[Effect of early-life antibiotic exposure on allergic symptoms in children aged 6-11 months and 18-23 months based a birth cohort study].

Objective: To analyze the associations between prenatal and 1-year-old exposure to antibiotics and allergic symptoms in children aged 6-11 months and 18-23 months. Methods: In this study, a prospective birth cohort study was adopted. A total of 2 122 pregnant women were enrolled in Maternal and Child Health Care Center of Ma'anshan from June 2015 to June 2016, and they were followed up from the beginning of pregnancy to children's 24 months of age. Excluding 564 pairs of mothers and children who were lost to follow-up or with incomplete information on the use of antibiotics and children's allergic symptoms, a total of 1 558 pairs of mothers and children were included in the analysis of this study. The parents and children's general demographic information, early-life antibiotic exposure and other data were collected, the information about allergic symptoms in children aged 6-11 months and 18-23 months were investigated by reference to the "International Study of Asthma and Allergies in Childhood (ISAAC)". The univariate and multivariate binary unconditional logistic regression model was used to was used to estimate associations between the effects of early-life antibiotic exposure on allergic symptoms in 2-year-old children. Results: The antibiotic usage rate of pregnant women during pregnancy was 3.4% (53), and the antibiotic usage rates of children between 0 to 2 months, 3 to 5 months, and 6 to 11 months were separately 15.2%(237), 15.5%(242) and 17.3%(269). The total prevalence of allergic diseases in children aged 6 to 11 months was 24.1% (375 children), and the total prevalence of allergic diseases in children aged 18 to 23 months was 22.0% (342 children). After adjust parental (maternal) education level, family monthly income per capita, parental (maternal) allergy history, parental (maternal) age at pregnancy, mother's Body Mass Index (BMI) before pregnancy, exposure to second-hand smoke during pregnancy, delivery method, child gender, birth weight, preterm birth, the use of antibiotics when children were 3-5 months old (RR=1.61,95%CI:1.19-2.17) and 6-11 months old (RR=1.43,95%CI:1.06-1.93) were the risk factors for allergic symptoms at 6-11 months of age; and the use of antibiotics when children were 0-2 months old (RR=1.41, 95%CI: 1.03-1.95), 3-5 months old (RR=1.54, 95%CI: 1.12-2.11) and 6-11 months old (RR=1.58, 95%CI: 1.17-2.14) were the risk factors for allergic symptoms at 18-23 months of age. Conclusion: Children's exposure to antibiotics within 1 year of age was a risk factor for allergic symptoms in children aged 6-11 months and 18-23 months, children should avoid unnecessary antibiotic use in infancy.

Risk factors for bacterial vaginosis: results from a cross-sectional study having a sample of 53,652 women.

The aim of this study was to estimate the risk factors of bacterial vaginosis (BV) among rural married women of childbearing age in Anhui Province of China. A cross-sectional study was conducted and the method of stratified cluster sampling was used to identify a sample of 53,652 married women aged 18-49 years. All women were asked to complete an interviewer-administered standardized questionnaire, covering sociodemographic characteristics, history of menstruation, marriage and procreation, sexual life, personal hygienic behaviors, and reproductive tract infections (RTIs) knowledge, followed by the gynecological examination and laboratory inspection. A total of 53,286 married women aged 18-49 years were included in this analysis. The prevalence of BV was 11.99 % (6,391/53,286). Risk factors for BV included the minority nationality, women's lower education levels, husband's elder age, over 35 days of menstrual cycle, less than 3 days of menstruation, dysmenorrhea, usage of an intrauterine device (IUD), lack of RTIs knowledge, higher frequency of washing genitals before having sex with husband and changing underwear, lower frequency of sexual intercourse per month, and suffering from other RTIs. The results suggest that BV can be affected by many factors among rural married women of reproductive age, so comprehensive, scheduled programs at healthcare educations should be provided for women in order to prevent BV.

