Development of Mutiplex Polymerase Chain Reaction for the Detection and Differentiation of Enteric Adenoviruses in Stool Samples

To find a technique of detecting and differentiating enteric adenoviruses (EAds) in clinical samples, a novel PCR approach was developed. EAds were able to be detected by use of a pair of subgroup F general primers (P1 and P2), and they were also be able to be differentiated from each other in the presence of another adenovirus type 40 (Ad40) specific primer (P3) in the same tube. Our results showed that there was one band for Ad41 and two bands for Ad40, respectively, on running-gel after PCR performance. PCR was performed on 40 specimens in parallel directly with dot-hybridization assay on the same diluted stool samples. 20 of 40 specimens were positive by hybridization (of them 12 were Ad41 and 8 were Ad40), whereas 26 were positive by PCR performance on the same samples with Ad41 18 and Ad40 8 positive as well. Our study indicated that this novel method could be used in clinical laboratory or in epidemic investigation for Eads

The preparation and expression of replication- deficient human interleukin-2 recombinant adenoviruses

Interleukin-2(IL-2) has been demonstrated to beone of the most effective target genes in cancerimmunogene therapy. There are more than 20 clinicalprotocols of cancer gene therapy introducing IL-2 intotumor patients to treat melanoma, renal carcinoma,prostate carcinoma, colon carcinoma,

COMBINED IL-2/IL-3 GENE THERAPY FOR G422 MOUSE GLIOBLASTOMA BY INTRATUMORAL INJECTION OF RECOMBINANT ADENOVIRUSES

Recombinant adenoviruses encoding murine IL 2 gene or IL 3 gene were directly injected into established subcutaneous tumor model of G422 glioblastoma cells. After treatment, the tumor size and survival of the glioblastoma bearing mice were observed. The splenic NK and CTL cytotoxicities were detected by standard 4 hour 51 Cr release assay. We also examined the histopathological changes of tumor by hematoxylin and eosin staining. The results showed that intratumoral injection of adenoviruses encoding murine IL 2 gene or IL 3 gene significantly inhibited the growth of G422 glioblastoma and prolonged the survival period of glioblastoma bearing mice. The CTL cytotoxicity of the gene therapy groups was significantly higher than that of the control groups, but NK activity remained unchanged, indicating that specific immunity contributes to the in vivo antitumor effect of the direct gene therapy. There were much more tumor necrosis and inflammatory cell infiltration in the tumor of the gene therapy groups. Combined IL 2/IL 3 gene therapy could induce higher level of CTL and enhance the therapeutic potential further. The results suggest that intratumoral injection of recombinant adenoviruses encoding certain kind of cytokines may be a useful approach in the treatment of a malignancy of the central nervous system.

Abnormal liver function in children hospitalized with acute respiratory infection of adenoviruses:a retrospective study

Human adenoviruses(HAdVs)can cause acute hepatitis in immunocompromised patients.However,it is unclear whether HAdVs are contributors to hepatitis in immunocompetent children.In this study,the liver function test(LFT)results were retrospectively analyzed among children hospitalized(age<14 years)between January 2016 and October 2019 for acute respiratory infection caused by adenoviruses.Alanine transaminase(ALT)and aspartate aminotransferase(AST)levels were elevated in 7.74%and 46.89%of patients,respectively.All patients with>2 folds of the upper limit of ALT or AST levels were infected with HAdV-7 or HAdV-55.Significantly higher levels of ALT,AST,γ-glutamyl transpeptidase(γ-GT),and lower albumin levels were observed in the HAdV-7 infection group than in the HAdV-3 infection group.HAdV-55 infection led to significantly higherγ-GT,total bilirubin,and direct bilirubin levels than the other infection types.The records of four patients with serial monitoring of the LFT results were further analyzed.Multiple indicators remained abnormal during the entire hospitalization in these patients.These results indicate that HAdV infection is often accompanied by abnormal liver function,and HAdV-7 and HAdV-55 might be under-recognized contributors to hepatitis among children.

