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OBJECTIVES: Dietary factors are of importance in the development of stomach cancer. This study aims to examine index-based dietary patterns associated with stomach cancer in a Chinese population. METHODS: Using data from a population-based case-control study conducted in Jiangsu Province, China, we included a total of 8,432 participants (1,900 stomach cancer cases and 6,532 controls). Dietary data collected by food frequency questionnaire was evaluated by modified Chinese Healthy Eating Index-2016 (mCHEI-2016) and the US Healthy Eating Index-2015 (HEI-2015). Multiple logistic regression analyses were applied to examine the association of mCHEI-2016 and HEI-2015 with stomach cancer while adjusting for potential confounders. The possible interactions between mCHEI-2016 or HEI-2015 and established risk factors were explored. RESULTS: Among non-proxy interviews, after adjusting for potential confounding factors, a higher score of sodium, reflecting lower intake per day, was inversely associated with stomach cancer (Odds Ratio [OR]= 0.95; 95% CI, 0.91–0.99 for mCHEI-2016; OR= 0.97; 95%CI, 0.94–0.99 for HEI-2015). No clear associations with stomach cancer were identified for total scores of HEI-2015 (OR= 0.98; 95% CI, 0.87–1.10 with a 10-point increase, p-trend = 0.98) and mCHEI-2016 (OR= 1.05; 95% CI, 0.94–1.17 with a 10-point increase, p-trend =0.22). However, the relation between stomach cancer and the mCHEI-2016 was modified by body mass index, with a possible inverse association in normal weight subjects. CONCLUSIONS: Our findings highlight that reduced intake of dietary sodium would prevent the development of stomach cancer. The data indicate a heterogeneity between normal weight and overweight’s dietary factors in relation to stomach cancer.

A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.

Although tobacco smoking has been reported as a risk factor for liver cancer, few studies have specifically explored the association among Chinese females and the potential interaction between smoking and other risk factors. A population-based case-control study was conducted and 2,011 liver cancer cases and 7,933 healthy controls were enrolled in Jiangsu, China from 2003 to 2010. Epidemiological data were collected, and serum hepatitis B surface antigen (HBsAg) and anti-HCV antibody were measured. Unconditional logistic regression was used to examine association and potential interaction, while semi-Bayes (SB) method was employed to make estimates more conservative. The prevalence of serum HBsAg positivity was 43.2% among cases and 6.5% among controls. The adjusted odds ratios (OR) for ever smoking were 1.62 (95% confidence interval [CI]: 1.33-1.96) among male and 0.82 (95% CI: 0.53-1.26) among female. Age at first cigarette, duration of smoking and pack-years of smoking were all significantly associated with liver cancer among men. Compared to HBsAg-negative never smokers, the adjusted ORs were 1.25 (95% CI: 1.03-1.52) for HBsAg-negative ever smokers, 7.66 (95% CI: 6.05-9.71) for HBsAg-positive never smokers, and 15.68 (95% CI: 12.06-20.39) for HBsAg-positive ever smokers. These different odds ratios indicated super-additive (RERI: 7.77, 95% CI: 3.81-11.73) and super-multiplicative interactions (ROR: 1.64, 95% CI: 1.17-2.30) between hepatitis B virus (HBV) infection and tobacco smoking. Most associations and interactions detected remained statistically significant after SB adjustments. Tobacco smoking and HBV infection positively interact in the development of liver cancer.

