Dexmedetomidine affects the NOX4/Nrf2 pathway to improve renal antioxidant capacity.

OBJECTIVES: The study aimed to investigate the protective effects of dexmedetomidine (DEX) on renal injury caused by acute stress in rats and explore the protective pathways of DEX on rat kidneys in terms of oxidative stress. METHODS: An acute restraint stress model was utilized, where rats were restrained for 3 hours after a 15-minute swim. Biochemical tests and histopathological sections were conducted to evaluate renal function, along with the measurement of oxidative stress and related pathway proteins. KEY FINDINGS: The open-field experiments validated the successful establishment of the acute stress model. Acute stress-induced renal injury led to increased NADPH oxidase 4 (NOX4) protein expression and decreased expression levels of nuclear transcription factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1). Following DEX treatment, there was a significant reduction in renal NOX4 expression. The DEX-treated group exhibited normalized renal biochemical results and less damage observed in pathological sections compared to the acute stress group. CONCLUSIONS: The findings suggest that DEX treatment during acute stress can impact the NOX4/Nrf2/HO-1/NQO1 signaling pathway and inhibit oxidative stress, thereby preventing acute stress-induced kidney injury. Additionally, DEX shows promise for clinical applications in stress syndromes.

In silico modeling for ex vivo placenta perfusion of nicotine.

Nicotine readily crosses the placenta to reach fetuses. However, membrane transporters, e.g., organic cation transporters (OCTs) play a role in the clearance of nicotine from the fetal to the maternal side, and this is rarely investigated clinically. In this work, we use an in silico model to simulate an ex vivo placenta perfusion experiment, which is the gold standard for measuring the transplacental permeability of compounds, including nicotine. The model consists of a system of seven ordinary differential equations (ODEs), where each equation represents the nicotine concentration in compartments that emulate the ex vivo experiment setup. The transport role of OCTs is simulated bi-directionally at the placenta's basal membrane (the fetal side). We show that the model can not only reproduce the actual ex vivo experiment results, but also predict the likely maternal and fetal nicotine concentrations when the OCT transporters are inhibited, which leads to a ∼12% increase in fetal nicotine concentration after 2 hours of OCT modulated nicotine perfusion. In conclusion, a first in silico model is proposed in this paper that can be used to simulate some subtle features of trans-placental properties of nicotine.

Effects of vitamin A on intramuscular fat development in beef cattle: A meta-analysis.

Vitamin A, a fat-soluble vitamin, is the basic substance required to maintain healthy vision and the main physiological functions of cattle. The results from previous studies regarding the effect of vitamin A on intramuscular fat varied. This meta-analysis aimed to generate a more comprehensive understanding of the relationship between vitamin A and intramuscular fat content and to provide potential clues for future research and commercial practice. Electronic databases such as MEDLINE and Ovid were systematically searched, and studies investigating the relationship between vitamin A and intramuscular fat content were included. Standardized mean differences (SMDs) in intramuscular fat percentage and intramuscular fat score, with their respective 95% confidence intervals (CIs), were calculated. The heterogeneity and publication bias were evaluated. A total of 152 articles were identified through searches of databases. Seven articles were confirmed for inclusion in this meta-analysis. The SMD of IMF percentage derived from the analysis was-0.78 (-2.68, 1.12) (Q = 246.84, p < 0.01). The SMD of the IMF score was 1.25 (-2.75, 5.25) (Q = 87.20, p < 0.01). Our meta-analysis indicates that the addition of vitamin A could decrease intramuscular fat in cattle steers.

Effects of Melt Hydrogenation on the Microstructure Evolution and Hot Deformation Behavior of TiBw/Ti-6Al-4V Composites.

