Inhibition of Epstein-Barr Virus Replication in Human Papillomavirus-Immortalized Keratinocytes.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OSCC). EBV-associated cancers harbor a latent EBV infection characterized by a lack of viral replication and the expression of viral oncogenes. Cellular changes promoted by HPV are comparable to those shown to facilitate EBV latency, though whether HPV-positive cells support a latent EBV infection has not been demonstrated. Using a model of direct EBV infection into HPV16-immortalized tonsillar cells grown in organotypic raft culture, we showed robust EBV replication in HPV-negative rafts but little to no replication in HPV-immortalized rafts. The reduced EBV replication was independent of immortalization, as human telomerase-immortalized normal oral keratinocytes supported robust EBV replication. Furthermore, we observed reduced EBV lytic gene expression and increased expression of EBER1, a noncoding RNA highly expressed in latently infected cells, in the presence of HPV. The use of human foreskin keratinocyte rafts expressing the HPV16 E6 and/or E7 oncogene(s) (HPV E6 and E7 rafts) showed that E7 was sufficient to reduce EBV replication. EBV replication is dependent upon epithelial differentiation and the differentiation-dependent expression of the transcription factors KLF4 and PRDM1. While KLF4 and PRDM1 levels were unaltered, the expression levels of KLF4 transcriptional targets, including late differentiation markers, were reduced in HPV E6 and E7 rafts compared to their levels in parental rafts. However, the HPV E7-mediated block in EBV replication correlated with delayed expression of early differentiation markers. Overall, this study reveals an HPV16-mediated block in EBV replication, through E7, that may facilitate EBV latency and long-term persistence in the tumor context.IMPORTANCE Using a model examining the establishment of EBV infection in HPV-immortalized tissues, we showed an HPV-induced interruption of the normal EBV life cycle reminiscent of a latent EBV infection. Our data support the notion that a persistent EBV epithelial infection depends upon preexisting cellular alterations and suggest the ability of HPV to promote such changes. More importantly, these findings introduce a model for how EBV coinfection may influence HPV-positive (HPV-pos) OSCC pathogenesis. Latently EBV-infected epithelial cells, as well as other EBV-associated head-and-neck carcinomas, exhibit oncogenic phenotypes commonly seen in HPV-pos OSCC. Therefore, an HPV-induced shift in the EBV life cycle toward latency would not only facilitate EBV persistence but also provide additional viral oncogene expression, which can contribute to the rapid progression of HPV-pos OSCC. These findings provide a step toward defining a role for EBV as a cofactor in HPV-positive oropharyngeal tumors.

Rhizosphere microbial communities associated to rose replant disease: links to plant growth and root metabolites.

Growth depression of Rosa plants at sites previously used to cultivate the same or closely related species is a typical symptom of rose replant disease (RRD). Currently, limited information is available on the causes and the etiology of RRD compared to apple replant disease (ARD). Thus, this study aimed at analyzing growth characteristics, root morphology, and root metabolites, as well as microbial communities in the rhizosphere of the susceptible rootstock Rosacorymbifera 'Laxa' grown in RRD-affected soil from two sites (Heidgraben and Sangerhausen), either untreated or disinfected by γ-irradiation. In a greenhouse bioassay, plants developed significantly more biomass in the γ-irradiated than in the untreated soils of both sites. Several plant metabolites detected in R. corymbifera 'Laxa' roots were site- and treatment-dependent. Although aloesin was recorded in significantly higher concentrations in untreated than in γ-irradiated soils from Heidgraben, the concentrations of phenylalanine were significantly lower in roots from untreated soil of both sites. Rhizosphere microbial communities of 8-week-old plants were studied by sequencing of 16S rRNA, ITS, and cox gene fragments amplified from total community DNA. Supported by microscopic observations, sequences affiliated to the bacterial genus Streptomyces and the fungal genus Nectria were identified as potential causal agents of RRD in the soils investigated. The relative abundance of oomycetes belonging to the genus Pythiogeton showed a negative correlation to the growth of the plants. Overall, the RRD symptoms, the effects of soil treatments on the composition of the rhizosphere microbial community revealed striking similarities to findings related to ARD.

