Bodil Norrild

Development of cytomegalovirus (CMV) disease may be predicted in HIV-infected patients by CMV polymerase chain reaction and the antigenemia test

Objective:Cytomegalovirus (CMV) is a frequent opportunistic viral pathogen in patients with AIDS leading to retinitis and other serious manifestations. CMV disease may be successfully treated. Prophylactic antiviral therapy has been shown to reduce the risk of CMV disease if initiated early. We evaluated PCR and the antigenemia test as methods for early detection of CMV disease. Methods:Two-hundred HIV-seropositive subjects with CD4 T-cell counts below 100 × 106/l were monitored with CMV polymerase chain reaction (PCR), the antigenemia test, blood cultures and CMV immunoglobulin (Ig) G and IgM titres every second month for 1 year. Results:Thirty-eight patients (19%) developed CMV disease. The PCR test detected CMV DNA a median of 46 days before onset of disease. This was earlier than the median of 34 days for the antigenemia test and a median of 1 day for CMV blood cultures. Univariate analysis showed that the CMV PCR, the antigenemia test and blood cultures all had significant predictive values for subsequent development of CMV disease with odds ratios (OR) of 30, 22 and 20. CMV serology had no predictive value. Multivariate analysis showed that the PCR method was superior to the other tests; OR: CMV PCR 10.0, antigenemia test 4.4 and CMV cultures 4.3. No clinical parameters had any significant predictive value in the stepwise multivariate model. Conclusions:The CMV PCR and the CMV antigenemia tests are both sensitive methods that may predict development of CMV disease up to several months prior to clinical disease. These methods make it possible to select patients at high risk for CMV disease and suitable for prophylactic therapy against CMV.

Different miRNA signatures of oral and pharyngeal squamous cell carcinomas: a prospective translational study.

Background:MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNA translation/decay, and may serve as biomarkers. We characterised the expression of miRNAs in clinically sampled oral and pharyngeal squamous cell carcinoma (OSCC and PSCC) and described the influence of human papilloma virus (HPV).Methods:Biopsies obtained from 51 patients with OSCC/PSCC and 40 control patients were used for microarray analysis. The results were correlated to clinical data and HPV status. Supervised learning by support vector machines was employed to generate a diagnostic miRNA signature.Results:One hundred and fourteen miRNAs were differentially expressed between OSCC and normal oral epithelium, with the downregulation of miR-375 and upregulation of miR-31 as the most significant aberrations. Pharyngeal squamous cell carcinoma exhibited 38 differentially expressed miRNAs compared with normal pharyngeal epithelium. Differences in the miRNA expression pattern of both normal epithelium and SCC were observed between the oral cavity compared with the pharynx. Human papilloma virus infection revealed perturbations of 21 miRNAs, most significantly in miR-127-3p and miR363. A molecular classifier including 61 miRNAs was generated for OSCC with an accuracy of 93%.Conclusion:MicroRNAs may serve as useful biomarkers in OSCC and PSCC. The influence of HPV on miRNA may provide a mechanism for the distinct clinical behaviour of HPV-infected tumours.

The role of miRNAs in human papilloma virus (HPV)-associated cancers: bridging between HPV-related head and neck cancer and cervical cancer

Background:Although the role of human papilloma virus (HPV) in cervical squamous cell carcinoma (CSCC) is well established, the role in head and neck SCC (HNSCC) is less clear. MicroRNAs (miRNAs) have a role in the cancer development, and HPV status may affect the miRNA expression pattern in HNSCC. To explore the influence of HPV in HNSCC, we made a comparative miRNA profile of HPV-positive (HPV+) and HPV-negative (HPV−) HNSCC against CSCC.Methods:Fresh frozen and laser microdissected-paraffin-embedded samples obtained from patients with HPV+/HPV− HNSCC, CSCC and controls were used for microarray analysis. Differentially expressed miRNAs in the HPV+ and HPV− HNSCC samples were compared with the differentially expressed miRNAs in the CSCC samples.Results:Human papilloma virus positive (+) HNSCC had a distinct miRNA profile compared with HPV− HNSCC. Significantly more similarity was seen between HPV+ HNSCC and CSCC than HPV− and CSCC. A set of HPV core miRNAs were identified. Of these especially the miR-15a/miR-16/miR195/miR-497 family, miR-143/miR-145 and the miR-106-363 cluster appear to be important within the known HPV pathogenesis.Conclusion:This study adds new knowledge to the known pathogenic pathways of HPV and substantiates the oncogenic role of HPV in subsets of HNSCCs.

