Variant-specific persistence of infections with human papillomavirus Types 31, 33, 45, 56 and 58 and risk of cervical intraepithelial neoplasia.

In our previous study of the etiologic role of oncogenic human papillomavirus (HPV) types other than HPV16 and 18, we observed a significantly higher risk of cervical intraepithelial neoplasia Grades 2-3 (CIN2/3) associated with certain lineages of HPV types 31/33/45/56/58 [called high-risk (HR) variants] compared with non-HR variants. This study was to examine whether these intra-type variants differ in persistence of the infection and persistence-associated risk of CIN2/3. Study subjects were women who had any of HPV types 31/33/45/56/58 newly detected during a 2-year follow-up with 6-month intervals. For each type, the first positive sample was used for variant characterization. The association of reverting-to-negativity with group of the variants and CIN2/3 with length of positivity was assessed using discrete Cox regression and logistic regression, respectively. Of the 598 newly detected, type-specific HPV infections, 312 became undetectable during follow-up. Infections with HR, compared with non-HR, variants were marginally more likely to become negative [adjusted hazard ratio = 1.3; 95% confidence interval (CI), 0.9-1.8]. The adjusted odds ratio associating with the development of CIN2/3 was 3.0 (95% CI, 1.2-7.4) for persistent infections with HR variants for 6 months and 10.0 (95% CI, 3.8-38.0) for persistent infections with HR variants for 12-18 months as compared with the first positive detection of HR variants. Among women with non-HR variants, there were no appreciable differences in risk of CIN2/3 by length of positivity. Findings suggest that the lineage-associated risk of CIN2/3 was not mediated through a prolonged persistent infection, but oncogenic heterogeneity of the variants.

Association of Human Papillomavirus 31 DNA Load with Risk of Cervical Intraepithelial Neoplasia Grades 2 and 3.

The association between human papillomavirus 31 (HPV31) DNA loads and the risk of cervical intraepithelial neoplasia grades 2 and 3 (CIN2-3) was evaluated among women enrolled in the atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) triage study (ALTS), who were monitored semiannually over 2 years and who had HPV31 infections detected at ≥1 visit. HPV31 DNA loads in the first HPV31-positive samples and in a random set of the last positive samples from women with ≥2 HPV31-positive visits were measured by a real-time PCR assay. CIN2-3 was histologically confirmed at the same time as the first detection of HPV31 for 88 (16.6%) of 530 women. After adjustment for HPV31 lineages, coinfection with other oncogenic types, and the timing of the first positive detection, the odds ratio (OR) per 1-log-unit increase in viral loads for the risk of a concurrent diagnosis of CIN2-3 was 1.5 (95% confidence interval [CI], 1.2 to 1.9). Of 373 women without CIN2-3 at the first positive visit who had ≥1 later visit, 44 had subsequent diagnoses of CIN2-3. The initial viral loads were associated with CIN2-3 diagnosed within 6 months after the first positive visit (adjusted OR, 1.5 [95% CI, 1.0 to 2.4]) but were unrelated to CIN2-3 diagnosed later. For a random set of 49 women who were tested for viral loads at the first and last positive visits, changes in viral loads were upward and downward among women with and without follow-up CIN2-3 diagnoses, respectively, although the difference was not statistically significant. Results suggest that HPV31 DNA load levels at the first positive visit signal a short-term but not long-term risk of CIN2-3.

Detection of Human Papillomavirus Infections at the Single-Cell Level.

OBJECTIVE: To explore the possibility of single-cell analysis of human papillomavirus (HPV) infection. METHODS: Two hundred and twenty cells were isolated by laser capture microdissection from formalin-fixed and paraffin-embedded cervical tissue blocks from 8 women who had HPV DNA detected in their cervical swab samples. The number of type-specific HPV copies in individual cells was measured by quantitative polymerase chain reaction with and without a prior reverse transcription. The cells were assayed and counted for more than once if the corresponding swab sample was positive for ≥2 HPV types. RESULTS: Infection with HPV16, HPV39, HPV51, HPV52, HPV58, HPV59 and HPV73 was detected in 12 (5.5%) of 220, 3 (9.4%) of 32, 3 (5.8%) of 52, 11 (22.9%) of 48, 9 (18.8%) of 48, 3 (9.4%) of 32 and none of 20 cells, respectively. The numbers of HPV genome copies varied widely from cell to cell. The coexistence of multiple HPV types was detected in 6 (31.6%) of 19 positive cells from 1 of the 6 women who had 2 or 3 HPV types detected in their swab samples. CONCLUSION: Given the heterogeneity of HPV status in individual cells, further clarification of HPV infection at the single-cell level may refine our understanding of HPV-related carcinogenesis.