Detecting episomal or integrated human papillomavirus 16 DNA using an exonuclease V-qPCR-based assay.

Screening for human papillomavirus (HPV) integration into host cell chromosomes typically requires large amounts of time and reagents. We developed a rapid and sensitive assay based on exonuclease V (ExoV) and quantitative polymerase chain reaction (qPCR) to determine HPV genome configurations in cell lines and tissues. We established the assay using genomic DNA from cell lines known to harbor integrated or episomal HPV16. DNA was incubated with ExoV, which is specific for linear DNA, and the DNA fraction resistant to digestion was measured by qPCR. The percent of DNA resistant to ExoV digestion was calculated relative to undigested DNA for determination of episomal or integrated HPV16. The ExoV assay was accurate, capable of distinguishing episomal from integrated HPV16 in cell lines and tissues. Future applications of the ExoV assay may include screening of HPV genome configurations in the progression of HPV-associated cancers.

Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.

Dendritic cells (DCs) are potent antigen-presenting cells that initiate protective T-cell immunity in mice. To study the immunogenicity of DCs in humans, we injected 9 healthy subjects subcutaneously with a control injection of autologous monocyte-derived, mature DCs, followed 4-6 weeks later by DCs pulsed with keyhole limpet hemocyanin (KLH), HLA-A*0201-positive restricted influenza matrix peptide (MP), and tetanus toxoid (TT). Four more subjects received these antigens without DCs. Injection of unpulsed DCs, or antigens alone, failed to immunize. Priming of CD4(+) T cells to KLH was observed in all 9 subjects injected with KLH-pulsed DCs, and boosting of TT-specific T-cell immunity was seen in 5 of 6 subjects injected with TT-pulsed DCs. Injection of antigen-pulsed DCs led to a severalfold increase in freshly isolated MP-specific, IFN-gamma-secreting CD8(+) T cells in all 6 HLA-A*0201-positive subjects, as early as 7 days after injection. When T cells were boosted in culture, there was an increase in MHC tetramer-binding cells and cytotoxic T cells after DC vaccination. These data provide the first controlled evidence of the immunogenicity of DCs in humans, and demonstrate that a single injection of mature DCs rapidly expands T-cell immunity.

Histone H1 represses transcription from minichromosomes assembled in vitro.

We have previously shown that transcription from a Xenopus 5S rRNA gene assembled into chromatin in vitro can be repressed in the absence of histone H1 at high nucleosome densities (one nucleosome per 160 base pairs of DNA) (A. Shimamura, D. Tremethick, and A. Worcel, Mol. Cell. Biol. 8:4257-4269, 1988). We report here that transcriptional repression may also be achieved at lower nucleosome densities (one nucleosome per 215 base pairs of DNA) when histone H1 is present. Removal of histone H1 from the minichromosomes with Biorex under conditions in which no nucleosome disruption was observed led to transcriptional activation. Transcriptional repression could be restored by adding histone H1 back to the H1-depleted minichromosomes. The levels of histone H1 that repressed the H1-depleted minichromosomes failed to repress transcription from free DNA templates present in trans. The assembly of transcription complexes onto the H1-depleted minichromosomes protected the 5S RNA gene from inactivation by histone H1.

Microbial changes linked to the accelerated degradation of the herbicide atrazine in a range of temperate soils.