Cloning of the human aspartoacylase cDNA and a common missense mutation in Canavan disease.

Canavan disease, an autosomal recessive leukodystrophy, is caused by deficiency of aspartoacylase and accumulation of N-acetylaspartic acid in brain. We have cloned the human aspartoacylase (ASP) cDNA spanning 1,435 basepairs, and show that the isolated cDNA expresses aspartoacylase activity in bacteria. Furthermore, an A to C base change, at nucleotide 854, has been found in 85% of the 34 Canavan alleles tested so far. This base change results in a missense Glu285Ala mutation that is predicted to be part of the catalytic domain of aspartoacylase. The data suggest that the catalytic centre of aspartoacylase involves a triad of Ser, His and Glu residues. Our findings have implications for diagnosis and screening of Canavan disease.

Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector.

Limb-girdle muscular dystrophies 2C-F represent a family of autosomal recessive diseases caused by defects in sarcoglycan genes. The cardiomyopathic hamster is a naturally occurring model for limb-girdle muscular dystrophy caused by a primary deficiency in delta-sarcoglycan. We show here that acute sarcolemmal disruption occurs in this animal model during forceful muscle contraction. A recombinant adeno-associated virus vector encoding human delta-sarcoglycan conferred efficient and stable genetic reconstitution in the adult cardiomyopathic hamster when injected directly into muscle. A quantitative assay demonstrated that vector-transduced muscle fibers are stably protected from sarcolemmal disruption; there was no associated inflammation or immunologic response to the vector-encoded protein. Efficient gene transduction with rescue of the sarcoglycan complex in muscle fibers of the distal hindlimb was also obtained after infusion of recombinant adeno-associated virus into the femoral artery in conjunction with histamine-induced endothelial permeabilization. This study provides a strong rationale for the development of gene therapy for limb-girdle muscular dystrophy.

New candidate vaccines against blood-stage Plasmodium falciparum malaria: prime-boost immunization regimens incorporating human and simian adenoviral vectors and poxviral vectors expressing an optimized antigen based on merozoite surface protein 1.

Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.

[Mobile phone use in early pregnant and infant sleep-wake behaviour in 6 months: a cohort study].

Objective: To describe the epidemiological characteristics of mobile phone use in early pregnancy, and to explore the relationship between pregnancy mobile use and infant sleep-wake behavior. Methods: During February 2015 to August 2016, 2 212 subjects who had their first antenatal examination at Maanshan Maternity and Child Health Hospital were recruited in this cohort study and followed until postpartum for 6 months. Information of phone use was collected through questionnaire in the third trimester. There were 1 779 pregnant reported hours of mobile phone use in the questionnaire. A total of 1 951 parent reported the night-wake times. Data on night-wake behavior in infants was collected during the 6 months study. Questionnaires were completed by parents when taking the physical examination. More than 3 times per night was defined as the night-wake frequency. Unconditional multivariate logistic regression was applied to analyze the association of pregnancy time of mobile phone use and the infant night-wake frequencies. Results: In this cohort study, the average age of 2 212 pregnant women was (26.95±3.82) years, with 1 983 of them were followed up to the time of delivery. The incidence of night-wake frequency was 28.3% (553/1 951) among these 6-month-old infants. After adjusted for feeding factors in the first trimester, frequencies of using the phone as "3 to 4 hour per day" and "5 hour and above per day" were both positively associated with the frequencies of night-wake behavior in infants. The adjusted OR (95%CI) were 1.49 (1.07-2.07) and 1.79 (1.31-2.46), respectively. Conclusions: The mobile phone use during pregnancy was associated with night-wake of infants. Mobile phone should be rationally used during pregnancy.

Single-dose protection against Plasmodium berghei by a simian adenovirus vector using a human cytomegalovirus promoter containing intron A.

