Targeting WDxR motif reprograms immune microenvironment and inhibits hepatocellular carcinoma progression.

The WD-repeat (WDR) family affects carcinogenesis, but its role in the immune microenvironment is poorly characterized. Although functional loss or gain of WDR6 does not markedly change in vitro proliferative and invasive capacity of HCC cells, its deficiency in hepa1-6 cells drastically inhibits the growth and lung metastasis of orthotopically implanted tumors in immune-competent C57BL/6J mice. Mechanistically, WDR6 targets tumor suppressor UVRAG to the CUL4A-DDB1-ROC1 E3 ubiquitin ligase complex through a unique WDxR motif and promotes its degradation. This upregulates chromatin accessibility at the TNFα locus by blocking autophagic degradation of p65, elevates intratumoral myeloid-derived suppressor cell (MDSC) number, and reduces CD8+ T cell infiltration, thereby promoting HCC progression. These immunosuppressive effects are reversed by TNFα blockade. TNFα recruits NF-κB to activate the transcription of WDR6, establishing a WDR6-TNFα loop. Clinically, the WDR6/UVRAG/NF-κB pathway is hyperactivated in HCC, predicting a poor prognosis. Importantly, a WDxR-like peptide disrupts the WDR6/UVRAG complex and enhances the efficiency of anti-PD-L1 against HCC with WDR6 dysregulation.

Human gingival mesenchymal stem cells improve movement disorders and tyrosine hydroxylase neuronal damage in Parkinson disease rats.

BACKGROUND AIMS: Gingival mesenchymal stem cells (GMSCs) demonstrate high proliferation, trilineage differentiation and immunomodulatory properties. Parkinson disease (PD) is the second most common type of neurodegenerative disease. This study aimed to explore the effect and mechanism of GMSC-based therapy in 6-hydroxydopamine-induced PD rats. METHODS: RNA sequencing and quantitative proteomics technology was used to validate the neuroprotective role of GMSCs therapeutic in 6-Hydroxydopamine -induced PD model in vitro and in vivo. Western blotting, immunofluorescence and real-time quantitative PCR verified the molecular mechanism of GMSCs treatment. RESULTS: Intravenous injection of GMSCs improved rotation and forelimb misalignment behavior, enhanced the anti-apoptotic B-cell lymphoma 2/B-cell lymphoma 2-associated X axis, protected tyrosine hydroxylase neurons, decreased the activation of astrocytes and reduced the astrocyte marker glial fibrillary acidic protein and microglia marker ionized calcium-binding adaptor molecule 1 in the substantia nigra and striatum of PD rats. The authors found that GMSCs upregulated nerve regeneration-related molecules and inhibited metabolic disorders and the activation of signal transducer and activator of transcription 3. GMSCs showed a strong ability to protect neurons and reduce mitochondrial membrane potential damage and reactive oxygen species accumulation. The safety of GMSC transplantation was confirmed by the lack of tumor formation following subcutaneous transplantation into nude mice for up to 8 weeks. CONCLUSIONS: The authors' research helps to explain the mechanism of GMSC-based therapeutic strategies and promote potential clinical application in Parkinson disease.

Transfer of Tumor-Bearing Mice Intestinal Flora Can Ameliorate Cognition in Alzheimer's Disease Mice.

BACKGROUND: Fecal microbiota transplant (FMT) is a potential treatment approach for many diseases. Alzheimer's disease (AD) and cancer have been proven to have a specific antagonistic relationship to FMT. OBJECTIVE: This article aims to explore whether intestinal flora transplantation from cancer individuals can ameliorate cognitive impairment. METHODS: Morris water maze and object recognition tests were performed to assess cognitive function after the fecal flora from tumor-bearing and WT mice were transplanted into AD mice by gavage. The effect of flora transplantation on AD was analyzed by thioflavin T staining, western blot, and 16S RNA sequencing. RESULTS: AD mice with FMT significantly improved short-term memory level and cognitive ability compared with Tg + NaCl group. Inflammatory factors in the plasma were regulated, and Aß plaques burden in the hippocampus and cortex were decreased. FMT in the tumor-bearing group showed a higher significant amelioration in symptoms compared to the healthy group. 16S RNA sequencing revealed that FMT treatments could reverse the increased Firmicutes and Prevotella and the decreased Bacteroidetes, Bacteroides, and Sutterella in AD mice. AD mice transplanted with tumor-bearing mice feces additionally increased the density of Oscillospira, Odoribacter, and AF12. Furthermore, the predicted functional analyses showed that the metabolism of inorganic and organic salts in the intestinal flora of AD mice was also reversed by FMT. CONCLUSION: Intestinal flora transplantation from tumor-bearing mice can ameliorate the cognitive impairment of AD mice.

