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author:("Feng, xinghua")
1.  HIV shedding in the oral cavity: an assessment of HIV type, immunovirologic, demographic and oral factors 
To quantify the prevalence and burden of HIV type 2 (HIV-2) and HIV-1 RNA in the oral cavity of antiretroviral therapy-naive HIV-infected Senegalese individuals and to identify correlates of oral HIV viral loads.
A cross-sectional study of 163 HIV-1 and 27 HIV-2-infected antiretroviral therapy-naive Senegalese adults.
Participants received clinical and oral exams and provided blood and oral wash samples for viral load and plasma CD4 count ascertainment. Logistic and interval regression models were used to identify univariate and multivariable associations between presence and level of oral HIV RNA and various immunovirologic, local and demographic factors.
Presence of detectable oral HIV RNA was less common in HIV-2-infected compared with HIV-1-infected study participants (33% vs 67%, OR 0.25, 95% CI 0.11 to 0.59). HIV type was no longer associated with oral shedding of HIV when plasma viral load was considered. Detection of oral HIV RNA was associated with increased plasma viral load in both HIV-1-infected and HIV-2-infected individuals (HIV-1, OR 1.89, 95% CI 1.24 to 2.61; HIV-2, OR 1.93, 95% CI 1.1 to 3.39). Oral HIV-1 detection was also associated with periodontal disease (OR 3.02, 95% CI 1.16 to 7.87).
Oral shedding of HIV-2 RNA is less common than HIV-1 RNA, a likely consequence of lower overall viral burden. Both systemic and local factors may contribute to shedding of HIV in the oral cavity.
PMCID: PMC4028315  PMID: 22250179
2.  Long-lasting complete regression of established mouse tumors by counteracting Th2 inflammation 
Mice with intraperitoneal ID8 ovarian carcinoma or subcutaneous SW1 melanoma were injected with monoclonal antibodies (mAbs) to CD137+PD-1+CTLA4 7-15 days following tumor initiation. Survival of mice with ID8 tumors tripled and >40% of mice with SW1 tumors remain healthy >150 days after later treatment and are probably cured. Therapeutic efficacy was associated with a systemic immune response with memory and antigen specificity and required CD4+ cells and involved CD8+ cells and NK cells to a less extent. The 3 mAb combination significantly decreased CD19+ cells at tumor sites, increased IFNγ and TNFα producing CD4+ and CD8+ T cells and mature CD86+ DC, and it increased the ratios of effector CD4+ and CD8+ T cells to CD4+Foxp3+ regulatory T cells and to CD11b+Gr-1+ myeloid suppressor cells. This is consistent with shifting the tumor microenvironment from an immunosuppressive Th2 to an immunostimulatory Th1 type and is further supported by PCR data. Adding an anti-CD19 mAb to the 3 mAb combination in the SW1 model further increased therapeutic efficacy. Data from ongoing experiments show that intratumoral injection of a combination of mAbs to CD137+PD-1+CLA4+CD19 can induce complete regression and dramatically prolong survival also in the TC1 carcinoma and B16 melanoma models, suggesting that the approach has general validity.
PMCID: PMC3685275  PMID: 23603859
ovarian carcinoma; melanoma; CTLA4; PD-1; CD137; CD19; inflammation
Journal of medical virology  2009;81(4):713-721.
Human papillomavirus (HPV) RNA levels may be a more sensitive early indicator of predisposition to carcinogenesis than DNA levels. We evaluated whether levels of HPV-16 and HPV-18 DNA and messenger RNA (mRNA) in newly detected infections are associated with cervical lesion development. Female university students were recruited from 1990-2004. Cervical samples for HPV DNA, HPV mRNA, and Papanicolaou testing were collected tri-annually, and women were referred for colposcopically-directed biopsy when indicated. Quantitative real-time polymerase chain reaction of L1 and E7 DNA and E7 mRNA was performed on samples from women with HPV-16 and HPV-18 infections that were incidently detected by consensus PCR. Adjusting for other HPV types, increasing E7 cervical HPV-16 mRNA levels at the time of incident HPV-16 DNA detection were associated with an increased risk of cervical intraepithelial neoplasia grade 2 to 3 (HR per 1 log10 increase in mRNA=6.36,95%CI=2.00-20.23). Increasing HPV-16 mRNA levels were also associated with an increased risk of cervical squamous intraepithelial lesions; the risk was highest at the incident positive visit and decreased over time. Neither HPV-16 E7 DNA levels nor HPV-18 E7 DNA nor mRNA levels were significantly associated with cervical lesion development. Report of >1 new partner in the past 8 months (relative to no new partners) was associated with increased HPV mRNA (viral level ratio [VLR]=10.05,95%CI=1.09-92.56) and increased HPV DNA (VLR=16.80,95%CI=1.46-193.01). In newly detected HPV-16 infections, increasing levels of E7 mRNA appear to be associated with an increased risk of developing cervical pre-cancer.
PMCID: PMC3984467  PMID: 19235870
HPV; viral load; mRNA; cervical pre-cancer
4.  An Automated High-Throughput Counting Method for Screening Circulating Tumor Cells in Peripheral Blood 
Analytical chemistry  2013;85(4):2465-2471.