Human adenovirus serotype 5 (AdH5) vector vaccines elicit strong immune responses to the encoded antigen and have been used in various disease models. We designed AdH5 vectors expressing antigen under the control of a human cytomegalovirus (HCMV) immediate-early promoter containing its intron A sequence. The transcriptional levels of antigen and immune responses to antigen for vectors with the HCMV promoter with the intron A sequence (LP) were greater than those for AdH5 vectors using the HCMV promoter sequence without intron A (SP). We compared an E1E3-deleted AdH5 adenoviral vector, which affords more space for insertion of foreign sequences, and showed it to be as immunogenic as an E1-deleted AdH5 vector. Neutralizing antibodies to AdH5 limit the efficacy of vaccines based on the AdH5 serotype, and simian adenoviral vectors offer an attractive option to overcome this problem. We constructed E1E3-deleted human and simian adenoviral vectors encoding the pre-erythrocytic-stage malarial antigen Plasmodium berghei circumsporozoite protein. We compared the immunogenicity and efficacy of AdC6, a recombinant simian adenovirus serotype 6 vector, in a murine malaria model to those of AdH5 and the poxviral vectors MVA and FP9. AdC6 induced sterile protection from a single dose in 90% of mice, in contrast to AdH5 (25%) and poxviral vectors MVA and FP9 (0%). Adenoviral vectors maintained potent CD8(+) T-cell responses for a longer period after immunization than did poxviral vectors and mainly induced an effector memory phenotype of cells. Significantly, AdC6 was able to maintain protection in the presence of preexisting immunity to AdH5.

[Potential interaction effect on attention-deficit/hyperactivity disorder between mother's educational level and preschoolers' dietary pattern].

Objective: To explore the interaction effect between mother's educational level and preschoolers' dietary pattern on attention-deficit/hyperactivity disorder (ADHD). Methods: In 2014, there were 16 439 children aged 3-6 years old from 91 kindergartens in Ma'anshan municipality of China. A semi-quantitative food frequency questionnaire and the 10-item Chinese version of the Conners' Abbreviated Symptom Questionnaire (C-ASQ) were administered to assess the usual dietary intake and symptoms on ADHD. Social-demographic information was collected through questionnaires. Unconditional logistic regression was used to analyze the multiplication interaction effect between mother's educational level and preschoolers' dietary pattern on ADHD. Excel software was used to analyze the additive interaction effect of mother's educational level and preschoolers'dietary pattern on ADHD. Results: Results showed that factors as: mother's low educational level[aOR=1.31 (1.13-1.52)], scores related to preschoolers in the top quintile of "food processing" [aOR=1.31 (1.16-1.48)] and "snack" [aOR=1.45 (1.29-1.63)]patterns showed greater odds while preschoolers in the top quintile of "vegetarian" [aOR=0.80 (0.71-0.90)]showed less odds for having ADHD symptoms. Both multiplication and additive interactions were observed between mothers with less education. The processed dietary patterns (OR=1.17, 95%CI: 1.11-1.25), relative excess risk of interaction (RERI), attributable proportion (AP) and the interaction index (SI) appeared as 0.21, 0.13 and 1.47, respectively. Multiplication interaction was observed between levels of mother's low education and the snack dietary pattern (OR=1.21, 95%CI: 1.14-1.29), with RERI, AP and SI as 0.49, 0.26 and 2.36, respectively. However, neither multiplication interaction or additive interaction was noticed between levels of mother's low education and the vegetarian dietary pattern (OR=0.97, 95%CI: 0.92-1.03), with RERI, AP and SI as 0.09, 0.05 and 1.15, respectively. Conclusions: Levels of mother's low education presented a risk factor for ADHD symptoms in preschool children. Both multiplication interaction and additive interaction were observed between mother's low education levels and the processed dietary pattern. Multiplication interaction was noticed between mother's education levels and the snack dietary pattern but not with the vegetarian dietary pattern.

Eradication of intraperitoneal and distant tumor by adenovirus-mediated interferon-beta gene therapy is attributable to induction of systemic immunity.