Lineages of oncogenic human papillomavirus types other than type 16 and 18 and risk for cervical intraepithelial neoplasia.

BACKGROUND: Data on clinical outcomes of infection with variants of oncogenic human papillomavirus (HPV) types other than HPV16 and HPV18 are rare. We investigated intratypic variations in non-HPV16/18 oncogenic types and their corresponding relationships with cervical intraepithelial neoplasia grades 2-3 (CIN2/3). METHODS: Study subjects were women who were positive for one or more of 11 non-HPV16/18 oncogenic types. Subjects were followed every six months for two years for detection of HPV and cervical lesions. Variant lineages were defined by sequencing the 3' part of the long control region and the entire E6/E7 region of HPV genome. Lineage-associated risk of CIN2/3 was assessed using logistic regression with generalized estimating equations. RESULTS: A total of 4591 type-specific HPV infections among 2667 women were included in the analysis. The increase in risk of CIN2/3 was statistically significant for women with HPV31 A or B compared with C variants, HPV33 A1 compared with B variants, HPV45 A3 or B2 compared with B1 variants, HPV56 B compared with A2 variants, and HPV58 A1 or A3 compared with C variants. For these five types, the adjusted odds ratio associated with CIN2/3 was 2.0 (95% confidence interval [CI] = 1.5 to 2.6) for infections with single-type high-risk (HR) variants, 1.7 (95% CI = 1.0 to 2.7) for infections with two or more types but only one HR variant, and 5.3 (95% CI = 3.1 to 8.4) for infections with HR variants of two or more types as compared with those with single-type non-HR variants. The likelihood of CIN2/3 was similar for women with HPV16 infection and for those with HPV58 A1 variant infection. CONCLUSIONS: These findings suggest that for a given HPV type, intratypic nucleotide changes may alter phenotypic traits that affect the probability of neoplasia.

Viral load and short-term natural history of type-specific oncogenic human papillomavirus infections in a high-risk cohort of midadult women.

Oncogenic human papillomavirus (HPV) viral load may inform the origin of newly detected infections and characterize oncogenic HPV natural history in midadult women. From 2007 to 2011, we enrolled 521 25-65-year-old-female online daters and followed them triannually with mailed health and sexual behavior questionnaires and kits for self-sampling for PCR-based HPV DNA testing. Samples from oncogenic HPV positive women were selected for type-specific DNA load testing by real-time PCR with adjustment for cellularity. Linear or logistic regression models were used to evaluate relationships between viral levels, health and sexual behavior, and longitudinal oncogenic HPV detection. Type-specific viral levels were borderline significantly higher in oncogenic HPV infections that were prevalent versus newly detected (p = 0.092), but levels in newly detected infections were higher than in infections redetected after intercurrent negativity (p < 0.001). Recent sex partners were not significantly associated with viral levels. Compared with prevalent infections detected intermittently, the likelihood of persistent (OR = 4.31, 95% CI: 2.20-8.45) or single-time (OR = 1.32, 95% CI: 1.03-1.71) detection increased per 1-unit increase in baseline log10 viral load. Viral load differences between redetected and newly detected infections suggest a portion of new detections were due to new acquisition, although report of recent new sex partners (a potential marker of new infection) was not predictive of viral load; oncogenic HPV infections in midadult women with new partners likely represent a mix of new acquisition and reactivation or intermittent detection of previous infection. Intermittent detection was characterized by low viral levels, suggesting that intermittent detection of persisting oncogenic HPV infection may be of limited clinical significance.

Evaluation of dry and wet transport of at-home self-collected vaginal swabs for human papillomavirus testing.