Although the major risk factors for liver cancer have been established, preventive factors for liver cancer have not been fully explored. We evaluated the association between raw garlic consumption and liver cancer in a large population-based case-control study in Eastern China. The study was conducted in Jiangsu, China, from 2003 to 2010. A total of 2011 incident liver cancer cases and 7933 randomly selected population-controls were interviewed. Epidemiological data including raw garlic intake and other exposures were collected, and serum markers of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection were assayed. Overall, eating raw garlic twice or more per week was inversely associated with liver cancer, with an adjusted odds ratio (aOR) of 0.77 (95% confidence interval (CI): 0.62&ndash, 0.96) compared to those ingesting no raw garlic or less than twice per week. In stratified analyses, high intake of raw garlic was inversely associated with liver cancer among Hepatitis B surface antigen (HBsAg) negative individuals, frequent alcohol drinkers, those having history of eating mold-contaminated food or drinking raw water, and those without family history of liver cancer. Marginal interactions on an additive scale were observed between low raw garlic intake and HBsAg positivity (attributable proportion due to interaction (AP) = 0.31, 95% CI: -0.01&ndash, 0.62) and heavy alcohol drinking (AP = 0.28, 95% CI: 0.00&ndash, 0.57). Raw garlic consumption is inversely associated with liver cancer. Such an association shed some light on the potential etiologic role of garlic intake on liver cancer, which in turn might provide a possible dietary intervention to reduce liver cancer in Chinese population.

Background & aims The potential interaction between family history of liver cancer and HBV infection on liver cancer has not been fully examined. Methods We conducted a population-based case-control study composed of 2011 liver cancer cases and 7933 controls in Jiangsu province, China from 2003 to 2010. Data on major risk or protective factors were collected and HBV/HCV sero-markers were assayed using blood samples. Semi-Bayes (SB) adjustments were applied to provide posterior estimates. Results Both family history of liver cancer (adjusted odds ratios [OR]: 4.32, 95% confidence intervals [CI]: 3.25-5.73) and hepatitis B surface antigen (HBsAg) positivity (adjusted OR: 9.94, 95% CI: 8.33-11.87) were strongly associated with liver cancer development. For individuals with different combinations of serological markers, the adjusted ORs were 8.45 (95% CI: 5.16-13.82) for HBsAg- and HBcAb-positive; 7.57 (95% CI: 4.87-11.77) for HBsAg-, HBeAg- and HBcAb-positive; and 3.62 (95% CI: 2.47-5.31) for HBsAg-, HBeAb- and HBcAb-positive, compared to all negatives in HBV serological markers. One log increase in HBV DNA level was associated with 17% increased risk (adjusted OR: 1.17, 95% CI: 1.03-1.32). The SB-adjusted OR of HBV-positive individuals with family history of liver cancer was 41.34 (95% posterior interval [PI]: 23.69-72.12) compared with those HBV-negative without family history. Relative excess risk due to additive interaction, the attributable proportion and synergy index were 73.13, 0.87 and 8.04 respectively. Adjusted ratio of OR for multiplicative interaction was 2.84 (95% CI: 1.41-5.75). Conclusions Super-additive and super-multiplicative interactions may exist between family history of liver cancer and HBV infection on the development of liver cancer.

To investigate the associations between dietary fatty acids and cholesterol consumption and stomach cancer (SC), we analyzed data from a population-based case-control study with a total of 1900 SC cases and 6532 controls. Dietary data and other risk or protective factors were collected by face-to-face interviews in Jiangsu Province, China, from 2003 to 2010. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using multiple unconditional logistic regression models and an energy-adjusted method. The joint associations between dietary factors and known risk factors on SC were examined. We observed positive associations between dietary saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and total cholesterol and the development of SC, comparing the highest versus lowest quarters. Increased intakes of dietary SFAs (p-trend = 0.005, aOR, 1.11, 95% CI, 1.01&ndash, 1.22 with a 7 g/day increase as a continuous variable) and total cholesterol (p-trend &lt, 0.001, aOR, 1.13, 95% CI, 1.06&ndash, 1.22 with a 250 mg/day increase as a continuous variable) were monotonically associated with elevated odds of developing SC. Our results indicate that dietary SFAs, MUFAs, and total cholesterol are associated with stomach cancer, which might provide a potential dietary intervention for stomach cancer prevention.