Accelerated degradation is the increased breakdown of a pesticide upon its repeated application, which has consequences for the environmental fate of pesticides. The herbicide atrazine was repeatedly applied to soils previously untreated with s-triazines for >5 years. A single application of atrazine, at an agriculturally relevant concentration, was sufficient to induce its rapid dissipation. Soils, with a range of physico-chemical properties and agricultural histories, showed similar degradation kinetics, with the half-life of atrazine decreasing from an average of 25 days after the first application to <2 days after the second. A mathematical model was developed to fit the atrazine-degrading kinetics, which incorporated the exponential growth of atrazine-degrading organisms. Despite the similar rates of degradation, the repertoire of atrazine-degrading genes varied between soils. Only a small portion of the bacterial community had the capacity for atrazine degradation. Overall, the microbial community was not significantly affected by atrazine treatment. One soil, characterised by low pH, did not exhibit accelerated degradation, and atrazine-degrading genes were not detected. Neutralisation of this soil restored accelerated degradation and the atrazine-degrading genes became detectable. This illustrates the potential for accelerated degradation to manifest when conditions become favourable. Additionally, the occurrence of accelerated degradation under agriculturally relevant concentrations supports the consideration of the phenomena in environmental risk assessments.

Prospective seroepidemiologic study of human papillomavirus infection as a risk factor for invasive cervical cancer.

BACKGROUND: Major risk factors for invasive cervical cancer include infection with human papillomavirus (HPV), infection with other sexually transmitted pathogens (e.g., Chlamydia trachomatis), and smoking. Since exposures to these risk factors can be related, the contribution of any single factor to cervical carcinogenesis has been difficult to assess. We conducted a prospective study to define the role of HPV infection in cervical carcinogenesis, with invasive cancer as an end point. METHODS: A nested case-control study within a joint cohort of 700,000 Nordic subjects was performed. The 182 women who developed invasive cervical cancer during a mean follow-up of 5 years were matched with 538 control women on the basis of age and time of enrollment. Serum samples taken at enrollment were analyzed for evidence of tobacco use (i.e., cotinine levels); for antibodies against HPV types 16, 18, and 33; and for antibodies against C. trachomatis. Relative risks (RRs) were estimated by use of conditional logistic regression. RESULTS: Presence of antibodies against HPV in serum (seropositivity) was associated with an increased risk of cervical cancer, and adjustment for smoking and for C. trachomatis seropositivity did not affect this finding (RR = 2.4; 95% confidence interval [CI] = 1.6-3.7). HPV16 seropositivity was associated primarily with an increased risk of squamous cell carcinoma (RR = 3.2; 95% CI = 1.7-6.2). In contrast, risk associated with HPV18 seropositivity tended to be higher for cervical adenocarcinoma (RR = 3.4; 95% CI = 0.8-14.9). In populations with a low prevalence of antibodies against C. trachomatis, the HPV16-associated risk of cervical cancer was very high (RR = 11.8; 95% CI = 3.7-37.0); in contrast, in populations with a high prevalence of antibodies against C. trachomatis, no excess risk was found. CONCLUSION: Past infection with HPV16 increases the risk of invasive cervical squamous cell carcinoma, most clearly seen in populations with a low prevalence of sexually transmitted diseases.

A prospective, seroepidemiological study of the role of human papillomavirus in esophageal cancer in Norway.

Infection with the human papillomavirus (HPV), notably HPV type 16, has been associated with esophageal cancer in seroepidemiological studies. To evaluate the consistency of the association, we performed a nested case-control study of HPV seropositivity and risk of esophageal cancer within a prospectively followed cohort of 300,000 Norwegian men and women who had donated blood samples to a serum bank. The data file of the serum bank was linked with the nationwide Cancer Registry of Norway to identify esophageal cancers diagnosed after donation of the serum sample. Fifty-seven cases and 171 matched controls were analyzed for antibodies to specific microorganisms, and odds ratios for developing esophageal cancer were calculated. There was an increased risk of developing esophageal cancer among HPV 16-seropositive subjects (odds ratio = 6.6; 95% confidence interval, 1.1-71) but not among Chlamydia trachomatis-seropositive subjects. Adjustment for the presence of serum cotinine, a marker of smoking habits, did not affect the estimates substantially. The seroepidemiological association between HPV 16 and esophageal cancer seems to be consistent in different countries.

Fenton's reagent for the rapid and efficient isolation of microplastics from wastewater.

Fenton's reagent was used to isolate microplastics from organic-rich wastewater. The catalytic reaction did not affect microplastic chemistry or size, enabling its use as a pre-treatment method for focal plane array-based micro-FT-IR imaging. Compared with previously described microplastic treatment methods, Fenton's reagent offers a considerable reduction in sample preparation times.

