Atrial Natriuretic Peptide Promotes Neurite Outgrowth and Survival of Cochlear Spiral Ganglion Neurons in vitro Through NPR-A/cGMP/PKG Signaling.

Sensorineural hearing loss (SNHL) is a dominant public health issue affecting millions of people around the globe, which is correlated with the irreversible deterioration of the hair cells and spiral ganglion neurons (SGNs) within the cochlea. Strategies using bioactive molecules that regulate neurite regeneration and neuronal survival to reestablish connections between auditory epithelium or implanted electrodes and SGN neurites would become attractive therapeutic candidates for SNHL. As an intracellular second messenger, cyclic guanosine-3',5'-monophosphate (cGMP) can be synthesized through activation of particulate guanylate cyclase-coupled natriuretic peptide receptors (NPRs) by natriuretic peptides, which in turn modulates multiple aspects of neuronal functions including neuronal development and neuronal survival. As a cardiac-derived hormone, atrial natriuretic peptide (ANP), and its specific receptors (NPR-A and NPR-C) are broadly expressed in the nervous system where they might be involved in the maintenance of diverse neural functions. Despite former literatures and our reports indicating the existence of ANP and its receptors within the inner ear, particularly in the spiral ganglion, their potential regulatory mechanisms underlying functional properties of auditory neurons are still incompletely understood. Our recently published investigation revealed that ANP could promote the neurite outgrowth of SGNs by activating NPR-A/cGMP/PKG cascade in a dose-dependent manner. In the present research, the influence of ANP and its receptor-mediated downstream signaling pathways on neurite outgrowth, neurite attraction, and neuronal survival of SGNs in vitro was evaluated by employing cultures of organotypic explant and dissociated neuron from postnatal rats. Our data indicated that ANP could support and attract neurite outgrowth of SGNs and possess a high capacity to improve neuronal survival of SGNs against glutamate-induced excitotoxicity by triggering the NPR-A/cGMP/PKG pathway. The neuroregenerative and neuroprotective effects of ANP/NPRA/cGMP/PKG-dependent signaling on SGNs would represent an attractive therapeutic candidate for hearing impairment.

Unusual presentation of a first branchial arch fistula with maxillofacial infection: a case report.

BACKGROUND: First branchial cleft anomaly (FBCA) is a rare congenital defect that arises due to incomplete closure of the ventral portion of the first and second branchial arches. There are variable complex clinical manifestations for patients with FBCA, which are prone to misdiagnosis and inadequate treatment. FBCAs usually involve the facial nerve with a consequent increased risk of facial nerve damage. Here, we present an unusual case of FBCA presenting with two preauricular pits in association with an abnormal maxillofacial cyst. CASE PRESENTATION: A 10-month-old girl presented to our department due to recurrent maxillofacial infections accompanied by swelling or abscess of the left cheek and purulent discharge from the preauricular pit for 4 months. A 3D-computed tomography (CT) fistulogram and magnetic resonance imaging (MRI) revealed two conjunctive tract lesions: one tract arose from the skin surface anteroinferior to the external auditory canal (EAC), through the deep lobe of the left parotid, and anteriorly extended to the left masseter; the other extended from the superficial lobe of the left parotid to the intertragic notch. After the maxillofacial infection was controlled by intravenous antibiotic administration, surgery was performed. Intraoperative tools, such as facial nerve monitors, microscopes, and methylene blue dyes, were used to facilitate the complete dissection and protection of the facial nerve. On follow-up over one year, the patient recovered well without facial palsy or recurrence. CONCLUSION: FBCA with maxillofacial cysts is rare and prone to misdiagnosis. Physicians should pay attention to this anatomic variant of FBCA with the fistula track located deep inside the facial nerve and projected medially to the masseter.

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.

[Effect of exogenous adrenomedullin on renal and hypothalamus adrenomedullin expression in rats early after mechanical renal trauma].