Garlic consumption has been inversely associated with esophageal cancer (EC), however, its interactions with tobacco smoking and alcohol consumption have never been evaluated in an epidemiological study. We evaluated the potential interactions between garlic intake and tobacco smoking as well as alcohol consumption in a population-based case-control study with 2,969 incident EC cases and 8,019 healthy controls. Epidemiologic data were collected by face-to-face interview using a questionnaire. The adjusted odds ratio (OR) and 95% confidence interval (CI) were estimated and additive and multiplicative interactions were evaluated using unconditional logistic regression models, adjusting for potential confounding factors. Semi-Bayes (SB) adjustments were used to reduce potential false-positive findings. EC was inversely associated with raw garlic intake (SB-adjusted OR for more than once a week = 0.68, 95% CI: 0.57–0.80) with a strong dose-response pattern in the overall analysis and in the stratified analyses by smoking and drinking. EC was positively associated with smoking and alcohol drinking with SB-adjusted OR of 1.73 (95% CI: 1.62–1.85) and 1.37 (95% CI: 1.28–1.46) in dose-response effects of increased intensity and longer duration of smoking/drinking. Moreover, garlic intake interacts with smoking [synergy index (S) = 0.83, 95% CI: 0.67–1.02; Ratio of ORs (ROR) = 0.88, 95% CI: 0.80–0.98] and alcohol drinking (S = 0.73, 95% CI: 0.57–0.93; ROR = 0.86, 95% CI: 0.77–0.95) both multiplicatively and additively. Our findings suggested that high intake of raw garlic may reduce EC risk and may interact with tobacco smoking and alcohol consumption which might shed a light on the development of EC as well as potential dietary intervention among high risk smokers and drinkers for EC prevention in Chinese population.

Inconsistent evidence has been reported on the role of female hormonal factors in the development of lung cancer. This population-based case-control study evaluated the main effect of menstrual/reproductive factors on the risk of lung cancer, and the effect modification by smoking status. Multivariable unconditional logistic regression models were applied adjusted for age, income, education, county of residence, body mass index, smoking status, pack-years of smoking, and family history of lung cancer. Among 680 lung cancer cases and 1,808 controls, later menopause (at >54 vs.

Alcohol drinking is a major risk factor for esophageal cancer (EC) and the metabolism of ethanol has been suggested to play an important role in esophageal carcinogenesis. Epidemiologic studies, including genome-wide association studies (GWAS), have identified single nucleotide polymorphisms (SNPs) in alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs) to be associated with esophageal cancer. Using a population-based case-control study with 858 EC cases and 1,081 controls conducted in Jiangsu Province, China, we aimed to provide further information on the association of ADH1B (rs1229984), ADH1C (rs698) and ALDH2 (rs671) polymorphisms with esophageal cancer in a Chinese population. Results showed that ADH1B (rs1229984) was associated with EC with odds ratios (ORs) of 1.34 (95% confidence interval: 1.08-1.66) for G-allele carriers compared to A/A homozygotes. No heterogeneity was detected on this association across different strata of alcohol drinking and tobacco smoking. Statistical interactions between ALDH2 (rs671) and alcohol drinking on EC susceptibility in both additive and multiplicative scales were observed. Compared to G/G homozygotes, A-allele carriers were positively associated with EC among moderate/heavy drinkers (OR=1.64, 1.12-2.40) and inversely associated with EC among never/light drinks (OR=0.75, 0.54-1.03). In addition, statistical interaction between ALDH2 and ADH1B polymorphisms on EC susceptibility among never/light drinkers was indicated. We did not observe association of ADH1C polymorphism with EC. In conclusion, our findings indicated that ADH1B (rs1229984) was associated with esophageal cancer independent of alcohol drinking and tobacco smoking status and alcohol drinking interacted with ALDH2 (rs671) on esophageal cancer susceptibility in this high-risk Chinese population.