Molecular structures of two human DNA topoisomerase I retrosequences.

We have isolated recombinant lambda-phage clones that contain sequences complementary to the 3' half of the cDNA encoding human topoisomerase I (hTOP1). These lambda clones belong to three distinct classes: class-I clones contain sequences from the active gene located on human chromosome 20. Class-II and class-III clones contain sequences corresponding to the cDNA encoding hTOP1 from nucleotide (nt) 2208 to 3434 and from nt 1639 to 3434, respectively. These sequences exhibit the characteristic features of retroposons or retrosequences. They are most likely derived from truncated mRNA transcripts of the active gene. We propose to designate the truncated hTOP1 sequence located on chromosome 1 as the pseudogene 1 (psi 1-hTOP1) and the sequence on chromosome 22 as the pseudogene 2 (psi 2-hTOP1). Pseudogene psi 1-hTOP1 has two unique properties: it is flanked by upstream sequences which display promoter activity in transient expression assays, and it contains an open reading frame which could code for the 211 C-terminal amino acids of hTOP1. Pseudogene psi 2-hTOP1 is located within an AluI repetitive element and is flanked on one side by a (CA)21 stretch.

Primary Hodgkin's disease of the CNS in an immunocompetent patient: a case study and review of the literature.

Primary Hodgkin's disease limited to the CNS is exceedingly rare. Little is known regarding etiologic risk factors, optimal management, and prognosis. A case of Hodgkin's disease confined to the CNS, with cerebrospinal fluid negative for cytology, is described in an immunocompetent patient previously treated for hyperthyroidism with 131I. The patient underwent craniotomy, with resection of two lesions in close proximity within the parenchyma of the temporoparietal lobe. Histopathology revealed classic nodular sclerosing Hodgkin's disease, without evidence of Epstein-Barr viral infection. Treatment included radiation to the whole brain with a boost to the tumor bed. The patient made a full neurologic recovery and remains free of disease recurrence 21 months after treatment. A literature review has identified only 9 additional cases. Seven of 8 evaluable patients remain alive and free of recurrence with a median follow-up of 13 months. The risk factors for this presentation remain undefined. Although follow-up is short, radiotherapy alone appears to provide excellent disease-free survival. Chemotherapy may be reserved for patients with positive cerebrospinal fluid, extracranial disease, or subsequent relapse.

Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood.

We have investigated an improved method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood. The procedure uses 1% human plasma in the place of 10% fetal calf serum and involves two steps. The first step or 'priming' phase is a 6-7 day culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or 'differentiation' phase requires the exposure to macrophage conditioned medium. This medium cannot be replaced by several known cytokines such as TNF-alpha, IL-1, IL-6, IL-12 and IL-15, and cannot be inhibited with neutralizing antibodies to IL-1, TNF-alpha, IL-6 or IL-12 alone, or in combination. Using this two-step approach, we obtain substantial yields. About 1-3 x 10(6) mature dendritic cells are generated from 40 ml of blood vs. < 0.1 x 10(6) from noncytokine treated blood. The dendritic cells derive from progenitors found primarily in a radioresistant population of CD14+ and adherent blood mononuclear cells and have all the features of mature cells. They include a stellate cell shape, nonadherence to plastic, and very strong T cell stimulatory activity. Strong APC function was evident for both the proliferation of allogeneic T cells in the MLR, and the generation by syngeneic T cells of class I restricted, CTL responses to influenza virus. A panel of dendritic cell restricted markers is also expressed, including CD83, p55, and perinuclear CD68. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. We suggest that these cells will be effective in vivo as adjuvants for active immunotherapy.

Dendritic cells generated from blood monocytes of HIV-1 patients are not infected and act as competent antigen presenting cells eliciting potent T-cell responses.