Malignant mesothelioma remains an incurable disease for which immune-modulatory therapies, such as exogenous cytokines, have shown some promise. One such cytokine, IFN-beta, has potent antiproliferative and immunostimulatory activity in vitro, but its in vivo use has been limited by toxicity. We thus conducted studies evaluating intracavitary delivery of a replication-deficient adenoviral (Ad) vector encoding for the murine IFN-beta gene (Ad.muIFN-beta) in mouse models of malignant mesothelioma. In contrast to multiple injections of recombinant protein, a single i.p. injection of Ad.muIFN-beta into animals with established tumors elicited remarkable antitumor activity leading to long-term survival in >90% of animals bearing either AB12 or AC29 i.p. mesotheliomas. A control adenovirus vector had minimal antitumor effect in vivo. Significant therapeutic effects were also seen in animals treated with large tumor burdens. Importantly, treatment of i.p. tumor also led to reduction of growth in tumors established at a distant site (flank). A number of experiments suggested that these effects were attributable to an acquired CD8(+) T-cell-mediated response including: (a) the induction of long-lasting antitumor immunity; (b) loss of efficacy of Ad.muIFN-beta in tumor-bearing, immune-deficient (SCID, SCID/beige) mice; (c) detection of high levels of specific antitumor cytolytic activity from unstimulated splenocytes harvested from Ad.muIFN-beta-treated animals that was abolished by CD8(+) T-cell depletion; and (d) abrogation of antitumor effects of Ad.muIFN-beta in tumor-bearing CD8(+) T-cell-depleted animals. These data show that intracavitary IFN-beta gene therapy using an adenoviral vector provides strong CD8(+) T-cell-mediated antitumor effects in murine models of mesothelioma and suggest that this may be a promising strategy for the treatment of localized tumors such as mesothelioma or ovarian cancer in humans.

A cell line for high-yield production of E1-deleted adenovirus vectors without the emergence of replication-competent virus.

Production of E1-deleted adenovirus vectors for gene therapy has been plagued by the emergence of replication-competent adenovirus. A number of investigators have minimized homologous sequences between the vector and transfected E1 DNA in an attempt to avoid replication-competent adenovirus. We describe a HeLa-based cell line called GH329 that stably expresses the E1 locus from a promoter derived from the phosphoglycerate kinase gene. Overlap sequences with a standard E1-deleted vector that retains a full pIX transcriptional unit have been eliminated at the 5' end and minimized at the 3' end. The GH329 cell line plaques and produces E1-deleted adenovirus as well as 293 cells. Replication-competent virus has emerged after 5 passages of vector on 293 cells but was not detected after 20 passages on GH329 cells.

Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column.

One of the most promising gene transfer vectors in human clinical trials is AAV2. The quality of the vector preparations is a key element in obtaining reliable and reproducible data in preclinical studies. However, established protocols either result in impure, low infectious virus (CsCl2 gradient centrifugation) or demand a high level of manual and technical skills (CsCl2 gradient centrifugation, iodixanol/heparin or HPLC purification). In this study, we present an easy-to-do single-step column purification (SSCP) of AAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation. Various vector preparations generated by our method reproducibly showed high titers, infectivity, and purity. In vivo, our single-step column-purified AAV2 vectors mediate significantly higher transduction efficiency compared with conventional protocols. Investigators still unsatisfied with previously published techniques or new to the field of AAV production may find in our method an interesting alternative.

Partial correction of murine hemophilia A with neo-antigenic murine factor VIII.