Stem Cells from Human Exfoliated Deciduous teeth Promote Hair Regeneration in Mouse.

Stem cells in different types may interact with each other to maintain homeostasis or growth and the interactions are complicated and extensive. There is increasing evidence that mesenchymal-epithelial interactions in early morphogenesis stages of both tooth and hair follicles show many similarities. In order to explore whether stem cells from one tissue could interact with cells from another tissue, a series of experiments were carried out. Here we successfully extracted and identified stem cells from human exfoliated deciduous teeth (SHED) of 8-12 years old kids, and then found that SHED could promote hair regeneration in a mouse model. In vitro, SHED shortened the hair regeneration cycle and promoted the proliferation and aggregation of dermal cells. In vivo, when SHED and skin cells of C57 mice were subcutaneously co-transplanted to nude mice, more hair was formed than skin cells without SHED. To further explore the molecular mechanism, epidermal and dermal cells were freshly extracted and co-cultured with SHED. Then several signaling molecules in hair follicle regeneration were detected and we found that the expression of Sonic Hedgehog (Shh) and Glioma-associated oncogene 1 (Gli1) was up-regulated. It seems that SHED may boost the prosperity of hairs by increase Shh/Gli1 pathway, which brings new perspectives in tissue engineering and damaged tissue repairing.

Metformin enhances the osteogenesis and angiogenesis of human umbilical cord mesenchymal stem cells for tissue regeneration engineering.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a potential clinical material in regenerative medicine applications. Metformin has shown safety and effectiveness as a clinical drug. However, the effect of metformin as a treatment on hUC-MSCs is unclear. Our research aimed to explore the effects of metformin on the osteogenesis, adipogenesis and angiogenesis of hUC-MSCs, and attempted to explain the molecular fluctuations of metformin through the mapping of protein profiles. Proliferation assay, osteogenic and adipogenic differentiation induction, cell cycle, flow cytometry, quantitative proteomics techniques and bioinformatics analysis were used to detect the influences of metformin treatment on hUC-MSCs. Our results demonstrated that low concentrations of metformin promoted the proliferation of hUC-MSCs, but high concentrations of metformin inhibited it. Metformin exhibited promotion of osteogenesis but inhibition of adipogenesis. Metformin treated hUC-MSCs up-regulated the expression of osteogenic marker ALP, OCN and RUNX2, but down-regulated the expression of adipogenic markers PPARγ and LPL. Proteomics analysis found that up-regulation of differentially expressed proteins in metformin treatment group involved the biological process of cell migration in Gene Ontology analysis. Metformin enhanced cell migration of HUVEC in a co-culture system, and hUC-MSCs treated with metformin exhibited stronger angiogenesis in vitro and in vivo compared to the hUC-MSCs group. The results of RT-qPCR revealed that the SCF and VEGFR2 were raised in metformin treatment. This study can promote the application of hUC-MSCs treated with metformin to tissue engineering for vascular reconstruction and angiogenesis.

The Application Potential and Advance of Mesenchymal Stem Cell-Derived Exosomes in Myocardial Infarction.

Myocardial infarction (MI) is a devastating disease with high morbidity and mortality caused by the irreversible loss of functional cardiomyocytes and heart failure (HF) due to the restricted blood supply. Mesenchymal stem cells (MSCs) have been emerging as lead candidates to treat MI and subsequent HF mainly through secreting multitudinous factors of which exosomes act as the most effective constituent to boost the repair of heart function through carrying noncoding RNAs and proteins. Given the advantages of higher stability in the circulation, lower toxicity, and controllable transplantation dosage, exosomes have been described as a wonderful and promising cell-free treatment method in cardiovascular disease. Nowadays, MSC-derived exosomes have been proposed as a promising therapeutic approach to improve cardiac function and reverse heart remodeling. However, exosomes' lack of modification cannot result in desired therapeutic effect. Hence, optimized exosomes can be developed via various engineering methods such as pharmacological compound preconditioned MSCs, genetically modified MSCs, or miRNA-loaded exosomes and peptide tagged exosomes to improve the targeting and therapeutic effects of exosomes. The biological characteristics, therapeutic potential, and optimizing strategy of exosomes will be described in our review.