Enumeration of circulating tumor cells (CTCs) has proved valuable for early detection and prognosis in cancer treatment. This paper describes an automated high-throughput counting method for CTCs based on microfluidics and line-confocal microscopy. Peripheral blood was directly labeled with multiple antibodies, each conjugated with a different fluorophore, pneumatically pumped through a microfluidic channel and interrogated by a line-confocal microscope. Based on the fluorescence signals and labeling schemes, the count of CTCs was automatically reported. Due to the high flow rate, 1 mL of whole blood can be analyzed in less than 30 minutes. We applied this method in analyzing CTCs from 90 stage IV breast-cancer patient samples, and performed a side-by-side comparison with the results of the CellSearch assay, which is the only method approved by U.S. Food and Drug Administration (FDA) at present for enumeration of CTCs. This method has a recovery rate for cultured breast cancer cells of 94% (n=9), with an average of 1.2 counts/mL of background level of detected CTCs from healthy donors. It detected CTCs from breast-cancer patients, ranging from 15 to 3375 counts/7.5 mL. Using this method, we also demonstrate the ability to enumerate CTCs from breast-cancer patients that were positive for Her2 or CD44+/CD24−, which is a putative cancer stem cell marker. This automated method can enumerate CTCs from peripheral blood with high throughput and sensitivity. It could potentially benefit the clinical diagnosis and prognosis of cancer.
PMCID: PMC3586433  PMID: 23387387
5.  Human papillomavirus DNA is rarely detected in colorectal carcinomas and not associated with microsatellite instability: The Seattle Colon Cancer Family Registry 
Persistent infection with oncogenic human papillomavirus (HPV) types-16 and -18 is an established cause of cervical and other cancers. Some studies report detection of oncogenic HPV DNA in colorectal carcinomas, with prevalence estimates as high as 84%. However, other studies report detecting no HPV DNA in colorectal tumors.
To evaluate the prevalence of HPV in colorectal cancer subsets, we conducted a case-case comparison study. This study included 555 cases of incident colorectal cancer from the Seattle Colon Cancer Family Registry (CCFR), ages 20-74, and diagnosed between 1998-2002. Standardized interviews were used to elicit demographics and risk factor data. Tumor DNA was assayed for HPV-16 and -18 DNA using real-time PCR. Microsatellite instability (MSI) status was assessed using a standard 10-marker panel and confirmed with immunohistochemical staining. Prevalence estimates were calculated for the overall sample, and stratified by patient and tumor characteristics. Fisher’s exact test was used to compare prevalence between strata.
HPV-16 DNA was detected in 2% of colorectal tumors, but no HPV-18 DNA was detected. HPV-16 prevalence did not vary between cases according to sex, age, race, smoking-status, or MSI-status (P>0.05). HPV-16 prevalence in rectal carcinomas was 5% compared to 1% in colon carcinomas (P=0.03).
Among a large sample of colorectal carcinomas, prevalence of HPV-16 and -18 was very low. Prior studies detecting high HPV prevalence in colorectal carcinomas are likely the result of contamination from the anal canal or clinical processing.
HPV is unlikely to play a large role in colorectal carcinogenesis.
PMCID: PMC3565050  PMID: 23250932
HPV; colorectal cancer; anatomic site; microsatellite instability
6.  tRNA genes rapidly change in evolution to meet novel translational demands 
eLife  2013;2:e01339.
Changes in expression patterns may occur when organisms are presented with new environmental challenges, for example following migration or genetic changes. To elucidate the mechanisms by which the translational machinery adapts to such changes, we perturbed the tRNA pool of Saccharomyces cerevisiae by tRNA gene deletion. We then evolved the deletion strain and observed that the genetic adaptation was recurrently based on a strategic mutation that changed the anticodon of other tRNA genes to match that of the deleted one. Strikingly, a systematic search in hundreds of genomes revealed that anticodon mutations occur throughout the tree of life. We further show that the evolution of the tRNA pool also depends on the need to properly couple translation to protein folding. Together, our observations shed light on the evolution of the tRNA pool, demonstrating that mutation in the anticodons of tRNA genes is a common adaptive mechanism when meeting new translational demands.
eLife digest
Genes contain the blueprints for the proteins that are essential for countless biological functions and processes, and the path that leads from a particular gene to the corresponding protein is long and complex. The genetic information stored in the DNA must first be transcribed to produce a messenger RNA molecule, which then has to be translated to produce a string of amino acids that fold to form a protein. The translation step is performed by a molecular machine called the ribosome, with transfer RNA molecules bringing the amino acids that are needed to make the protein.
The information in messenger RNA is stored as a series of letters, with groups of three letters called codons representing the different amino acids. Since there are four letters—A, C, G and U—it is possible to form 64 different codons. And since there are only 20 amino acids, two or more different codons can specify the same amino acid (for example, AGU and AGC both specify serine), and two or more different transfer RNA molecules can take this amino acid to the ribosome. Moreover, some codons are found more often than others in the messenger RNA molecules, so the genes that encode the related transfer RNA molecules are more common than the genes for other transfer RNA molecules.