We have previously reported a factor VIII knockout (FVIII KO) mouse model for hemophilia A. Here we demonstrate the presence of nonfunctional heavy chain factor VIII protein in the mouse, making it an excellent model for cross-reacting material (CRM)-positive hemophilia A patients, who express normal levels of a dysfunctional FVIII protein. We attempted to correct these mice phenotypically by transduction of wild-type mouse factor VIII cDNA delivered in an E1/E3-deleted adenoviral vector by tail vein injection. All treated mice displayed initial high-level FVIII expression that diminished after 1 month. Ten of 12 mice administered between 6 x 10(9) and 1 x 10(11) particles/mouse along with anti-CD4 antibody showed long-term FVIII activity (0.03-0.05 IU/ml, equivalent to 3-5% of normal FVIII) that corrected the phenotype. Wild-type murine FVIII was a neo-antigen to the KO mice, generating both cytotoxic and humoral immune responses. Immune suppression with anti-CD4 antibody abrogated these immune responses. These data demonstrate that despite the presence of endogenous FVIII protein the immune system still recognizes a species-specific transgene protein as a neo-antigen, eliciting a cytotoxic T cell response. This phenomenon may exist in the treatment of other genetic disorders by gene therapy.

Evaluating the potential of germ line transmission after intravenous administration of recombinant adenovirus in the C3H mouse.

The goal of this study is to assess the likelihood that an adenoviral vector disseminated to gonads will be transmitted to offspring. This study is based on the observation that systemically administered vector can be detected in both ovaries and testes, using sensitive nested PCR techniques. Although the extent of vector dissemination to gonads is extremely small, as it is detectable only by nested PCR, it is unclear where it is located within these tissues and whether the DNA is capable of integration and transmission to offspring. A protocol was developed in C3H mice to address this question. Both male and female C3H mice were injected with a high dose of H5.001CBhOTC, an E1- and E4-deleted vector expressing human ornithine transcarbamylase. This dose of vector was sufficient to target 80% of hepatocytes (Gao et al., J. Virol. 1996; 70:8934-8943) and disseminate, at low levels, to both ovaries and testes in 94% of animals as determined by PCR. Vector-administered animals and controls were mated and 814 offspring were evaluated for germ line transmission of the adenoviral vector by DNA hybridization of total cellular DNA extracted from the fetus. Southern blot analysis showed no evidence of germ line transmission in 578 offspring of crosses in which either one or both parents received recombinant adenovirus.

Muscle-specific promoters may be necessary for adeno-associated virus-mediated gene transfer in the treatment of muscular dystrophies.

Recombinant adeno-associated virus (rAAV) vectors allow efficient gene transfer and expression in the muscle; therefore, rAAVs represent a potential gene therapy vector for muscular dystrophies. For further investigations, we used a mouse muscular dystrophy model (gsg(-/-) mice) gamma-sarcoglycan, a subunit of the dystrophin-glycoprotein complex, is missing. gsg(-/-) mice develop progressive dystrophy representative of a severe human phenotype disease. We previously showed high levels and stable expression of gamma-sarcoglycan in myofibers after direct muscle injection into gsg(-/-) mice of a recombinant AAV vector (AAV.dMCK.gSG) carrying the gamma-sarcoglycan cDNA driven by a muscle-specific promoter (truncated version of muscle creatine kinase). Here, we show that when gamma-sarcoglycan expression is driven by the ubiquitous cytomegalovirus (CMV) promoter (AAV.CMV.gSG), lower levels of transgene expression are observed and are associated with a humoral response to gamma-sarcoglycan. When using an rAAV vector, expressing the highly immunogenic product gamma-galactosidase under the CMV promoter (AAV.CMV.LacZ), we measured a strong cellular and humoral immune response to the transgene after intramuscular injection into gsg(-/-) mice. This study suggests that restriction of transgene expression to the muscle is an important criterion for the treatment of muscular dystrophies and will aid in the design of protocols for gene therapy.

High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus.

Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV. We describe a novel method for producing AAV that addresses these problems. A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters. Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the E1 region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV. Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia. This method of AAV production should be useful in scaling up for studies in large animals, including humans.

Selective gene transfer into the liver of non-human primates with E1-deleted, E2A-defective, or E1-E4 deleted recombinant adenoviruses.

