Adoptive Cellular Gene Therapy for the Treatment of Experimental Autoimmune Polychondritis Ear Disease.

In the past, the clinical therapy for autoimmune diseases, such as autoimmune polychondritis ear disease, was mostly limited to nonspecific immunosuppressive agents, which could lead to variable responses. Currently, gene therapy aims at achieving higher specificity and less adverse effects. This concept utilizes the adoptive transfer of autologous T cells that have been retrovirally transduced ex vivo to express and deliver immunoregulatory gene products to sites of autoimmune inflammation. In the animal model of collagen-induced autoimmune polychondritis ear disease (CIAPED), the adoptive transfer of IL-12p40-expressing collagen type II (CII)-specific CD4+ T-cell hybridomas resulted in a significantly lower disease incidence and severity compared with untreated or vector-only-treated animals. In vivo cell detection using bioluminescent labels showed that transferred CII-reactive T-cell hybridomas accumulated in the inflamed earlobes of the mice with CIAPED. In vitro analysis demonstrated that IL-12p40-transduced T cells did not affect antigen-specific T-cell activation or systemic anti-CII Ab responses. However, IL-12p40-transduced T cells suppressed IFN-γ and augmented IL-4 production, indicating their potential to act therapeutically by interrupting Th1-mediated inflammatory responses via augmenting Th2 responses. These results indicate that the local delivery of IL-12p40 by T cells could inhibit CIAPED by suppressing autoimmune responses at the site of inflammation.

Increased serum levels of high-mobility group box 1 protein and the location characteristics in the patients of intracranial aneurysms.

OBJECTIVE: Inflammation-related factors play a crucial role in intracranial aneurysms (IA) initiation, progression, and rupture. High mobility group box 1 (HMGB-1) serves as an alarm to drive the pathogenesis of the inflammatory disease. This study aimed to evaluate the role of HMGB-1 in IA and explore the correlation with other inflammatory-related factors. METHODS: A total of twenty-eight adult male Japanese white rabbits were included in with elastase-induced aneurysms, n = 18) and the control group (normal rabbits, n = 10). To assess the expression of HMGB-1, both reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) was performed on serum samples obtained from human subjects (10 patients with IA and 10 healthy donors) as well as from rabbits (aneurysm group and control group). Immunohistochemistry and immunofluorescence were employed to evaluate the expression levels of elastic fibers, HMGB-1, tumor necrosis factor-alpha (TNF-α), and triggering receptor expressed on myeloid cells-1 (TREM-1). RESULTS: The expression of HMGB-1 was found to be significantly higher in the IA group compared to the control group, both at the mRNA and protein levels (P < 0.0001). Similar findings were observed in the rabbit aneurysm model group compared to the control group (P < 0.0001). HMGB-1 expression was observed to be more abundant in the inner wall of the aneurysm compared to the external wall, whereas in the control group, it was rarely scattered. Additionally, the localization patterns of TNF-α and TREM-1 exhibited similar characteristics to HMGB-1. CONCLUSION: Our findings demonstrate that HMGB-1 is highly expressed in both IA patients and rabbit aneurysm models. Furthermore, the similar localization patterns of HMGB-1, TNF-α, and TREM-1 suggest their potential involvement in the inflammatory processes associated with IA. These results highlight the potential of HMGB-1 as a novel therapeutic target for IA.

Cryosurgery and rhTNF-α play synergistic effects on a rat cortex C6 glioma model.

Glioma, a type of brain tumor originating from glioma cells, varies widely in aggressiveness and causes serious symptoms, but the treatments are limited. Studies have shown that cryosurgery has multiple effects on tumor treatments, and administration of human tumor necrosis factor-alpha (rhTNF-α) arguments the anti-tumor effect of cryotherapy in breast and prostate cancers. To test the hypothesis that cryosurgery and rhTNF-α play synergistic effects against brain tumors, we established a brain glioma model on rat cortex regions following different treatments: the G1 group was sham-operated; the G2 group was treated with cryosurgery; the G3 group was treated with rhTNF-α; and G4 group received combined treatment with cryosurgery and rhTNF-α. Tumor sizes were measured by magnetic resonance imaging; DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay); P21(WAF1/CIP1) and proliferating cell nuclear antigen (PCNA) expression levels were scored using immunohistochemical staining. G2 and G4 rats had significantly longer survival time than did G1 rats. Tumor sizes in each group were significantly decreased as compared with those in G1 rats. PCNA-positive cells were significantly decreased in G2, G3 and G4 rats as compared with G1 rats. In contrast, DNA fragmentation and P21(WAF1/CIP1)-positive cells were significantly increased in each treatment group. Importantly, a combined treatment enhanced the effects of cryosurgery. Combined treatment with cryosurgery and rhTNF-α may have a synergistic effect on glioma tumor therapy, enhancing the inhibition of proliferation and the induction of apoptosis.

