Baat Gene Knockout Alters Post-Natal Development, the Gut Microbiome, and Reveals Unusual Bile Acids in Mice.

Bile acids (BAs) are steroid detergents in bile that contribute to fat absorption, cell signaling, and microbiome interactions. The final step in their synthesis is amino acid conjugation with either glycine or taurine in the liver by the enzyme bile acid-CoA:amino acid N-acyltransferase (BAAT). Here, we describe the microbial, chemical, and physiological consequences of Baat gene knockout. Baat-/- mice were underweight after weaning but quickly exhibited catch-up growth. At three weeks of age, KO animals had increased phospholipid excretion and decreased subcutaneous fat pad mass, liver mass, glycogen staining in hepatocytes, and hepatic vitamin A stores, but these were less marked in adulthood. Additionally, KO mice had an altered microbiome in early life. Their BA pool was highly enriched in cholic acid but not completely devoid of conjugated BAs. KO animals had 27-fold lower taurine-conjugated BAs than wild type in their liver but similar concentrations of glycine-conjugated BAs and higher microbially conjugated BAs. Furthermore, the BA pool in Baat-/- was enriched in a variety of unusual BAs that were putatively sourced from cysteamine conjugation with subsequent oxidation and methylation of the sulfur group mimicking taurine. Antibiotic treatment of KO mice indicated the microbiome was not the likely source of the unusual conjugations, instead, the unique BAs in KO animals were likely derived from the peroxisomal acyltransferases Acnat1 and Acnat2, which are duplications of Baat in the mouse genome that are inactivated in humans. This study demonstrates that BA conjugation is important for early life development of mice.

Elevated phosphorylation of EGFR in NSCLC due to mutations in PTPRH.

The role of EGFR in lung cancer is well described with numerous activating mutations that result in phosphorylation and tyrosine kinase inhibitors that target EGFR. While the role of the EGFR kinase in non-small cell lung cancer (NSCLC) is appreciated, control of EGFR signaling pathways through dephosphorylation by phosphatases is not as clear. Through whole genome sequencing we have uncovered conserved V483M Ptprh mutations in PyMT induced tumors. Profiling the downstream events of Ptprh mutant tumors revealed AKT activation, suggesting a key target of PTPRH was EGFR tyrosine 1197. Given the role of EGFR in lung cancer, we explored TCGA data which revealed that a subset of PTPRH mutant tumors shared gene expression profiles with EGFR mutant tumors, but that EGFR mutations and PTPRH mutations were mutually exclusive. Generation of a PTPRH knockout NSCLC cell line resulted in Y1197 phosphorylation of EGFR, and a rescue with expression of wild type PTPRH returned EGFR phosphorylation to parental line values while rescue with catalytically dead PTPRH did not. A dose response curve illustrated that two human NSCLC lines with naturally occurring PTPRH mutations responded to EGFR tyrosine kinase inhibition. Osimertinib treatment of these tumors resulted in a reduction of tumor volume relative to vehicle controls. PTPRH mutation resulted in nuclear pEGFR as seen in immunohistochemistry, suggesting that there may also be a role for EGFR as a transcriptional co-factor. Together these data suggest mutations in PTPRH in NSCLC is inhibitory to PTPRH function, resulting in aberrant EGFR activity and ultimately may result in clinically actionable alterations using existing therapies.

ELOVL4-Mediated Production of Very Long-Chain Ceramides Stabilizes Tight Junctions and Prevents Diabetes-Induced Retinal Vascular Permeability.

Tight junctions (TJs) involve close apposition of transmembrane proteins between cells. Although TJ proteins have been studied in detail, the role of lipids is largely unknown. We addressed the role of very long-chain (VLC ≥26) ceramides in TJs using diabetes-induced loss of the blood-retinal barrier as a model. VLC fatty acids that incorporate into VLC ceramides are produced by elongase elongation of very long-chain fatty acids protein 4 (ELOVL4). ELOVL4 is significantly reduced in the diabetic retina. Overexpression of ELOVL4 significantly decreased basal permeability, inhibited vascular endothelial growth factor (VEGF)- and interleukin-1ß-induced permeability, and prevented VEGF-induced decrease in occludin expression and border staining of TJ proteins ZO-1 and claudin-5. Intravitreal delivery of AAV2-hELOVL4 reduced diabetes-induced increase in vascular permeability. Ultrastructure and lipidomic analysis revealed that ω-linked acyl-VLC ceramides colocalize with TJ complexes. Overall, normalization of retinal ELOVL4 expression could prevent blood-retinal barrier dysregulation in diabetic retinopathy through an increase in VLC ceramides and stabilization of TJs.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Imbalances in Mobilization and Activation of Pro-Inflammatory and Vascular Reparative Bone Marrow-Derived Cells in Diabetic Retinopathy.

