Diffuse gastrointestinal bleeding and BK polyomavirus replication in a pediatric allogeneic haematopoietic stem cell transplant patient.

Patients undergoing haematopoietic stem cell transplantation (HSCT) are at high risk of severe gastrointestinal bleeding caused by infections, graft versus host disease, and disturbances in haemostasis. BK polyomavirus (BKPyV) is known to cause hemorrhagic cystitis, but there is also evidence of BKV shedding in stool and its association with gastrointestinal disease. We report putative association of BKPyV replication with high plasma viral loads in a pediatric HSCT patient developing hemorrhagic cystitis and severe gastrointestinal bleeding necessitating intensive care. The observation was based on chart review and analysis of BKPyV DNA loads in plasma and urine as well as retrospective BKPyV-specific IgM and IgG measurements in weekly samples until three months post-transplant. The gastrointestinal bleeding was observed after a >100-fold increase in the plasma BKPyV loads and the start of hemorrhagic cystitis. The BKPyV-specific antibody response indicated past infection prior to transplantation, but increasing IgG titers were seen following BKPyV replication. The gastrointestinal biopsies were taken at a late stage of the episode and were no longer informative of BK polyomavirus involvement. In conclusion, gastrointestinal complications with bleeding are a significant problem after allogeneic HSCT to which viral infections including BKPyV may contribute.

A case of primary JC polyomavirus infection-associated nephropathy.

A 15-year-old boy with a posterior urethral valve received a deceased donor kidney transplant (KT) in March 2011. Basiliximab induction followed by tacrolimus-based triple medication was used as immunosuppression. Eleven months after KT, the graft function deteriorated and the biopsy demonstrated interstitial nephritis suggestive of acute rejection. BK polyomavirus (BKPyV) surveillance in urine and plasma was negative. The patient received methylprednisolone pulses and anti-thymocyte globulin. Immunohistochemistry was positive for simian virus 40 (SV40) large T-antigen (LTag) in the biopsies, and quantitative polymerase chain reaction for JC polyomavirus (JCPyV) indicated high viral loads in urine and borderline levels in plasma. Immunosuppression was reduced and follow-up biopsies showed tubular atrophy and interstitial fibrosis. Two years after KT, antibody-mediated rejection resulted in graft loss and return to hemodialysis. Retrospective serologic work-up indicated a primary JCPyV infection with seroconversion first for IgM, followed by IgG, but no indication of BKPyV infection. In the SV40 LTag positive biopsies, JCPyV deoxyribonucleic acid (DNA) with archetype noncoding control region was detected, while BKPyV DNA was undetectable. To the best of our knowledge, this is the first reported case of primary JCPyV infection as the cause of PyV-associated nephropathy in KT.

Altered expression of ezrin, E-Cadherin and ß-Catenin in cervical neoplasia.

High-grade cervical squamous intraepithelial lesions (CIN) as well as squamous cell carcinoma and adenocarcinoma of the cervix are associated with persistent high-risk human papillomavirus (HPV) infection. A number of cellular events play a role in HPV pathogenesis and in the development of cervical lesions, including alterations in cell adhesion and motility. The crucial plasma membrane - cytoskeleton linker protein ezrin of the Ezrin-Radixin-Moesin (ERM) protein family is involved in the regulation of cell morphology, cell adhesion and invasion. Based on our previous work on ERM proteins we sought out to study the expression of ezrin in cervical premalignant lesions. We also studied the expression of E-cadherin and ß-catenin, which play an important role in epithelial cell adhesion. We observed intensifying expression of ezrin along with progressing grade of neoplasia. Ezrin staining was found to colocalize with p16 staining in high-risk HPV associated lesions. Expression of E-cadherin and ß-catenin was found to be altered along with the severity of the lesion, similar to ezrin. Enhanced expression of ezrin in cervical HPV associated lesions suggests a role in the development of cervical neoplasia and cancer. Further clinical evaluation should reveal the feasibility of ezrin as a biomarker for the progression of cervical lesions.

Colposcopy referral rate can be reduced by high-risk human papillomavirus triage in the management of recurrent atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion cytology in Finland.