Item does not contain fulltext Although the association for esophageal cancer with tobacco smoking and alcohol drinking has been well established, the risk appears to be less strong in China. To provide more evidence on the effect of smoking and alcohol consumption with esophageal cancer in China, particularly among Chinese women, a population-based case-control study has been conducted in Jiangsu, China, from 2003 to 2007. A total of 1,520 cases and 3,879 controls were recruited. Unconditional multivariate logistic regression analysis was applied. Results showed that the odds ratio (OR) and confidence interval (CI) for ever smoking and alcohol drinking were 1.57 (95% CI: 1.34-1.83) and 1.50 (95% CI: 1.29-1.74). Dose-response relationships were observed with increased intensity and longer duration of smoking/drinking. Risk of smoking and alcohol drinking at the highest joint level was 7.32 (95% CI: 4.58-11.7), when compared to those never smoked and never drank alcohol. Stratifying by genders, smoking and alcohol drinking increased the risk among men with an OR of 1.74 (95% CI: 1.44-2.09) and 1.76 (95% CI: 1.48-2.09); however, neither smoking nor alcohol consumption showed a significant association among women. In conclusion, smoking and alcohol drinking were associated with esophageal cancer risk among Chinese men, but not among Chinese women.

Author(s): Wang, N.; Jin, T.; Trivedi, P.; Setubal, J. C.; Tang, J.; Machado, M. A.; Triplett, E.; Coletta-Filho, H. D.; Cubero, J.; Deng, X.; Wang, X.; Zhou, C.; Ancona, V.; Lu, Z.; Dutt, M.; Borneman, J.; Rolshausen, P. E.; Roper, C.; Vidalakis, G.; Capote, N.; Catara, V.; Pietersen, G.; Al-Sadi, A. M.; Srivastava, A.; Graham, J. H.; Leveau, J.; Ghimire, S. R.; Verniere, C.; Zhang, Y.

Background Although the International Agency for Research on Cancer (IARC) has classified various indoor air pollutants as carcinogenic to humans, few studies evaluated the role of household ventilation in reducing the impact of indoor air pollutants on lung cancer risk. Objectives To explore the association between household ventilation and lung cancer. Methods A population-based case-control study was conducted in a Chinese population from 2003 to 2010. Epidemiologic and household ventilation data were collected using a standardized questionnaire. Unconditional logistic regression was employed to estimate adjusted odds ratios (ORadj) and their 95% confidence intervals (CI). Results Among 1,424 lung cancer cases and 4,543 healthy controls, inverse associations were observed for good ventilation in the kitchen (ORadj = 0.86, 95% CI: 0.75, 0.98), bedroom (ORadj = 0.90, 95% CI: 0.79, 1.03), and both kitchen and bedroom (ORadj = 0.87, 95% CI: 0.75, 1.00). Stratified analyses showed lung cancer inversely associated with good ventilation among active smokers (ORadj = 0.85, 95% CI: 0.72, 1.00), secondhand smokers at home (ORadj = 0.77, 95% CI: 0.63, 0.94), and those exposed to high-temperature cooking oil fumes (ORadj = 0.82, 95% CI: 0.68, 0.99). Additive interactions were found between household ventilation and secondhand smoke at home as well as number of household pollutant sources. Conclusions A protective association was observed between good ventilation of households and lung cancer, most likely through the reduction of exposure to indoor air pollutants, indicating ventilation may serve as one of the preventive measures for lung cancer, in addition to tobacco cessation.

The Rep proteins encoded by the adeno-associated virus type 2 (AAV) play a crucial role in the rescue, replication, and integration of the viral genome. In the absence of a helper virus, little expression of the AAV Rep proteins occurs, and the AAV genome fails to undergo DNA replication. Since previous studies have established that expression of the Rep78 and Rep68 proteins from the viral p5 promoter is controlled by the Rep-binding site (RBS) and the YY1 factor-binding site (YBS), we constructed a number of recombinant AAV plasmids containing mutations and/or deletions of the RBS and the YBS in the p5 promoter. These plasmids were transfected in HeLa or 293 cells and analyzed for the potential to undergo AAV DNA rescue and replication. Our studies revealed that (i) a low-level rescue and autonomous replication of the wild-type AAV genome occurred in 293 but not in HeLa cells; (ii) mutations in the RBS resulted in augmented expression from the p5 promoter, leading to more efficient rescue and/or replication of the AAV genome in 293 but not in HeLa cells; (iii) little rescue and/or replication occurred from plasmids containing mutations in the YBS alone in the absence of coinfection with adenovirus; (iv) expression of the adenovirus E1A gene products was insufficient to mediate rescue and/or replication of the AAV genome in HeLa cells; (v) autonomously replicated AAV genomes in 293 cells were successfully encapsidated in mature progeny virions that were biologically active in secondary infection of HeLa cells in the presence of adenovirus; and (vi) stable transfection of recombinant AAV plasmids containing a gene for resistance to neomycin significantly affected stable integration only in 293 cells, presumably because rescue and autonomous replication of the AAV genome from these plasmids occurred in 293 cells but not in HeLa or KB cells. These data suggest that in the absence of adenovirus, the AAV Rep protein-RBS interaction plays a dominant role in down-regulating viral gene expression from the p5 promoter and that perturbation in this interaction is sufficient to confer autonomous replication competence to AAV in 293 cells.

