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.

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.

Hair regrowth in alopecia areata patients following Stem Cell Educator therapy.

BACKGROUND: Alopecia areata (AA) is one of the most common autoimmune diseases and targets the hair follicles, with high impact on the quality of life and self-esteem of patients due to hair loss. Clinical management and outcomes are challenged by current limited immunosuppressive and immunomodulating regimens. METHODS: We have developed a Stem Cell Educator therapy in which a patient's blood is circulated through a closed-loop system that separates mononuclear cells from the whole blood, allows the cells to briefly interact with adherent human cord blood-derived multipotent stem cells (CB-SC), and returns the "educated" autologous cells to the patient's circulation. In an open-label, phase 1/phase 2 study, patients (N = 9) with severe AA received one treatment with the Stem Cell Educator therapy. The median age was 20 years (median alopecic duration, 5 years). RESULTS: Clinical data demonstrated that patients with severe AA achieved improved hair regrowth and quality of life after receiving Stem Cell Educator therapy. Flow cytometry revealed the up-regulation of Th2 cytokines and restoration of balancing Th1/Th2/Th3 cytokine production in the peripheral blood of AA subjects. Immunohistochemistry indicated the formation of a "ring of transforming growth factor beta 1 (TGF-ß1)" around the hair follicles, leading to the restoration of immune privilege of hair follicles and the protection of newly generated hair follicles against autoimmune destruction. Mechanistic studies revealed that co-culture with CB-SC may up-regulate the expression of coinhibitory molecules B and T lymphocyte attenuator (BTLA) and programmed death-1 receptor (PD-1) on CD8ß(+)NKG2D(+) effector T cells and suppress their proliferation via herpesvirus entry mediator (HVEM) ligands and programmed death-1 ligand (PD-L1) on CB-SCs. CONCLUSIONS: Current clinical data demonstrated the safety and efficacy of the Stem Cell Educator therapy for the treatment of AA. This innovative approach produced lasting improvement in hair regrowth in subjects with moderate or severe AA. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01673789, 21 August 2012.

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.

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.

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.

Expression of a murine homolog of apoptosis-inducing human IL-24/MDA-7 in murine tumors fails to induce apoptosis or produce anti-tumor effects.

Expression of human interleukin (IL)-24 in tumors achieved anti-tumor effects through apoptosis. IL-24 also induced secretion of proinflammatory cytokines, suggesting the role in immunity. We showed that murine IL-24 transcripts started from the second initiation codon and that expressed mIL-24 in tumors failed to induce apoptosis. Proliferation of murine cells expressing mIL-24 was the same as that of the parent cells and inoculation of the mIL-24-expressing tumors into syngeneic mice did not produce anti-tumor effects. Secretory mIL-24 did not induce the expression of the IL-6, TNF-α or IFN-γ gene in spleen cells. Expression of mIL-24 receptor subunits, IL-22R and IL-20R1, was undetectable in spleen cells even though they were stimulated by anti-CD3, anti-CD40 antibody or concanavalin A. Transduction of murine tumors with adenoviruses expressing the human IL-24 gene however suppressed the viability and decreased the tumor growth. These data suggest that mIL-24 is functionally irrelevant to the human counterpart.

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.

A possible anticancer agent, type III interferon, activates cell death pathways and produces antitumor effects.

Recently identified interleukin-28 and -29 belong to a novel type III interferon (IFN) family, which could have distinct biological properties from type I and II IFNs. Type I IFNs, IFN-α/ß, have been clinically applied for treating a certain kind of malignancies for over 30 years, but a wide range of the adverse effects hampered the further clinical applications. Type III IFNs, IFN-λs, have similar signaling pathways as IFN-α/ß and inhibits proliferation of tumor cells through cell cycle arrest or apoptosis. Restricted patterns of type III IFN receptor expression in contrast to ubiquitously expressed IFN-α/ß receptors suggest that type III IFNs have limited cytotoxicity to normal cells and can be a possible anticancer agent. In this paper, we summarize the current knowledge on the IFN-λs-mediated tumor cell death and discuss the functional difference between type I and III IFNs.

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.

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.

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.

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.

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.

The secreted form of p28 subunit of interleukin (IL)-27 inhibits biological functions of IL-27 and suppresses anti-allogeneic immune responses.

Interleukin-27 (IL-27) is a new IL-12-related heterodimeric cytokine comprising a novel p28 molecule and the Epstein-Barr-virus-induced gene 3 (EBI3) molecules. It augments initiation of T helper type 1-mediated immunity by enhancing the proliferation and cytokine production of T cells. In this study, we examined whether a secreted form of IL-27 subunits would inhibit IL-27-mediated immunological responses. COS-7 cells transduced with the mouse (m) p28 gene secreted a monomeric mp28 protein; however, those transduced with the mEBI3 gene did not detect a mEBI3 protein in the culture supernatants. The secreted mp28 prevented the IL-27-mediated signal transduction and activator of transcription 1 phosphorylation and subsequently inhibited the IL-27-mediated intercellular adhesion molecule-1 induction and interferon-gamma production in CD4(+) T cells. We generated mp28-expressing murine carcinoma Colon 26 cells and inoculated a mixture of the mp28- and mIL-27-expressing Colon 26 cells into syngeneic BALB/c mice. Simultaneous production of mp28 and mIL-27 from Colon 26 cells suppressed IL-27-mediated anti-tumour effects in the mice. We examined the p28-mediated immune suppression by inoculating mp28-expressing myoblasts into allogeneic mice. Forced production of mp28 suppressed the allogeneic cytotoxic T-lymphocyte induction and subsequently retarded the graft rejection. Furthermore, production of both mp28 and mp40, which inhibits the functions of IL-12 and IL-23, prolonged the graft survival longer than the grafts expressing either mp28 or mp40. We propose that p28 can be a regulatory subunit for IL-27-mediated cellular immune responses and a possible therapeutic agent to suppress unfavourable immune responses.