Environmental pressures mean that organisms must adapt to survive, with some genes and proteins increasing in importance, and others becoming less important. Clearly the relative numbers of the different transfer RNA molecules will also need to change to reflect these evolutionary changes, but the details of how this happens were not understood.
Now Yona et al. have explored this issue by studying yeast cells that lack a gene for one of the less common transfer RNA molecules (corresponding to the codon AGG, which specifies the amino acid arginine). At first this mutation resulted in slower growth of the yeast cells, but after being allowed to evolve over 200 generations, the rate of growth matched that of a normal strain with all transfer RNA genes. Yona et al. found that the gene for a more common transfer RNA molecule, corresponding to the codon AGA, which also specifies arginine, had mutated to AGG. As a result, the mutated yeast was eventually able to produce proteins as quickly as wild type yeast. Moreover, further experiments showed that the levels of some transfer RNAs are kept deliberately low in order to slow down the production of proteins so as to ensure that the proteins assume their correct structure.
But does the way these cells evolved in the lab resemble what happened in nature? To address this question Yona et al. examined a database of transfer RNA sequences from more than 500 species, and found evidence for the same codon-based switching mechanism in many species across the tree of life.
PMCID: PMC3868979  PMID: 24363105
tRNA genes; anticodon; translation; evolution; protein folding stress; S. cerevisiae
7.  Th2 type inflammation promotes the gradual progression of HPV-infected cervical cells to cervical carcinoma 
Gynecologic oncology  2012;127(2):412-419.
To investigate the role of immunological parameters in tumorigenesis of cervical cancer in women infected with high risk human papillomavirus (hr-HPV), and determine whether key findings with human material can be recapitulated in the mouse TC1 carcinoma model which expresses hr-HPV epitopes.
Epithelial and lymphoid cells in cervical tissues were analyzed by immunohistochemistry and serum IL10 levels were determined by ELISA. Tumor draining lymph nodes were analyzed in the mouse TC1 model by flow cytometry.
The mucosa was infiltrated by CD20+ and CD138+ cells already at cervical intraepithelial neoplasia 1 (CIN1) and infiltration increased in cervical intraepithelial neoplasia 3 (CIN3)/carcinoma in situ (CIS) and invasive cervical cancer (ICC), where it strongly correlated with infiltration by CD32B+ and FoxP3+ lymphocytes. GATA3+ and T-bet+ lymphoid cells were increased in ICC compared to normal, and expression in epithelial cells of the Th2 inflammation-promoting cytokine TSLP and of IDO1 was higher in CIN3/CIS and ICC. As a corollary, serum levels of IL10 were higher in women with CIN3/CIS or ICC than in normals. Finally we demonstrated in the mouse TC1 carcinoma, which expresses hr-HPV epitopes, an increase of cells expressing B cell or plasma cell markers or Fc receptors in tumor-draining than distal lymph nodes or spleen.
hr-HPV initiates a local Th2 inflammation at an early stage, involving antibody forming cells, and fosters an immunosuppressive microenvironment that aids tumor progression.
PMCID: PMC3472044  PMID: 22828962
8.  Prevalence and risk factors for oncogenic HPV infections in high-risk mid-adult women 
Sexually transmitted diseases  2012;39(11):848-856.
The epidemiology of high-risk (hr) HPV infections in mid-adult women with new sex partners is undefined.
We analyzed baseline data from 518 25–65 year old female online daters. Women were mailed questionnaires and kits for self-collecting vaginal specimens for PCR-based hrHPV testing. Risk factors for infection were identified using Poisson regression models to obtain prevalence ratios (PRs).
The prevalence of hrHPV infection was 35.9%. In multivariate analysis restricted to sexually active women, the likelihood of hrHPV infection was associated with abnormal Pap test history (PR=1.42, 95% CI:1.10–1.84), lifetime number of sex partners >14 (relative to 1–4; PR=2.13, 95% CI:1.13–4.02 for 15–24 partners and PR=1.91, 95% CI:1.00–3.64 for ≥25 partners), male partners with ≥1 concurrent partnership (PR=1.34, 95% CI:1.05–1.71) and male partners whom the subject met online (PR=1.39, 95% CI:1.08–1.79). Age was inversely associated with infection only in women who were sexually inactive (PR=0.67 per 5-year age difference, adjusted for Pap history and lifetime number of partners). Compared to sexually inactive women, the likelihood of infection increased with increasing risk level, (from low-risk to high-risk partners) (p<.0001 by trend test). In multivariate analysis, infection with multiple versus single hrHPV types was inversely associated with ever having been pregnant (PR=0.64, 95% CI:0.46–0.90) and recent consistent condom use (PR=0.56, 95% CI:0.32–0.97), and positively associated with genital wart history (PR=1.43, 95% CI:1.03–1.99).
Measures of both cumulative and recent sexual history were associated with prevalent hrHPV infection in this high-risk cohort of mid-adult women.