The aim of this study was to establish whether a combination of high-risk human papillomavirus (hrHPV) testing and cervical cytology could reduce colposcopy referral among women with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion cytology. We randomized 598 women in the Helsinki area, Finland into three study groups. Different strategies of hrHPV testing, cytology and colposcopy with biopsy were used; subsequent hrHPV test results and cytological findings were compared with histology. The rates of hrHPV positivity and CIN2+ were compared. In total, 62.5% of all samples were hrHPV-positive. Altogether 45 (12.7%) CIN2 or worse (CIN2+) lesions were found in study groups A and B. Among hrHPV-positive women the rate of CIN2+ was 19.0% (n = 43), in contrast with 1.6% (n = 2) among hrHPV-negative women (relative risk = 12.2, 95% confidence interval [CI] 3.6-81.1, P < 0.001). Among all hrHPV-negative women whose cytological findings were normal or ASCUS, dysplastic lesions were uncommon (n = 4/119, 3.4%), and all were CIN1. If these women had not been referred to colposcopy, the number of colposcopies would have been reduced by 33.6%. We conclude that hrHPV testing combined with repeat cervical cytology had a high negative predictive value in patients with recurrent low-grade cervical cytology. This could reduce the referral rate to colposcopy without jeopardizing patient safety.

Genes involved in cell adhesion, cell motility and mitogenic signaling are altered due to HPV 16 E5 protein expression.

We investigated the effects of the human papillomavirus type 16 E5 oncogene on cellular gene expression in human epithelial cells using cDNA microarray. In a genome-wide microarray assay, the expression of 179 genes was found to be significantly altered due to E5 expression. The expression of lamin A/C was downregulated at protein level. The expression of protein kinase C-delta and phosphoinositide-3-kinase proteins was found to be upregulated. We also observed increased motility of E5-expressing cells. We conclude that the E5 protein affects several cellular pathways involved in cell adhesion, cell motility and mitogenic signaling. These alterations may together lead to inhibition of apoptosis and facilitate the establishment of persistent infection in the epithelium.

Repeated human papillomavirus DNA findings among female university students.

In a previous study, we found a high (33%) human papillomavirus (HPV) DNA prevalence among first year university students in the Helsinki metropolitan area. We have now performed HPV rescreening among first-round HPV-positive students using a liquid-based hybridization assay. A total of 212 students participated in rescreening, and 82 (38.7%) of 212 were found to be positive for HPV DNA. Low-risk (lr) HPV DNA was repeatedly found in 16.8% of the patients who had been lr positive in the first screening round. High-risk (hr) HPV DNA was repeatedly found in 33.3% of the patients. Although HPV typing in these samples has not been carried out yet, we conclude that repeatedly positive HPV DNA findings were strikingly common. hrHPV DNA was repeatedly found twice as often as lrHPV DNA. HPV DNA prevalence was higher among oral contraceptive users than among patients using other contraception.

Human papillomavirus type 16 E5 protein colocalizes with the antiapoptotic Bcl-2 protein.

Human papillomavirus type 16 E5 protein contributes to cellular transformation by increasing the mitogenic stimulus from growth factor receptors to the nucleus. In order to study the biological mechanisms of the E5 protein we performed site-directed mutagenesis of the E5 gene. Wild-type as well as mutant E5 proteins were transiently expressed in human cervical epithelial cells, and cell morphology, expression of proteins involved in cell adhesion, and localization of the different proteins were studied. Little differences in cell morphology or expression kinetics were observed between the different E5 proteins, except for relocalization of a mutant E5 protein where a hydrophobic leucine membrane anchor was mutated to positively charged amino acids. This mutant E5 protein localized to lamellipodia, which are motility-associated structures at the leading edge of motile cells. In our experimental conditions, 100% of E5-expressing epithelial cells died by four days of expression, possibly due to toxicity or disturbance of the membrane compartment by the E5 protein. Most interestingly, a remarkable colocalization of the E5 protein with the Bcl-2 antiapoptotic protein on intracellular membranes was established.

Malignancies after heart transplantation: presence of Epstein-Barr virus and cytomegalovirus.

The presence of Epstein-Barr virus (EBV), human papilloma virus (HPV), and cytomegalovirus (CMV) was studied in 20 patients who developed malignancies after heart transplantation in the Helsinki University Central Hospital. The tumors were analyzed for the presence of HPV by polymerase chain reaction and for EBV by in situ hybridization. Clinical CMV infection was verified by immunochemical quantitation of CMV antigen in peripheral blood cells. HPV was detected in one of the eight epithelial malignant tumors studied. Three of the six lymphomas were positive for EBV. Two (67%) of 3 patients with EBV-positive lymphomas and one (33%) of the other three lymphomas but only 2 (14%) of 14 patients who developed other malignancies had a history of a manifest post-transplantation CMV infection prior to the development of malignancy. These results confirm the presence of EBV in lymphomas of heart transplant recipients and suggest that CMV might have a contributory role in the development of EBV-associated lymphomas.