Synthetic mRNA-based gene therapy for glioblastoma: TRAIL-mRNA synergistically enhances PTEN-mRNA-based therapy.

Glioblastoma (GBM) is characterized as having high molecular heterogeneity and complexity, which can be well revealed by genomic study. A truly effective treatment for GBM should flexibly address its heterogeneities, complexity, and strong drug resistance. This study was performed to explore the effectiveness of an mRNA-based therapeutic strategy using in vitro synthesized PTEN-mRNA and TRAIL-mRNA in tumor cells derived from PTEN-deletion patients. The PTEN gene alterations were revealed by whole-exome sequencing of three paired clinical GBMs and selected as the therapy target. Patient-derived primary glioblastoma stem cells (GBM2) and a DBTRG-cell-derived xenograft were used to detect mRNA's cytotoxicity in vitro and tumor suppression in vivo. Following the successful in vitro synthesis of PTEN-mRNA and TRAIL-mRNA, the combinational treatment of PTEN-mRNA and TRAIL-mRNA significantly suppressed tumor growth compared with treatment with PBS (96.4%), PTEN-mRNA (89.7%), and TRAIL-mRNA (84.5%). The combinational application of PTEN-mRNA and TRAIL-mRNA showed synergistic inhibition of tumor growth, and the JNK pathway might be the major mechanism involved. This study provided a basis for an mRNA-based therapeutic strategy to be developed into an effective patient-tailored treatment for GBM.

FOXN3 inhibits cell proliferation and invasion via modulating the AKT/MDM2/p53 axis in human glioma.

This study aimed to evaluate the biological role of forkhead box N3 (FOXN3) in human glioma and clarify the possible molecular mechanisms. FOXN3 expression patterns in clinical tissue specimens were characterized via qPCR and Western blotting. Kaplan-Meier survival curve was applied to assess the correlation between FOXN3 expression and overall survival. Effects of FOXN3 over-expression and depletion on glioma cell proliferation, apoptosis, migration and invasion were assessed by CCK8, colony formation assay, flow cytometry, scratch wound healing assay and Transwell invasion assay, respectively. Moreover, the involvement of AKT/murine double minute 2 (MDM2)/p53 pathway was evaluated. Additionally, tumor transplantation model assay was performed to determine the effects of FOXN3 over-expression on glioma cell growth in vivo. Results showed that FOXN3 was significantly down-regulated in glioma tissues compared with normal tissues. Patients with lower FOXN3 expression exhibited a shorter overall survival time. Gain- and loss-of-function analyses demonstrated that FOXN3 over-expression significantly suppressed proliferation, survival and motility of glioma cells, whereas FOXN3 knockdown remarkably promoted glioma cell proliferation, survival and motility. Furthermore, FOXN3 over-expression inhibited the activation of AKT/MDM2/p53 signaling pathway in glioma cells, while FOXN3 depletion facilitated its activation. Additionally, tumor xenograft assays revealed that FOXN3 over-expression retarded glioma cell growth in vivo. Collectively, these findings indicate that FOXN3 inhibits cell growth and invasion through inactivating the AKT/MDM2/p53 signaling pathway and that FOXN3-AKT/MDM2/p53 axis may represent a novel therapeutic target for glioma patients.

Case Report: Primary Pulmonary Angiosarcoma With Brain Metastasis.