In this study, Ti-6Al-4V matrix composites reinforced with TiB ceramic whiskers were in situ synthesized and hydrogenated using the melt hydrogenation technique (MHT). The effects of MHT on the microstructure evolution and hot compression behavior of the composites were investigated by optical microscopy (OM), electron backscatter diffraction (EBSD), and transmission electron microscopy (TEM). Hot compression tests were performed at strain rates of 0.1/s, 0.01/s, and 0.001/s and temperatures of 800 °C, 850 °C, and 900 °C; the hot workability of composites significantly improved after hydrogenation, for example, the 900 °C peak flow stress of hydrogenated composites (43 MPa) decreased by 53.76% compared with that of unhydrogenated ones (93 MPa) at a strain rate of 0.01/s. Microstructural observations show that MHT can effectively facilitate the dispersion of TiB whiskers and induce the α/ß lath refinement of the matrix in our as-cast hydrogenated composite. During hot compression, MHT effectively promoted the as-cast composite microstructure refinement, accelerated the dynamic recrystallization (DRX) generation, and reduced the stress concentration at the interface between the reinforcement and matrix; in turn, the hydrogenated composites presented low peak stress during hot compression.

Effects of Plasma Exchange Combined with Immunoglobulin Therapy on Consciousness, Immune Function, and Prognosis in Patients with Myasthenia Gravis Crisis: A Prospective Randomized Test.

Background: Myasthenia gravis (MG) is an acquired autoimmune disease. The main clinical features of MG are skeletal muscle fatigue and pathological fatigue, which worsen at night or after fatigue, such as dyspnea, dysphagia, and systemic weakness. Plasma exchange (PE) is often used in patients with acute exacerbation of MG. Intravenous immunoglobulin (IVIG) is a collection of immunoglobulins from thousands of donors. IVIG can replace a variety of immunosuppressants or PE. However, the effect of PE or IVIG on patients' consciousness, immune function, and prognosis is not clear. Objective: A prospective randomized test of the effects of PE combined with immunoglobulin on consciousness, immune function, and prognosis in patients with myasthenia gravis crisis (MGC). Methods: Sixty patients with MGC treated from February 2019 to April 2021 were enrolled in our hospital. The cases who received PE were set as the PE group, and those who received PE combined with immunoglobulin were set as the PE+immunoglobulin group. The efficacy, clinical score, state of consciousness, immune function, acetylcholine receptor antibody (AChR-Ab), lymphocyte (LYM), albumin (ALB) levels, and the incidence of adverse reactions were compared. Results: The improvement rate was 100.005% in the treatment group and 83.33% in the PE group. After treatment, the clinical score of the PE+immunoglobulin group was lower than that of the PE group, and the clinical relative score of the PE+immunoglobulin group was higher than that of the PE group (P < 0.05). The number of conscious people in the PE+immunoglobulin group was more than that in the PE group (P < 0.05). Immunoglobulin A, immunoglobulin M, immunoglobulin G, and immunoglobulin G in the PE+immunoglobulin group were higher than those in the PE group (P < 0.05). The levels of AChR-Ab and ALB in the PE+immunoglobulin group were higher than those in the PE group, while the level of LYM in the PE+immunoglobulin group was lower than that in the PE group. The incidence of skin system, gastrointestinal system, nervous system, and systemic damage in the PE+immunoglobulin group was lower than that in the PE group (P < 0.05). Conclusion: The treatment of MGC with PE combined with immunoglobulin can not only effectively enhance the consciousness and immune function of patients but also effectively promote the prognosis, and the safety of treatment can be guaranteed.

microRNA-23a promotes cell growth and metastasis in gastric cancer via targeting SPRY2-mediated ERK signaling.

[This retracts the article DOI: 10.3892/ol.2018.8374.].

Metformin Ameliorates Diabetic Cardiomyopathy by Activating the PK2/PKR Pathway.