Poor antibody response against human papillomavirus in adult-onset laryngeal papillomatosis.

To investigate whether adult-onset laryngeal papillomatosis induces serum antibodies to the human papillomavirus (HPV), 60 patients underwent a clinical examination, and HPV DNA from their laryngeal biopsy was assayed by PCR and HPV serology with virus-like particles as the antigen. Patients and controls (n = 53) showed no differences in their HPV 6 and 16 antibodies. Patients more often had HPV 11 antibodies, female patients more often than female controls or male patients. Of the female patients, 5 of 15 had a history of genital condylomas and, at the follow-up visit, 5 of 9 had cervical cytology consistent with genital HPV infection. The fact that HPV antibodies did not correlate with clinical features of the laryngeal disease or with HPV DNA detected in the larynx, suggests that HPV antibodies in female patients were induced by genital rather than laryngeal HPV infection. The high prevalence of abnormal Pap smears indicates that gynaecological examination of female adult-onset laryngeal papilloma patients is warranted.

Human papillomavirus type 16 E5 protein localizes to the Golgi apparatus but does not grossly affect cellular glycosylation.

The E5 protein of papillomaviruses is a strongly hydrophobic membrane protein that can associate with the 16 kDa protein subunit of the vacuolar proton ATPase in endosomes and the Golgi apparatus resulting in raise of intraorganelle pH. We demonstrate that E5 of human papillomavirus type 16 (HPV16) when transfected into human keratinocytes localizes to the Golgi. Using FACS analysis and western blotting with a variety of lectins as well as analysing the sialylation status of a specific cell surface glycoprotein CD95 (APO-1/Fas), we show that HPV16 E5 does not grossly affect cellular glycosylation, a main Golgi function.

HLA-DQ alleles and human papillomavirus DNA in adult-onset laryngeal papillomatosis.

Sixty-two patients with histologically confirmed adult-onset laryngeal papilloma were clinically examined; their HLA class II DQA1 and DQB1 alleles and the presence and type of human papillomavirus (HPV) in their laryngeal papilloma biopsies were determined by polymerase chain reaction-based methods. No differences in the DQA1 or DQB1 frequencies appeared between the patients as a group and the reference population. When the patients were divided into groups according to number of laryngeal procedures performed, no HLA association was noticed with any group, nor did the presence of HPV-6 or HPV-11 DNA in the laryngeal specimen correlate with HLA type. A suggestive association was found between the DQB1 *0501 allele and the 16 patients whose laryngeal biopsy was HPV-negative, but because of the small series, additional patients need to be studied. Earlier, the DQB1 *0501 allele was reported to be protective against cervical cancer, another HPV-associated disease.

Anti-proliferative effect of retinoids and interferon-alpha-2a on vaginal cell lines derived from squamous intra-epithelial lesions.

A panel of retinoids (all-trans-, 13-cis-, 19-cis retinoic acid and acitretin), and interferon-alpha-2a was tested for the capacity to modulate the proliferation of UT-DEC-1 (HPV-33-positive) and UT-DEC-2 (HPV-16-positive) cell lines derived from vaginal intra-epithelial neoplasias (VAIN). At concentrations 10(-6) to 10(-8) M, all retinoids inhibited the growth of early-passage UT-DEC cell lines, but also of normal vaginal keratinocytes and fibroblasts. The inhibition was significantly reduced in late-passage UT-DEC cells. The effect on proliferation was essentially equal for all retinoids in high (1.8 mM)-Ca2+ medium, but decreased markedly in low (0.09 mM)-Ca2+ medium. Interferon-alpha-2a at 1000 IU/ml had an additive growth-inhibitory effect in the low- and in the high-Ca2+ medium. No consistent decrease in HPV E6-E7 mRNA levels could be associated either with retinoid or with interferon effect in either cell line. The expression of TGFbeta1 and TGFbeta2 mRNA increased 2- to 3-fold by 10(-6) M 13-cis-RA treatment in early- and in late-passage cells of both cell lines. TGFbeta1 at 0.1 to 1.0 ng/ml also inhibited the proliferation of both cell lines, and was more effective at early passage, but the inhibition was not dependent on calcium concentration. Neutralizing anti-TGFbeta antibodies partially relieved the proliferation inhibition by 13-cis-RA. The results show that the calcium-associated regulation of growth by the tested retinoids was seen in normal vaginal cells and in early pre-neoplastic cells, but was significantly reduced in cells with higher-grade phenotype, while also suggesting that the loss of responsiveness to retinoids and TGFbeta may play a role in the progression of squamous intra-epithelial neoplasia.