OBJECTIVE: To observe the effects of exogenous adrenomedullin (ADM) on endogenous expression of ADM in the kidney and hypothalamus of rats early after mechanical renal trauma. METHODS: Adult Wistar rats were randomized into 4 groups (n=32), namely the control group, renal impact trauma group, preventive ADM injection group, and therapeutic ADM injection group. In the latter two groups, ADM (0.1 nmol/kg) was administrated by intraperitoneal injection 10 min before and 10 min after renal trauma. The rats were executed at 1, 6, 12, and 24 h after the trauma to examine the expression of ADM in the kidney and hypothalamus. RESULTS: In preventive ADM injection group, the renal expression of ADM increased significantly at 1 h after the trauma (P<0.05) and tended to further increase with time till 24 h when its expression recovered the normal level. In the therapeutic ADM injection group, strong renal ADM positivity was found at 1 and 6 h after the injury (P<0.05) followed by gradual decrease till recovering the normal level at 24 h. Low renal ADM expression was detected, which was the strongest at 1 and 12 h (P<0.05) and became normal at 24 h. The time course of ADM expression in the hypothalamus was similar to that in the kidney in the therapeutic ADM injection group, and in the preventive injection group, the strongest ADM expression in the hypothalamus occurred at 6 and 24 h, and the lowest expression occurred at 12 h (P<0.05). The trauma group showed significantly decreased ADM expression in the hypothalamus compared with the control group (P<0.05). CONCLUSION: The hypothalamic ADM expression can upregulate renal ADM expression. ADM maintains the relative stability of the internal environment and physiological activity by local and systemic positive and negative feedback mechanisms.

Assessment of islet graft survival using a 3.0-Tesla magnetic resonance scanner.

Some studies have recently described a magnetic resonance (MR) method for detection of iron-labeled islets transplanted into the liver. The aim of this work was to assess the survival of islet graft using a clinical 3.0-T scanner. Islets from Lewis rats were cultured in the presence of iron oxide nanoparticles. One thousand iron-labeled islets were transplanted into the portal vein of diabetic rats. Blood glucose levels were measured daily through day 14 post-transplantation. MR imaging of the same section of the liver was performed on 1, 3, 7, 10, and 14 days post-transplantation. The labeled islets were visualized by MR as distinct hypointensive spots distributed in the liver. There was a linear correlation between the relative value of delta R2* relaxometry multiplied by the cubic diameter (relative value of the iron volume, Ir) and blood glucose level on 14 days post-transplantation in allograft and isograft (P<0.05). The relative value of delta R2* relaxometry, diameter, and number of hypointensive spots could be calculated to assess the survival of the iron-labeled islet grafts. Assessment of iron-labeled islet grafts using a clinical 3.0-T magnetic resonance scanner represents a useful method that has potential for clinical use.

Antitumor immunity by a dendritic cell vaccine encoding secondary lymphoid chemokine and tumor lysate on murine prostate cancer.

AIM: To investigate the antitumor immunity by a dendritic cell (DC) vaccine encoding secondary lymphoid chemokine gene and tumor lysate on murine prostate cancer. METHODS: DC from bone marrow of C57BL/6 were transfected with a plasmid vector expressing secondary lymphoid chemokine (SLC) cDNA by Lipofectamine 2,000 liposome and tumor lysate. Total RNA extracted from SLC+lysate-DC was used to verify the expression of SLC by reverse transcriptase-polymerase chain reaction (RT-PCR). The immunotherapeutic effect of DC vaccine on murine prostate cancer was assessed. RESULTS: We found that in the prostate tumor model of C57BL/6 mice, the administration of SLC+lysate-DC inhibited tumor growth most significantly when compared with SLC-DC, lysate-DC, DC or phosphate buffer solution (PBS) counterparts (P < 0.01). Immunohistochemical fluorescent staining analysis showed the infiltration of more CD4(+), CD8(+) T cell and CD11c(+) DC within established tumor treated by SLC+lysate-DC vaccine than other DC vaccines (P < 0.01). CONCLUSION: DC vaccine encoding secondary lymphoid chemokine and tumor lysate can elicit significant antitumor immunity by infiltration of CD4(+), CD8(+) T cell and DC, which might provide a potential immunotherapy method for prostate cancer.

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

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.

More than chemotaxis: a new anti-tumor DC vaccine modified by rAAV2-SLC.