Diabetic cardiomyopathy (DCM) is a complication of diabetes that can cause damage to myocardial structure and function. Metformin (Met) is a widely used type 2 diabetes treatment drug that exerts cardioprotective effects through multiple pathways. Prokineticin 2 (PK2) is a small-molecule secreted protein that plays pivotal parts in cardiomyocyte survival and angiogenesis. However, the role of Met in regulating the PK2 signaling pathway in DCM remains unclear. This experiment explored the effects of Met on high glucose (HG)-induced injury through the PK2/PKR pathway in vivo and in vitro. Cardiomyocytes isolated from adult or AKT-knockout mice were treated with HG (33 mmol/L) and PK2 or AKT1/2 kinase inhibitor (AKT inhibitor). Heart contraction properties based on cell shortening were evaluated; these properties included the resting cell length, peak shortening (PS), maximum speed of shortening/relengthening (±dL/dt), time to 90% relengthening (TR90), and time to peak shortening (TPS). Mice with streptozotocin-induced diabetes were treated with Met to evaluate cardiac function, myocardial structure, and the PK2/PKR and AKT/GSK3ß pathways. Moreover, H9c2 cardiomyocytes were exposed to HG in the absence or presence of Met with or without the PK2 antagonist PKRA7 or the AKT inhibitor, and apoptotic proteins such as Bax and Bcl-2 and the PK2/PKR and AKT/GSK3ß pathways were evaluated using western blot analysis. The prolongation of TR90 and decreases in PS and ±dL/dt caused by HG were ameliorated by PK2 in cardiomyocytes, but the effects of PK2 were ameliorated or negated by the AKT inhibitor and in AKT-knockout mice. Diabetic mice showed metabolic abnormalities, aberrant myocardial enzyme levels, declines in myocardial systolic and diastolic function associated with myocardial fibrosis, and pronounced apoptosis, but these effects were greatly rescued by Met treatment. Moreover, PK2, PKR1, and PKR2 expression and p-AKT/AKT and p-GSK3ß/GSK3ß ratios were decreased in diabetic mice, and these decreases were attenuated by Met. Likewise, H9c2 cells exposed to HG showed reduced PK2/PKR expression and decreased p-AKT/AKT and p-GSK3ß/GSK3ß ratios, and these effects were nullified by Met. In addition, the effects of Met on cardiomyocytes exposed to HG were abolished after intervention with PKRA7 or the AKT inhibitor. These results suggest that Met can activate the PK2/PKR-mediated AKT/GSK3ß pathway, thus improving cardiac function and alleviating apoptosis in DM mice.

Protective Effects of Ozone Oxidative Postconditioning on Long-term Injury After Renal Ischemia/Reperfusion in Rat.

BACKGROUND: Renal ischemia/reperfusion (I/R) injury can cause serious kidney damage (eg, acute aortic injury, chronic fibrosis). Some postconditioning treatments have been reported to protect from I/R effects. However, their mechanisms remain unclear. Here, we focused on potential protective effects of ozone on tubulointerstitial fibrosis after renal I/R injury in rats. METHODS: Adult male rats were randomly divided into 4 groups with (1) sham-without I/R; (2) I/R-by clamping renal pedicle for 45 minutes; (3) I/R with ozone oxidative postconditioning (OzoneOP) following a 10-day reperfusion; and (4) I/R with oxygen oxidative postconditioning (OxygenOP) following a 10-day reperfusion. The kidneys were collected at 2 time points post I/R 10 days (at early phase) and 12 weeks (at late phase) and then analyzed for renal function, tissue fibrosis, and serum creatinine and urea nitrogen levels by staining and colorimetric methods. Additionally, expression levels of related fibrotic factors, such as α-smooth muscle actin, transforming growth factor ß1, and phospho-Smad2, were assayed by immunochemistry staining. RESULTS: OzoneOP treatment downregulated the α-smooth muscle actin, transforming growth factor ß1, and phospho-Smad 2 protein expression in rats subjected to I/R at 10 days and 12 weeks. Moreover, it improved renal dysfunction and attenuated the patchy tubulointerstitial fibrosis. CONCLUSION: Ourdata indicate that I/R-induced renal damage might cause severe tubulointerstitial fibrosis at the late phase, and OzoneOP treatment may inhibit this fibrotic development.

microRNA-23a promotes cell growth and metastasis in gastric cancer via targeting SPRY2-mediated ERK signaling.