PMCID: PMC3476060  PMID: 23064533
HPV; human papilloma virus; mid-adult; prevalence; risk factors
9.  Disclosure of Genital Human Papillomavirus Infection to Female Sex Partners by Young Men 
Sexually Transmitted Diseases  2012;39(8):583-587.
A survey was administered to male university students testing positive for high-risk human papillomavirus. Disclosure was more likely in men with fewer partners, in main partnerships, and in longer partnerships. Disclosure was associated with discussing the Pap test/HPV vaccine with female partners and not associated with a worsening relationship.
PMCID: PMC3398400  PMID: 22797688
human papillomavirus; male; disclosure; sexually transmitted infection; sexual partnerships
10.  Co-administration of epithelial junction opener JO-1 improves the efficacy and safety of chemotherapeutic drugs 
Epithelial junctions between tumor cells inhibit the penetration of anti-cancer drugs into tumors. We previously reported on recombinant adenovirus serotype 3 derived protein (JO-1), which triggers transient opening of intercellular junctions in epithelial tumors through binding to desmoglein 2 (DSG2), and enhances the anti-tumor effects of several therapeutic monoclonal antibodies. The goal of this study was to evaluate whether JO-1 co-therapy can also improve the efficacy of chemotherapeutic drugs.
Experimental Design
The effect of intravenous application of JO-1 in combination with several chemotherapy drugs including paclitaxel/Taxol™, nanoparticle albumin bound paclitaxel/Abraxane™, liposomal doxorubicin/Doxil™ and irinotecan/Camptosar™, was tested in xenograft models for breast, colon, ovarian, gastric and lung cancer. Because JO-1 does not bind to mouse cells, for safety studies with JO-1, we also used human DSG2 (hDSG2) transgenic mice with tumors that overexpressed human DSG2.
JO-1 increased the efficacy of chemotherapeutic drugs, and in several models overcame drug resistance. JO-1 treatment also allowed for the reduction of drug doses required to achieve anti-tumor effects. Importantly, JO-1 co-admininstration protected normal tissues, including bone marrow and intestinal epithelium, against toxic effects that are normally associated with chemotherapeutic agents. Using the hDSG2 transgenic mouse model, we demonstrated that JO-1 predominantly accumulates in tumors. Except for a mild, transient diarrhea, intravenous injection of JO-1 (2mg/kg) had no critical side effects on other tissues or hematological parameters in hDSG2-transgenic mice.
Our preliminary data suggest that JO-1 co-therapy has the potential to improve the therapeutic outcome of cancer chemotherapy.
PMCID: PMC3547677  PMID: 22535153
11.  Epithelial junction opener JO-1 improves monoclonal antibody therapy of cancer 
Cancer research  2011;71(22):7080-7090.
The efficacy of monoclonal antibodies (mAbs) used to treat solid tumors is limited by intercellular junctions which tightly link epithelial tumor cells to each another. In this study, we define a small, recombinant adenovirus serotype 3-derived protein, termed junction opener 1 (JO-1), which binds to the epithelial junction protein desmoglein 2 (DSG2). In mouse xenograft models employing Her2/neu- and EGFR-positive human cancer cell lines, JO-1 mediated cleavage of DSG2 dimers and activated intracellular signaling pathways which reduced E-cadherin expression in tight junctions. Notably, JO-1-triggered changes allowed for increased intratumoral penetration of the anti-Her2/neu mAb trastuzumab (Herceptin) as well as improved access to its target receptor, Her2/neu, which is partly trapped in tight junctions. This effect translated directly into increased therapeutic efficacy of trastuzumab in mouse xenograft models using breast, gastric, and ovarian cancer cells that were Her2/neu-positive. Furthermore, combining JO-1 with the EGFR-targeting mAb cetuximab (Erbitux) greatly improved therapeutic outcomes in a metastatic model of EGFR-positive lung cancer. Taken together, our findings offer preclinical proof of concept to employ JO-1 in combination treatments which enhance the efficacy of trastuzumab treatment, by generating a transient degradation of tumor stroma proteins that can elicit eradication of tumors.
PMCID: PMC3217128  PMID: 21990319
12.  Circumcision and acquisition of HPV infection in young men 
Sexually transmitted diseases  2011;38(11):1074-1081.
The role of circumcision in male HPV acquisition is not clear.
Male university students (18–20 years of age) were recruited from 2003–2009 and followed tri-annually. Shaft/scrotum, glans, and urine samples were tested for 37 alpha HPV genotypes. Cox proportional hazards methods were used to evaluate the association between circumcision and HPV acquisition. Logistic regression was used to assess whether number of genital sites infected at incident HPV detection or site of incident detection varied by circumcision status.
In 477 men, rates of acquiring clinically-relevant HPV types (high-risk types plus types 6 and 11) did not differ significantly by circumcision status (hazard ratio [HR] for uncircumcised relative to circumcised subjects: 0.9[95%CI:0.7–1.2]). However, compared to circumcised men, uncircumcised men were 10.1 (95%CI:2.9–35.6) times more likely to have the same HPV type detected in all 3 genital specimens than in a single genital specimen and were 2.7 (95%CI:1.6–4.5) times more likely to have an HPV-positive urine or glans specimen at first detection.