Secondary lymphoid tissue chemokine (SLC) is strongly expressed in secondary lymphoid organs. Its ability to facilitate chemotaxis of both dendritic cells (DC) and T cells makes it a promising candidate for cancer therapy. In this study, we modified a BMDC vaccine by incorporating the SLC mature peptide gene. The efficacy of this vaccine was evaluated using a mouse hepatocellular carcinoma (HCC) model, with rAAV2 as the gene delivery vector. The rAAV2 encoding SLC (rAAV2-SLC) transfected immature BMDCs at high efficiency and the anti-tumor effects of SLC gene modified BMDCs (rAAV2-SLC/BMDC) were evaluated. In addition, rAAV2-SLC/BMDC vaccine injected directly into tumors attracted more CD4(+) and CD8(+) T lymphocytes into tumors and showed stronger anti-tumor effects than footpad delivery. Moreover, we found that the phenotypic expression of MHC II, the secretion of IL-12 and IFN-gamma, and T cell stimulation were increased in vitro following treatment with rAAV2-SLC/BMDC vaccine and these responses were inhibited by PTX. In vivo, PTX also inhibited the anti-tumor effects of the vaccine. The results suggest that the expression of SLC by rAAV2-SLC/BMDC plays more than a chemotactic role in anti-tumor responses, thus these studies further demonstrate that SLC has potential to be valuable in cancer therapy.

[Effects and mechanism of hyperglycemia on development and maturation and immune function of human monocyte derived dendritic cells].

OBJECTIVE: Dendritic cells play an important role in the pathogenesis of atherosclerosis. To explore the effects of hyperglycemia on the maturation and immune function of human monocyte derived dendritic cells (MDCs). METHODS: Immature MDCs were cultured in RPMI1640 medium with either 5.5 mmol/L D-glucose (NG), 25 mmol/L D-glucose (HG) or 5.5 mmol/L D-glucose + 19.5 mmol/L mannitol (HM) in the absence or presence of 30 mmol/L N-acetylcysteine [NAC, a reactive oxygen species inhibitor (ROS)] for 48 hours. FACS was used to investigate the MDCs immunophenotypic expression. Immune function was evaluated by allogeneic mixed T lymphocyte reaction and measurement of cytokine levels from culture supernatants. Intracellular ROS production in MDCs was also measured by 2', 7'-dichlorodihydrofluorescein (DCF, 10 micromol/L) fluorescence using confocal laser-scanning microscopy techniques. RESULTS: Compared with NG and HM treated MDCs, the expression of maturation markers such as CD1a, HLA-DR, CD83, CD86 were significantly upregulated, allogeneic T cells proliferation as well as the cytokines secretions (IL-2, IL-12, IL-10 and IFN-gamma) significantly increased in HG treated MDCs. Intracellular ROS production in MDCs was also significantly increased and all these stimulatory effects of HG could be partially attenuated by NAC. CONCLUSION: High glucose promote the maturation of MDCs and augment their capacity to stimulate T-cell proliferation and cytokine secretions at least in part through enhancing intracellular ROS generation. These stimulating effects of high glucose on MDCs maturation may be one of the mechanisms of accelerated atherosclerosis found in patients with diabetes.

A novel strategy to compensate the disadvantages of live vaccine using suicide-gene system and provide better antitumor immunity.

Fusing dendritic cells (DCs) with tumor cells is a powerful vaccine to increase tumor immunogenicity. To develop more effective and safer therapeutic vaccine, we fused rat bone marrow-derived DCs with ovarian tumor cell line NuTu-19 modified by suicide gene (HSV1-TK gene) to obtain live vaccine against ovarian cancer. Our data showed that immunization of rats with such live vaccine solicited stronger ovarian tumor-specific cytotoxic T lymphocyte responses and induced immunopreventive and immunotherapeutic effects against parental tumor cells in vivo. Live vaccine could be induced to death after ganciclovir administration in vitro and in vivo. Our researches suggest that live vaccine modified with suicide gene might be effective and controllable in the therapy of ovarian cancer.

[Suicidal cancer vaccine enhances anti-tumor immunotherapeutic effect and its safety in the treatment of ovarian cancer].

