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Cellular and viral microRNAs (miRNAs) are the transcriptional products of RNA polymerase II and are regulated by transcriptional factors for their differential expression. The altered expression of miRNAs in many cancer types has been explored as a marker for possible diagnosis and therapy. We report in this study that oncogenic human papillomaviruses (HPVs) induce aberrant expression of many cellular miRNAs and that HPV18 infection produces no detectable viral miRNA. Thirteen abundant host miRNAs were specifically regulated by HPV16 and HPV18 in organotypic raft cultures of foreskin and vaginal keratinocytes as determined by miRNA array in combination with small RNA sequencing. The increase of miR-16, miR-25, miR-92a, and miR-378 and the decrease of miR-22, miR-27a, miR-29a, and miR-100 could be attributed to viral oncoprotein E6, E7, or both, all of which are known to target many cellular transcription factors. The examination of 158 cervical specimens, including 38 normal, 52 cervical intraepithelial neoplasia (CIN), and 68 cervical cancer (CC) tissues, for the expression of these eight miRNAs showed a remarkable increase of miR-25, miR-92a, and miR-378 with lesion progression but no obvious change of miR-22, miR-29a, and miR-100 among the HPV-infected tissues. Further analyses indicate that an expression ratio ≥1.5 of miR-25/92a group over miR-22/29a group could serve as a cutoff value to distinguish normal cervix from CIN and from CIN to CC.

High-risk HPV infections lead to development of cervical cancer. This study identified the differential expression of 16 novel genes (LY6K, FAM83A, CELSR3, ASF1B, IQGAP3, SEMA3F, CLDN10, MSX1, CXCL5, ASRGL1, ELAVL2, GRB7, KHSRP, NOVA1, PTBP1, and RNASEH2A) in HPV-infected cervical tissue samples and keratinocytes. Eight of these genes (CDKN2A, ELAVL2, GRB7, HSPB1, KHSRP, NOVA1, PTBP1, and RNASEH2A) encode RNA-binding proteins. Further studies indicated that both HPV16 and HPV18 infections lead to the aberrant expression of selected RBP-encoding genes. We found that viral E6 and E7 decrease NOVA1 expression but that E7 increases RNASEH2A expression via E2F1. The altered expression of these genes may be utilized as biomarkers for high-risk (HR)-HPV carcinogenesis and progression. RNA-binding proteins (RBPs) control mRNA processing, stability, transport, editing, and translation. We recently conducted transcriptome analyses comparing normal (i.e., healthy) cervical tissue samples with human papillomavirus (HPV)-positive cervical cancer tissue samples and identified 614 differentially expressed protein-coding transcripts which are enriched in cancer-related pathways and consist of 95 known RBPs. We verified the altered expression of 26 genes with a cohort of 72 cervical samples, including 24 normal cervical samples, 25 cervical intraepithelial neoplasia grade 2 (CIN2) and CIN3 samples, and 23 cervical cancer tissue samples. LY6K (lymphocyte antigen 6 complex locus K), FAM83A (family member with sequence similarity 83), CELSR3, ASF1B, IQGAP3, SEMA3F, CLDN10, MSX1, CXCL5, ASRGL1, ELAVL2, GRB7, KHSRP, NOVA1, PTBP1, and RNASEH2A were identified as novel candidate genes associated with cervical lesion progression and carcinogenesis. HPV16 or HPV18 infection was found to alter the expression of 8 RBP genes (CDKN2A, ELAVL2, GRB7, HSPB1, KHSRP, NOVA1, PTBP1, and RNASEH2A) in human vaginal and foreskin keratinocytes. Both viral E6 and E7 decreased NOVA1 expression, but only E7 increased the expression of RNASEH2A in an E2F1-dependent manner. Proliferating cell nuclear antigen (PCNA) directs RNASEH2 activity with respect to DNA replication by removing the RNA primers to promote Okazaki fragment maturation, and two factors are closely associated with neoplasia progression. Therefore, we predict that the induction of expression of RNASEH2A via viral E7 and E2F1 may promote DNA replication and cancer cell proliferation. IMPORTANCE High-risk HPV infections lead to development of cervical cancer. This study identified the differential expression of 16 novel genes (LY6K, FAM83A, CELSR3, ASF1B, IQGAP3, SEMA3F, CLDN10, MSX1, CXCL5, ASRGL1, ELAVL2, GRB7, KHSRP, NOVA1, PTBP1, and RNASEH2A) in HPV-infected cervical tissue samples and keratinocytes. Eight of these genes (CDKN2A, ELAVL2, GRB7, HSPB1, KHSRP, NOVA1, PTBP1, and RNASEH2A) encode RNA-binding proteins. Further studies indicated that both HPV16 and HPV18 infections lead to the aberrant expression of selected RBP-encoding genes. We found that viral E6 and E7 decrease NOVA1 expression but that E7 increases RNASEH2A expression via E2F1. The altered expression of these genes may be utilized as biomarkers for high-risk (HR)-HPV carcinogenesis and progression.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3′ untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