The adeno-associated virus 2 (AAV), a single-stranded DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral DNA synthesis is the rate-limiting step. We hypothesized that a host-cell protein interacts with the single-stranded D sequence within the inverted terminal repeat structure of the AAV genome and prevents the viral second-strand DNA synthesis. Indeed, a cellular protein has been identified that interacts specifically and preferentially with the D sequence at the 3′ end of the AAV genome. This protein, designated the single-stranded D-sequence-binding protein (ssD-BP), is phosphorylated at tyrosine residues and blocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis. Inhibition of cellular protein tyrosine kinases by genistein results in dephosphorylation of the ssD-BP, leading not only to significant augmentation of transgene expression from recombinant AAV but also to autonomous replication of the wild-type AAV genome. Dephosphorylation of the ssD-BP also correlates with adenovirus infection, or expression of the adenovirus E4orf6 protein, which is known to induce AAV DNA replication and gene expression. Thus, phosphorylation state of the ssD-BP appears to play a crucial role in the life cycle of AAV and may prove to be an important determinant in the successful use of AAV-based vectors in human gene therapy.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.

The adeno-associated virus type 2 (AAV) genome contains inverted terminal repeats (ITRs) of 145 nucleotides. The terminal 125 nucleotides of each ITR form palindromic hairpin (HP) structures that serve as primers for AAV DNA replication. These HP structures also play an important role in integration as well as rescue of the proviral genome from latently infected cells or from recombinant AAV plasmids. Each ITR also contains a stretch of 20 nucleotides, designated the D sequence, that is not involved in HP structure formation. We have recently shown that the D sequence plays a crucial role in high-efficiency rescue, selective replication, and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein (D-BP), specifically interacts with this sequence (X.-S. Wang, S. Ponnazhagan, and A. Srivastava, J. Virol. 70:1668-1677, 1996). We have now performed mutational analyses of the D sequences to evaluate their precise role in viral DNA rescue, replication, and packaging. We report here that 10 nucleotides proximal to the HP structure in each of the D sequences are necessary and sufficient to mediate high-efficiency rescue, replication, and encapsidation of the viral genome in vivo. In in vitro studies, the same 10 nucleotides were found to be required for specific interaction with D-BP, but viral Rep protein-mediated cleavage at the functional terminal resolution site is independent of these sequences. These data suggest that AAV replication and terminal resolution functions can be uncoupled and that the lack of efficient replication of AAV DNA may not be a consequence of impaired resolution of the viral ITRs. These studies further illustrate that the D sequence-D-BP interaction plays an important role in the AAV life cycle and indicate that it may be possible to develop the next generation of AAV vectors capable of encapsidating larger pieces of DNA.