Our objective was to compare human papillomavirus (HPV) detection in paired self-collected vaginal samples transported by overnight mail in liquid specimen transport medium (STM) (wet) or in dry tubes (dry). Women aged 18-24 years were recruited online to self-collect vaginal swab samples at home for HPV testing and 159 women returned paired wet and dry samples. Dry samples were rehydrated with STM upon arrival at the laboratory. HPV was detected by the Roche Linear Array HPV genotyping test (37 genotypes) and Kappa and McNemar statistics were used to compare wet versus dry samples for detecting HPV. Of the subjects tested in this study, 51 % were HPV-positive (in either sample) and 40 % were positive for high-risk HPV. A total of 216 type-specific infections were detected among the 80 HPV-positive women. Almost perfect agreement was observed between paired samples for detecting any HPV (subject-level positive agreement: 91.9 %, κ: 0.85) or type-specific HPV (positive agreement across types: 90.1 %, κ: 0.90). Similar agreement between sample types was seen when testing for high-risk types and 81.9 % of all type-specific infections were detected in both samples. Among discordant pairs, wet samples were 3.3 times more likely to be positive for type-specific HPV than dry samples (P = 0.02). However, in 63.6 % of wet-positive/dry-negative discordant pairs analysed for viral load, type-specific HPV was either undetectable or detected at a low level (<100 copies) in the wet samples, suggesting that the majority of infections missed by using dry samples are less likely to be clinically relevant. Our results indicate that dry transport is a feasible option for transporting at-home self-collected vaginal samples for HPV DNA testing.

Association of human papillomavirus type 31 variants with risk of cervical intraepithelial neoplasia grades 2-3.

Although the lineages of human papillomavirus type 31 (HPV31) variants are recognized, their clinical relevance is unknown. The purpose of our study was to examine risk of cervical intraepithelial neoplasia Grades 2-3 (CIN2/3) by HPV31 variants. Study subjects were women who participated in the atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion Triage Study and who had HPV31 infections detected at one or more visits. They were followed semi-annually over 2 years for detection of HPV DNA and cervical lesion. HPV31 isolates were characterized by DNA sequencing and assigned into 1 of 3 variant lineages. CIN2/3 was histologically confirmed in 127 (27.0%) of the 470 HPV31-positive women, 83 diagnosed at the first HPV31-positive visit and 44 thereafter. The odds ratio for the association of 2-year cumulative risk of CIN2/3 was 1.7 (95% CI: 1.0-2.9) for infections with A variants and 2.2 (95% CI: 1.2-3.9) for infections with B variants as compared to those with C variants. Among women without CIN2/3 at the first HPV31-positive visit, the risk of subsequent CIN2/3 was 2.2-fold greater for those with A variants (95% CI: 1.0-4.8) and 2.0-fold greater for those with B variants (95% CI: 0.9-4.9) as compared to those with C variants. Similar associations were observed when CIN3 was used as the endpoint. The findings from our study help to tag HPV31 variants that differ in risk of CIN2/3 and to explain in part why some HPV31 infections regress spontaneously and others lead to disease progression.

Inverse association between methylation of human papillomavirus type 16 DNA and risk of cervical intraepithelial neoplasia grades 2 or 3.

BACKGROUND: The clinical relevance of human papillomavirus type 16 (HPV16) DNA methylation has not been well documented, although its role in modulation of viral transcription is recognized. METHODS: Study subjects were 211 women attending Planned Parenthood clinics in Western Washington for routine Papanicolaou screening who were HPV16 positive at the screening and/or subsequent colposcopy visit. Methylation of 11 CpG dinucleotides in the 3' end of the long control region of the HPV16 genome was examined by sequencing the cloned polymerase chain reaction products. The association between risk of CIN2/3 and degree of CpG methylation was estimated using a logistic regression model. RESULTS: CIN2/3 was histologically confirmed in 94 (44.5%) of 211 HPV16 positive women. The likelihood of being diagnosed as CIN2/3 increased significantly with decreasing numbers of methylated CpGs (meCpGs) in the 3' end of the long control region (P(for trend) = 0.003). After adjusting for HPV16 variants, number of HPV16-positive visits, current smoking status and lifetime number of male sex partners, the odds ratio for the association of CIN2/3 with ≥4 meCpGs was 0.31 (95% confidence interval, 0.12-0.79). The proportion of ≥4 meCpGs decreased appreciably as the severity of the cervical lesion increased (P(for trend) = 0.001). The inverse association remained similar when CIN3 was used as the clinical endpoint. Although not statistically significant, the ≥4 meCpGs-related risk reduction was more substantial among current, as compared to noncurrent, smokers. CONCLUSION: Results suggest that degree of the viral genome methylation is related to the outcome of an HPV16 cervical infection.