The genes encoding the enzymes for metabolising alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) - exhibit genetic polymorphism and ethnic variations. Although the ALDH2*2 variant allele has been widely accepted as protecting against the development of alcoholism in Asians, the association of the ADH1B*2 variant allele with drinking behaviour remains inconclusive. The goal of this study was to determine whether the polymorphic ADH1B and ALDH2 genes are associated with stroke in male Han Chinese with high alcohol consumption. Sixty-five stroke patients with a history of heavy drinking (HDS) and 83 stroke patients without such a history (NHDS) were recruited for analysis of the ADH1B and ALDH2 genotypes from the stroke registry in the Tri-Service General Hospital, Taipei, Taiwan, between January 2000 and December 2001. The allelotypes of ADH1B and ALDH2 were determined using the polymerase chain reaction-restriction fragment length polymorphism method. The HDS patients (3 per cent) showed a significantly lower ALDH2*2 allele frequency than NHDS patients (27 per cent) (p < 0.001). After controlling for age, patients with HDS were associated with a significantly higher occurrence of cigarette smoking (p < 0.01) and liver dysfunction (p < 0.01). Multiple logistic regression analyses revealed that the ALDH2*2 variant allele was an independent variable exhibiting strong protection (odds ratio 0.072; 95 per cent confidence interval 0.02-0.26) against HDS after adjustment for hypertension, diabetes mellitus, smoking status and liver dysfunction. By contrast, allelic variations in ADH1B exerted no significant effect on HDS. The present study indicated that, unlike ALDH2*2, ADH1B*2 appears not to be a significant negative risk factor for high alcohol consumption in male Han Chinese with stroke.

Human papillomaviruses (HPVs) amplify in differentiated strata of a squamous epithelium. The HPV E7 protein destabilizes the p130/retinoblastoma susceptibility protein family of tumor suppressors and reactivates S-phase reentry, thereby facilitating viral DNA amplification. The high-risk HPV E6 protein destabilizes the p53 tumor suppressor and many other host proteins. However, the critical E6 targets relevant to viral DNA amplification have not been identified, because functionally significant E6 mutants are not stably maintained in transfected cells. Using Cre-loxP recombination, which efficiently generates HPV genomic plasmids in transfected primary human keratinocytes, we have recapitulated a highly productive infection of HPV-18 in organotypic epithelial cultures. By using this system, we now report the characterization of four HPV-18 E6 mutations. An E6 null mutant accumulated high levels of p53 and amplified very poorly. p53 siRNA or ectopic WT E6 partially restored amplification, whereas three missense E6 mutations that did not effectively destabilize p53 complemented the null mutant poorly. Unexpectedly, in cis , two of the missense mutants amplified, albeit to a lower extent than the WT and only in cells with undetectable p53. These observations and others implicate p53 and additional host proteins in regulating viral DNA amplification and also suggest an inhibitory effect of E6 overexpression. We show that high levels of viral DNA amplification are critical for late protein expression and report several previously undescribed viral RNAs, including bicistronic transcripts predicted to encode E5 and L2 or an alternative form of E1^E4 and L1.

