CD25 mAbs was an effective treatment for HCC, which was correlated with the altered tumor microenvironment and systemically optimized percentages of Tregs, CD8+ T cells and CD4+ T cells in peripheral immune organs.

The combined treatment of amyloid-β1-42-stimulated bone marrow–derived dendritic cells plus splenocytes from young mice prevents the development of Alzheimer's disease in APPswe/PSENldE9 mice

Anti-amyloid-β (Aβ) immunotherapy is a potential therapeutic strategy to reduce amyloid plaques and amyloid-associated pathologies in Alzheimers disease (AD). Immune senescence with aging has also played a crucial role in AD pathogenesis and influences the effect of anti-Aβ immunotherapy. In this study, a combined treatment of Aβ₁₋₄₂-bone marrow-derived dendritic cells (BMDCs) with intraperitoneal injection of splenocytes from young mice was designed as a novel immunotherapy for AD in APPswe/PSEN1de9 transgenic mice models. The results showed that the combined treatment not only elevated the level of anti-Aβ antibodies but also reduced amyloid plaques in brain and finally ameliorated deterioration of spatial learning and memory in AD mice. Additionally, the results revealed an increase of CD68 positive microglial cells in the vicinity of amyloid plaques in the mouse brain, which was responsible for the enhanced phagocytosis of Aβ plaques. In conclusion, the Aβ₁₋₄₂-BMDCs plus splenocytes treatment improved the phagocytosis of microglia and prevented AD pathology more effectively. This combined immunotherapy provided a promising treatment in preventing the progression of AD in clinical studies in the near future.

Chemokines and their receptors play important roles in the development of hepatocellular carcinoma

The chemokine system consists of four different subclasses with over 50 chemokines and 19 receptors. Their functions in the immune system have been well elucidated and research during the last decades unveils their new roles in hepatocellular carcinoma (HCC). The chemokines and their receptors in the microenvironment influence the development of HCC by several aspects including: inflammation, effects on immune cells, angiogenesis, and direct effects on HCC cells. Regarding these aspects, pre-clinical research by targeting the chemokine system has yielded promising data, and these findings bring us new clues in the chemokine-based therapies for HCC.

Tetraspanin-8 promotes hepatocellular carcinoma metastasis by increasing ADAM12m expression

Recent evidence indicates that tetraspanin-8 (TSPAN8) promotes tumor progression and metastasis. In this study, we explored the effects of TSPAN8 and the molecular mechanisms underlying hepatocellular carcinoma (HCC) metastasis using various HCC cell lines, tissues from 149 HCC patients, and animal models of HCC progression. We showed that elevated expression of TSPAN8 promoted HCC invasion in vitro and metastasis in vivo, but did not influence HCC cell proliferation in vitro. Increased TSPAN8 expression in human HCC was predictive of poor survival, and multivariate analyses indicated TSPAN8 expression to be an independent predictor for both postoperative overall survival and relapse-free survival. Importantly, TSPAN8 enhanced HCC invasion and metastasis by increasing ADAM12m expression. We therefore conclude that TSPAN8 and ADAM12m may be useful therapeutic targets for the prevention of HCC progression and metastasis.

SLC/CCR7 Stimulates the Proliferation of BMDCs by the pNF-κB p65 Pathway

The chemokine receptor CCR7 is highly expressed in dendritic cells (DCs), T cells, and other immune effector cells. One of the high‐affinity ligand that can bind to CCR7 is the secondary lymphoid tissue chemokine (SLC). The SLC/CCR7 axis plays an important role in the immune system by inducing the chemotaxis and migration of immune effector cells. In this study, we examined the effect of SLC at different concentrations (0, 50, 100, 200, 300, and 400 ng/mL) on the proliferation of bone‐marrow‐derived dendritic cells (BMDCs). ELC (CCL19), another high‐affinity ligand for CCR7, was used as the control at the same time. We found that SLC directly stimulated the proliferation of BMDCs and enhanced the antigen‐presenting function and CCR7 expression. Western blot analysis showed that pNF‐κBp65 was involved in this mechanism. We also found that the NF‐κB inhibitor PDTC could specifically block the proliferation and CCR7 expression of BMDCs induced by SLC or ELC (200 ng/mL). The results suggested that there were cross‐talk signals between the chemotaxis and proliferation of BMDCs involving the SLC/CCR7 axis. Anat Rec, 2010.