Primary pulmonary angiosarcoma (PPA) is a rare malignant vascular tumor, of which early diagnosis is challenging due to lack of specific clinical manifestations and a low level of suspicion. Here, we report a case of PPA presented with advanced brain metastasis. A 21-year-old patient with 1 week history of headache and mild cough was hospitalized for a head injury. Head MRI showed multiple intracranial lesions with brain edema. Chest CT displayed bilateral pulmonary infiltrates with mediastinal lymph node enlargement. After 2 months of anti-tuberculosis treatment, the patient was readmitted for persistent headache and cough with occasional hemosputum along with worsening pulmonary and intracranial lesions. Despite seizure prophylaxis and control of intracranial pressure and brain edema, his symptoms progressively aggravated, accompanied by cough with bloody sputum, frequent epileptic seizures, and hypotension. He eventually developed coma and died within 3 months of onset of symptoms. An autopsy confirmed PPA with brain metastasis.

CXCR4 and SDF-1 production are stimulated by hepatocyte growth factor and promote glioma cell invasion.

BACKGROUND: Hepatocyte growth factor (HGF) and its receptor play an important role in the formation and progression of glioma and can promote tumor proliferation. In this study, we investigated the ability of HGF to promote the proliferation and invasion of U251n cells; we also tested the effects of HGF on stromal cell-derived factor 1 (SDF1) and CXCR4 mRNA expression. METHODS: We measured the effect of HGF on the proliferation of U251n cells using enzyme-linked immunosorbent assays (ELISAs) to detect incorporated bromodeoxyuridine (BrdU) as a marker of DNA synthesis. The effects of HGF and SDF-1 on U251n cell invasion and proliferation were measured using the inhibitors K252a to c-Met and AMD3100 to CXCR4. SDF-1 and CXCR4 mRNA and protein expression were measured using quantitative polymerase chain reaction (PCR) and fluorescence-activated cell sorter (FACS) analysis. Small interfering (si)RNAs were also used to down-regulate HGF and c-Met expression in U251n cells. RESULTS: HGF significantly increased U251n cell proliferation and invasion in a dose-dependent manner; K252a blocked this. AMD3100 blocked invasion but not proliferation. CXCR4 and SDF-1 mRNAs were up-regulated when cells were treated with HGF. CXCR4 and SDF-1 mRNA levels and HGF and c-Met protein levels were down-regulated after cells were transfected with siRNAs. CONCLUSIONS: HGF has a direct effect on glioma cell proliferation and invasion. HGF up-regulates SDF-1 and CXCR4 mRNA expression and contributes to cell invasion.

RNA interference targeting EphA2 inhibits proliferation, induces apoptosis, and cooperates with cytotoxic drugs in human glioma cells.

BACKGROUND: Overexpression of EphA2 was detected in low- and high-grade glioma. To examine the role of EphA2 in human glioma cells, we studied its effects on proliferation and apoptosis using gene silencing through RNA interference. METHODS: One siRNA targeting EphA2 gene was synthesized in vitro and was transfected into the glioma U251n cells. Expression of EphA2 proteins was detected by Western blots and immunofluorescence. Cell apoptosis and mitochondrial membrane potential were analyzed by flow cytometry and annexin-V/fluorescein isothiocyanate/propidium iodide, respectively. Caspase-3 activity was measured by a spectrofluorometer. MTT assay was used to examine changes in cell proliferation. RESULTS: After treatment with sequence-specific siRNA targeting EphA2, the protein level of the transfected group decreased significantly. As compared to non-siRNA transfected cells, the transfected group showed lower proliferation, higher apoptosis, and loss of mitochondrial membrane potential. Caspase-3 activity increased in cells treated with siRNA and downregulated when treated with caspase-3 inhibitor. And the effects were clearly additive when siRNA transfected cells treated with the anticancer agents. CONCLUSIONS: The results suggest that EphA2-siRNA inhibit U251n cell proliferation and induce their apoptosis. It is possible that EphA2 via mitochondrial and caspase-3 inhibits U251n cell apoptosis. And EphA2-siRNA transfection enhances U251n cells' sensitivity to chemotherapy. EphA2 may be an effective therapeutic target in patients with glioma. Silencing the receptor EphA2 gene is a novel approach for the containment of growth and migration of tumor in patients with malignant glioma.

Experimental study on treatment of glioma by embryonic neural stem cell transplantation in rats.