Plasma Metabonomics of Human Adenovirus-infected Patients with Pneumonia and Upper Respiratory Tract Infection

Objective Human adenovirus(HAdV)infection is common and can develop to serious conditions with high mortality,yet the mechanism of HAdV infection remains unclear.In the present study,the serum metabolite profiles of HAdV-7-infected patients with pneumonia or upper respiratory tract infection(URTI)were explored.Methods In total,35 patients were enrolled in the study following an outbreak of HAdV-7 in the army,of whom 14 had pneumonia and 21 had URTI.Blood samples were collected at the acute stage and at the recovery stage and were analyzed by untargeted metabolomics.Results Over 90% of the differential metabolites identified between the pneumonia patients and URTI patients were lipids and lipid-like molecules,including glycerophospholipids,fatty acyls,and sphingolipids.The metabolic pathways that were significantly enriched were primarily the lipid metabolism pathways,including sphingolipid metabolism,glycerophospholipid metabolism,and linoleic acid metabolism.The sphingolipid metabolism was identified as a significantly differential pathway between the pneumonia patients and URTI patients and between the acute and recovery stages for the pneumonia patients,but not between the acute and recovery stages for the URTI patients.Ceramide and lactosylceramide,involved in sphingolipid metabolism,were significantly higher in the pneumonia patients than in the URTI patients with good discrimination abilities[area under curve(AUC)0.742 and 0.716,respectively;combination AUC 0.801].Conclusion Our results suggested that HAdV modulated lipid metabolism for both the patients with URTI and pneumonia,especially the sphingolipid metabolism involving ceramide and lactosylceramide,which might thus be a potential intervention target in the treatment of HAdV infection.

High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria

Background Suicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene （tk）-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses. Methods pAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E. coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease Pad and polymerase chain reaction （PCR）, plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with Pacl and sequently transfected into human embryo kidney 293 cells （HEK293） using Lipofectamine 2000. Results Comet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk. Conclusion The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.

Oncolytic adenoviruses:A thorny path to glioma cure

Glioblastoma Multiforme(GBM)is a rapidly progressing brain tumor.Despite the relatively low percentage of cancer patients with glioma diagnoses,recent statistics indicate that the number of glioma patients may have increased over the past decade.Current therapeutic options for glioma patients include tumor resection,chemotherapy,and concomitant radiation therapy with an average survival of approximately 16 months.The rapid progression of gliomas has spurred the development of novel treatment options,such as cancer gene therapy and oncolytic virotherapy.Preclinical testing of oncolytic adenoviruses using glioma models revealed both positive and negative sides of the virotherapy approach.Here we present a detailed overview of the glioma virotherapy field and discuss auxiliary therapeutic strategies with the potential for augmenting clinical efficacy of GBM virotherapy treatment.

Lung-Targeted Transgene Expression of Nanocomplexed Ad5 Enhances Immune Response in the Presence of Preexisting Immunity

Recombinant adenovirus serotype 5(Ad5)vector has been widely applied in vaccine development targeting infectious diseases,such as Ebola virus disease and coronavirus disease 2019(COVID-19).However,the high prevalence of preexisting anti-vector immunity compromises the immunogenicity of Ad5-based vaccines.Thus,there is a substantial unmet need to minimize preexisting immunity while improving the insert-induced immunity of Ad5 vectors.Herein,we address this need by utilizing biocompatible nanoparticles to modulate Ad5–host interactions.We show that positively charged human serum albumin nanoparticles((+)HSAnp),which are capable of forming a complex with Ad5,significantly increase the transgene expression of Ad5 in both coxsackievirus–adenovirus receptor-positive and-negative cells.Furthermore,in charge-and dose-dependent manners,Ad5/(+)HSAnp complexes achieve robust(up to227-fold higher)and long-term(up to 60 days)transgene expression in the lungs of mice following intranasal instillation.Importantly,in the presence of preexisting anti-Ad5 immunity,complexed Ad5-based Ebola and COVID-19 vaccines significantly enhance antigen-specific humoral response and mucosal immunity.These findings suggest that viral aggregation and charge modification could be leveraged to engineer enhanced viral vectors for vaccines and gene therapies.

May 2022 acute hepatitis outbreak,is there a role for COVID-19 and other viruses?

There has been an increasing number of reported cases of acute hepatitis of unknown origin in previously healthy children since first reported on March 31,2022.This clinical syndrome is identified by jaundice and markedly elevated liver enzymes with increased aspartate transaminase and/or alanine aminotransa-minase(greater than 500 IU/L).We conducted an inclusive literature review with respect to acute hepatitis outbreaks in children using the search terms acute hepatitis,outbreak,children,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),coronavirus disease 2019(COVID-19),and adenovirus.According to the cumulative data presented in four main studies,the median age is 4 years,with a male predominance(1.3:1).Jaundice was the most common clinical manifestation(69%),followed by vomiting(63%),anorexia(52.9%),diarrhea(47.2%),abdominal pain(39%),pyrexia(33.3%),pale stool(30%),and dark urine(30%).Coryza and lethargy were reported in 16.6%,while pruritus was reported in 2%of cases.Acute liver failure was observed in 25%of cases.The exact mechanism of this acute hepatitis outbreak is still not entirely clear.Adenoviruses and SARS-CoV-2 were detected in a significant number of patients.Coinfection with adenovirus and SARS-CoV-2 could be a possible underlying mechanism.However,other possible infections and mechanisms must be considered in the pathogenesis of this condition.Acute hepatitis of unknown origin in children has been a serious problem since the start of the COVID-19 pandemic but has not yet been sufficiently addressed.Many questions remain regarding the underlying mechanisms leading to acute liver failure in children,and it is likely that extensive future research is needed.