The adeno-associated virus 2 (AAV)-based vector system has been suggested for its potential use in human gene therapy because the wild-type (wt) AAV genome appears to integrate into the human chromosomal DNA in a site-specific manner. We systematically investigated the integration patterns of the recombinant AAV genomes lacking one or both the viral coding sequences. Four recombinant AAV genomes were constructed containing the genes for resistance to tetracycline (TcR) and the herpesvirus thymidine kinase (TK) promoter-driven gene for resistance to neomycin (neoR; vTc.Neo), the genes for resistance to ampicillin (ApR) and TK-neoR (vAp.Neo), the genes for AAV replication (rep) genes and TK-neoR (vRep.Neo), and the AAV capsid (cap) genes and TK-neoR (vCap.Neo). The integration pattern of each of the recombinant AAV genomes in individual clonal isolates of the human nasopharyngeal carcinoma cell line (KB) analyzed on Southern blots using a neo-specific DNA probe was distinctly different. In addition, in none of the clones examined was the proviral genome covalently linked to the previously described AAV right-junction (Rt.Jn.) human chromosomal DNA fragment, the putative specific-site of integration for the wt AAV genome. Furthermore, whereas a 276-bp DNA fragment could be readily amplified from each of these clones, using a neo-specific primer-pair by polymerase chain reaction (PCR), no amplified DNA product was obtained using the neo- and the Rt.Jn. primer-pair under identical conditions. Fluorescence in situ hybridization (FISH) analyses further revealed the lack of integration of the recombinant AAV into human chromosome 19, even in the presence of a functional rep gene as determined by rescue of the recombinant AAV genome in the presence of adenovirus. These data suggest that the recombinant AAV genomes integrate at sites that are different from that characterized for the wt AAV genome. These studies may have implications in the development of the AAV-based vector system for its potential use in human gene therapy.

The adeno-associated virus 2 (AAV) contains a single-stranded DNA genome of which the terminal 145 nucleotides are palindromic and form T-shaped hairpin structures. These inverted terminal repeats (ITRs) play an important role in AAV DNA replication and resolution, since each of the ITRs contains a terminal resolution site (trs) that is the target site for the AAV rep gene products (Rep). However, the Rep proteins also interact with the AAV DNA sequences that lie outside the ITRs, and the ITRs also play a crucial role in excision of the proviral genome from latently infected cells or from recombinant AAV plasmids. To distinguish between Rep-mediated excision of the viral genome during rescue from recombinant AAV plasmids and the Rep-mediated resolution of the ITRs during AAV DNA replication, we constructed recombinant AAV genomes that lacked either the left or the right ITR sequence and one of the Rep-binding sites (RBSs). No rescue and replication of the AAV genome occurred from these plasmids following transfection into adenovirus type 2-infected human KB cells, as expected. However, excision and abundant replication of the vector sequences was clearly detected from the plasmid that lacked the AAV left ITR, suggesting the existence of an additional putative excision site in the left end of the AAV genome. This site was precisely mapped to one of the AAV promoters at map unit 5 (AAV p5) that also contains an RBS. Furthermore, deletion of this RBS abolished the rescue and replication of the vector sequences. These studies suggest that the Rep-mediated cleavage at the RBS during viral DNA replication may, in part, account for the generation of the AAV defective interfering particles.

Adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus, is gaining attention as a vector for its potential use in human gene therapy. However, few studies have examined the safety and the efficacy of this vector system in vivo. We report here that recombinant AAV vectors, when directly injected intravenously in mice, accumulated predominantly in liver cells, suggesting that AAV may possess in vivo organ-tropism for liver. The transduced lacZ reporter gene was expressed in hepatocytes in the liver and, at the level examined, did not appear to induce any detectable cytotoxic T lymphocyte response against βGal. AAV-mediated transduction of murine hematopoietic progenitor cells ex vivo followed by transplantation into lethally irradiated syngeneic mice also revealed high-efficiency gene transfer into progeny cells without any observable cytotoxicity or deleterious effect. The transduced reporter gene sequences were also expressed in mice in vivo. The AAV-based vectors may thus prove useful as a potentially safe alternative to the more commonly used retrovirus- and adenovirus-based vector systems.