The neural stem cells in Wistar rats were cultured in vitro, purified, and transplanted into C6 glioma model in order to observe their biological characters and provide a basic foundation for treatment of neurological diseases by neural stem cell transplantation. The cells at hippocampal area from gestation 15-day rats were cultured in vitro, and frozen and preserved in liquid nitrogen. C6 tumor-bearing models (n=25) and neural stem cells transplantation models (n=35) were established. When the tumor grew to 3 to 4 weeks, 5 rats in each group were randomly selected for MRI examination. At different intervals, the rats were perfused and sampled for HE staining, GFAP and BrdU immunohistochemical staining. The results showed that after resuscitation of neural stem cells at 1-4 passages, the cell viability was 40%-63% with the difference being not significant. The cells could proliferate, passage, and most cells transplanted into glioma model survived. The mean survival time in neural stem cell transplantation group and control was 4.28 and 3.88 weeks respectively, and the average tumor size in the former was smaller than in the latter. It was concluded that embryonic neural stem cells in rats could proliferate and differentiate, and after resuscitation the biological characteristic and viability of the cells were not influenced. Neural stem cells had inhibitory effects on the growth of glioma cells and could prolong the survival of rat model.

The Tumor-promoting Effects of FAM92A1-289 in Cervical Carcinoma Cells.

BACKGROUND/AIM: FAM92A1-289 is recognized as one of the newly-discovered putative oncogenes. This study was performed to reveal its oncogenic functions in human cervical carcinoma cells. MATERIALS AND METHODS: The FAM92A1-289+ cell line was established with knock-in technique and selected by puromycin-resistance screening. Scratch assay, methylthiazol tetrazolium assay, colony forming assay and xenograft test were used to examine cell migration, cell proliferation, cell viability and tumor formation, respectively. RESULTS: FAM92A1-289+ cells showed higher migration rate (p<0.05), higher cell viability (p<0.01), higher colony formation and tumor growth. The FAM92A1-289 protein was pulled-down by antibodies against proliferating cell nuclear antigen (PCNA) in the co-immunoprecipitation assay. CONCLUSION: The up-regulated expression of FAM92A1-289 could facilitate cell migration, boost cell proliferation and promote colony formation in vitro and tumor growth in vivo. The interaction between FAM92A1-289 and PCNA was verified by co-immunoprecipitation. This study provided functional evidence for FAM92A1-289 to be developed as a therapeutic target for cancer treatment.

The application of mRNA-based gene transfer in mesenchymal stem cell-mediated cytotoxicity of glioma cells.

Since the tumor-oriented homing capacity of mesenchymal stem cells (MSCs) was discovered, MSCs have attracted great interest in the research field of cancer therapy mainly focused on their use as carries for anticancer agents. Differing from DNA-based vectors, the use of mRNA-based antituor gene delivery benefits from readily transfection and mutagenesis-free. However, it is essential to verify if mRNA transfection interferes with MSCs' tropism and their antitumor properties. TRAIL- and PTEN-mRNAs were synthesized and studied in an in vitro model of MSC-mediated indirect co-culture with DBTRG human glioma cells. The expression of TRAIL and PTEN in transfected MSCs was verified by immunoblotting analysis, and the migration ability of MSCs after anticancer gene transfection was demonstrated using transwell co-cultures. The viability of DBTRG cells was determined with bioluminescence, live/dead staining and real time cell analyzer. An in vivo model of DBTRG cell-derived xenografted tumors was used to verify the antitumor effects of TRAIL- and PTEN-engineered MSCs. With regard to the effect of mRNA transfection on MSCs' migration toward glioma cells, an enhanced migration rate was observed with MSCs transfected with all tested mRNAs compared to non-transfected MSCs (p<0.05). TRAIL- and PTEN-mRNA-induced cytotoxicity of DBTRG glioma cells was proportionally correlated with the ratio of conditioned medium from transfected MSCs. A synergistic action of TRAIL and PTEN was demonstrated in the current co-culture model. The immunoblotting analysis revealed the apoptotic nature of the cells death in the present study. The growth of the xenografted tumors was significantly inhibited by the application of MSCPTEN or MSCTRAIL/PTEN on day 14 and MSCTRAIL on day 28 (p<0.05). The results suggested that anticancer gene-bearing mRNAs synthesized in vitro are capable of being applied for MSC-mediated anticancer modality. This study provides an experimental base for further clinical anticancer studies using synthesized mRNAs.

