Human Papillomavirus L1 Capsid Research Articles

Utility of human papillomavirus L1 capsid protein and HPV test as prognostic markers for cervical intraepithelial neoplasia 2+ in women with persistent ASCUS /LSIL cervical cytology.

Objective: Efficient and highly predictive biomarkers reflecting the prognosis of persistent atypical squamous cells of unknown significance(ASCUS) and low grade squamous intraepithelial lesion(LSIL)s are unavailable and need to be developed urgently. We aimed to develop a predictive model for diagnosis of cervical intraepithelial neoplasia(CIN)2+ by analyzing the immunocytochemical expression of the HPV L1 capsid protein in patients with persistent ASCUS and LSIL with a high risk of HPV infection.Methods: Cervical cytology samples comprising (70 ASCUS and 215 LSIL Pap smears) were analyzed. Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed. Expression levels of HPV L1 capsid protein in cervical cytology samples were measured, and the correlation between HPV L1 expression and cervical pathologic diagnosis was evaluated. The risk for CIN2+ was calculated using the results of immunocytochemistry and the HPV DNA test.Results: Negative results for HPV L1 immunochemistry test were more frequently observed in CIN2+, and expression of the HPV L1 capsid protein was higher in CIN1 or cervicitis (Fisher's exact test, p<0.05). Diagnosis rates for CIN2+ were highest for the combination of HPV L1 capsid protein immunocytochemistry, cytology and HPV test when compared with other combinations (Akaike information criterion (AIC): 191.7, Schwarz criterion(SC): 206.3, p<0.001).Conclusion: Absence of HPV L1 capsid expression and presence of HPV type 16 or 18 infection are reliable predictors of progression to CIN2+ in patients showing persistent ASCUS and LSIL.

Clinical significance of CK7, HPV-L1, and koilocytosis for patients with cervical low-grade squamous intraepithelial lesions: a retrospective analysis

Most cervical low-grade squamous intraepithelial lesions (LSILs) do not progress to high-grade squamous intraepithelial lesions (HSILs); however, reliable biomarkers that predict LSIL progression are lacking. We investigated the association of cytokeratin 7 (CK7), human papillomavirus-L1 capsid protein (HPV-L1), and koilocytosis with clinical outcomes of patients with LSIL. CK7, HPV-L1, Ki67, and p16-INK4A expression was determined in 72 cervical LSIL and 28 HSIL biopsy samples; koilocytosis was evaluated by reviewing biopsy slides. Fifty patients with LSIL received follow-up. CK7, HPV-L1, and koilocytosis were detected in 48.6%, 44.4%, and 52.0% of LSIL tissues and in 78.6%, 10.7%, and 64.3% of HSIL tissues, respectively. Lesion grade was correlated directly with CK7 expression (P=.007) and inversely with HPV-L1 expression (P=.004). CK7 expression in LSILs was correlated inversely with HPV-L1 expression and directly with p16-INK4A and Ki67 status. Furthermore, koilocytosis was significantly associated with HPV-L1 and p16-INK4A expression. Progression to cervical intraepithelial lesions of grades ≥2 occurred in 34% of cases. CK7 negativity and HPV-L1 positivity were significantly associated with lower HSIL progression rates. HPV-L1-positive and CK7-negative LSILs showed significantly lower progression rates compared with HPV-L1-negative and CK7-positive LSILs (6.3% v-positive cases showed a significantly lower progression rate (17.6%) compared with nonkoilocytic and HPV-L1-negative cases (50%). CK7-negative, HPV-L1-positive, and koilocytic LSILs showed a progression rate of 7.7%. Koilocytosis and p16-INK4A were not significantly associated with clinical outcomes. Hence, evaluating HPV-L1, CK7, and koilocytosis profiles combined may be more reliable for LSIL prognostication.