microRNAs (miRs) serve important roles in various human cancer types. Recently, miR-23a has been indicated as an oncogene in gastric cancer, but the underlying mechanism remains unclear. In the present study, reverse transcription-quantitative polymerase chain reaction and western blot analysis was used to explore the effects of miR-23a in gastric cancer. Additionally, cell proliferation, migration and invasion were examined using an MTT assay, wound healing assay and Transwell assay, respectively. Furthermore, a luciferase reporter gene assay was used to confirm the target association. It was determined that miR-23a was significantly upregulated in gastric cancer tissues and cell lines compared with adjacent tissues, and a normal gastric epithelial cell line. Furthermore, its upregulation was significantly associated with cancer progression and poor prognosis of patients. Knockdown of miR-23a caused a notable reduction in the proliferation, migration and invasion of gastric cancer AGS cells. Sprouty homolog 2 (SPRY2) was then predicted to be target gene of miR-23a. A luciferase reporter gene assay data demonstrated that miR-23a has the ability to directly bind to the 3'-untranslational region of SPRY2 mRNA. Further investigation demonstrated that SPRY2 was significantly downregulated in gastric cancer tissues and cell lines, and the protein expression of SPRY2 was negatively regulated by miR-23a in AGS cells. Furthermore, knockdown of SPRY2 reduced the suppressive effects of miR-23a inhibition in AGS cell proliferation, migration and invasion. In addition, the activity of extracellular signal-regulated kinase (ERK) signaling was also inhibited by the miR-23a/SPRY2 knockdown in AGS cells. The present study indicated that miR-23a serves a promoting role in gastric cancer via targeting SPRY2 and downstream ERK signaling.

MicroRNA-219 exerts a tumor suppressive role in glioma via targeting Sal-like protein 4.

MicroRNAs (miRs) serve important roles in the development and progression of various human cancer types, including glioma. Recently, miR-219 has been suggested to function as a tumor suppressor in glioma; however, the underlying mechanism remains largely unknown. The aim of this study was to investigate the regulatory mechanism of miR-219 in the malignant phenotypes of glioma cells. Quantitative polymerase chain reaction (qPCR) and western blotting were conducted to examine the mRNA and protein expression. An MTT assay, wound healing assay and Transwell assay were used to study cell proliferation, migration and invasion. The qPCR data indicated that the expression of miR-219 was significantly decreased in glioma tissues compared with normal brain tissues. In addition, a low expression of miR-219 was identified to be associated with an advanced pathological grade. In vitro experiments demonstrated that miR-219 was also downregulated in several common glioma cell lines, including A172, U87, U251 and U373, when compared with that in normal astrocytes. Ectopic expression of miR-219 caused a significant decrease in U87 cell proliferation, migration and invasion. Luciferase reporter assay data indicated that Sal-like protein 4 (SALL4) was a direct target gene of miR-219, while the protein expression of SALL4 was negatively regulated by miR-219 in U87 cells. Furthermore, SALL4 was significantly upregulated in glioma tissues and cell lines, and upregulation of SALL4 was associated with a higher pathological grade. Furthermore, overexpression of SALL4 significantly attenuated the suppressive effects of miR-219 on U87 cell proliferation, migration and invasion, suggesting that miR-219 serves a suppressive role in glioma growth and metastasis via targeting SALL4. Therefore, the present study highlighted the clinical significance of the miR-219/SALL4 axis in glioma.

Ischemic postconditioning inhibits the renal fibrosis induced by ischemia-reperfusion injury in rats.

OBJECTIVE: To investigate whether ischemic postconditioning effects on the development of tubulointerstitial fibrosis follow acute renal ischemia-reperfusion. METHODS: Rat models of warm renal I/R were established by clamping left pedicles for 45 minutes after right nephrectomy, both with and without treatment with ischemic postconditioning, and then reperfused for up to 12 weeks. Hematoxylin-eosin (H&E) and Masson's trichrome staining were used to assess renal fibrosis. The expression spot and protein levels of α-smooth muscle actin (α-SMA), transforming growth factor-ß1 (TGF-ß1), and phospho-Smad2 were also analyzed. RESULTS: Our data showed that patchy inflammation and tubulointerstitial fibrosis were found 12 weeks later in rats subjected to I/R alone or with postconditioning. Tubulointerstitial fibrosis worsened further in rats subjected to 45-minute ischemia-reperfusion, accompanied by the increased expressions of α-SMA, TGF-ß1, and phospho-Smad2 at the end of 12 weeks. In contrast, the above histologic changes and molecular expressions were significantly attenuated in rats of ischemic postconditioning group. CONCLUSION: The results indicated that 45-minute I/R injury may cause tubulointerstitial fibrosis in the long term, and ischemic postconditioning has beneficial effects on renal fibrosis. Its mechanisms may involve inhibition of the TGF-ß1/phospho-Smad2 pathway to exert protective effects.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6 percent, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4 percent) and CD86 (80.13 ± 2.81 percent)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells.