Correlation between human papillomavirus and p16 overexpression in oropharyngeal tumours: a systematic review

Background:A significant proportion of squamous cell carcinomas of the oropharynx (OP-SCC) are related to human papillomavirus (HPV) infection and p16 overexpression. This subgroup proves better prognosis and survival but no evidence exists on the correlation between HPV and p16 overexpression based on diagnostic measures and definition of p16 overexpression. We evaluated means of p16 and HPV diagnostics, and quantified overexpression of p16 in HPV-positive and -negative OP-SCCs by mode of immunohistochemical staining of carcinoma cells.Methods:PubMed, Embase, and the Cochrane Library were searched from 1980 until October 2012. We applied the following inclusion criteria: a minimum of 20 cases of site-specific OP-SCCs, and HPV and p16 results present. Studies were categorised into three groups based on their definition of p16 overexpression: verbal definition, nuclear and cytoplasmatic staining between 5 and 69%, and ⩾70% staining.Results:We identified 39 studies with available outcome data (n=3926): 22 studies (n=1980) used PCR, 6 studies (n=688) used ISH, and 11 studies (n=1258) used both PCR and ISH for HPV diagnostics. The methods showed similar HPV-positive results. Overall, 52.5% of the cases (n=2062) were HPV positive. As to p16 overexpression, 17 studies (n=1684) used a minimum of 5–69% staining, and 7 studies (n=764) used ⩾70% staining. Fifteen studies (n=1478) referred to a verbal definition. Studies showed high heterogeneity in diagnostics of HPV and definition of p16. The correlation between HPV positivity and p16 overexpression proved best numerically in the group applying ⩾70% staining for p16 overexpression. The group with verbal definitions had a significantly lower false-positive rate, but along with the group applying 5–69% staining showed a worse sensitivity compared with ⩾70% staining.Conclusions:There are substantial differences in how studies diagnose HPV and define p16 overexpression. Numerically, p16 staining is better to predict the presence of HPV (i.e. larger sensitivity), when the cutoff is set at ⩾70% of cytoplasmatic and nuclear staining.

Identification and preliminary mapping with monoclonal antibodies of a herpes simplex virus 2 glycoprotein lacking a known type 1 counterpart

The properties of herpes simplex virus 2 (HSV-2)-specific proteins reactive with monoclonal antibody H966 derived from mice immunized with HSV-2 strain G are reported. The reactive proteins contained in infected cell lysates subjected to electrophoresis in denaturing gels and transferred to nitrocellulose sheets form a relatively sharp band characteristic of Mr 124,000 proteins and a diffuse, more slowly migrating band. Antigens reactive with H966 were detected on the surface of viable, unfixed cells. The electrophoretic mobility of the H966-reactive proteins made in the presence of tunicamycin was more rapid than that of the proteins made in the absence of the drug. Direct evidence that the HSV-2-specific antigen was a glycoprotein emerged from purification of [14C]glucosamine-labeled proteins with similar electrophoretic mobilities by immunoabsorption to H966 bound to Sepharose beads. Analyses of the reactivity of HSV-1 X HSV-2 recombinants indicated the gene specifying the glycoprotein maps in the S component of the DNA. The glycoprotein detected by H966 has no known counterpart in HSV-1 and corresponds to the glycoprotein previously designated as gC of HSV-2 and reported to map to the right of gC specified by HSV-1. Inasmuch as an HSV-2 gene colinear with HSV-1 gC has been reported to specify a glycoprotein currently designated as gC of HSV-2 by Para et al. [J. Virol. 45, 1223-1227 (1983)], the glycoprotein identified by H966 should be designated as gG.

Immunochemistry of Herpes Simplex Virus Glycoproteins

Herpes simplex viruses (HSV) are large DNA viruses belonging to the Herpesvirus family. Like all other herpesviruses, the virions of HSV are made up of a DNA core, an icosahedral capsid consisting of 162 capsomers, an ill-defined structural component designated the tegument, and a trilaminar membrane containing numerous short spikes, designated the envelope (Fig. 1) (Roizman and Furlong 1974).