Clinical significance of HPV L1 capsid protein detection in cervical exfoliated cells in high-risk HPV positive women

To explore the clinical significance of human papillomavirus L1 capsid protein detection in cervical exfoliated cells in high-risk HPV positive women. From November 2012 to June 2013, 386 high-risk HPV positive (detected by hybrid capture II) cases were enrolled as eligible women from Huzhou Maternity & Child Care Hospital and Women's Hospital, School of Medicine, Zhejiang University. All eligible women underwent liquid-based cytology (ThinPrep) followed by colposcopy. Biopsies were taken if indicated. Cervical exfoliated cells were collected for HPV L1 capsid protein detection by immunocytochemistry. Expression of HPV L1 capsid protein in groups with different histological diagnosis were compared, and the role of HPV L1 capsid protein detection in cervical exfoliated cells in cervical lesions screening was accessed. Total 386 enrolled eligible women were finally diagnosed histologically as follwed: 162 normal cervix, 94 low-grade squamous intraepithelial lesion (LSIL), 128 high-grade squamous intraepithelial lesion (HSIL) and 2 squamous cervical cancer (SCC). The positive expression rate of HPV L1 in HSIL+ (HSIL or worse) group was significantly lower than that in LSIL- (LSIL or better) group (19.2% vs 66.4%, P=0.000). While identifying HSIL+ in HPV positive cases and compared with cytology, HPV L1 detection resulted in significant higher sensitivity (80.77% vs 50.77%, P=0.000) and negative predictive value (NPV; 87.18% vs 76.47%, P=0.004), significant lower specificity (66.41% vs 81.25%, P=0.000), and comparable positive predictive value (PPV; 54.97% vs 57.89%, P=0.619). To identify HSIL+ in HPV-positive/cytology-negative women, the sensitivity, specificity, PPV, and NPV of HPV L1 detection were 87.50%, 61.54%, 41.18%, and 94.12% respectively, while 80.00%, 86.36%, 80.00% and 86.36% respectively in HPV-positive/atypical squamous cell of undetermined significance (ASCUS) women. HPV L1 capsid detection in cervical exfoliated cells have a role in cervical lesions screening in high-risk HPV positive women, and may be a promising triage for high-risk HPV-positive/cytology-negative or ASCUS women.

Application of a novel cell-permeable peptide-driven protein delivery in mouse blastocysts

Cell-permeable peptides (CPPs) mediate the delivery of macromolecules into cells. However, whether CPPs are usable in mammalian oocytes and embryos for the modulation of protein expression has not been widely investigated. We have previously designed a novel 12-mer CPP from the conserved region of the human papillomavirus L1 capsid protein. In this study, we tested whether this peptide, LDP12, effectively delivers a protein cargo to mouse oocytes and preimplantation embryos. We prepared a LDP12-EGFP fusion protein having LDP12 as an N-terminal tag. This fusion protein readily enters HeLa cells, a cervical cancer cell line. The entry of LDP12-EGFP was partially blocked by amiloride, while cytochalasin D or methyl-β-cyclodextrin slightly increased the uptake. LDP12-EGFP shows efficient transduction in mouse blastocysts, but not in oocytes, two-cell-stage, or morula-stage-preimplantation embryos. LDP12-mediated delivery of EGFP-LC3, a widely used marker of autophagic activation, is successful in HeLa cells and mouse blastocysts, as it enters cells and exhibits a signature punctate pattern. The lipidation of EGFP-LC3 also normally occurs after transduction, suggesting that the transduced protein retains the functional characteristics. Collectively, we show that LDP12-driven protein delivery is a fast and convenient method applicable to mouse blastocysts and reproductive cancer cells.

LDP12, a novel cell-permeable peptide derived from L1 capsid protein of the human papillomavirus

We designed a novel cell-permeable peptide, LDP12, from the human papillomavirus L1 capsid protein. In this work, we examined the mechanism of cellular entry by LDP12 and its efficacy as a potential carrier of protein cargoes. The 12-mer peptide linked to FITC freely enters various types of mammalian cells within a few minutes via an endocytic pathway, as its entry is blocked at a low temperature. Several inhibitors which block certain pathway of endocytosis were used to determine which pathway is utilized by LDP12. We found that methyl-β-cyclodextrin specifically blocks LDP12 entry, suggesting that lipid raft-mediated pathway is involved. Intraluminal injection of LDP12-FITC into the mouse uterus shows that this peptide could penetrate the uterine tissue and stay as long as 24h. Furthermore, LDP12 with a cysteamide group at the C-terminus successfully carries purified protein cargoes into mammalian cells including rat cortical neurons. Collectively, LDP12 could be utilized as a method of protein therapeutics targeting various mammalian cell types.