Preclinical studies were designed to investigate the feasibility and safety of recombinant adenoviruses transduced into the hepatic artery of nonhuman primates. The vectors used are recombinant adenoviruses deleted in E1 and contain either a temperature-sensitive mutation in the E2a gene, which encodes a defective DNA-binding protein at nonpermissive temperatures, or a deletion of the E4 region, including open reading frame (ORF) 6. Six 8- to 10-kg baboons underwent femoral artery cannulation, and angiographic techniques were used to introduce vector selectively into either a portion of the right lobe of the liver via a branch of the right hepatic artery or the common hepatic artery. Necropsies were performed at 4, 29, or 61 days. Serial sequential liver biopsies were performed in the baboons that survived 29 or 61 days. In the 2 baboons with vector transduction into the right hepatic artery, X-Gal histochemical analysis of the liver showed evidence of quantitatively increased gene transfer in the targeted lobe; however, gene transfer was present throughout the liver. Quantitative analysis of histopathology showed that portal inflammation was present throughout both livers transduced with the highest dose of vector. No differences were seen in the level of portal inflammation in targeted and untargeted lobes despite the observed qualitative and quantitative differences in gene expression. Southern blot analysis of total cellular DNA isolated from targeted and nontargeted lobes showed similar levels of viral DNA throughout the liver. Polymerase chain reaction (PCR) analysis was able to detect viral DNA sequence in gonads and brain as well as many other tissues in baboons treated with high-dose vector. In baboons treated with lower doses of an E1-E4 deleted vector expressing the human ornithine transcarbamylase (OTC) gene, DNA was detectable by nested PCR in liver but not gonads at days 29 and 61. The data suggest that intraarterial administration of recombinant adenoviral E1-E4 deleted vector is feasible and safe. At high doses of vector, widespread dissemination of vector DNA is seen. At low doses, hepatic gene transfer is not associated with vector DNA dissemination to gonads.

Evaluation of an E1E4-deleted adenovirus expressing the herpes simplex thymidine kinase suicide gene in cancer gene therapy.

Studies with first-generation adenoviral vectors have uncovered limitations that include finite transgene persistence, potential hepatotoxicity, and contamination with replication-competent adenovirus (RCA). To address these limitations within the context of cancer suicide gene therapy, a new adenoviral vector was developed containing the herpes simplex virus type 1 thymidine kinase (HSV tk) gene inserted in the E1 region of a recombinant vector containing deletions in the E1 and E4 regions of the Ad5 genome. The HSV tk minigene was placed under transcriptional control of a Rous sarcoma virus (RSV) promoter. This new E1E4-deleted vector was compared with the first-generation E1E3-deleted Ad.RSVtk vector. Generation of replication-competent adenovirus during production was eliminated. Using semiquantitative immunoblotting, the two vectors produced equivalent amounts of the expected 44-kDa tk-encoded protein in three different cell lines tested. The ability of the E1E4-deleted vector to sensitize tumor cells to ganciclovir (GCV) using in vitro assays and mixing studies was comparable to that of the E1E3-deleted vector. In vivo bystander effects were investigated using mixing studies in a syngeneic flank tumor model and demonstrated no difference between vectors in either immunocompetent or immunodeficient mice. To test the efficiency of these vectors in treating tumors in clinically relevant models, virus was injected intraperitoneally into tumor-bearing SCID mice and intrapleurally in a syngeneic rat mesothelioma model. After treatment of animals with ganciclovir, both vectors were roughly equivalent in their ability to increase mean survival (from approximately 40 to approximately 70 days) and markedly reduce tumor burden. Finally, formal toxicology studies were performed and showed similar amounts of local inflammation without systemic toxicity. In summary, this series of in vitro and in vivo experiments indicates that the performance of the recombinant E1E4-deleted adenoviral vector was virtually identical to that of the E1E3-deleted vector. Since the E1E4 vector has a much lower rate of recombination during production and has been shown to be less hepatotoxic in animal models, this new vector should prove superior to the first-generation Ad.HSVtk vectors in clinical cancer gene therapy trials.