儿童重症腺病毒肺炎诊疗进展

腺病毒（Adenovirus,ADV）是儿童急性呼吸道感染的常见病原,引起的儿童肺炎约占3%~5%,特别是6个月~2岁的婴幼儿,致病原因与此阶段腺病毒特异抗体缺乏有关。腺病毒肺炎有1/3发展为重症肺炎,约占重症肺炎的19.3%。

Molecular Characterization of Human Respiratory Adenovirus Infection in Children from November 2016 to October 2017 in Xining City, China

Human adenoviruses (HAdVs) belong to the genus Mastadenovirus, family Adenoviridae, and can cause respiratory tract and gastrointestinal tract infections, as well as ocular infections in humans[1]. Till date, a total of 84 unique genotypes of AdVs have been identified and classified as human Mastadenovirus species A to G, and specific types are often associated with certain clinical symptoms, epidemiological settings, and demographic risk groups[2]. Among these species, members of the species HAdV-B (HAdV-3, HAdV-7, HAdV-11, HAdV-16, HAdV-21, HAdV-34, HAdV-35, HAdV-50, etc), HAdV-C (HAdV-1, HAdV-2, HAdV-5, and HAdV-6), and HAdV-E (HAdV-4) have been generally associated with respiratory infections[3]. HAdV infections are mild and self-limited in healthy individuals, whereas they can result in high mortality rates in immunocompetent and immunocompromized patients[4].

Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

BACKGROUND： Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene. OBJECTIVE： To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN： Gene directed cloning. SETTING： Central Laboratory of Northern China Coal Medical College. MATERIALS： DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University. METHODS： NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid （pAAV-NT-3）. pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10：1 and the mixture was used to infect HT 1080 cells. X-gal stain was used to measure virus titer. MAIN OUTCOME MEASURES： Size of targeted gene fragments, validity of vehicle construction and virus titer. RESULTS： Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified. β-galactoside staining in situ indicated that LacZ genes were expressed in human fibrosarcoma cells （HT1080） and the recombinant virus titer was measured as 1 ×10^12 virus particles per milliliter. CONCLUSION： Total-length cDNA fragment of NT-3 gene, which is obtained from pCDNA3-NT-3 plasmids, is closely matched to polyclone enzyme-shearing sites of adeno-associated viral vectors, while the combination can be used to construct recombinant adeno-associated viral vectors expression in hNT-3 gene.

缺氧诱导因子-1α和BCL-2/腺病毒E1B19 kDa相关蛋白3在中耳胆脂瘤表达及意义

目的探讨缺氧诱导因子-1α(hypoxia inducible factor-1,HIF-1α)和BCL-2/腺病毒E1B19KDa相关蛋白3(Bcl2/adenovirus E1B 19 kD interacting protein 3,BNIP3)在中耳胆脂瘤中的表达及胆脂瘤上皮的凋亡情况。方法采用免疫组织化学方法检测30例中耳胆脂瘤标本与18例外耳道皮肤标本中HIF-1α和BNIP3蛋白的表达情况,使用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling,Tunel)检测20例中耳胆脂瘤标本和18例外耳道皮肤标本的凋亡情况。使用Pearson相关分析检验HIF-1α和BNIP3蛋白之间的相关性。结果 HIF-1α在胆脂瘤组和对照组的平均光密度分别为0.16±0.07和0.08±0.03,两组比较差异有统计学意义(t=4.279,P<0.01);BNIP3在胆脂瘤组和对照组的平均光密度分别为0.16±0.08和0.11±0.06,两组比较差异有统计学意义(t=2.463,P=0.0185);经pearson相关分析,在胆脂瘤上皮中,HIF-1α和BNIP3之间呈正相关(r=0.418,P=0.003);Tunel染色中,凋亡指数在胆脂瘤组和对照组分别为(52.8±12.5)%和(9.99±2.97)%,两组比较差异有统计学意义(t=14.166,P<0.01)。结论 HIF-1α和BNIP3在中耳胆脂瘤中的异常表达可能与胆脂瘤的高凋亡特性有关。