Correlation between immunocytochemistry of human papilloma virus L1 capsid protein and behavior of low‐grade cervical cytology in Korean women

The aim of this study was to evaluate the behavior of low-grade squamous intraepithelial lesion (LSIL) in Korean women infected with human papillomavirus (HPV) in relation to the immunocytochemical detection of the HPV L1 capsid protein. From January 2006 to December 2007, a total of 353 immunocytochemistry tests were performed on specimens from HPV-infected patients with LSIL. Due to exclusions, the study population was reduced to 318. Subjects were monitored at 4-6 month intervals. The regression, persistence, and progression of the cytologic abnormalities of the 318 cases were compared with the results of HPV L1 capsid protein immunocytochemical detection. Of the 137 patients negative for the HPV L1 capsid protein, 38 (27.7%) showed progression to high-grade lesions, 50 (36.5%) showed persistence, and 49 (35.8%) showed regression to normal cytological features. In contrast, of the remaining 181 patients positive for the HPV L1 capsid protein, 15 (8.3%) showed progression to high-grade lesions, 74 (40.9%) showed persistence, and 92 (50.8%) showed regression. The results of immunocytochemical testing for the HPV L1 capsid protein show a linear association with the progression or regression behavior of low-grade cervical cytology in patients infected with HPV (linear by linear association test, P<0.05). Immunocytochemical detection of HPV L1 was significantly related with the biological patterns of LSIL in Korean women. Hence, immunocytochemistry for the detection of HPV L1 is beneficial in providing further information for LSIL.

Diagnostic value of p16INK4A, Ki‐67, and human papillomavirus L1 capsid protein immunochemical staining on cell blocks from residual liquid‐based gynecologic cytology specimens

This study was conducted to evaluate the reliability and role of cell block preparations in the diagnosis of neoplastic and preneoplastic lesions of the cervix and to improve the value of cell block preparations in diagnosing and predicting the prognosis of cervical lesions through immunostaining of p16INK4A (p16), Ki-67, and human papillomavirus (HPV) L1 capsid protein (HPV L1). In total, 138 specimens were diagnosed on liquid-based cytology (LBC) and cell block preparations, and 63 specimens were subjected subsequently to tissue follow-up and immunostaining for p16, Ki-67, and HPV L1 on cell block sections. In 42 specimens that were diagnosed as low-grade squamous intraepithelial lesion, high-grade squamous intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC) on cell blocks, 38 specimens (90.5%) were confirmed by histopathologic reports, and there was slightly better than 81.6% agreement between LBC and tissue follow-up. Immunointensity and cells that were positive for p16 were enhanced according to increased pathologic grade and differed statistically between cervical intraepithelial neoplasia 1 (CIN-1) and CIN-2/CIN-3 as well as SCC. The positive rates of HPV L1 decreased gradually according to the severity of cervical neoplasia, and HPV L1/p16 expression patterns were related to the severity of cervical lesions. The cell block preparation technique was complementary to LBC, and the authors concluded that the application of LBC combined with cell block preparations may improve the diagnostic accuracy of cytology. Immunostaining for p16 and Ki-67 on cell block preparations can help to improve the diagnostic accuracy of HSIL and SCC. A combined expression pattern of p16 and HPV L1 may serve as a valuable index for predicting prognosis and follow-up of cervical dysplastic lesions.