Proteomic profile of human stem cells from dental pulp and periodontal ligament.

Background The study of molecular profiling of dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) contributes to understanding the high proliferation ability and multi-lineage differentiation potential. Objectives The aim of the study was to compare the protein abundance and specific markers of DPSCs and PDLSCs by protein profiles. Material and methods The DPSCs and PDLSCs extracted from the same tooth were lysed with 3 biological replicates and the protein was collected. Two-dimensional electrophoresis technology and TMT proteomics were used to separate and identify proteins. The data are available via ProteomeXchange with identifier PXD021997. The RT-qPCR detection of mRNA expression revealed a special marker for distinguishing two kinds of dental stem cells. Results Compared with PDLSCs, 962 differential proteins (DAPs) were up-regulated, and 127 were down-regulated in DPSCs. In the up-regulated DAPs, two high-scoring sub-networks were detected for neural-related molecules, which encode cell vesicle transport and mitochondrial energy transfer to regulate cell proliferation and secretion factors. A large number of cell adhesion molecules were distinguished among the highly expressed molecules of PDLSCs, supporting that stem cells provide cell attachment functions. It was interpreted ENPL, HS90A and HS90B were highly expressed in DPSCs, while CKB was highly abundant in PDLSCs. Another cell group confirmed that these molecules can be used as special biomarkers to identify and distinguish between DPSCs and PDLSCs. Conclusions This study can promote the basic research and clinical application of dental stem cells. Significance The high-throughput protein profiles were tested by combining two-dimensional gel proteomics and TMT-based proteomics. The proteomics of DPSCs and PDLSCs without individual difference demonstrated an accurate and comprehensive molecular expression profiles and interpretation of neural application potential, this study promotes the basic research of dental stem cells and clinical application.

Calreticulin as a special marker to distinguish dental pulp stem cells from gingival mesenchymal stem cells.

The construction of protein abundance profiles helps to interpret the clinical applications of stem cells. Dental pulp stem cells (DPSCs) and gingival mesenchymal stem cells (GMSCs) can be isolated from teeth and used as a highly convenient clinical potential material. Here, we aimed to explore commonalities and differences of DPSCs and GMSCs at the protein level. TMT-based quantitative proteomics and two-dimensional gel electrophoresis technology were used in combination to describe the protein profile of DPSCs and GMSCs extracted from the same donor. A total of 2821 proteins were identified by LC-MS/MS, of which 248 differentially abundant proteins (DAPs) were highly expressed in GMSCs while 782 proteins were highly expressed in DPSCs. The biological functions and molecular pathways of DAPs were annotated with GO enrichment and KEGG analysis. The relationship between molecular abundance and cell characteristics including source, proliferation, angiogenesis and inflammation were connected by WGCNA. Special markers, including Calreticulin (CALR), Annexin A5 (ANXA5) and Rho GDP dissociation inhibitor alpha (GDIR1), were proposed to distinguish DPSCs from GMSCs. Our results provide a molecular basis for in-depth understanding of the protein composition and special functions of dental stem cells, and promote the potential clinical application.

hUC-MSCs ameliorated CUMS-induced depression by modulating complement C3 signaling-mediated microglial polarization during astrocyte-microglia crosstalk.