Protective effect of garlic on the development of cancer has been reported in the in vitro and in vivo experimental studies; however, few human epidemiologic studies have evaluated the relationship. A population-based case–control study has been conducted in a Chinese population from 2003 to 2010, with the aim to explore the association between raw garlic consumption and lung cancer. Epidemiologic data were collected by face-to-face interviews using a standard questionnaire among 1,424 lung cancer cases and 4,543 healthy controls. Unconditional logistic regression was used to estimate adjusted ORs and their 95% confidence intervals (CI), and to evaluate ratio of ORs (ROR) for multiplicative interactions between raw garlic consumption and other risk factors. After adjusting for potential confounding factors, raw garlic consumption of 2 times or more per week is inversely associated with lung cancer (OR = 0.56; 95% CI, 0.44–0.72) with a monotonic dose–response relationship (Ptrend < 0.001). Furthermore, strong interactions at either additive and/or multiplicative scales were observed between raw garlic consumption and tobacco smoking [synergy index (SI) = 0.70; 95% CI, 0.57–0.85; and ROR = 0.78; 95% CI, 0.67–0.90], as well as high-temperature cooking oil fume (ROR = 0.77; 95% CI, 0.59–1.00). In conclusion, protective association between intake of raw garlic and lung cancer has been observed with a dose–response pattern, suggesting that garlic may potentially serve as a chemopreventive agent for lung cancer. Effective components in garlic in lung cancer chemoprevention warrant further in-depth investigation. Cancer Prev Res; 6(7); 711–8. ©2013 AACR.

Adeno-associated virus type 2 (AAV), a single-stranded, DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have recently documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays an important role in AAV-mediated transgene expression (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997) and that a strong correlation exists between the phosphorylation state of the ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing et al., J. Virol. 72:1593–1599, 1998). In this report, we document that treatment of cells with specific inhibitors of the epidermal growth factor receptor protein tyrosine kinase (EGF-R PTK) activity, such as tyrphostin, leads to significant augmentation of AAV transduction efficiency, and phosphorylation of the ssD-BP is mediated by the EGF-R PTK. Treatment of cells with EGF results in phosphorylation of the ssD-BP, whereas treatment with tyrphostin causes dephosphorylation of the ssD-BP and consequently leads to increased expression of the transgene. Furthermore, AAV transduction efficiency inversely correlates with expression of the EGF-R in different cell types, and stable transfection of the EGF-R cDNA causes phosphorylation of the ssD-BP, leading to significant inhibition in AAV-mediated transgene expression which can be overcome by the tyrphostin treatment. These data suggest that the PTK activity of the EGF-R is a crucial determinant in the life cycle of AAV and that further studies on the interaction between the EGF-R and the ssD-BP may yield new insights not only into its role in the host cell but also in the successful use of AAV vectors in human gene therapy.

The p Sub 201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077–3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence and helper plasmids lacking the adenovirus ITRs led to complete elimination of replication-competent wt AAV-like particles in recombinant vector stocks. These strategies should be useful in producing clinical-grade AAV vectors suitable for human gene therapy.

Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3′ end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the β-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34 + primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1 + lin − primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.

Although the adeno-associated virus type 2 (AAV) is known to possess a broad host range that transcends the species barrier, we suggested in an earlier study that AAV infection of human cells is receptor mediated (S. Ponnazhagan et al., J. Gen. Virol. 77:1111-1122, 1996). In the present studies, we investigated the ability of AAV to infect primary human hematopoietic progenitor cells capable of multilineage differentiation. Bone marrow-derived CD34+ cells from 12 hematologically normal volunteer donors were infected with a recombinant AAV containing the beta-galactosidase gene under the control of the cytomegalovirus immediate-early promoter (vCMVp-lacZ). Whereas 15 to 80% of the cells from approximately 50% of the donors showed various levels of lacZ gene expression, the expression was undetectable in cells from the remaining donors. However, if cells from both sets of donors were stimulated with various combinations of cytokines to induce differentiation into myeloid and lymphoid lineages following AAV infection, then the level of expression of the transduced gene increased up to 20-fold over a period of 14 days. The results of virus-binding assays suggested that the observed difference between the two groups was due to the differential susceptibility of CD34+ cells to AAV infection rather than to differences in transcription and translation of the transduced gene. To corroborate these results, CD34+ cells from the two donor groups, KB (human nasopharyngeal carcinoma) cells, and M07e (human megakaryocytic leukemia) cells were infected with vCMVp-lacZ. KB cells served as a positive control for AAV infection, and M07e cells served as a negative control. Whereas abundant hybridization to the single-stranded viral DNA on Southern blots was detected in KB and CD34+ cells that were positive for lacZ gene expression, little activity was detected in M07e and CD34+ cells that did not show expression of the lacZ gene. These results suggest that the levels of expression of the putative cellular receptor for AAV vary widely in CD34+ cells from different donors. These studies have implications for the potential use of AAV vectors in human gene therapy involving primary human primitive hematopoietic stem and progenitor cells.