•Gardasil-immunized sera or IgG possesses strong extracellular neutralization.•FcRn expressed on primary human keratinocytes (PHKs) mediates the uptake of IgG.•Bound or intracellular Gardasil-immunized IgG does not neutralize HPV virions.•Neutralization window of the immune sera is limited to 4h in early passage PHKs.•Growth characteristics of PHKs affect the neutralization window from 4h to 16h. The prevalent human papillomaviruses (HPVs) infect human epithelial tissues. Infections by the mucosotropic HPV genotypes cause hyperproliferative ano-genital lesions. Persistent infections by high-risk (HR) HPVs such as HPV-16, HPV-18 and related types can progress to high grade intraepithelial neoplasias and cancers. Prophylactic HPV vaccines are based on DNA-free virus-like particles (VLPs) composed of the major capsid protein L1 of HPV-16, -18, -6 and -11 (Gardasil) or HPV-16 and -18 (Cervarix). Sera from vaccinated animals effectively prevent HPV pseudovirions to infect cell lines and mouse cervical epithelia. Both vaccines have proven to be highly protective in people. HPV pseudovirions are assembled in HEK293TT cells from matched L1 and L2 capsid proteins to encapsidate a reporter gene. Pseudovirions and genuine virions have structural differences and they infect cell lines or primary human keratinocytes (PHKs) with different efficiencies. In this study, we show that sera and isolated IgG from women immunized with Gardasil prevent authentic HPV-18 virions from infecting PHKs, whereas non-immune sera and purified IgG thereof are uniformly ineffective. Using early passage PHKs, neutralization is achieved only if immune sera are added within 2–4h of infection. We attribute the timing effect to a conformational change in HPV virions, thought to occur upon initial binding to heparan sulfate proteoglycans (HSPG) on the cell surface. This interpretation is consistent with the inability of immune IgG bound to or taken up by PHKs to neutralize the virus. Interestingly, the window of neutralization increases to 12–16h in slow growing, late passage PHKs, suggestive of altered cell surface molecules. In vivo, this window might be further lengthened by the time required to activate the normally quiescent basal cells to become susceptible to infection. Our observations help explain the high efficacy of HPV vaccines.

To determine whether a vaccine consisting of an idiotope peptide mimic of the third complementarity-determining region of the immunoglobulin heavy chain (CDR-H3) is an effective substitute for its parent idiotype. Such peptide vaccines could ultimately be used for targeting pathological B lymphocytes. Hen egg lysozyme (HEL) conjugates of the Fab' fragment of monoclonal anti-fluorescein antibody 9-40 (Fab'-HEL) or a peptide mimic of the 9-40 CDR-H3 (referred to as the \"B epitope\" or \"Bep,\" the conjugate is referred to as \"Bep-HEL\") were injected into separate cohorts of B10.A mice. Two additional control cohorts were injected with either the Bep peptide alone or a noncovalent mixture of Bep and HEL. Sera were assayed for both anti-idiotope and anti-idiotype activity by enzyme-linked immunosorbant assay (ELISA). Primary, secondary, and tertiary immune responses were examined. Both the Bep-HEL idiotope and the Fab-HEL idiotype immunogens elicited homologous, allogenic immune responses. No cross-reactivity was observed between anti-idiotope and anti-idiotype responses after primary immunization. With secondary immunization, 50% of mice immunized with the Bep-HEL conjugate exhibited a cross-reactive anti-idiotype response. Conversely, 100% of mice immunized with the Fab'-HEL conjugate exhibited a marginal, but statistically significant cross-reactive anti-idiotope response. Upon tertiary immunization, 100% of mice immunized with Bep-HEL exhibited a cross-reactive anti-idiotype response, and 55.6% of mice immunized with the Fab'-HEL conjugate exhibited a cross-reactive anti-idiotope response. Covalent coupling of a xenogenic carrier protein to an idiotype immunogen or its peptide mimic significantly enhances the intensity of homologous, allogenic anti-idiotype or anti-idiotope immune responses. Multiple immunizations are necessary to induce cross-reactivity between the peptide mimic and its parent idiotype.

This work examines the effectiveness of synthetic peptide immunogens derived from immunodominant T-cell epitopes as replacements for their intact parent protein in vaccines. Fluorescein was conjugated to hen egg lysozyme (FL-HEL, positive control) and three synthetic peptide immunogens: (a) murine B10.A (H-2a) immunodominant T-cell epitope of HEL [FL-(T-cell epitope)]; (b) multiple antigenic peptide (MAP) multimer of this epitope ([FL-(T epitope)]n-MAP, n = 2-4); and (c) negative control MAP with T-cell epitope residues replaced with glycine [(FL-Gly18)4-MAP]. The dose response of each immunogen was examined over a 300-fold range in B10.A mice. The immune response was monitored using antifluorescein ELISA assays. FL-(T epitope)'s immune response correlated positively with dose, with maximum response comparable to that of [FL-(T epitope)]n-MAP, or FL-HEL. This trend was consistent across 1 degrees, 2 degrees, and 3 degrees responses, although interanimal variability was higher in the latter two because of an all-or-none response in mice immunized with this peptide. [FL-(T epitope)]n-MAP's immune response was consistently high and nearly dose independent, a trend observed across 1 degrees, 2 degrees, and 3 degrees responses. FL-HEL's immune response correlated negatively to dose in the 1 degrees response but was nearly dose independent in the 2 degrees and 3 degrees responses. The magnitude of these latter responses was comparable to that observed for [FL-(T epitope)]n-MAP. (FL-Gly18)4-MAP did not elicit an immune response except at the highest dose. This trend was consistent across 1 degrees, 2 degrees, and 3 degrees responses. The monomeric epitope was 300-fold less potent than its parent carrier protein, but increasing immunogen valency using MAP technology compensated totally for reduced potency. (FL-Gly18)4-MAP's lack of response at all but the highest dose strongly suggests that a specific immunodominant T-cell epitope sequence for HEL is necessary for successful peptide mimicry of HEL. This work also demonstrates the importance of quality assessment of commercial MAP core resins.