BACKGROUND: Major depressive disorder (MDD) has been shown to be related to immune inflammation and the complement system. Previous studies have suggested that human umbilical cord mesenchymal stem cells (hUC-MSCs) play an important role in inflammatory diseases. METHODS: hUC-MSCs were administered into chronic unpredictable mild stress model (CUMS) mice through the tail vein once a week for 4 weeks. After the administration of hUC-MSCs, the depression-like and anxiety-like phenotypes, neuronal histopathology, synaptic-related protein expression and inflammatory index of the mice were assessed. Microglial M1/M2 polarization and the expression of C3a in astrocytes and C3aR in microglia was detected by immunofluorescence co-localization. Then, CUMS mice were injected with a C3aR antagonist, and the expression of C3a and C3aR and microglial polarization were observed. RESULTS: Based on the sucrose preference and tail suspension tests, hUC-MSCs ameliorated the depression-like behaviors of CUMS mice. Additionally, the anxiety-like behaviors of CUMS mice in the open-field and plus-maze tests were improved after the administration of hUC-MSCs. hUC-MSCs altered microglia polarization by alleviating complement C3a-C3aR signaling activation, which decreased pro-inflammatory factor levels and increased anti-inflammatory factor levels, alleviating neuronal damage and synaptic deficits. CONCLUSION: hUC-MSCs have therapeutic effects on anxiety-like and depressive-like phenotypes caused by CUMS. They can alter the polarization of microglia by inhibiting C3a-C3aR signaling to reduce neuroinflammation.

Peripheral vessel and air bronchograms for detecting the pathologic patterns of subsolid nodules.

PURPOSE: To assess the relationships of subsolid nodules (SSNs) with peripheral vessels and aerated bronchi using computed tomography (CT), and to correlate the imaging features with the benign/malignant pathological diagnoses. METHODS: This study retrospectively analyzed data from 83 patients with a solitary SSN (January 2008 to December 2016). SSNs were imaged (LightSpeed 64-slice spiral CT, General Electric, USA), their mean diameter determined, and the relationship with peripheral vessels (types I-IV) and aerated bronchi (types I-V) were classified. Pathologic diagnoses were obtained from the surgical specimens. RESULTS: SSNs were diagnosed as benign (n = 29), pre-invasive (n = 9), micro-invasive adenocarcinoma (n = 7) and invasive adenocarcinoma (n = 38). SSN size, peripheral vessel class and aerated bronchus class differed between pathologic types (P < 0.05). For benign SSNs, peripheral vessel type II (58.6%) was most common, followed by III (20.7%) and IV (6.9%). Aerated bronchus type V (65.5%) was most frequent, followed by IV (27.6%); type I aerated bronchus was not observed. No cases of micro-invasive or invasive adenocarcinoma were peripheral vessel type I or aerated bronchus type V. For invasive adenocarcinoma, 92.1% were peripheral vessel types III + IV while 71.8% were aerated bronchus types I + II. CONCLUSIONS: SSN pathologic types differ with regard to peripheral vessel and aerated bronchus types. Type I peripheral vessel and type V aerated bronchus (both least involved) suggest a benign lesion, whereas type III/IV peripheral vessel and type I/II aerated bronchus (both most involved) suggest malignancy.

Vitamin C Prevents Hydrocortisone-Induced Injury in HMEC-1 through Promoting Bestrophin-3 Expression.

OBJECTIVE: To investigate the protective effects and underlying mechanisms of Vitamin C (VC) on hydrocortisone (HC)-induced cell injury in human microvascular endothelial cells (HMEC). METHODS: Cell viability was measured by CCK-8 assay and the expression of Best-3 was detected by Western blotting assay. The experiment was divided into normal control, HC injury group, VC treatment groups, HC + Best-3 siRNA group, HC + VC + Best-3 siRNA group, HC + pcDNA3.1 Best-3 group, and HC + VC + pcDNA3.1 Best-3 group. RESULTS: HC inhibited HMEC-1 cell viability was balanced with lower expression of Best-3 in a dose-dependent manner. Conversely, VC promoted HMEC-1 cell viability was paralleled to higher expression of Best-3 in a dose-dependent manner. Silencing Best-3 with Best-3 siRNA inhibited HMEC-1 cell viability, however, over-expression of Best-3 with pcDNA3.1 Best-3 promoted HMEC-1 cell viability. Moreover, VC and over-expression of Best-3 prevented HC-induced HMEC-1 cell apoptosis; however, silencing Best-3 further enhanced HC-induced HMEC-1 cell apoptosis. HC reduced Best-3 expression, which was alleviated by VC treatment. HC treatment decreased Bcl-2 expression, facilitated Bax expression. Both of VC and over-expression of Best-3 promoted Bcl-2 expression and decreased Bax expression. Additionally, VC and Best-3 expression have a synergistic effect. CONCLUSIONS: VC can efficiently attenuate HC-induced HMEC-1 cell injury, which may be related to promote Best-3 expression.