OBJECTIVE: To study the anti-tumor immunotherapeutic effect induced by the suicidalcancer vaccine FC/TK, and to evaluate the safety of this vaccine. METHODS: The suicidal cancer vaccine, named FC/TK, was prepared by fusion of suicide gene (HSVI,-TK gene) -modified ovarian carcinoma NuTu-19 cells with rat bone marrow-derived dendritic cells (DCs). The morphology of FC/TK was evaluated by scanning electron microscopy. The stimulatory effect of FC/TK on T cells was determined by T cell proliferation assay. In immunotherapeutic studies in vivo, Fischer344 rats were injected subcutaneously with NuTu-19 cells, followed by treatment of FC/TK on days 7 and 14, compared to controls treated with irradiated FC/TK, FC or PBS, respectively. Tumor incidence and volume were measured in 90 days after challenge. To determine the killing effect of FC/TK in vivo, TUNEL assays were applied to detect apoptotic cell death in spleen of vaccinated rats with prodrug ganciclovir administration. RESULTS: FC/TK cells were of irregular shape with surface membrane processes. Compared to the control groups, FC/TK significantly promoted T cell proliferation (P <0.01). The rats vaccinated with FC/TK and FC significantly inhibited the tumor growth compared to rats vaccinated with irradiated FC/TK (P <0.05) or with PBS ( P <0.01). The immunotherapeutic effect induced by FC/TK was similar to that using FC. Fluorescence microscopy showed that fluorescein-stained FC/TK cells migrated into spleen also showed to be TUNEL-positive, suggesting that the FC/TK cells were killed by ganciclovir in vivo. CONCLUSION: Our data indicate that suicidal cancer vaccine is an effective and safe therapy for ovarian carcinoma and may serve as a broadly applicable approach for other cancer vaccines in the future.

[Herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV(1)-TK/GCV) system as an effective "in vivo death switch" of live tumor vaccines].

BACKGROUND & OBJECTIVE: Live tumor vaccines could achieve better immunotherapeutic effects than irradiated tumor vaccines; however, the tumorigenicity is the crucial drawback incurred by the current procedures for vaccine preparation. This study was to explore the application of herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV1-TK/GCV) system as "in vivo death switch" to control the survival status of cancer vaccines under certain circumstances. METHODS: Suicide gene HSV(1)-TK was transferred into ovarian cancer cell line NuTu-19 with a retrovirus vector, followed by G418 selection to obtain HSV(1)-TK-transfected NuTu-19 cells (NuTu-19/TK). Dendritic cells (DCs) derived from Fischer344 rat bone marrow were fused with NuTu-19/TK cells to construct live tumor vaccine FC/TK. The expression of HSV(1)-TK in FC/TK cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The cytotoxic efficacy of GCV on FC/TK cells was evaluated by XTT assay. The apoptosis of FC/TK cells was analyzed by flow cytometry and Hoechst33258 staining after GCV treatment. Rats were vaccinated with FC/TK cells and divided into 2 groups: GCV group (5 rats) were intraperitoneally treated with GCV for 7 days, control group (5 rats) were treated with normal saline. The tumorigenesis and tumor metastasis in the rats were observed 90 days after inoculation. RESULTS: HSV1-TK was specifically expressed in FC/TK cells. GCV showed in vitro cytotoxicity to FC/TK cells in a dose-dependent manner, and 86.25% of the FC/TK cells were killed by GCV at a concentration of 100 microg/ml; the apoptosis rate of FC/TK cells was over 80%, and apoptotic morphology, including cell shrinkage, chromatin condensation, was observed. In the rat models, the tumor was developed at the injection site in 3 rats of control group, while no tumor was observed in the rats of GCV group. CONCLUSION: HSV(1)-TK/GCV could act as the "death switch" of tumor vaccines by triggering apoptosis of tumor vaccines both in vitro and in vivo.

[Effects of hepatectomized rat serum on the transdifferentiation of adult rat bone marrow cells into hepatocyte-like cells].

OBJECTIVES: To study the effects of hepatectomized rat serum and hepatocyte growth factor (HGF) on the transdifferentiation of adult rat bone marrow stem cells (ABMSCs) into hepatic parenchymal cells. METHODS: The serum was collected from the rats 24 hours after being subjected to subtotal hepatectomy. ABMSCs were collected and cultured in DMEM/F12 (1:1) containing the hepaetectomized rat serum or HGF. The differentiated hepatocyte-like cells were labeled with CM-DiI and administrated by tail vein injection into the isogeneic rats. The cultured and injected cells were both identified by immunocytochemistry and cultured cells were assayed using RT-PCR and Western blot. RESULTS: Hepatectomized rat serum and HGF were demonstrated to have the effect of inducing transdifferentiation of ABMSCs into hepatocyte-like cells in vitro. The differentiated cells expressed albumin mRNA and albumin after 7 days++'s co-incubation. Albumin-expressing and CM-DiI positive hepatocyte-like cells were characterized in livers and spleens of the rats injected with the cultivated cells. CONCLUSION: ABMSCs could transdifferentiate into hepatic parenchymal cells by hepatectomized rat serum or HGF.