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMax™ Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD(450) = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

[Prostate cancer cell vaccine transfected with 4-1BBL induces anti-tumor immunity in vitro].

OBJECTIVE: To explore the anti-tumor immunity in vitro induced by prostate cancer cell vaccine transfected with recombinant adenovirus encoding 4-1BBL in mice. METHODS: The replication-deficient adenovirus AdEasy-1 system was used to construct recombinant adenovirus Ad-m4-1BBL and Ad-eGFP. The prostate cancer cell RM-1 of mice was transfected with Ad-m4-1BBL and Ad-eGFP, and treated with mitomycin (MMC) to produce TCV, TCV-Ad-eGFP and TCV-Ad-m4-1BBL, followed by co-culture with syngeneic murine spleen cells. Then the cytotoxic activity of the lymphocytes against RM-1 cells was analyzed with CCK-8 solution, and IL-2 and INF-gamma were detected by ELISA. RESULTS: The 4-1BBL protein was highly expressed in the TCV-Ad-m4-1BBL of the 4-1BBL-transfected mice. TCV-Ad-m4-1BBL significantly increased the expressions of IL-2 ([180.24 +/- 2.22] pg/ml) and INF-gamma ([1512.46 +/- 23.64] pg/ml) as compared with TCV and TCV-Ad-eGFP (P < 0.05), and induced higher RM-1 cell specific cytotoxicity ([34.24 +/- 2.64]%) than the latter two ([9.82 +/- 1.48]%) and ([14.65 +/- 3. 21]%), (P < 0.05). But none of them exhibited significant cytotoxicity against hepatocellular carcinoma Hepal-6. CONCLUSION: The m4-1BBL-expressing prostate cancer cell vaccine can effectively induce anti-tumor immune responses.

Ischemic postconditioning attenuates renal ischemic/reperfusion injury in mongrel dogs.

OBJECTIVE: To investigate the effect of Postcond on renal ischemia-reperfusion (I/R) injury in a canine model. I/R injury is the most common cause of renal dysfunction. Ischemic postconditioning (Postcond) is a phenomenon by which intermittent interruptions of blood flow in the early phase of reperfusion can protect organs from I/R injury. MATERIAL AND METHODS: Forty adult male mongrel dogs were randomly divided into five groups of eight dogs each. Animals underwent 60 minutes of renal pedicle occlusion followed by reperfusion for 72 hours. Postcond was performed by 15-second, 30-second, or 1-minute I/R for six or three cycles. Blood and urine were collected at different reperfusion time points (24, 48, and 72 hours), and blood urea nitrogen (BUN), creatinine (Cr) levels, urine N-acetyl-ß-D-glucosaminidase (NAG), and Cr levels were assayed. Kidney samples were harvested after I/R, and renal superoxide dismutase (SOD), malondialdehyde (MDA), and myeloperoxidase (MPO) concentrations were measured, respectively. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay in the tissue samples. RESULTS: Compared with the sham group, I/R resulted in renal dysfunction, decreased SOD levels, increased MDA and MPO levels, and increased apoptosis indexes. However, Postcond attenuated the aforementioned effects, the protection of which in the Postcond of 15-second reperfusion/ischemia for six cycles was the most notable. CONCLUSIONS: Postcond exerts protective effects on renal (I/R) injury. It may be a promising strategy against I/R injury in clinical practice. Its mechanisms may involve reduction of lipid peroxidation and cellular apoptosis.

Antitumor immune response induced by DNA vaccine encoding human prostate-specific membrane antigen and mouse 4-1BBL.