The genes encoding the enzymes for metabolising alcohol dehydrogenase 1B ( ADH1B ) and aldehyde dehydrogenase 2 ( ALDH2 ) -- exhibit genetic polymorphism and ethnic variations. Although the ALDH2*2 variant allele has been widely accepted as protecting against the development of alcoholism in Asians, the association of the ADH1B*2 variant allele with drinking behaviour remains inconclusive. The goal of this study was to determine whether the polymorphic ADH1B and ALDH2 genes are associated with stroke in male Han Chinese with high alcohol consumption. Sixty-five stroke patients with a history of heavy drinking (HDS) and 83 stroke patients without such a history (NHDS) were recruited for analysis of the ADH1B and ALDH2 genotypes from the stroke registry in the Tri-Service General Hospital, Taipei, Taiwan, between January 2000 and December 2001. The allelotypes of ADH1B and ALDH2 were determined using the polymerase chain reaction-restriction fragment length polymorphism method. The HDS patients (3 per cent) showed a significantly lower ALDH2*2 allele frequency than NHDS patients (27 per cent) ( p < 0.001). After controlling for age, patients with HDS were associated with a significantly higher occurrence of cigarette smoking ( p < 0.01) and liver dysfunction ( p < 0.01). Multiple logistic regression analyses revealed that the ALDH2*2 variant allele was an independent variable exhibiting strong protection (odds ratio 0.072; 95 per cent confidence interval 0.02-0.26) against HDS after adjustment for hypertension, diabetes mellitus, smoking status and liver dysfunction. By contrast, allelic variations in ADH1B exerted no significant effect on HDS. The present study indicated that, unlike ALDH2*2 , ADH1B*2 appears not to be a significant negative risk factor for high alcohol consumption in male Han Chinese with stroke.

Infections by the high-risk human papillomavirus type 18 (HPV-18) or type 16 (HPV-16) can lead to cancers of the anogenital tract. Because the propagation of HPVs is restricted to differentiated human squamous epithelia, experimental studies of the productive program are conducted in organotypic cultures. The current methods are of low efficiency and time consuming and have relied on immortalized epithelial cell lines that do not support efficient viral DNA amplification or virion production, compromising HPV genetic analyses and obscuring normal virus-host cell interactions. I used Cre-loxP site-specific recombination to develop a relatively simple, fast and highly efficient system to produce HPV-18 genomic plasmids in primary human keratinocytes (PHKs). The transfected primary human keratinocytes can be quickly developed into organotypic raft cultures, obviating the need for immortalized cells or the immortalization function of the high-risk HPV types. Upon keratinocyte differentiation and early viral gene expression, the HPV-18 replicons amplified to high copy numbers in raft cultures. The major capsid protein (L1) expression was widely detected in superficial cells and in cornified layers on and after day 14 or 16. Electron microscopy examination revealed HPV-18 particles were packed in paracryatalling arrays in shrunken nuclei with condensed chromatins. These virions infected PHKs efficiently and were passaged in fresh PHK raft cultures, a feat not achieved until now. Thus, this system should be a useful tool to study HPV mutants or low-risk HPV genotypes. I further verified the utility of this system in HPV genetic analyses. Indeed, I was able to analyze the phenotypes of the HPV-18 E6*I and E6-null mutant replicons which had not been studied previously in raft cultures due to their inability to immortalize PHKs or failure to be maintained in transfected PHKs as plasmids. The mutant genomes were highly deficient in L1 production but were complemented in trans by a retrovirus expressing wild type HPV-18 E6. In support of translating basic scientific capabilities into clinical outcomes, this system should also be highly informative for testing agents that can prevent or treat human papillomavirus infections. Keywords. Organotypic raft cultures of primary human keratinocytes, Cre-loxP site specific recombination, HPV-18 infection cycle, viral DNA amplification, HPV-18 E6*I and E6-null mutants, genetic complementation in trans.