Efficacy, safety and influencing factors of intra-calf muscular injection of bone marrow mononuclear cells in the treatment of type 2 diabetes mellitus-induced lower extremity vascular disease.

The efficacy, safety and impact of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) associated with the intra-calf muscular injection of bone marrow mononuclear cells (BMMCs) in the treatment of type 2 diabetes mellitus (T2DM)-induced lower extremity vascular disease (LEVD) were evaluated. Patients with T2DM-LEVD were randomly divided into a control group and BMMCs group to assess the efficacy and safety of the treatment; serum VEGF and bFGF levels were detected. The BMMCs group was divided into superior genicular artery (SGA) and inferior genicular artery (IGA) subgroups as well as low-dose and high-dose subgroups for the comparison of efficacy indices. The BMMCs group exhibited significantly improved indices (P<0.05) compared with the control group and no fatalities or cancer occurred. There were no significant changes in serum VEGF and bFGF levels (P>0.05). The claudication distance in the IGA subgroup was significantly greater that in the SGA subgroup (P<0.05); the low-dose subgroup and the high-dose subgroup did not demonstrate any significant differences in each index (P>0.05). BMMC treatment for T2DM-LEVD was found to be safe and effective and had no significant impact on serum VEGF and bFGF levels in the short term; However, the degree of LEVD may affect its efficacy.

FOXK1 facilitates cell proliferation through regulating the expression of p21, and promotes metastasis in ovarian cancer.

Ovarian cancer is one of the most common cancer in the world. FOX family plays essential function in multiple cancers. In our work, FOXK1 was found to up-regulate in ovarian cancer tissue samples and cell lines; moreover, the expression of FOXK1 was correlated with tumor size, metastasis and poorly prognosis. To evaluate the function of FOXK1 in ovarian cancer, we performed colony formation analysis, CCK-8 assay and cell cycle analysis to determine the effect of FOXK1 on cell proliferation and cell cycle. We found that FOXK1 obviously improved the ability of cell proliferation through promoting cell cycle. Furthermore, ChIP assay and luciferase reporter assay indicated that FOXK1 facilitated cell cycle through regulating the expression of p21, but FOXK1 had no effect on cell apoptosis. In addition, wound healing assay and transwell invasion analysis demonstrated that FOXK1 promoted migration and invasion in ovarian cancer. In conclusion, our work indicate FOXK1 plays a key function in the ovarian cancer, it promotes cell proliferation and metastasis. FOXK1 serves as a novel molecular therapy target in ovarian cancer.

Protective Effects of Chronic Intermittent Hypobaric Hypoxia Pretreatment against Aplastic Anemia through Improving the Adhesiveness and Stress of Mesenchymal Stem Cells in Rats.

Aplastic anemia (AA) is a common malignant blood disease, and chronic intermittent hypobaric hypoxia (CIHH) has a beneficial effect against different diseases. The aim of the present study was to investigate the protective effect of CIHH against AA and underlying mechanisms. 5-Fluorouracil and busulfan treatment induced AA model in rats with reduction of hematological parameters and bone marrow tissue injury and decrease of the colony numbers of progenitor cells. CIHH pretreatment significantly reduced the incidence rate of AA and alleviated above symptoms in AA model. The adhesive molecules of bone marrow mesenchymal stem cells (BMMSCs) in AA model, VLA-4, VCAM-1, and ICAM-1 were upregulated, and those of CD162 and CD164 were downregulated by CIHH pretreatment. The expressions of HIF-1α and NF-κB in BMMSCs were also decreased through CIHH pretreatment. Overall, the results demonstrated for the first time that CIHH has an anti-AA effect through improving the adhesiveness and stress of mesenchymal stem cells in rats. CIHH could be a promising and effective therapy for AA.

The Preclinical Research Progress of Stem Cells Therapy in Parkinson's Disease.