While the likelihood of HPV acquisition did not differ by circumcision status, uncircumcised men were more likely than circumcised men to have infections detected at multiple genital sites, which may have implications for HPV transmission.
PMCID: PMC3210112  PMID: 21992987
HPV; human papilloma virus; circumcision; epidemiology; risk factors
13.  MicroRNA expression in ovarian carcinoma and its correlation with clinicopathological features 
MicroRNA (miRNA) expression is known to be deregulated in ovarian carcinomas. However, limited data is available about the miRNA expression pattern for the benign or borderline ovarian tumors as well as differential miRNA expression pattern associated with histological types, grades or clinical stages in ovarian carcinomas. We defined patterns of microRNA expression in tissues from normal, benign, borderline, and malignant ovarian tumors and explored the relationship between frequently deregulated miRNAs and clinicopathologic findings, response to therapy, survival, and association with Her-2/neu status in ovarian carcinomas.
We measured the expression of nine miRNAs (miR-181d, miR-30a-3p, miR-30c, miR-30d, miR-30e-3p, miR-368, miR-370, miR-493-5p, miR-532-5p) in 171 formalin-fixed, paraffin-embedded ovarian tissue blocks as well as six normal human ovarian surface epithelial (HOSE) cell lines using Taqman-based real-time PCR assays. Her-2/neu overexpression was assessed in ovarian carcinomas (n = 109 cases) by immunohistochemistry analysis.
Expression of four miRNAs (miR-30c, miR-30d, miR-30e-3p, miR-370) was significantly different between carcinomas and benign ovarian tissues as well as between carcinoma and borderline tissues. An additional three miRNAs (miR-181d, miR-30a-3p, miR-532-5p) were significantly different between borderline and carcinoma tissues. Expression of miR-532-5p was significantly lower in borderline than in benign tissues. Among ovarian carcinomas, expression of four miRNAs (miR-30a-3p, miR-30c, miR-30d, miR-30e-3p) was lowest in mucinous and highest in clear cell samples. Expression of miR-30a-3p was higher in well-differentiated compared to poorly differentiated tumors (P = 0.02), and expression of miR-370 was higher in stage I/II compared to stage III/IV samples (P = 0.03). In multivariate analyses, higher expression of miR-181d, miR-30c, miR-30d, and miR-30e-3p was associated with significantly better disease-free or overall survival. Finally, lower expression of miR-30c, miR-30d, miR-30e-3p and miR-532-5p was significantly associated with overexpression of Her-2/neu.
Aberrant expression of miRNAs is common in ovarian tumor suggesting involvement of miRNA in ovarian tumorigenesis. They are associated with histology, clinical stage, survival and oncogene expression in ovarian carcinoma.
PMCID: PMC3449188  PMID: 22925189
miRNA; Ovarian tumor; Her2/neu; Survival
14.  Serum Antibody Response Following Genital α9 Human Papillomavirus Infection in Young Men 
The Journal of Infectious Diseases  2011;204(2):209-216.
Background. Although the prevalence of human papillomavirus (HPV) genital infection is similarly high in males and females, seroprevalence is lower in males. This study assessed rates and determinants of seroconversion after detection of genital HPV infection in young men.
Methods. We investigated HPV type-specific seroconversion in a cohort of heterosexual male university students who had an α9 HPV type (HPV-16, -31, -33, -35, -52, -58, or -67) detected in the genital tract (n = 156). HPV DNA and antibodies were detected and typed using liquid bead-based multiplex assays. We calculated seroconversion using Kaplan–Meier survival analysis. Cox proportional hazards models with generalized estimating equations were used to examine associations with seroconversion.
Results. Within 24 months of detecting genital HPV infection, type-specific seroconversion ranged from 4% for HPV–52 to 36% for HPV-31. HPV-16 seroconversion at 24 months was 13% (95% confidence interval [CI], 7%–25%). Among incident HPV infections, ever cigarette smoking and infection site(s) (shaft/scrotum and glans/urine vs shaft/scrotum or glans/urine only) were positively associated with type-specific seroconversion.
Conclusions. For each of the α9 HPV types, type-specific seroconversion within 24 months was observed in 36% or less of infected men. Seroconversion might be related to cigarette smoking and genital site(s) infected.
PMCID: PMC3114468  PMID: 21673030
15.  Analysis of Tp53 Codon 72 Polymorphisms, Tp53 Mutations, and HPV Infection in Cutaneous Squamous Cell Carcinomas 
PLoS ONE  2012;7(4):e34422.
Non-melanoma skin cancers are one of the most common human malignancies accounting for 2–3% of tumors in the US and represent a significant health burden. Epidemiology studies have implicated Tp53 mutations triggered by UV exposure, and human papilloma virus (HPV) infection to be significant causes of non-melanoma skin cancer. However, the relationship between Tp53 and cutaneous HPV infection is not well understood in skin cancers. In this study we assessed the association of HPV infection and Tp53 polymorphisms and mutations in lesional specimens with squamous cell carcinomas.