A high and increasing HPV prevalence in tonsillar cancers in Eastern Denmark, 2000-2010: the largest registry-based study to date.

The aim was to explore whether the incidence of tonsillar squamous cell carcinomas (TSCCs) increased in Eastern Denmark, 2000–2010, and whether human papillomavirus (HPV) could explain the increase, and to assess the association of HPV prevalence with gender, age, and origin (i.e., the certainty of tonsillar tumor origin). We applied HPV DNA PCR and p16 immunohistochemistry to all TSCCs registered in the Danish Head and Neck Cancer Group (DAHANCA) and in the Danish Pathology Data Bank (n = 632). Pathologists reviewed and subdivided the tumors into two groups: specified and nonspecified TSCCs. Approximately 10% of HPV‐positive tumors was genotyped by amplicon next‐generation sequencing. The overall crude incidence of TSCCs increased significantly (2.7% per year) and was explained by an increasing incidence of HPV‐positive TSCCs (4.9% per year). The overall HPV prevalence was 58%, with HPV16 being the predominant HPV type. In multivariate analysis, the HPV prevalence was associated with age (<55 vs. >60 years) (OR, 1.72; 95% CI 1.13–2.63) and origin (nonspecified vs. specified TSCCs) (OR, 0.15; 95% CI 0.11–0.22). The association of HPV prevalence with origin increased over time in specified TSCCs (OR per year, 1.10; 95% CI 1.01–1.19), whereas no change over time was observed among nonspecified TSCCs (OR per year, 0.99; 95% CI 0.90–1.08). In conclusion, the observed increase in the number of HPV‐positive TSCCs can explain the increasing number of TSCCs in Eastern Denmark, 2000–2010. HPV prevalence was associated with younger age (<55 years) and a high certainty of tonsillar tumor origin.

The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element

The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3′ UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA.

Human papillomavirus in normal conjunctival tissue and in conjunctival papilloma. Types and frequencies in a large series.

Aim: To examine conjunctival papilloma and normal conjunctival tissue for the presence of human papillomavirus (HPV). Methods: Archival paraffin wax-embedded tissue from 165 conjunctival papillomas and from 20 histological normal conjunctival biopsy specimens was analysed for the presence of HPV by PCR. Specimens considered HPV positive using consensus primers, but with a negative or uncertain PCR result using type-specific HPV probes, were analysed with DNA sequencing. Results: HPV was present in 86 of 106 (81%) β-globin-positive papillomas. HPV type 6 was positive in 80 cases, HPV type 11 was identified in 5 cases and HPV type 45 was present in a single papilloma. All the 20 normal conjunctival biopsy specimens were β-globin positive and HPV negative. Conclusion: There is a strong association between HPV and conjunctival papilloma. The study presents the largest material of conjunctival papilloma investigated for HPV and the first investigation of HPV in normal conjunctival tissue. HPV types 6 and 11 are the most common HPV types in conjunctival papilloma. This also is the first report of HPV type 45 in conjunctival papilloma.

Expression of CPEB, GAPDH and U6snRNA in cervical and ovarian tissue during cancer development.

Persistent infection with high‐risk human papillomavirus (HPV) and expression of the proteins E6 and E7 is a prerequisite for development of cervical cancer. The distal non‐coding part of E6/E7 messengers from several HPV types is able to downregulate synthesis of a reporter gene through mechanisms with involvement of cytoplasmic polyadenylation elements (CPEs) in the messengers. We here show that the mRNA levels of one of the four known CPE‐binding proteins (CPEBs), the CPEB3, were downregulated in HPV‐positive cervical cancers, whereas in ovarian cancer the CPEB1 mRNA level was downregulated. In addition, we showed that the RNA levels of the widely used reference marker GAPDH were upregulated in both cancer forms, and the level of the reference marker U6snRNA was upregulated in cervical cancers. Moreover, a possible correlation between the degree of U6snRNA upregulation and cervical cancer propagation was shown. These changes observed in CPEB1 and CPEB3 might indicate regulatory functions of CPEBs in cancer development of HPV‐positive and HPV‐negative tumors, respectively, and the U6snRNA, GAPDH mRNA and CPEB1 mRNA levels may be useful as tumor markers for genital cancers although further investigations are needed.