Human papillomavirus type 33 DNA and E6-E7 transcripts in late passages of the UT-DEC-1 vaginal keratinocyte cell line.

Transcription of human papillomavirus (HPV) type 33 early region was analysed in the UT-DEC-1 keratinocyte cell line, which has been derived from a HPV-33-containing mild vaginal dysplasia. Fifteen cDNA clones from transcripts from the E6-E7 open reading frames were constructed and analysed. Most clones represented viral transcripts spliced within the E6 open reading frame, probably encoding the E7 protein. Interestingly, a less abundant unspliced transcript species with coding capacity for the full length E6 protein was found, reported here for the first time for the malignancy-associated HPV type 33.

The human papillomavirus type 16 E5-protein modulates ligand-dependent activation of the EGF receptor family in the human epithelial cell line HaCaT.

The E5 open reading frame of the human papillomavirus type 16 encodes a transmembrane protein associated with the Golgi, ER, and plasma membranes. We have analyzed the effect of E5 expression on the activation of the EGF receptor family. We find that expression of the E5-protein strongly enhances EGFR activation in a ligand-dependent manner. This activation takes place immediately after addition of ligand, demonstrating that increased tyrosine phosphorylation cannot solely be due to an impaired downregulation of the receptors. Furthermore, this activation is not a result of impaired activity of EGFR-specific phosphatase through the E5-protein, as demonstrated by using inhibitors specifically blocking EGFR activation. In addition, treatment with EGF results in an enhanced activation of the ErbB2 receptor in E5-expressing cells. This superactivation must be a result of heterodimer formation between EGFR and ErbB2, since EGF is not a ligand for ErbB2. Finally, treatment of E5-expressing cells with HB-EGF shows no increased phosphorylation of the ErbB4 receptor, suggesting a specific effect of E5 on the activation of the different members of the EGFR family.

Enhancement of EGF- and PMA-mediated MAP kinase activation in cells expressing the human papillomavirus type 16 E5 protein.

In this report we demonstrate that cells expressing the human papillomavirus type 16 E5 open reading frame (HPV16-E5) show a greatly enhanced transcription of the immediate early genes after EGF or PMA treatment compared to control cells. This enhancement is due to amplification of the signal transduction pathways in response to growth factors or phorbol esters. Upon short-time EGF treatment of the E5-expressing cells we observed an increase in the activation of EGF receptors, resulting in a stronger activation of MAP kinases ERK1/2 compared to control-transfected cells. We also observed that in E5-expressing cells, treatment with PMA results in an increase in membrane-associated PKC activity, and a superactivation of the ERK1/2 MAP kinases. This superactivation is PKC-dependent, since pretreatment of the cells with the PKC inhibitor Ro 31-8220 inhibits MAP kinase activation and early gene transcription almost completely. Furthermore, treatment with genistein strongly reduces the PMA-mediated superactivation of ERK1/2 kinases, demonstrating a PKC-mediated, tyrosine kinase-dependent pathway in the superinduction of MAP kinase activation. Thus, HPV16-E5 effects superactivation of MAP kinases over at least two different pathways, a PKC-mediated, and another, receptor tyrosine-kinase mediated, PKC-independent one.

Human papillomavirus and Epstein-Barr virus in epithelial carcinomas of the head and neck region.

BACKGROUND: Human papillomavirus (HPV) DNA has been detected in carcinomas of the upper aerodigestive tract. However, studies of the subject show considerable variation in their results, and the causal relationship between HPV and squamous cell carcinomas of the head and neck area still remains to be determined. Epstein-Barr virus (EBV) is consistently detected in nasopharyngeal carcinoma lesions, but little is known about its association with other carcinomas of the head and neck region. The present study was carried out on the role of HPV and EBV in epithelial carcinomas of the upper aerodigestive tract. MATERIALS AND METHODS: The study material comprised 79 frozen biopsy samples from epithelial head and neck carcinomas. DNA was extracted from frozen biopsy samples for analysis using Southern blot hybridization (SBH) and polymerase chain reaction (PCR) to detect HPV DNA. RESULTS: HPV DNA was detected in 13 samples (16.5%) by SBH under low stringency conditions and in three samples (3.8%) by PCR using general primers targeting the HPV L1 region. HPV seemed to have affinity for labial carcinomas: four of the six samples (66.7%) were HPV DNA positive. The detection rate of HPV diminished from the labial and oral epithelium towards the laryngeal region. In SBH, EBV DNA was not found in any of the biopsy samples. CONCLUSIONS: HPV seems to be involved in multifactorial carcinogenesis in the head and neck epithelium, but the association is not as evident as that found in genital carcinomas. The results suggest that EBV is not associated with sporadic nasopharyngeal carcinomas.