We studied 55 cases of histologically confirmed cutaneous squamous cell carcinoma and 41 controls for the presence of HPV infection and Tp53 genotype (mutations and polymorphism).
We found an increased number of Tp53 mutations in the squamous cell carcinoma samples compared with perilesional or control samples. There was increased frequency of homozygous Tp53-72R polymorphism in cases with squamous cell carcinomas, while the Tp53-72P allele (Tp53-72R/P and Tp53-72P/P) was more frequent in normal control samples. Carcinoma samples positive for HPV showed a decreased frequency of Tp53 mutations compared to those without HPV infection. In addition, carcinoma samples with a Tp53-72P allele showed an increased incidence of Tp53 mutations in comparison carcinomas samples homozygous for Tp53-72R.
These studies suggest there are two separate pathways (HPV infection and Tp53 mutation) leading to cutaneous squamous cell carcinomas stratified by the Tp53 codon-72 polymorphism. The presence of a Tp53-72P allele is protective against cutaneous squamous cell carcinoma, and carcinoma specimens with Tp53-72P are more likely to have Tp53 mutations. In contrast Tp53-72R is a significant risk factor for cutaneous squamous cell carcinoma and is frequently associated with HPV infection instead of Tp53 mutations. Heterozygosity for Tp53-72R/P is protective against squamous cell carcinomas, possibly reflecting a requirement for both HPV infection and Tp53 mutations.
PMCID: PMC3335843  PMID: 22545084
Characterizing short-term detection patterns of young women’s incident alpha-genus human papillomavirus (HPV) infections may further understanding of HPV transmission.
Between 2000–2007, we followed 18–22 year old female university students with triannual HPV DNA and Papanicolau testing. Using Kaplan-Meier methods, we estimated: duration of detectable, type-specific incident infections; time to re-detection (among infections that became undetectable); and time to cervical lesion development after incident infection. We evaluated risk factors for short-term persistent versus transient infection with logistic regression.
303 incident type-specific infections were detected in 85 sexually active women. Median time to first negative test after incident infection was 9.4 (95%CI:7.8–11.2) months; 90.6% of infections became undetectable within two years. 19.4% of infections that became undetectable were re-detected within one year. Cervical lesions were common, and 60% were positive for multiple HPV types in concurrent cervical swabs. Incident HPV detection in the cervix only (versus the vulva/vagina only or both sites) was associated with short-term transience.
While most incident infections became undetectable within two years, re-detection was not uncommon. Cervical lesions were a common early manifestation of HPV infection.
It remains unclear whether potentially modifiable risk factors can be identified to reduce infection duration (and transmission likelihood).
PMCID: PMC3078690  PMID: 21173170
human papillomavirus; incidence; duration; persistence; women; epidemiology
17.  Expression of Mir-21 and Mir-143 in Cervical Specimens Ranging from Histologically Normal through to Invasive Cervical Cancer 
PLoS ONE  2011;6(12):e28423.
MicroRNA expression is severely disrupted in carcinogenesis, however limited evidence is available validating results from cell-line models in human clinical cancer specimens. MicroRNA-21 (mir-21) and microRNA-143 (mir-143) have previously been identified as significantly deregulated in a range of cancers including cervical cancer. Our goal was to investigate the expression patterns of several well-studied microRNA species in cervical samples and compare the results to cell line samples.
Methodology/Principal Findings
We measured the expression of mir-21 and mir-143 in 142 formalin-fixed, paraffin embedded (FFPE) cervical biopsy tissue blocks, collected from Dantec Oncology Clinic, Dakar, Senegal. MicroRNA expression analysis was performed using Taqman-based real-time PCR assays. Protein immunohistochemical staining was also performed to investigate target protein expression on 72 samples. We found that mir-21 expression increased with worsening clinical diagnosis but that mir-143 was not correlated with histology. These observations were in stark contrast to previous reports involving cervical cancer cell lines in which mir-143 was consistently down-regulated but mir-21 largely unaffected. We also identified, for the first time, that cytoplasmic expression of Programmed Cell Death Protein 4 PDCD4; a known target of mir-21) was significantly lower in women with invasive cervical carcinoma (ICC) in comparison to those with cervical intraepithelial neoplasia (2–3) or carcinoma in situ (CIN2-3/CIS), although there was no significant correlation between mir-21 and PDCD4 expression, despite previous studies identifying PDCD4 transcript as a known mir-21 target.
Whilst microRNA biomarkers have a number of promising features, more studies on expression levels in histologically defined clinical specimens are required to investigate clinical relevance of discovery-based studies. Mir-21 may be of some utility in predictive screening, given that we observed a significant correlation between mir-21 expression level and worsening histological diagnosis of cervical cancer.
PMCID: PMC3237431  PMID: 22194833
18.  Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays 
BMC Molecular Biology  2011;12:49.
The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP) and methylated DNA (MeDIP) represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription.
To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease.
The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation to pursue mechanistic, functional and diagnostic information at genes of interest in health and disease.