Parkinson's disease (PD) is a type of degenerative disorder of the basal ganglia, causing tremor at rest, muscle rigidity hypokinesia, and dementia. The effectiveness of drug treatments gradually diminishes because the conversion to dopamine within the brain is increasingly disrupted by the progressive degeneration of the dopaminergic terminals. After long-term treatment, most patients with PD suffer from disability that cannot be satisfactorily controlled. To solve these issues, stem cells have recently been used for cell therapy of PD. In this review, the characteristics of different stem cells and their therapeutic effects on PD treatment will be discussed.

Effect of IL-17 in the development of colon cancer in mice.

Cytokine therapy is commonly used for tumor immunotherapy. Although early studies focused directly on the tumor, current investigations are more attentive of the tumor microenvironment. Various immune cells and related cytokines in the tumor microenvironment play an important role in the occurrence and development of tumor. Interleukin (IL)-17 is the characteristic cytokine produced by Th17 cells. IL-17 has been associated with various immune responses. The results of previous studies showed that IL-17 can significantly reduce the size of transplanted tumors in tumor-bearing mice, albeit it has no effect on the survival time of mice. By investigating the effect of IL-17 in the number and distribution of lymphocyte infiltration in tumor tissues, the expression of cytokines and transcription factors associated with the subsets of CD4+T cells in tumor tissues, the distribution of subsets of spleen lymphocyte in tumor-bearing mice, a preliminary investigation of the possible antitumor mechanism of IL-17 was performed. In conclusion, the antitumor effect of IL-17 gene transfection in the colon cancer of mice may be associated with the mechanisms whereby IL-17 gene transfection can change the distribution of different subsets of spleen lymphocytes in mice. IL-17 gene transfection can increase the number of lymphocyte infiltration in tumor tissues. IL-17 gene transfection can promote the high expression of interferon-γ in tumor tissue, while reducing the expression of IL-10 and IL-13 factors, thus exerting an antitumor effect.

Expression of activation-induced cytidine deaminase is associated with a poor prognosis of diffuse large B cell lymphoma patients treated with CHOP-based chemotherapy.

PURPOSE: Activation-induced cytidine deaminase (AID) is involved in somatic hypermutation and class switch recombination processes in the antibody formation. The AID activity induces gene mutations and could be associated with transformation processes of B cells. Nevertheless, the relation between AID expression and the prognosis of B cell lymphoma patients remains uncharacterized. METHODS: We examined expression levels of the AID gene in 89 lymph node specimens from lymphoma and non-lymphoma patients with Northern blot analysis and investigated an association with their survival. RESULTS: The AID gene was preferentially expressed in B cell lymphoma in particular in diffuse large B cell lymphoma and follicular lymphoma. We confirmed AID protein expression in the mRNA-positive but not in the negative specimens with Western blot analysis and immunohistochemical staining. Survival of the patients treated with cyclophosphamide-/doxorubicin-/vincristine-/prednisone-based chemotherapy demonstrated that the prognosis of diffuse large B cell patients was unfavorable in the mRNA-positive group compared with the negative group, and that AID expression levels were correlated with the poor prognosis. In contrast, AID expression was not linked with the prognosis of follicular lymphoma patients. CONCLUSIONS: AID expression is a predictive marker for an unfavorable outcome in DLBCL patients treated with the chemotherapy.

A combinatory use of adenoviruses expressing melanoma differentiation-associated gene-7 and replication-competent adenoviruses produces synergistic effects on pancreatic carcinoma cells.

Type 5 adenoviruses expressing mda-7 gene (Ad-mda-7) induced cell death in various kinds of human tumors, but pancreatic carcinoma cells were relatively resistant to Ad-mda-7-mediated cytotoxicity. We then examined whether infection of Ad-mda-7 together with replication-competent Ad produced combinatory cytotoxic effects. We prepared replication-competent Ad, defective of the E1B55kDa gene or activated by a transcriptional regulatory region of the midkine or the survivin gene of which the expression was up-regulated in human tumors. Type 5 Ad bearing the exogenous regulatory region were further modified by replacing the fiber-knob region with that of type 35 Ad. Pancreatic carcinoma cells were infected with replication-incompetent Ad-mda-7 and the replication-competent Ad. Combinatory effects were examined with the CalcuSyn software and cell cycle analyses. Ad-mda-7 and the replication-competent Ad achieved cytotoxicity to pancreatic carcinoma. A combinatory use of Ad-mda-7 and either Ad defective of the E1B55kDa gene or Ad activated by the regulatory region produced synergistic cytotoxic effects. Cell cycle analyses demonstrated that the combination increased sub-G1 populations. These data collectively suggest that expression of MDA-7 augments cytotoxicity of replication-competent Ad and achieves adjuvant effects on Ad-mediated cell death.