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.

Cancer therapy with local oncolysis and topical cytokine secretion.

Direct destruction of targeted tumors and subsequent induction of systemic immunity is not pertinent to gene therapy but gene therapy is probably the most suitable therapeutic modality to achieve the local and systemic anti-tumor effects. Current strategies for cancer gene therapy in fact consist of direct inhibition of tumor growth and activation of systemic host defense mechanisms. We have been working on development of oncolytic adenoviruses and cytokine-mediated activation of host immune systems to produce better therapeutic effects. The adenoviruses in which the E1A expression is controlled by an exogenous regulatory region are preferentially cytotoxic to target tumor cells depending on the specificity of the regulatory region and cytokines that differentiate naive T cells into T helper type 1 cells can amplify immune responses generated. Combination of the two strategies has an advantage. Tumor destruction by oncolytic viruses does not impair immune systems in contract to chemotherapy and radiotherapy but enable to produce anti-tumor responses against putative tumor antigens that are subsequently released from the destroyed tumor. In this process, dendritic cells play a pivotal role since they act as professional antigen presenting cells and are involved in an initial phase of immune responses, either activation of immunity or induction of immune tolerance. Antigen loading with subsequent appropriate activation of dendritic cells is thereby crucial for activated anti-tumor responses, which possibly eliminate even distant metastatic foci. Combinatory gene therapy with oncolytic viruses and activation of host immune system thereby can evoke immune responses against all the tumor antigens expressed by the process of "antigen-spreading" mechanisms.

Silencing of polo-like kinase (Plk) 1 via siRNA causes inhibition of growth and induction of apoptosis in human esophageal cancer cells.

Esophageal cancer ranks among one of the most frequent causes of cancer death in the world. Polo-like kinase 1 (Plk1) is overexpressed in human tumors and has prognostic value in many cancers including esophageal cancer, indicating its potential as a therapeutic target. In this study, we investigated the therapeutic potential of Plk1 in esophageal cancer using the technique of RNA silencing via small interfering RNA (siRNA). Synthetic siRNA duplexes against Plk1 were introduced into 4 esophageal cancer cell lines, which subsequently resulted in a significant inhibition in Plk1 expression in the cells. We found that the targeted depletion of Plk1 caused a dramatic mitotic catastrophe (mitotic cell cycle arrest as well as defects in several mitotic events such as incomplete separation of sister chromatids and failure of cytokinesis) followed by massive apoptotic cell death, and eventually resulted in a significant decrease in growth and viability of all 4 esophageal cancer cell lines studied. In addition, our results also indicated that the mitotic arrest induced by Plk1 depletion is mediated by the inactivation of the cdc2/cyclin B1 complex. Taken together, our study strongly suggests that Plk1 may serve as a potential therapeutic target in human esophageal cancer.

[Enhanced expression of CD40L cDNA on ovarian cancer cell line OVHM induces the secretion of Th1 cytokines from dendritic cells].

OBJECTIVE: To examine whether the enhanced expression of CD40L cDNA on murine ovarian cancer (OVHM) cells could induce the secretion of Th1 cytokines from dendritic cells (DC). METHODS: OVHM cells were transfected with the full-length mouse CD40L cDNA by lipofectamine 2000 and then G418 resistant cells as positive cells were selected. They were examined for their expression of CD40L with flow cytometry. Bone marrow cells were firstly depleted of erythrocytes, macrophages, T and B cells with PE-conjugated magnetic beads, and then cultured in 10% FCS RPMI 1640 medium supplemented with recombinant mouse GM-CSF and IL-4 for 10 days. PKH67-labeled tumor cells were cultured with DC, and then the stained cells were analyzed for the expression of MHC-I, MHC-II, CD80, CD86, CCR7 in DC with flow cytometry. The expression of p40, p19, p35, p28, EBI3 subunits, IL-18, IFN-gamma, Mig gene in cocultured DC-tumor cells were detected by RT-PCR. RESULTS: The CD40L cDNA was successfully transfected into OVHM cells. Bone marrow-derived DCs, when cultured with CD40L/OVHM, formed clusters with the tumors and showed an upregulated expression of MHC- I, MHC-II, CD80, CD86, CCR7. Expression of the IL-12, IL-23, IL-27, IL-18, interferon-gamma (IFN-gamma) and Mig (monokine induced by IFN-gamma) genes was induced in the DCs that were cultured with CD40L/OVHM but not with OVHM cells. CONCLUSION: These data directly showed that the expression of CD40L on ovarian cancer cells facilitates the interaction between DCs and tumors, enhances the maturation of DCs, induces secretion of Th1 cytokines, especially for IL-12, IL-23 and IL-27, which maybe one of the possible antitumor mechanism for CD40L-transfected ovarian cancer cell line.