Diabetic retinopathy is a sight-threatening complication of diabetes, affecting 65% of patients after 10 years of the disease. Diabetic metabolic insult leads to chronic low-grade inflammation, retinal endothelial cell loss and inadequate vascular repair. This is partly due to bone marrow (BM) pathology leading to increased activity of BM-derived pro-inflammatory monocytes and impaired function of BM-derived reparative circulating angiogenic cells (CACs). We propose that diabetes has a significant long-term effect on the nature and proportion of BM-derived cells that circulate in the blood, localize to the retina and home back to their BM niche. Using a streptozotocin mouse model of diabetic retinopathy with GFP BM-transplantation, we have demonstrated that BM-derived circulating pro-inflammatory monocytes are increased in diabetes while reparative CACs are trapped in the BM and spleen, with impaired release into circulation. Diabetes also alters activation of splenocytes and BM-derived dendritic cells in response to LPS stimulation. A majority of the BM-derived GFP cells that migrate to the retina express microglial markers, while others express endothelial, pericyte and Müller cell markers. Diabetes significantly increases infiltration of BM-derived microglia in an activated state, while reducing infiltration of BM-derived endothelial progenitor cells in the retina. Further, control CACs injected into the vitreous are very efficient at migrating back to their BM niche, whereas diabetic CACs have lost this ability, indicating that the in vivo homing efficiency of diabetic CACs is dramatically decreased. Moreover, diabetes causes a significant reduction in expression of specific integrins regulating CAC migration. Collectively, these findings indicate that BM pathology in diabetes could play a role in both increased pro-inflammatory state and inadequate vascular repair contributing to diabetic retinopathy.

Role of Acid Sphingomyelinase in Shifting the Balance Between Proinflammatory and Reparative Bone Marrow Cells in Diabetic Retinopathy.

The metabolic insults associated with diabetes lead to low-grade chronic inflammation, retinal endothelial cell damage, and inadequate vascular repair. This is partly due to the increased activation of bone marrow (BM)-derived proinflammatory monocytes infiltrating the retina, and the compromised function of BM-derived reparative circulating angiogenic cells (CACs), which home to sites of endothelial injury and foster vascular repair. We now propose that a metabolic link leading to activated monocytes and dysfunctional CACs in diabetes involves upregulation of a central enzyme of sphingolipid signaling, acid sphingomyelinase (ASM). Selective inhibition of ASM in the BM prevented diabetes-induced activation of BM-derived microglia-like cells and normalized proinflammatory cytokine levels in the retina. ASM upregulation in diabetic CACs caused accumulation of ceramide on their cell membrane, thereby reducing membrane fluidity and impairing CAC migration. Replacing sphingomyelin with ceramide in synthetic membrane vesicles caused a similar decrease in membrane fluidity. Inhibition of ASM in diabetic CACs improved membrane fluidity and homing of these cells to damaged retinal vessels. Collectively, these findings indicate that selective modulation of sphingolipid metabolism in BM-derived cell populations in diabetes normalizes the reparative/proinflammatory cell balance and can be explored as a novel therapeutic strategy for treating diabetic retinopathy.

Increased metastasis with loss of E2F2 in Myc-driven tumors.

In human breast cancer, mortality is associated with metastasis to distant sites. Therefore, it is critical to elucidate the biological mechanisms that underlie tumor progression and metastasis. Using signaling pathway signatures we previously predicted a role for E2F transcription factors in Myc induced tumors. To test this role we interbred MMTV-Myc transgenic mice with E2F knockouts. Surprisingly, we observed that the loss of E2F2 sharply increased the percentage of lung metastasis in MMTV-Myc transgenic mice. Examining the gene expression profile from these tumors, we identified genetic components that were potentially involved in mediating metastasis. These genes were filtered to uncover the genes involved in metastasis that also impacted distant metastasis free survival in human breast cancer. In order to elucidate the mechanism by which E2F2 loss enhanced metastasis we generated knockdowns of E2F2 in MDA-MB-231 cells and observed increased migration in vitro and increased lung colonization in vivo. We then examined genes that were differentially regulated between tumors from MMTV-Myc, MMTV-Myc E2F2-/-, and lung metastases samples and identified PTPRD. To test the role of PTPRD in E2F2-mediated breast cancer metastasis, we generated a knockdown of PTPRD in MDA-MB-231 cells. We noted that decreased levels of PTPRD resulted in decreased migration in vitro and decreased lung colonization in vivo. Taken together, these data indicate that E2F2 loss results in increased metastasis in breast cancer, potentially functioning through a PTPRD dependent mechanism.