Human papillomavirus type 16 E5 protein (review).

The association of certain human papillomavirus (HPV) types with malignancies of the anogenital tract is well established. The virus type most frequently associated with cellular transformation is HPV 16, as has been shown in epidemiological studies. Its transforming capacity has also been demonstrated in many in vitro cell transformation experiments. The most potent oncogenes of HPV 16 are the E6 and E7 proteins, but the E5 protein, whose homologue is the main oncogene of bovine papillomavirus, has recently been identified as an oncogene also for HPV. On the basis of epidemiological and clinical data from tumor material as well as from in vitro data it has been suggested, that the HPV 16 E5 protein would have a function at the early stages of cervical carcinogenesis. The E5 protein enhances growth factor-mediated signal transduction to the nucleus and consequently augments cellular proliferation. Expression of the E5 protein enables the infected cell to escape growth control provided by surrounding cells by inhibiting gap junctional intercellular communication in epithelial cells. This viral oncogene seems to interfere with the control mechanisms of cellular growth and proliferation and thus facilitate the function of the E6 and E7 proteins and further steps towards epithelial cell transformation.

Genomic cloning and characterization of the nonoccupied allele corresponding to the integration site of human papillomavirus type 16 DNA in the cervical cancer cell line SiHa.

Human papillomavirus (HPV) type 16 DNA sequences have been found integrated into the host cell genome in a large number of cervical tumors and cell lines derived therefrom. In this study, we have cloned and analyzed the nonoccupied allele corresponding to the integration site of HPV-16 in the cervical cancer cell line SiHa. Our mapping analyses revealed an approximately 7.8-kb deletion of cellular DNA upon viral integration. Computer analysis of 2.3 kb of DNA sequences from the deleted genomic region as well as 1.0 kb of sequences upstream of the viral integration site showed no significant homology to any known human sequences. DNase I mapping experiments on native chromatin demonstrated the existence of two hypersensitive sites in both the HPV-16-containing and nonoccupied alleles located approximately 1.1 and 1.7 kb upstream of the viral integration site. This suggests that viral integration occurred close to putative regulatory sequences and that recombination with host cellular DNA was not followed by a reorganization of the chromatin structure upstream of the integration site. Nuclear run-on and RT-PCR experiments showed HPV-specific transcription spanning the E2, E4, E5, and L1/L2 open reading frames (ORFs) located upstream of the HPV-16 regulatory region (URR). Taken together, our data suggest that the cellular DNA region upstream of the HPV-16 integration site in the SiHa cell line contains regulatory elements affecting transcription of HPV-16 ORFs located upstream of the HPV-16 URR.

A comparison of human papillomavirus detection rates by dot blot assay from smear and biopsy specimens with regard to human papillomavirus type and histologic diagnosis.

Colposcopic biopsy and cervical smear sampling techniques for human papillomavirus (HPV) DNA dot hybridization were compared to reveal differences related to the level of the histopathologic detection of HPV type 16. The authors used a previously published dot blot assay to analyze 814 pairs of concurrent biopsy and smear DNA specimens for the presence of DNA of HPV 6, 11, 16, and 18. The overall HPV detection rate was 38%, the most prevalent type being HPV 16 (39% of all HPV-positive cases). In detection and typing of HPV DNA, a 81% concordance (658 of 814 pairs) was noted between the smear and biopsy specimens, with a significant correlation in detection of any of the HPV types in the specimens (kappa, .609). The rate of smear-negative cases among all biopsy-positive cases was similar for HPV 11 and HPV 16 (41% and 42%, respectively). Further analysis of distribution of the smear-negative and biopsy-positive cases among different histopathologic levels of disease showed no significant difference between neoplastic and nonneoplastic lesions for either virus type. In 56 cases, only the smear specimen was positive for DNA of the studied HPV types. Both biopsy and smear specimens should be used for HPV detection in cervical dysplasias.