OBJECTIVES: To determine whether a novel DNA vaccine encoding truncated human prostate-specific membrane antigen (tPSMA) can be enhanced by a genetically enhanced adjuvant (4-1BB ligand [4-1BBL]). METHODS: A eukaryotic expression plasmid pDC316-tPSMA-internal ribosome entry site-mouse 4-1BBL (pDC316-tPSMA-IRES-m4-1BBL) was constructed. The efficacy of vaccination using pDC316-tPSMA-IRES-m4-1BBL was compared with pDC316-tPSMA in terms of the antigen-specific cytotoxic T lymphocyte activity and antitumor immunity to RM-1-tPSMA in a murine tumor model. RESULTS: pDC316-tPSMA-IRES-m4-1BBL induced potent cytotoxicity against RM-1-tPSMA cells expressing tPSMA (42.6% specific killing) compared with pDC316-tPSMA vaccinated mice (24.8% killing) and mice not vaccinated (10.8% killing; P <.01). Moreover, the vaccination of mice with pDC316-tPSMA-IRES-m4-1BBL induced a potent protective antitumor immunity to RM-1-tPSMA in a subcutaneous tumor model. CONCLUSIONS: These results suggest that a specific antitumor immune response is enhanced by DNA vaccines expressing PSMA and 4-1BBL. This approach could offer a new strategy for treating carcinoma of the prostate after standard therapy.

Anti-tumor immune response induced by dendritic cells transduced with truncated PSMA IRES 4-1BBL recombinant adenoviruses.

Up-regulation of receptor-ligand pairs during interaction of a peptide-bound MHC complex on dendritic cells (DCs) with cognate TCR may amplify, sustain, and drive diversity in the ensuing T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily contribute to this costimulatory molecule signaling. In the present study, we used replication deficient adenoviruses to introduce a tumor-associated Ag (a truncated human prostate-specific membrane antigen (tPSMA)) and the T cell costimulatory molecule 4-1BBL into murine DCs, and observed the ability of these recombinant DCs to elicit tPSMA-directed T-cell responses in vitro and anti-tumor immunity to RM-1-tPSMA in a murine tumor model. Infection of DCs with Ad-tPSMA-IRES-m4-1BBL induced tPSMA-specific proliferative responses and up-regulated CD80 and CD86 s signaling molecules. The cytotoxic T lymphocytes activated by the Ad-tPSMA-IRES-m4-1BBL-transfected DCs showed significantly higher IFN-gamma production and cytotoxicity against the RM-1 cells transfected with tPSMA. Moreover, vaccination of mice with Ad-tPSMA-IRES-m4-1BBL-transfected DCs induced a potent protective and therapeutic anti-tumor immunity to RM-1-tPSMA in a tumor model. These results demonstrated that development of DCs engineered to express tPSMA and 4-1BBL by recombinant adenovirus-mediated gene transfer may offer a new strategy for prostate cancer immunotherapy.

The Change of Immunoactivity of Dendritic Cells Induced by Mouse 4-1BBL Recombinant Adenovirus

PURPOSE: The purpose of this study is to construct a recombinant adenovirus vector carrying mouse 4-1BBL and observe its effects in dendritic cells. MATERIALS AND METHODS: Mouse 4-1BBL cDNA was taken from the plasmid pcDNA3-m4-1BBL and subcloned into adenovirus shuttle plasmid pAdTrack-CMV, and then transformed into competent BJ5183 with plasmid pAdEasy-1. After recombination in E. coli, Ad-4-1BBL was packaged and amplified in HEK 293 cells. The expression of 4-1BBL in Ad-4-1BBL-transfected mouse prostate cancer cell line RM-1 was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. After the co-culture of dendritic cells (DCs) with Ad-4-1BBL-transfected RM-1 cells, interleukin (IL)-6 and IL-12 production were assessed by enzyme-linked immunosorbent assay (ELISA) and co-stimulatary moleculs (CD80 and CD86) on DCs were analyzed by flow cytometry. RESULTS: The levels of IL-6 (3,960 pg/mL) and IL-12 (249 pg/mL) production in Ad-m4-1BBL-pulsed DCs were more than those in none-pulsed DCs. The differences were statistically significant (p < 0.05). The expression of co-stimulatary molecules (CD80 and CD86) was up-regulated in Ad-m4-1BBL-pulsed DCs. CONCLUSION: The results indicated the recombinant mouse 4-1BBL can effectively activate DCs.