Assessing the in vivo efficacy of doxorubicin loaded hyaluronan nanoparticles.

Magnetic nanoparticles are attractive platforms for biomedical applications including diagnosis and treatment of diseases. We have shown previously that hyaluronan-coated superparamagnetic iron oxide nanoparticles (HA-SPIONs) enhanced the efficacy of the conjugated anticancer drug doxorubicin (DOX) in vitro against drug-sensitive and drug-resistant human ovarian cancer cells. In this manuscript, we report our findings on the efficacy of DOX loaded HA-SPIONs in vivo using subcutaneous and intraperitoneal SKOV-3 ovarian tumor models in nude mice. The accumulation of the nanoparticles in subcutaneous tumors following an intravenous nanoparticle administration was confirmed by magnetic resonance imaging, and its distribution in the tumors was evaluated by confocal microscopy and Prussian blue staining. DOX delivered by nanoparticles accumulated at much higher levels and distributed wider in the tumor tissue than intravenously injected free DOX, leading to significant reduction of tumor growth. The IVIS Spectrum for in vivo bioluminescence imaging was used to aid in therapy assessment of the DOX-loaded nanoparticles on intraperitoneal ovarian tumors formed by firefly luciferase expressing human ovarian SKOV-3 cells. DOX-loaded HA-SPIONs significantly reduced tumor growth, delayed tumor development, and extended the survival of mice. Thus, utilizing HA-SPIONs as drug delivery vehicles constitutes a promising approach to tackle CD44 expressing ovarian cancer.

Reprint of BMCL_19101.

The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice.

Prevalence and risk factors for oncogenic human papillomavirus infections in high-risk mid-adult women.

BACKGROUND: The epidemiology of high-risk (hr) human papillomavirus (HPV) infections in mid-adult women with new sex partners is undefined. METHODS: We analyzed baseline data from 518 25- to 65-year-old women online daters. Women were mailed questionnaires and kits for self-collecting vaginal specimens for polymerase chain reaction-based hrHPV testing. Risk factors for infection were identified using Poisson regression models to obtain prevalence ratios (PRs). RESULTS: 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 Papanicolaou test history (PR = 1.42, 95% confidence interval [CI]: 1.10-1.84), lifetime number of sex partners >14 (compared with 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 Papanicolaou history and lifetime number of partners). Compared with sexually inactive women, the likelihood of infection increased with increasing risk level (from low-risk to hr partners; P < 0.0001 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). CONCLUSIONS: Measures of both cumulative and recent sexual history were associated with prevalent hrHPV infection in this hr cohort of mid-adult women.

Rates and determinants of oral human papillomavirus infection in young men.

BACKGROUND: Little is known about the rates and determinants of oral human papillomavirus (HPV) infection, an infection that is etiologically linked with oropharyngeal cancers. METHODS: A cohort of male university students (18-24 years) was examined every 4 months (212 men, 704 visits). Oral specimens were collected via gargle/rinse and swabbing of the oropharynx. Genotyping for HPV-16 and 36 other α-genus types was performed by polymerase chain reaction-based assay. Data on potential determinants were gathered via clinical examination, in-person questionnaire, and biweekly online diary. Hazards ratios (HR) were used to measure associations with incident infection. RESULTS: Prevalence of oral HPV infection at enrollment was 7.5%, and 12-month cumulative incidence was 12.3% (95% confidence interval [CI], 7.0, 21.3). Prevalence of oral HPV-16 was 2.8% and 12-month cumulative incidence was 0.8% (95% CI, 0.1%-5.7%). None of the incident oral HPV infections and 28.6% of the prevalent oral HPV infections were detected more than once. In a multivariate model, incident oral HPV infection was associated with recent frequency of performing oral sex (≥1 per week: HR, 3.7; 95% CI, 1.4-9.8), recent anal sex with men (HR, 42.9; 95% CI, 8.8-205.5), current infection with the same HPV type in the genitals (HR, 6.2; 95% CI, 2.4-16.4), and hyponychium (HR, 11.8, 95% CI, 4.1-34.2). CONCLUSIONS: Although nearly 20% of sexually active male university students had evidence of oral HPV infection within 12 months, most infections were transient. Human papillomavirus type 16 was not common. Sexual contact and autoinoculation appeared to play independent roles in the transmission of α-genus HPV to the oral cavity of young men.