Cerebral small vessel disease and Alzheimer's disease.

Cerebral small vessel disease (CSVD) is a group of pathological processes with multifarious etiology and pathogenesis that are involved into the small arteries, arterioles, venules, and capillaries of the brain. CSVD mainly contains lacunar infarct or lacunar stroke, leukoaraiosis, Binswanger's disease, and cerebral microbleeds. CSVD is an important cerebral microvascular pathogenesis as it is the cause of 20% of strokes worldwide and the most common cause of cognitive impairment and dementia, including vascular dementia and Alzheimer's disease (AD). It has been well identified that CSVD contributes to the occurrence of AD. It seems that the treatment and prevention for cerebrovascular diseases with statins have such a role in the same function for AD. So far, there is no strong evidence-based medicine to support the idea, although increasing basic studies supported the fact that the treatment and prevention for cerebrovascular diseases will benefit AD. Furthermore, there is still lack of evidence in clinical application involved in specific drugs to benefit both AD and CSVD.

TRAIL-engineered bone marrow-derived mesenchymal stem cells: TRAIL expression and cytotoxic effects on C6 glioma cells.

BACKGROUND: TNF-related apoptosis-inducing ligand (TRAIL) is considered as a tumor cell-specific cytotoxic agent. Through the aid of mesenchymal stem cells (MSCs), TRAIL is capable of inducing apoptosis of tumor cells in tumor sites. The present study was performed to investigate the cytotoxic effects of TRAIL-engineered MSCs on glioblastoma cells (C6) in vitro. MATERIALS AND METHODS: An expression vector of secreting form of TRAIL was used to engineer MSCs. The cytotoxic effects of TRAIL-transfected MSCs on C6 cells were invstigated using the MTT method and Hochest33258 staining after co-culture of the two cell types. RESULTS: TRAIL and control plasmid transfection of MSCs showed no significant effect on MSC's viability (p>0.05). A significant inhibition of C6 cells was observed when they were co-cultured with TRAIL-engineered MSCs (63.7%±0.12, p<0.05). CONCLUSION: Mesenchymal stem cells were very well tolerant to the transfection of TRAIL-bearing vectors. The cytotoxic effects of TRAIL-engineered MSCs on C6 cells indicates the therapeutic potential of this strategy for treatment of glioblastoma patients.

A protocol for islet isolation from mouse pancreas.

Mouse islets are commonly used in diabetes-related studies. Adequate amounts of good quality islets are prerequisites for a reliable investigation. We describe a protocol for islet isolation from mouse pancreas. Three major manipulations are employed in the islet isolation procedure: in situ pancreas perfusion with collagenase, pancreas digestion and islet purification. The whole procedure takes 30-45 min for each individual mouse. By using this protocol, a reasonable number of islets can be obtained in a relatively short period of time. This protocol has been proven to be practicable and reproducible. It can be easily followed by individuals who do not have previous experience in the related research field.

Do immunotherapy and beta cell replacement play a synergistic role in the treatment of type 1 diabetes?

Type 1 diabetes (T1D) is the result of the autoimmune response against pancreatic insulin-producing ss-cells. Its ultimate consequence is beta-cell insufficiency-mediated dysregulation of blood glucose control. In terms of T1D treatment, immunotherapy addresses the cause of T1D, mainly through re-setting the balance between autoimmunity and regulatory mechanisms. Regulatory T cells play an important role in this immune intervention. An alternative T1D treatment is beta-cell replacement, which can reverse the consequence of the disease by replacing destroyed beta-cells in the diabetic pancreas. The applicable insulin-producing cells can be directly obtained from islet transplantation or generated from other cell sources such as autologous adult stem cells, embryonic stem cells, and induced pluripotent stem cells. In this review, we summarize the recent research progress and analyze the possible advantages and disadvantages of these two therapeutic options especially focusing on the potential synergistic effect on T1D treatment. Exploring the optimal combination of immunotherapy and beta-cell replacement will pave the way to the most effective cure for this devastating disease.