Insulin gene is a target in activin receptor-like kinase 7 signaling pathway in pancreatic beta-cells.

Activins regulate pancreatic development, differentiation and insulin secretion. Activin receptor-like kinase 7 (ALK7) has been identified as a receptor for Nodal and Activin AB and B, and is expressed in pancreatic islets and beta-cell lines. In this study, human insulin promoter was activated by Smad2, Smad3 and the pancreatic and duodenal homeobox factor-1 (PDX-1) in the ALK7 pathway. A conserved Smad binding element was related to the promoter activation. Phosphorylated Smad2/Smad3 and PDX-1 were bound to insulin gene with Nodal and Activin AB, and the phosphorylated Smad2/Smad3 interacted with PDX-1. These results indicate that one of the direct target genes of Nodal and Activin AB signals is the insulin gene in pancreatic beta-cells and that PDX-1 is directly involved in the ALK7-Smad pathway.

[Study on surface infrared radiation spectrums of danzhong (CV 17) in the patient of hyperplasia of mammary glands].

OBJECTIVE: To compare differences of surface infrared radiation spectrums between Danzhong (CV 17) A high-sensitive PHE and non-acupoint control point in the patient of hyperplasia of mammary glands. METHODS: 201 surface infrared spectrograph was used to detect infrared radiation spectrums of Danzhong (CV 17) and non-acupoint control point at 1.5-16.0 microm wave band in the patient of hyperplasia of mammary glands. RESULTS: The shape of the infrared spectrums of Danzhong (CV 17) was similar to that of the non-acupoint control point, but with lower radiation intensity. Of the 59 wavelength spots detected, 13 (6.75-8.25 microm, 9.00 microm, 9.25 microm, 9.75 microm, 13.25-13.75 microm) had significant differences in infrared radiation intensity between Danzhong (CV 17) and non-acupoint control point (P<0.05). CONCLUSION: The intensity of infrared radition of Danzhong (CV 17) is lower and Qimen (LR 14) is higher than that of the non-acupoint control point in the patient with hyperplasia of mammary glands, showing that different channels are at different states of deficiency and excess under pathological condition of hyperplasia of mammary glands.

Long-term treatment with rosiglitazone and metformin reduces the extent of, but does not prevent, islet amyloid deposition in mice expressing the gene for human islet amyloid polypeptide.

Islet amyloid deposition in type 2 diabetes is associated with reduced beta-cell mass. Therefore, interventions aimed at reducing islet amyloid formation may help preserve beta-cell mass in type 2 diabetes. Rosiglitazone and metformin act by different mechanisms to improve insulin sensitivity and thereby reduce beta-cell secretory demand, resulting in decreased release of insulin and islet amyloid polypeptide (IAPP), the unique constituent of islet amyloid deposits. We hypothesized that this reduced beta-cell secretory demand would lead to reduced islet amyloid formation. Human IAPP (hIAPP) transgenic mice, a model of islet amyloid, were treated for 12 months with rosiglitazone (1.5 mg.kg(-1).day(-1), n = 19), metformin (1 g.kg(-1).day(-1), n = 18), or control (n = 17). At the end of the study, islet amyloid prevalence (percent islets containing amyloid) and severity (percent islet area occupied by amyloid), islet mass, beta-cell mass, and insulin release were determined. Islet amyloid prevalence (44 +/- 8, 13 +/- 4, and 11 +/- 3% for control, metformin-, and rosiglitazone-treated mice, respectively) and severity (9.2 +/- 3.0, 0.22 +/- 0.11, and 0.10 +/- 0.05% for control, metformin-, and rosiglitazone-treated mice, respectively) were markedly reduced with both rosiglitazone (P < 0.001 for both measures) and metformin treatment (P < 0.001 for both measures). Both treatments were associated with reduced insulin release assessed as the acute insulin response to intravenous glucose (2,189 +/- 857, 621 +/- 256, and 14 +/- 158 pmol/l for control, metformin-, and rosiglitazone-treated mice, respectively; P < 0.05 for metformin vs. control and P < 0.005 for rosiglitazone vs. control), consistent with reduced secretory demand. Similarly, islet mass (33.4 +/- 7.0, 16.6 +/- 3.6, and 12.2 +/- 2.1 mg for control, metformin-, and rosiglitazone-treated mice, respectively) was not different with metformin treatment (P = 0.06 vs. control) but was significantly lower with rosiglitazone treatment (P < 0.05 vs. control). When the decreased islet mass was accounted for, the islet amyloid-related decrease in beta-cell mass (percent beta-cell mass/islet mass) was ameliorated in both rosiglitazone- and metformin-treated animals (57.9 +/- 3.1, 64.7 +/- 1.4, and 66.1 +/- 1.6% for control, metformin-, and rosiglitazone-treated mice, respectively; P < 0.05 for metformin or rosiglitazone vs. control). In summary, rosiglitazone and metformin protect beta-cells from the deleterious effects of islet amyloid, and this effect may contribute to the ability of these treatments to alleviate the progressive loss of beta-cell mass and function in type 2 diabetes.