PMCID: PMC3247195  PMID: 22098709
19.  Magnetic Resonance Imaging of Bone Marrow Cell-Mediated Interleukin-10 Gene Therapy of Atherosclerosis 
PLoS ONE  2011;6(9):e24529.
A characteristic feature of atherosclerosis is its diffuse involvement of arteries across the entire human body. Bone marrow cells (BMC) can be simultaneously transferred with therapeutic genes and magnetic resonance (MR) contrast agents prior to their transplantation. Via systemic transplantation, these dual-transferred BMCs can circulate through the entire body and thus function as vehicles to carry genes/contrast agents to multiple atherosclerosis. This study was to evaluate the feasibility of using in vivo MR imaging (MRI) to monitor BMC-mediated interleukin-10 (IL-10) gene therapy of atherosclerosis.
For in vitro confirmation, donor mouse BMCs were transduced by IL-10/lentivirus, and then labeled with a T2-MR contrast agent (Feridex). For in vivo validation, atherosclerotic apoE−/− mice were intravenously transplanted with IL-10/Feridex-BMCs (Group I, n = 5) and Feridex-BMCs (Group II, n = 5), compared to controls without BMC transplantation (Group III, n = 5). The cell migration to aortic atherosclerotic lesions was monitored in vivo using 3.0T MRI with subsequent histology correlation. To evaluate the therapeutic effect of BMC-mediated IL-10 gene therapy, we statistically compared the normalized wall indexes (NWI) of ascending aortas amongst different mouse groups with various treatments.
Principal Findings
Of in vitro experiments, simultaneous IL-10 transduction and Feridex labeling of BMCs were successfully achieved, with high cell viability and cell labeling efficiency, as well as IL-10 expression efficiency (≥90%). Of in vivo experiments, MRI of animal groups I and II showed signal voids within the aortic walls due to Feridex-created artifacts from the migrated BMCs in the atherosclerotic plaques, which were confirmed by histology. Histological quantification showed that the mean NWI of group I was significantly lower than those of group II and group III (P<0.05).
This study has confirmed the possibility of using MRI to track, in vivo, IL-10/Feridex-BMCs recruited to atherosclerotic lesions, where IL-10 genes function to prevent the progression of atherosclerosis.
PMCID: PMC3168522  PMID: 21915349
20.  DNA hypermethylation of tumors from non-small cell lung cancer (NSCLC) patients is associated with gender and histologic type 
We previously identified a number of genes which were methylated significantly more frequently in the tumor compared to the non-cancerous lung tissues from non-small cell lung cancer (NSCLC) patients. Detection of methylation profiles of genes in NSCLC could provide insight into differential pathways to malignancy and lead to strategies for better treatment of individuals with NSCLC.
We determined the DNA methylation status of 27 genes using quantitative MethyLight assays in lung tumor samples from 117 clinically well-characterized NSCLC patients.
Hypermethylation was detected in one of more of the genes in 106 (91%) of 117 cases and was detected at high levels (Percentage of Methylation Reference (PMR)≥4%) in 79% of NSCLC cases. Methylation of APC, CCND2, KCNH5 and, RUNX was significantly more frequent in adenocarcinomas compared to squamous cell carcinomas (SCC), while methylation of CDKN2A was more common in SCC. Hypermethylation of KCNH5, KCNH8, and RARB was more frequent in females compared to males. Hypermethylation of APC and CCND2 was inversely associated with proliferation score assessed by Ki-67 level.
Our findings of differential gene hypermethylation frequencies in tumor tissues from patients with adenocarcinoma or squamous cell cancers and in females compared to males suggests that further investigation is warranted in order to more fully understand the potential disparate pathways and/or risk factors for NSCLC associated with histologic type and gender.
PMCID: PMC2888601  PMID: 19945765
hypermethylation; lung cancer; gender; histology
Little is known about detection of genital human papillomavirus (HPV) types in women’s fingertips. The study objectives were to determine the presence of genital HPV types in fingertip samples and agreement between fingertip and genital samples for detecting HPV.
At tri-annual visits, genital and fingertip samples were collected from female university students and tested for 37 HPV genotypes by PCR-based assay. Type-specific concordance between paired fingertip and genital samples was evaluated using a kappa statistic for percent positive agreement (“kappa +”). Paired samples with type-specific concordant fingertip and genital results were selected for variant characterization.
A total of 357 fingertip samples were collected from 128 women. HPV prevalence in fingertip samples was 14.3%. Although percent positive agreement between fingertips and genitals for detecting type-specific HPV was low (17.8%; kappa+=0.17, 95%CI:0.10–0.25), 60.4% of type-specific HPV detected in the fingertips was detected in a concurrent genital sample. All but one of 28 paired concordant samples were positive for the same type-specific variant in the fingertip and genital sample. Re-detection of HPV types at the subsequent visit was more common in genital samples (73.3%) than in fingertip samples (14.5%) (p<.001).
Detection of genital HPV types in the fingertips was not uncommon. While impossible to distinguish between deposition of DNA from the genitals to the fingertips and true fingertip infection, the rarity of repeat detection in the fingertips suggests that deposition is more common.