一起鸡心包积液病例的诊治

鸡包涵体肝炎（Inclusion Body Hepatitis,IBH）是由Ⅰ群禽腺病毒（Fowl adenovirusⅠ,FAV-Ⅰ）引起的一种急性传染病。该病最早发生在巴基斯坦卡拉奇市安卡拉地区,所以也称之为安卡拉病[1]。该病特征性病理变化为心包囊积液和肝炎。目前该病已在我国多地流行,可造成肉鸡、蛋鸡乃至水禽发病死亡,已严重影响养禽业健康发展[2-3]。

GROWTH INHIBITION OF HUMAN LARYNGEAL CANCER CELL WITH THE ADENOVIRUS-MEDIATED p53 GENE

Objective: In most laryngeal cancers, the function of p53 gene is down regulated. To explore the potential use of p53 in gene therapy of laryngeal cancer, by introducing wild-type p53 into laryngeal cancer cell line via a recombinant adenoviral vector, Ad5CMV-p53 and analyzing its effects on cell and tumor growth. Methods: A human laryngeal cancer, cell line Hep-2 was used. Recombinant cytomegalovirus-promoted adenoviruses containing human wild-type p53 cDNA was transiently introduced into Hep-2 line. The growth suppression of the Hep-2 cells and established s.c. squamous, carcinoma model was examined. The p53 protein expression was detected using immunohistochemical analysis. Results: The transduction efficiencies of Hep-2 cell line were 100% at a multiplicity of 100 or greater. The p53 protein expression peaked on day 2 after infection and lasted far 5 days. In vitro growth assays revealed cell death following Ad5CMV-p53 infected. In vivo studies, Ad5CMV-p53 inhibited the tumorigenicity of Hep-2 cell, and in nude mice with established s.c. squamous, carcinoma, nodules showed that tumor volumes were significantly reduced in mice that received peritumoral infiltration of Ad5CMV-p53. Conclusion: Adenovirus-mediated antitumor therapy carrying the p53 gene is an efficient method to inhibit laryngeal cancer growth. Transfection of laryngeal cancer cells with the wild-type p53 gene via Ad5CMV-p53 is a potential novel approach to the therapy of laryngeal cancer.

The Molecular Epidemiological Study on Enteric Adenovirus in Stool Specimens Collected from Wuhan Area by Using Digoxigenin Labeled DNA Probes

A summer-autumn (1994) molecular epidemiological study of enteric adenoviruses (EAds) in stool specimens collected in Wuhan area was conducted by using Digoxigenin-labelled DNA probes specific to EAd40, and EAd41, respectively.44 of 602 specimens were positive, among which 23 cases were identified as EAd40, 14 were EAd41, infection and 7 were dual infection. The ratio of males to females for the positive specimens was 1. 44. The infection rate of EAd40 and EAd41 each displayed no marked difference in seasons (summer and autumn) and similar age distribution was found between them. All of the two types of EAds in-fections predominated in patients with diarrhea under 3 years old- The results indicated that the Digoxigenin probe could detect DNA quantities as low as 1 pgwith satis factory specificity and the technique can be used for both clinical and ex-perimental purposes.

Inhibition of Dual Specific Oncolytic Adenovirus on Esophageal Cancer via Activation of Caspases by a Mitochondrial-dependent Pathway

We investigated the anti-tumor effects of dual cancer specific-oncolytic adenovirus Ad-VP on esophageal cancer（EC）. The anti-tumor activity of Ad-VP was compared with that of the control recombinant adenoviruses （Ad-GP, Ad-Apoptin, Ad-EGFP） in human esophageal cancer cell EC-109 and human normal liver cell L02 in vitro. In 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assays, the growth of EC-109 cells was slightly inhibited by Ad-GP, Ad-Apoptin and Ad-EGFE However, Ad-VP induced a significant cytotoxic effect. Infection of EC-109 cells with Ad-VP resulted in a significant induction of apoptosis of them in vitro, detected by 4＇,6-diamidino-2-phenylindole（DAPI） or acridine orange and ethidium bromide staining. The results of Western blot and flow cytometric assay indicate the loss of mitochondrial membrane potential（Aψm）, the release of eytochrome c and the activation of caspase-3, 6 and 7 in Ad-VP infected EC-109 cells. In contrast, all these assays show almost no effects of the recombinant adenoviruses on L02 cells. These results demonstrate that the treatment of tumors with Ad-VP selectively inhibits tumor growth and induces apoptosis of esophageal cancer cells. Ad-VP may provide a novel and powerful strategy for cancer gene therapy.