Chimpanzee-origin adenovirus vectors as vaccine carriers.

Vaccines based on replication-defective adenoviral vectors are being developed for infectious agents and tumor-associated antigens. Early work focused on vaccines derived from a common human serotype of adenovirus, that is, adenovirus of the serotype 5 (AdHu5). Neutralizing antibodies against AdHu5 virus, present in a large percentage of the human population, dampen the efficacy of vaccines based on this carrier. To circumvent this problem, we generated vectors derived from chimpanzee adenoviruses. Here we describe some basic parameters of vectors derived from chimpanzee adenoviruses C68 and C7, including growth characteristics, yields of infectious particles, effects of additional deletions in E3 and E4 and lengths of the inserted foreign sequence as they relate to the suitability for their eventual development as vaccine carriers for clinical use.

Enrichment of middle repetitive element Bm-1 transcripts in translationally active RNA fractions of the silkmoth, Bombyx mori.

The Bm-1 repetitive element family represents a group of transcribed repetitive sequences in the genome of the silkmoth Bombyx mori. In the Bm-5 and BmN permanent cell lines studied here, alpha-amanitin inhibition and nuclear 'run-on' experiments demonstrated that approximately 80% of the Bm-1 transcripts are produced by RNA polymerase II. Bm-1 transcripts are dramatically enriched in poly A+ and polysomal RNA fractions compared to total RNA in these two cell lines. In the Bm-5 cell line, from total to poly A+ and polysomal RNA fractions, Bm-1 transcripts are enriched approximately 4 and 2 times, respectively, while in the BmN cell line these same fractions are enriched about 2 and 19 times compared to total RNA. This suggests that the Bm-1 transcripts may be involved in post-transcriptional processes or control of translation. Our data also revealed less size heterogeneity of Bm-1 transcripts in polysomal as compared to nuclear fractions. In the Bm-5 and BmN cell lines, the size of most transcripts containing Bm-1 sequences increases from approximately 1700 nt in the nucleus to 3000 nt in the polysomal fraction, both fractions with RNA much larger than the Bm-1 consensus sequence (250 bp). This raises the possibility that some Bm-1 elements are transcribed as part of larger transcripts containing mRNA by way of 'read-through', and may be involved in post-transcriptional regulation of gene expression as cis and/or trans acting elements.

CD40 ligand-dependent activation of cytotoxic T lymphocytes by adeno-associated virus vectors in vivo: role of immature dendritic cells.

Recombinant adeno-associated virus type 2 (rAAV) is being explored as a vector for gene therapy because of its broad host range, good safety profile, and persistent transgene expression in vivo. However, accumulating evidence indicates that administration of AAV vector may initiate a detectable cellular and humoral immune response to its transduced neo-antigen in vivo. To elucidate the cellular basis of the AAV-mediated immune response, C57BL/6 mouse bone marrow-derived immature and mature dendritic cells (DCs) were infected with AAV encoding beta-galactosidase (AAV-lacZ) and adoptively transferred into mice that had received an intramuscular injection of AAV-lacZ 10 days earlier. Unexpectedly, C57BL/6 mice but not CD40 ligand-deficient (CD40L(-/-)) mice adoptively transferred with AAV-lacZ-infected immature DCs developed a beta-galactosidase-specific cytotoxic T-lymphocyte (CTL) response that markedly diminished AAV-lacZ-transduced gene expression in muscle fibers. In contrast, adoptive transfer of AAV-lacZ-infected mature DCs failed to elicit a similar CTL response in vivo. Our findings indicate, for the first time, that immature DCs may be able to elicit a CD40L-dependent T-cell immunity to markedly diminish AAV-lacZ transduced gene expression in vivo when a sufficient number of DCs capturing rAAV vector and/or its transduced gene products is recruited.