Genetic background determines the extent of islet amyloid formation in human islet amyloid polypeptide transgenic mice.

Genetic background is important in determining susceptibility to metabolic abnormalities such as insulin resistance and beta-cell dysfunction. Islet amyloid is associated with reduced beta-cell mass and function and develops in the majority of our C57BL/6J x DBA/2J (F(1)) male human islet amyloid polypeptide (hIAPP) transgenic mice after 1 yr of increased fat feeding. To determine the relative contribution of each parental strain, C57BL/6J (BL6) and DBA/2J (DBA2), to islet amyloid formation, we studied male hIAPP mice on each background strain (BL6, n = 13; and DBA2 n = 11) and C57BL/6J x DBA/2J F(1) mice (n = 17) on a 9% (wt/wt) fat diet for 1 yr. At the end of 12 mo, islet amyloid deposition was quantified from thioflavin S-stained pancreas sections. The majority of mice in all groups developed islet amyloid (BL6: 91%, F(1): 76%, DBA2: 100%). However, the prevalence (%amyloid-positive islets; BL6: 14 +/- 3%, F(1): 44 +/- 8%, DBA2: 49 +/- 9%, P < 0.05) and severity (%islet area occupied by amyloid; BL6: 0.03 +/- 0.01%, F(1): 9.2 +/- 2.9%, DBA2: 5.7 +/- 2.3%, p < or = 0.01) were significantly lower in BL6 than F(1) and DBA2 mice. Increased islet amyloid severity was negatively correlated with insulin-positive area per islet, in F(1) (r(2) = 0.75, P < 0.001) and DBA2 (r(2) = 0.87, P < 0.001) mice but not BL6 mice (r(2) = 0.07). In summary, the extent of islet amyloid formation in hIAPP transgenic mice is determined by background strain, with mice expressing DBA/2J genes (F(1) and DBA2 mice) being more susceptible to amyloid deposition that replaces beta-cell mass. These findings underscore the importance of genetic and environmental factors in studying metabolic disease.

Naked DNA transfer of Factor VIII induced transgene-specific, species-independent immune response in hemophilia A mice.

The development of antibodies to a previously unexpressed protein product may limit the success of human gene therapy approaches. We inserted B-domain-deleted factor VIII (FVIII) cDNA of human, canine, or murine origin into the multiple cloning site of a liver-specific vector, pBS-HCRHPI-A, to yield plasmids pBS-HCRHPI-FVIIIA, pBS-HCRHPI-cFVIIIA, and pBS-HCRHPI-mFVIIIA, respectively. Fifty micrograms of each plasmid in 2 ml of solution was rapidly injected into the tail vein of three groups of hemophilia A mice. Factor VIII levels ranging from 3 to 12 IU/ml were obtained from all three groups (normal is 1 IU/ml in human plasma) 3 days after treatment. These initial very high levels of functional human, canine, or murine factor VIII, however, fell gradually to undetectable levels within 2-3 weeks, and their disappearance correlated with the generation of high-titer, inhibitory anti-FVIII antibodies. Notably, this immune response occurred independent of the species of origin of the exogenous factor VIII. Antibody titers to factor VIII were detected beginning at 2 weeks, reached a plateau and remained at high levels for over 6 months. The majority of anti-hFVIII IgG was IgG1 isotype specific, suggesting a humoral response mediated by Th2-induced signals. Consistent with this idea, in a separate group of mice treated with pBS-HCRHPI-FVIIIA, transient immunosuppression by cyclophosphamide significantly delayed (5/6) or abolished (1/6) inhibitory antibody formation against the transgene.