Protective effect of thodioloside and bone marrow mesenchymal stem cells infected with HIF-1-expressing adenovirus on acute spinal cord injury

Rhodioloside has been shown to protect cells from hypoxia injury,and bone marrow mesenchymal stem cells have a good effect on tissue repair.To study the effects of rhodioloside and bone marrow mesenchymal stem cells on spinal cord injury,a rat model of spinal cord injury was established using the Infinite Horizons method.After establishing the model,the rats were randomly divided into five groups.Rats in the control group were intragastrically injected with phosphate buffered saline(PBS)(5μL).PBS was injected at 6 equidistant points around 5 mm from the injury site and at a depth of 5 mm.Rats in the rhodioloside group were intragastrically injected with rhodioloside(5 g/kg)and intramuscularly injected with PBS.Rats in the mesenchymal stem cell(MSC)group were intramuscularly injected with PBS and intramuscularly with MSCs(8×10^6/mL in a 50-μL cell suspension).Rats in the Ad-HIF-MSC group were intragastrically injected with PBS and intramuscularly injected with HIF-1 adenovirus-infected MSCs.Rats in the rhodioloside+Ad-HIF-MSC group were intramuscularly injected with MSCs infected with the HIF-1 adenovirus and intragastrically injected with rhodioloside.One week after treatment,exercise recovery was evaluated with a modified combined behavioral score scale.Hematoxylin-eosin staining and Pischingert’s methylene blue staining were used to detect any histological or pathological changes in spinal cord tissue.Levels of adenovirus IX and Sry mRNA were detected by real-time quantitative polymerase chain reaction and used to determine the number of adenovirus and mesenchymal stem cells that were transfected into the spinal cord.Immunohistochemical staining was applied to detect HIF-1 protein levels in the spinal cord.The results showed that:(1)compared with the other groups,the rhodioloside+Ad-HIF-MSC group exhibited the highest combined behavioral score(P<0.05),the most recovered tissue,and the greatest number of neurons,as indicated by Pischingert’s methylene blue staining.(2)Compared with the PBS group,HIF-1 protein expression was greater in the rhodioloside group(P<0.05).(3)Compared with the Ad-HIF-MSC group,Sry mRNA levels were higher in the rhodioloside+Ad-HIF-MSC group(P<0.05).These results confirm that rhodioloside combined with bone marrow mesenchymal stem cells can promote the recovery of spinal cord injury and activate the HIF-1 pathway to promote the survival of bone marrow mesenchymal stem cells and repair damaged neurons within spinal cord tissue.This experiment was approved by the Animal Ethics Committee of Gansu University of Traditional Chinese Medicine,China(approval No.2015KYLL029)in June 2015.

Gene transfer and therapy with adenoviral vector in rats with diethylnitrosamine-induced hepatocellular carcinoma

Viral-mediated gene transfer of thymidine kinase ofherpes simplex virus (HSV-tk) has been used to confercytotoxic sensitivity to ganciclovir (GCV) in a variety oftnmor cells. HSV-tk converts GCV into a phosphorylatedcompound which is toxic for dividing cells by blockingDNA synthesis. Our previous study has shown

Recombinant adenovirus vector Ad-hIL-10 protects grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats

BACKGROUND: Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. This study was designed to investigate the protective effects of adenovirus-mediated human IL-10 (Ad-hIL-10) gene transfer on protecting grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats. METHODS: Adenoviruses encoding hIL-10 or beta-galactosidase (Ad-lacZ) were injected via the superior mesenteric vein into prospective donor animals. The donor liver was harvested 48 hours after transduction, and stored for 12 hours at 4 degrees C in lactated Ringer's solution prior to transplantation. The rats were divided into saline, Ad-lacZ, and Ad-hIL-10 groups. Liver function test, histopathological examination, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting were performed at 24 hours after transplantation in the three groups. RESULTS: Liver function (ALT and AST) was significantly improved, and the Suzuki score was significantly decreased in the Ad-hIL-10 group. The levels of hepatic TNF-alpha, MIP-2, ICAM-1 mRNA, and NF-kappa B protein in the Ad-hIL-10 group were significantly decreased. The expression of hIL-10 mRNA was detected by RT-PCR in Ad-hIL-10-treated grafts but not in controls treated with saline or Ad-lacZ. CONCLUSIONS: Donor pretreatment with Ad-hIL-10 down-regulates the expression of proinflammatory cytokines TNF-alpha, MIP-2, and ICAM-1 mRNA. hIL-10 protects against hepatic cold ischemia-reperfusion injury, at least in part, by suppressing NF-kappa B activation and subsequent expression of proinflammatory mediators. (Hepatobiliary Pancreat Dis Int 2010; 9: 144-148)