Human Papillomavirus L1 Capsid Protein and Human Papillomavirus Type 16 as Prognostic Markers in Cervical Intraepithelial Neoplasia 1

The aim of the study was to determine whether human papillomavirus (HPV) L1 capsid protein and the HPV genotype can predict the disease course as prognostic markers for cervical intraepithelial neoplasia 1 (CIN1). Immunohistochemical staining was performed for HPV L1 capsid protein in 101 women who had been confirmed to have CIN1 by histologic examination and HPV high-risk infection by HPV genotyping. The disease course was analyzed by follow-up histologic examination according to the HPV L1 capsid protein and HPV genotype over a minimum of 12 months. The CIN1 regressed spontaneously in 60.4% of the women; most cases of regression occurred within 1 year (90.9% of regression cases). The HPV L1 capsid protein-positive patients had a spontaneous regression rate of 72.7% (48/66) and a rate of persistent disease or progression to higher grade disease of 27.3% (18/66). The HPV L1 capsid protein-negative women had a regression rate of 37.1% (13/35) and a rate of persistent disease or progression of 62.9% (22/35; P < 0.001). The HPV-16-infected patients had a regression rate of 38.6% (17/44) and a rate of persistent disease or progression of 61.4% (27/44), whereas the non-HPV-16-infected patients had a regression rate of 77.2% (44/57) and a rate of persistent disease or progression of 22.8% (13/57; P < 0.001). The HPV L1 protein expression is closely related to spontaneous disease regression, but HPV-16 infection is related to persistent disease or progression to high-grade lesions in patients with CIN1.

Detection of human papillomavirus L1 capsid protein expression in liquid-based cytology samples with abnormal cytology.

To investigate the possibility of detection of the human papillomavirus (HPV) L1 capsid protein to predict the course of mild or moderate cervical intraepithelial neoplasia (CIN). Immunocytochemical analysis using antibody against HPV L1 capsid protein was carried out on 274 samples obtained from women performed TriPath Pap tests, positive for high-risk HPV DNA detected by hybrid capture II (HC-II) or cytologic diagnosed atypical squamous cells of undetermined significance (ASCUS) or more severe. For cytological diagnosed, there were ASCUS 105 cases, low-grade squamous intraepithelial lesions (LSIL) 119 cases, atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H) 9 cases, high-grade squamous intraepithelial lesions (HSIL) 36 cases, and squamous cell carcinoma (SCC) 5 cases. But for the pathologic diagnosed, there were chronic cervicitis 96 cases, CINI 85 cases, CINII 55 cases, CIN III 32 cases, and SCC 6 cases. Of the 274 cases, HPV L1 capsid protein was positive in 69.8% (67/96) of cervicitis, 83.5% (71/85) of CINI, 41.8% (23/55) of CINII, 3.1% (1/32) of CINIII and 0(0/6) of SCC. Cytologic diagnosis revealed a higher expression rate in LSIL (75.6%, 90/119) than that in ASCUS (63.8%, 67/105) or in HSIL + SCC (9.8%, 4/41; all P < 0.01). Of 71 cases with ASCUS and LSIL without treated, none of HPV L1 positive cases (0/55) progressed in cytology, while 19% (3/16) of HPV L1 negative cases progressed to ASC-H, HSIL (P < 0.01). The expression rates of HPV L1 protein in liquid-based cell specimen is decreased as the cytopathology diagnosis severe degree, which may imply the histopathology diagnosis of cervix, predict the progression of cervical lesion, and help to treat the cases with ASCUS and LSIL.

Up-regulated expression of Sp1 protein coincident with a viral protein in human and mouse differentiating keratinocytes may act as a cell differentiation marker

Sp1 is a transcription factor that regulates expression of mammalian and viral genes and is involved in different facets of cellular functions in eukaryotic cells. Here, we investigated Sp1 expression in primary mouse and human keratinocyte (KC) culture using quantitative reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence microscopy. Expression of Sp1 was post-transcriptionally up-regulated with increasing time in primary KC cultures. Sp1 expression, coincident with expression of human papillomavirus L1 capsid protein and involucrin, was associated with cell differentiation in vitro and in vivo in human and mouse skins. Immunoprecipitation experiments showed that Sp1 and L1 could be bound in a complex. Calcium (Ca 2+) and all- trans retinoic acid are the positive modulators of KC differentiation, which positively and negatively regulated Sp1 and L1 expression. The data suggest that coincident expression of Sp1 with L1 proteins in differentiating KCs is mediated by a calcium-dependent signaling pathway.