Expression from the human parvovirus B19p6 promoter fused to the firefly luciferase (‘Luc’) reporter gene was evaluated in a non-erythroid human nasopharyngeal carcinoma cell line, KB, and a human megakaryocytic leukaemia cell line, MB-02, known to become permissive for B19 replication following erythroid-differentiation. The B19p6-Luc construct was introduced into KB and MB-02 cells, both in undifferentiated and differentiated states, either via DNA-mediated transfection, or via infection with recombinant adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus known to possess a broad host-range. Although Luc activity was readily detected in KB cells following transfection of the B19p6-Luc plasmid DNA, no expression from the B19p6 promoter was observed following infection with recombinant virus. In addition, transfection of the reporter plasmid resulted in high-level expression of Luc in differentiated but not in undifferentiated MB-02 cells. However, no Luc activity was detected, even in differentiated MB-02 cells, following infection with recombinant virus. Further studies with an additional recombinant as well as wild-type (wt) AAV revealed that MB-02 cells were non-permissive for AAV infection. A second human megakaryocytic leukaemia cell line, M07e, was likewise resistant to infection by recombinant as well as wt AAV. Taken together, these studies identify the first human cell type that cannot be infected by AAV. They indicate that expression from the B19p6 promoter, in the context of an AAV genome, is restricted to primary human haematopoietic cells, perhaps because parvoviral DNA replication and transcription are intrinsically coupled.

The adeno-associated virus type 2 (AAV) genome can be successfully rescued from recombinant plasmids following transfection in adenovirus-infected human cells. However, following rescue, the AAV genome undergoes preferential replication and encapsidation, whereas little replication and packaging of the vector DNA sequences occur. In view of the crucial role in the rescue, replication, and packaging of the proviral genome played by the AAV inverted terminal repeats (ITRs), which consist of a palindromic hairpin (HP) structure and a 20-nucleotide stretch, designated the D-sequence, that is not involved in the HP-formation, we evaluated the involvement of the individual ITRs as well as their components in the selective viral DNA replication and encapsidation. A number of recombinant AAV plasmids that contained deletions-substitutions in different regions of the individual ITRs were constructed and examined for their potential to allow rescue, replication, and/or packaging in adenovirus-infected human cells in vivo. The results reported here document that (ii) two HP structures and one D-sequence are sufficient for efficient rescue and preferential replication of the AAV DNA, (ii) two HP structures alone allow a low-level rescue and replication of the AAV DNA, but rescue and replication of the vector DNA sequences also occur in the absence of the D-sequences, (iii) one HP structure and two D-sequences, but not one HP structure and one D-sequence, also allow rescue and replication of the AAV as well as the vector DNA sequences, (iv) one HP structure alone or two D-sequences, but not one D-sequence alone, allow replication of the full-length plasmid DNA, but no rescue of the AAV genome occurs, (v) no rescue-replication occurs in the absence of the HP structures and the D-sequences, (vi) in the absence of the D-sequences, the HP structures are insufficient for successful encapsidation of the AAV genomes, and (vii) the AAV genomes containing only one ITR structure can be packaged into biologically active virions. Thus, the D-sequence plays a crucial role in the efficient rescue and selective replication and encapsidation of the AAV genome. Furthermore, the D-sequence specifically interacts with a hitherto unknown host-cell protein that we have designated the D-sequence-binding protein (D-BP). These studies illustrate that the D-sequence-D-BP interaction constitutes an important step in the AAV life cycle.

The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.