Attenuation of collagen-induced arthritis by orally available imidazoline-based NF-κB inhibitors.

The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice.

Circumcision and acquisition of human papillomavirus infection in young men.

BACKGROUND: The role of circumcision in male HPV acquisition is not clear. METHODS: Male university students (aged 18-20 years) were recruited from 2003 to 2009 and followed up triannually. Shaft/scrotum, glans, and urine samples were tested for 37 α human papillomavirus (HPV) genotypes. Cox proportional hazards methods were used to evaluate the association between circumcision and HPV acquisition. Logistic regression was used to assess whether the number of genital sites infected at incident HPV detection or site of incident detection varied by circumcision status. RESULTS: 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 for uncircumcised relative to circumcised subjects: 0.9 [95% confidence interval{CI}: 0.7-1.2]). However, compared with 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. CONCLUSIONS: Although 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.

Early natural history of incident, type-specific human papillomavirus infections in newly sexually active young women.

BACKGROUND: Characterizing short-term detection patterns of young women's incident α-genus human papillomavirus (HPV) infections may further our understanding of HPV transmission. METHODS: Between 2000 and 2007, we followed 18- to 22-year-old female university students with triannual HPV DNA and Papanicolaou testing. Using Kaplan-Meier methods, we estimated duration of detectable, type-specific incident infections; time to redetection (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. RESULTS: Three hundred three 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 2 years. About 19.4% of infections that became undetectable were redetected within 1 year. Cervical lesions were common and 60% were positive for multiple HPV types in concurrent cervical swabs. Incident HPV detection in the cervix only (vs. the vulva/vagina only or both sites) was associated with short-term transience. CONCLUSIONS: Although most incident infections became undetectable within 2 years, redetection was common. Cervical lesions were a common early manifestation of HPV infection. IMPACT: It remains unclear whether potentially modifiable risk factors can be identified to reduce infection duration (and transmission likelihood).

Development of genital warts after incident detection of human papillomavirus infection in young men.

Determining the rate at which men develop genital warts after infection with alpha genus human papillomavirus (HPV) types will provide important information for the design of prevention strategies. We conducted a cohort study of 18-21-year-old men who underwent triannual genital examinations. The 24-month cumulative genital wart incidence was 57.9% (95% confidence interval [CI], 38.1%-79.1%) among 46 men with incident detection of HPV-6 or HPV-11 infection, 2.0% (95% CI, 0.5%-7.9%) among 161 men with incident detection of infection with other HPV types, and 0.7% (95% CI, 0.2%-2.8%) among 331 men who tested negative for HPV. Our results suggest that genital warts are common after HPV-6 or HPV-11 infection in young men.

Detection of genital HPV types in fingertip samples from newly sexually active female university students.

BACKGROUND: Little is known about detection of genital human papilloma virus (HPV) types in women's fingertips. The study objectives were to determine the presence of genital HPV types in fingertip samples and the agreement between fingertip and genital samples for detecting HPV. METHODS: At triannual 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 kappa statistics for percent positive agreement (kappa+). Paired samples with type-specific concordant fingertip and genital results were selected for variant characterization. RESULTS: 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% confidence interval, 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. Redetection of HPV types at the subsequent visit was more common in genital samples (73.3%) than in fingertip samples (14.5%; P < 0.001). CONCLUSIONS: Detection of genital HPV types in the fingertips was not uncommon. Although 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. IMPACT: Finger-genital transmission is plausible but unlikely to be a significant source of genital HPV infection.

Rac1 and Cdc42 are regulators of HRasV12-transformation and angiogenic factors in human fibroblasts.