Finger-genital transmission is plausible, but unlikely to be a significant source of genital HPV infection.
PMCID: PMC2901391  PMID: 20570905
human papillomavirus; fingertip; genital; women; epidemiology
22.  DNA methylation changes in normal liver tissues and hepatocellular carcinoma with different viral infection 
Hepatocellular carcinoma (HCC) is known to be associated with both HBV and HCV and HVC. While epigenetic changes have been previously reported to be associated with hepatocellular carcinoma (HCC), whether the epigenetic profile of HBC associated HCC differs from that of HCV associated HCC is unclear. We analyzed DNA methylation of ten genes (APC, CCND2, CDKN2A, GSTP1, HOXA9, RARB, RASSF1, RUNX, SFRP1, and TWIST1) using MethyLight assays on 65 archived liver tissue blocks. Three genes (APC, CCND2, and GSTP1) were frequently methylated in normal liver tissues. Five genes (APC, CDKN2A, HOXA9, RASSF1, and RUNX) were significantly more frequently methylated in malignant liver tissues than normal liver tissues. Among HCC cases, HOXA9, RASSF1 and SFRP1 were methylated more frequently in HBV positive HCC cases, while CDKN2A were significantly more frequently methylated in HCV positive HCC cases. Our data support the hypothesis that HCC resulting from different viral etiologies are associated with different epigenetic changes.
PMCID: PMC2848881  PMID: 20079733
hypermethylation; HBV; HCV; hepatocellular carcinoma
23.  Hypermethylation of CCND2 May Reflect a Smoking-Induced Precancerous Change in the Lung 
Journal of Oncology  2011;2011:950140.
It remains unknown whether tobacco smoke induces DNA hypermethylation as an early event in carcinogenesis or as a late event, specific to overt cancer tissue. Using MethyLight assays, we analyzed 316 lung tissue samples from 151 cancer-free subjects (121 ever-smokers and 30 never-smokers) for hypermethylation of 19 genes previously observed to be hypermethylated in nonsmall cell lung cancers. Only APC (39%), CCND2 (21%), CDH1 (7%), and RARB (4%) were hypermethylated in >2% of these cancer-free subjects. CCND2 was hypermethylated more frequently in ever-smokers (26%) than in never-smokers (3%). CCND2 hypermethylation was also associated with increased age and upper lobe sample location. APC was frequently hypermethylated in both ever-smokers (41%) and never-smokers (30%). BVES, CDH13, CDKN2A (p16), CDKN2B, DAPK1, IGFBP3, IGSF4, KCNH5, KCNH8, MGMT, OPCML, PCSK6, RASSF1, RUNX, and TMS1 were rarely hypermethylated (<2%) in all subjects. Hypermethylation of CCND2 may reflect a smoking-induced precancerous change in the lung.
PMCID: PMC3090638  PMID: 21577262
24.  Evaluation of Transported Dry and Wet Cervical Exfoliated Samples for Detection of Human Papillomavirus Infection▿  
Journal of Clinical Microbiology  2010;48(9):3068-3072.
We determined the feasibility of human papillomavirus (HPV) detection in cervical exfoliated cells collected as dry swab samples. Both dry cervical swab and specimen transport medium (STM) cervical swab samples were collected from 135 patients attending either colposcopy or women's clinics in Guayaquil, Ecuador, who had a cytology diagnosis within 6 months. HPV was detected by dot blot hybridization and genotyped by the liquid bead microarray assay (LBMA). Overall, 23.1% of dry samples were positive for any high-risk HPV types, and 24.6% of STM samples were positive for any high-risk HPV types. Of 125 paired samples, the type-specific high-risk HPV proportion positive agreement was 60.7% (kappa, 0.69; 95% confidence interval [CI], 0.53 to 0.82). Of six women with cytological evidence of invasive cervical cancer, high-risk HPV DNA was detected in three of their STM samples and in five of their dry samples. Dry samples were more likely to be insufficient for HPV testing than STM samples. Consistent with this observation, the amount of genomic DNA quantitated with the β-actin gene was almost 20 times lower in dry samples than in STM samples when detected by the real-time TaqMan assay; however, HPV DNA viral loads in dry samples were only 1.6 times lower than those in matched STM samples. We concluded that exfoliated cervical cells could be collected as dry swab samples for HPV detection.
PMCID: PMC2937663  PMID: 20610686
25.  Risk of Female Human Papillomavirus Acquisition Associated with First Male Sex Partner 
The Journal of infectious diseases  2008;197(2):279-282.
To quantify the risk of human papillomavirus (HPV) acquisition associated with a first male sex partner and to identify associated risk factors, we analyzed data from women who were enrolled before or within 3 months of first intercourse with a male partner and were censored at the report of a second partner. The 1-year cumulative incidence of first HPV infection was 28.5% (95% confidence interval, 20.6%–38.6%) and increased to almost 50% by 3 years. The risk was increased when the first male partner was sexually experienced. Our results indicate a high risk of HPV infection in young women who have had just 1 male sex partner.
PMCID: PMC2875685  PMID: 18179386

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