Expression characteristic and significance of interleukin-6, nuclear factor kappa beta, and bone formation markers in rat models of osteoporosis.

The goal of this study was to investigate the expression levels of interleukin 6 (IL-6), nuclear factor kappa beta (NF-kappabeta), bone-specific alkaline phosphatase (BALP), and bone osteocalcin (BGP) in rats with osteoporosis and their significance in the pathogenesis of osteoporosis. In all, 60 adult female SD rats were divided randomly into 3 groups of 20 rats each: normal control group (control), sham-operated group (sham), and ovariectomized group (OVX). In 2, 3, 4, 5, and 6 months after surgery, 4 rats were randomized from each group for assays of BMD, IL-6, BALP, and BGP. Then, the rats were sacrificed for the detection of IL-6 and NF-kappabeta expression levels in bone tissue by quantitative real-time RT-PCR analysis. Compared with the sham (0.097 +/- 0.04 g/cm2, 0.097 +/- 0.01 g/cm2, 0.095 +/- 0.07 g/cm2) and control group (0.107 +/- 0.01 g/cm2, 0.103 +/- 0.07 g/cm2, 0.108 +/- 0.06 g/cm2), the BMD of rats in the OVX group was reduced remarkably in 4, 5, and 6 months (0.082 +/- 0.05 g/cm2, 0.073 +/- 0.02 g/cm2, 0.061 +/- 0.05 g/cm2, respectively; P < 0.01); the serum IL-6 level increased significantly from 2 to 6 months after surgery (P < 0.01); and the serum levels of BALP and BGP were greater at 4, 5, and 6 months (P < 0.05). The quantitative real-time RT-PCR analysis demonstrated that IL-6 and NF-kappabeta mRNA levels in OVX group increased in a time-dependent manner. Moreover, the IL-6, NF-kappabeta, BALP, and BGP levels were correlated negatively with the BMD. Meanwhile, a positive correlation was observed between IL-6 and NF-kappabeta. In conclusion, the expression levels of IL-6, NF-kappabeta, and bone formation markers may increase significantly in the osteoporosis rats. These molecules could play a role in the pathogenesis.

[Effect of cryotherapy and 5-fluorouracil on apoptosis of G422 glioma cells].

BACKGROUND & OBJECTIVE: It was unknown whether the combination of cryotherapy and 5-fluorouracil (5-FU) could produce a synergistic effect, though both of them could induce apoptosis of glioma cells. The aim of this study was to observe the effect of cryotherapy and 5-fluorouracil on apoptosis of G422 glioma cells and to investigate the potential mechanism. METHODS: (1)Experimental mice models with G422 glioma were established. Cryotherapy (-180 degrees C,90s),chemotherapy (5-FU, 18 mg/kg), and cryochemotherapy were performed on them,respectively.(2)TUNEL was used to determine the apoptosis of glioma cells in each group at different time points.(3)The expression of the p53 and the HSP90alpha gene of the frozen glioma were analyzed by immunohistochemical staining. RESULTS: Cryotherapy or 5-FU induced apoptosis of G422 glioma cells, while the combination of cryotherapy and 5-FU remarkably increased the apoptosis rate. The numbers of apoptotic cells of combination group were 30.6+/-11.7, 86.4+/-21.5, 128.1+/-4.1, 237.0+/-30.1, 72.8+/-23.0 at 6h, 12h, 24h, 48h, 72h after cryochemotherapy, respectively, which were significantly higher than those of other groups. There was no expression of HSP90alpha and p53 in the center of freezing area. The p53 and the HSP90alpha proteins increased obviously in the periphery area especially at 48h after freezing compared with control group [(73.1+/-9.3)% vs (60.6+/-9.9)%, (35.6+/-6.6)% vs (13.7+/-6.5)%](P< 0.01). The expression of p53 was positively correlated with expression of HSP90alpha in frozen glioma (r=0.3610, P< 0.01). CONCLUSION: Combination of cryotherapy with 5-FU could enhance the apoptosis of G422 glioma cells presumably through modulating the HSP90alpha and p53 expression pattern. Cryochemotherapy showed better effect than that of cryotherapy or chemotherapy alone.