Fas ligand DNA enhances a vaccination effect by coadministered DNA encoding a tumor antigen through augmenting production of antibody against the tumor antigen.

Interaction of Fas and Fas ligand (FasL) plays an important role in the regulation of immune responses by inducing apoptosis of activated cells; however, a possible role of FasL in DNA vaccination has not been well understood. We examined whether administration of DNA encoding FasL gene enhanced antitumor effects in mice that were vaccinated with DNA expressing a putative tumor antigen gene, ß-galactosidase (ß-gal). Growth of ß-gal-positive Colon 26 tumors was retarded in the syngeneic mice immunized with ß-gal and FasL DNA compared with those vaccinated with ß-gal or FasL DNA. We did not detect increased numbers of ß-gal-specific CD8(+) T cells in lymph node of mice that received combination of ß-gal and FasL DNA, but amounts of anti-ß-gal antibody increased with the combination but not with ß-gal or FasL DNA injection alone. Subtype analysis of anti-ß-gal antibody produced by the combination of ß-gal and FasL DNA or ß-gal DNA injection showed that IgG2a amounts were greater in mice injected with both DNA than those with ß-gal DNA alone, but IgG2b amounts were lower in both DNA-injected than ß-gal DNA-injected mice. These data suggest that FasL is involved in boosting humoral immunity against a gene product encoded by coinjected DNA and enhances the vaccination effects.

Mesenchymal stem cells are efficiently transduced with adenoviruses bearing type 35-derived fibers and the transduced cells with the IL-28A gene produces cytotoxicity to lung carcinoma cells co-cultured.

BACKGROUND: Transduction of human mesenchymal stem cells (MSCs) with type 5 adenoviruses (Ad5) is limited in the efficacy because of the poor expression level of the coxsackie adenovirus receptor (CAR) molecules. We examined a possible improvement of Ad-mediated gene transfer in MSCs by substituting the fiber region of type 5 Ad with that of type 35 Ad. METHODS: Expression levels of CAR and CD46 molecules, which are the major receptors for type 5 and type 35 Ad, respectively, were assayed with flow cytometry. We constructed vectors expressing the green fluorescent protein gene with Ad5 or modified Ad5 bearing the type 35 fiber region (AdF35), and examined the infectivity to MSCs with flow cytometry. We investigated anti-tumor effects of MSCs transduced with interleukin (IL)-28A gene on human lung carcinoma cells with a colorimetric assay. Expression of IL-28A receptors was tested with the polymerase chain reaction. A promoter activity of transcriptional regulatory regions in MSCs was determined with a luciferase assay and a tumor growth-promoting ability of MSCs was tested with co-injection of human tumor cells in nude mice. RESULTS: MSCs expressed CD46 but scarcely CAR molecules, and subsequently were transduced with AdF35 but not with Ad5. Growth of MSCs transduced with the IL-28A gene remained the same as that of untransduced cells since MSCs were negative for the IL-28A receptors. The IL-28A-transduced MSCs however suppressed growth of lung carcinoma cells co-cultured, whereas MSCs transduced with AdF35 expressing the ß-galactosidase gene did not. A regulatory region of the cyclooygenase-2 gene possessed transcriptional activities greater than other tumor promoters but less than the cytomegalovirus promoter, and MSCs themselves did not support tumor growth in vivo. CONCLUSIONS: AdF35 is a suitable vector to transduce MSCs that are resistant to Ad5-mediated gene transfer. MSCs infected with AdF35 that activate an exogenous gene by the cytomegalovirus promoter can be a vehicle to deliver the gene product to targeted cells.