Marked prolongation of murine cardiac allograft survival using recipient immature dendritic cells loaded with donor-derived apoptotic cells.

We investigated whether recipient dendritic cells (DCs), pretreated with nuclear factor-kappaB oligodeoxyribonucleotide decoy (NF-kappaB ODN decoy) and loaded with ultraviolet B-irradiated donor apoptotic splenocytes (Apo-SCs), were able to induce murine cardiac allograft tolerance. Heterotopic vascularized heart transplantation was performed from BALB/c to C57BL/6 mice, and recipients (C57BL/6) were given one injection of recipient DCs pretreated with NF-kappaB ODN decoy and loaded with donor (BALB/c) apoptotic SCs (decoy Apo-SCs DCs) through the portal vein at 7 days, before heart transplantation in the absence of immunosuppression. The cardiac allograft survival time and the expressive levels of intragraft cytokine genes [interleukin (IL)-2, IL-10, and interferon-gamma] were evaluated. Our results indicated that injection of decoy Apo-SCs DCs significantly prolonged vascularized heart allograft survival and led to skewing of intragraft cytokine expression towards T helper 2 (IL-10). The mechanisms can be useful for therapy of allograft rejection with minimization of systemic immunosuppression.

[Controlled live dendritic cell vaccine mediates potent antitumor immune responses].

OBJECTIVE: To investigate the efficiency of antitumor immune responses induced by a controlled live dendritic cell (DC) vaccine. METHODS: DC precursors were isolated from Fischer 344 rat bone marrow and cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4. The rat ovarian tumor cell line NuTu-19 was genetically modified by retroviral-mediated suicide gene (HSV(1)-TK), and the positive clones were selected using G418. Live DC vaccine was then fused with DC and NuTu-19/TK cell by polyethylene glycol. The characteristics of live DC vaccine were assayed with flow cytometry and confocal laser scanning microscopy. The specific expression of HSV(1)-TK gene in live DC vaccine was evaluated by RT-PCR and western blot. The sensitivity of live DC vaccine to ganciclovir (GCV) was evaluated by methylthiazoletetrazolium assay. In vivo, rats vaccinated twice with live DC vaccine were compared to those vaccinated with killed DC vaccine, unfused DC and NuTu-19/TK cell or phosphate buffered saline. Seven days following the last immunization, the rats were sacrificed to test the specific cytotoxic T lymphocyte (CTL) activity by lactate dehydrogenase release assay, or challenged with NuTu-19 and tumor incidence was observed. RESULTS: The fusion efficiency was approximately (23 +/- 14). Live DC vaccine displayed an up-regulated expression of major histocompatibility complex (MHC)-IIOX6 (87.6 +/- 3.4)%, costimulatory molecule B(1 - 2) (71.1 +/- 9.3)%, integrin OX 62 (68.0 +/- 7.4)%, and adhesion ICAM-1 (77.1 +/- 2.0)%, and specifically expressed HSV(1)-TK gene. Our data showed that spleen T lymphocytes from rats vaccinated with live vaccine displayed enhanced CTL activity (61.8 +/- 8.3)% contrast to that of rats vaccinated with killed vaccines (26.0 +/- 3.8)% (P < 0.05). Compared to the control groups, rats immunized with live DC vaccine demonstrated a significant delay in tumor development [(39 +/- 8) d vs (70 +/- 16) d], reduced tumor incidence (100% vs 80%) and decreased tumor volume [(806 +/- 553) mm(3) vs (89 +/- 53) mm(3), P < 0.05]. Seventy-three percent of TK-transduced live DC vaccine was killed after 7 of GCV treatment by a functional assay. CONCLUSION: The results demonstrate that DC live vaccine modified by HSV(1)-TK gene can induce efficient protective immunity and be killed by GCV.