BACKGROUND: The activities of Rac1 and Cdc42 are essential for HRas-induced transformation of rodent fibroblasts. What is more, expression of constitutively activated mutants of Rac1 and/or Cdc42 is sufficient for their malignant transformation. The role for these two Rho GTPases in HRas-mediated transformation of human fibroblasts has not been studied. Here we evaluated the contribution of Rac1 and Cdc42 to maintaining HRas-induced transformation of human fibroblasts, and determined the ability of constitutively activated mutants of Rac1 or Cdc42 to induce malignant transformation of a human fibroblast cell strain. METHODS: Under the control of a tetracycline regulatable promoter, dominant negative mutants of Rac1 and Cdc42 were expressed in a human HRas-transformed, tumor derived fibroblast cell line. These cells were used to determine the roles of Rac1 and/or Cdc42 proteins in maintaining HRas-induced transformed phenotypes. Similarly, constitutively active mutants were expressed in a non-transformed human fibroblast cell strain to evaluate their potential to induce malignant transformation. Affymetrix GeneChip arrays were used for transcriptome analyses, and observed expression differences were subsequently validated using protein assays. RESULTS: Expression of dominant negative Rac1 and/or Cdc42 significantly altered transformed phenotypes of HRas malignantly transformed human fibroblasts. In contrast, expression of constitutively active mutants of Rac1 or Cdc42 was not sufficient to induce malignant transformation. Microarray analysis revealed that the expression of 29 genes was dependent on Rac1 and Cdc42, many of which are known to play a role in cancer. The dependence of two such genes, uPA and VEGF was further validated in both normoxic and hypoxic conditions. CONCLUSION(S): The results presented here indicate that expression of both Rac1 and Cdc42 is necessary for maintaining several transformed phenotypes in oncogenic HRas transformed human cells, including their ability to form tumors in athymic mice. Our data also indicate that expression of either activated Rac1 or Cdc42 alone is not sufficient for malignant transformation of human fibroblasts, although each is required for specific transformed phenotypes. Furthermore, our study elucidates that the expression of several highly significant cancer related genes require the activities of Rac1 and/or Cdc42 which may also play a critical role in cellular transformation.

Comparison of incident cervical and vulvar/vaginal human papillomavirus infections in newly sexually active young women.

Vulvar/vaginal human papillomavirus (HPV) infections may precede cervical infections, and certain low-risk types may display vaginal tropism. We evaluated whether incident infections in young women display site-specific preferences by HPV risk group or phylogenetic species. Although incident infections were more likely to be detected in the vulva/vagina than in the cervix (odds ratio, 4.38 [95% confidence interval, 2.51-7.63]), the majority were first detected at both sites. Low- or undetermined-risk types were more likely than high-risk types to be first detected in the vulva/vagina (P = .03). Site-by-species differences were not statistically significant. Our results suggest that low- or undetermined-risk HPV types preferentially infect vaginal epithelium.

Persistence of genital human papillomavirus infection in a long-term follow-up study of female university students.

BACKGROUND: Little is known about the epidemiology of human papillomavirus (HPV) infections that persist for more than a few years. METHODS: Four to 12 years after participation in a longitudinal study of incident HPV infection, a cohort of former university students returned for a follow-up visit that included HPV genotyping of cervical and vulvovaginal swab specimens and collection of colposcopy-directed biopsy specimens. RESULTS: Of 147 women with HPV infection detected during their undergraduate years, 24 (16.3%) were positive for 1 or more of the same HPV types at follow-up. Overall, 27 (4.8%) of 567 type-specific HPV infections persisted, and DNA sequence analyses of a subset revealed that all were variant specific. Long-term HPV persistence was positively associated with frequent but sporadic detection of the same HPV type early during the course of the infection and with abnormal Pap tests and genital warts; it was negatively associated with marriage and was not associated with the number of intercurrent sex partners. CONCLUSIONS: HPV variant and behavioral risk factor analyses indicated that long-term detection of the same HPV type was more consistent with viral persistence than with reinfection. Although long-term persistence was not common, it was associated with detection of HPV-related pathologies.

The biphasic role of the hypoxia-inducible factor prolyl-4-hydroxylase, PHD2, in modulating tumor-forming potential.

Hypoxia is a common feature of solid tumors. The cellular response to hypoxic stress is controlled by a family of prolyl hydroxylases (PHD) and the transcription factor hypoxia-inducible factor 1 (HIF1). To investigate the relationship between PHD and HIF1 activity and cellular transformation, we characterized the expression levels of PHD isoforms across a lineage of cell strains with varying transformed characteristics. We found that PHD2 is the primary functional isoform in these cells and its levels are inversely correlated to tumor-forming potential. When PHD2 levels were altered with RNA interference in nontumorigenic fibroblasts, we found that small decreases can lead to malignant transformation, whereas severe decreases do not. Consistent with these results, direct inhibition of PHD2 was also shown to influence tumor-forming potential. Furthermore, we found that overexpression of PHD2 in malignant fibroblasts leads to loss of the tumorigenic phenotype. These changes correlated with HIF1alpha activity, glycolytic rates, vascular endothelial growth factor expression, and the ability to grow under hypoxic stress. These findings support a biphasic model for the relationship between PHD2 activity and malignant transformation.