The CTL response to HIV-I can be vigorous, but antigen presenting cell requirements have not been studied in detail. To approach this question, we have examined the dendritic cell populations that can be obtained from the blood of HIV-1 infected individuals. We studied 13 asymptomatic patients, who spanned a wide range of plasma viremia and CD4 counts. We show here that sizeable numbers of mature dendritic cells can be generated from nonproliferating progenitors in the blood of HIV + patients using a recently developed approach. The procedure involves two steps. The first step or 'priming' phase is a 7 day culture of T-cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or 'differentiation' phase requires the exposure to monocyte conditioned medium. The yields of DCs from HIV + individuals were comparable to normal blood donors, 0.4 - 3 x 10(6) mature dendritic cells from 50 ml of blood. Strong APC function was evident for both the proliferation of allogeneic T-cells in the MLR, and the generation by syngeneic T-cells of class I restricted, CTL responses to influenza virus. A panel of dendritic cell restricted markers are expressed, including CD83, p55, and perinuclear CD68. By semi-quantitative PCR analysis, the cytokine derived cells did not express HIV-1 DNA. We suggest that these blood derived dendritic cells will be effective for studies of immune responses to HIV-1 antigens and may be considered as adjuvants for active immunotherapy.

The effect of tear depth on the propagation of aortic dissections in isolated porcine thoracic aorta.

An in vitro model was used to determine the influence of tear depth on the propagation pressure of aortic dissections. Saline was injected into the media of segments of 20 porcine thoracic aortas to create blebs. A circumferential slit was made on the intimal side of each bleb, connecting the true lumen to the false lumen. Each aorta was then pressurized under no-flow conditions until propagation in either the anterograde or retrograde direction occurred. Histological sections of each principal propagating edge were used to determine depth of tear, measured as the ratio of elastin layers in the intimal flap to the elastin layers in the intact wall. Propagation occurred for tear depths ranging from 0.44 to 0.89, with dissections closest to the adventitia (with tear depths near 1) requiring the lowest pressures. Propagation pressure (P) depends on the number of elastin layers (L) in the outer wall of a dissection, P = 0.44 L + 25(kPa), r2 = 0.465, p = 0.003 and also on tear depth (d): P = -58 d + 81(kPa), r2 = 0.547, p < 0.001. Various in vivo factors are discussed which may affect these experimentally determined relationships.

Dendritic cells as immunogens for human CTL responses.

The cellular requirements for generating potent human CD8+ CTLs to influenza A virus in vitro have been defined. Furthermore, we have developed improved methods for generating large numbers of DCs from non-proliferating progenitors. These developments have enabled the design of new strategies to elicit CTLs in vivo. For example, together with IL-12, antigen-pulsed DCs may be a useful approach for boosting CTL responses against infectious agents and malignancies. Our results also reopen the potential use of inactivated virus preparations as immunogens for CTL responses.

Adrenal histoplasmosis: diagnosis by fine-needle aspiration biopsy.

A 54-year-old man presented with a 6-month history of fever, night sweats, and weight loss. He had hepatosplenomegaly, and bilateral adrenal masses were discovered on computed tomographic (CT) scan. CT-guided fine-needle aspiration biopsy (FNAB) of the right adrenal mass demonstrated purulent material. Special stains done on this material showed organisms with morphologic features of Histoplasma capsulatum. The patient was started on antifungal therapy and discharged. FNAB of the adrenal gland is an effective method in the diagnosis of unusual infectious diseases. Special stains for micro-organisms proved helpful in the initial diagnosis of histoplasmosis.

Prospective seroepidemiological study of role of human papillomavirus in non-cervical anogenital cancers.

OBJECTIVE: To evaluate the association between infection with the major oncogenic types of human papillomavirus and the risk of developing non-cervical anogenital cancers in a cohort followed up prospectively. DESIGN: Data from two large serum banks to which about 700,000 people had donated serum samples were followed up for a mean of 8 years. People who developed non-cervical anogenital cancers during follow up were identified by registry linkage with the nationwide cancer registries in Finland and Norway. Within this cohort a nested case-control study was conducted based on the serological diagnosis of infection with human papillomavirus types 16, 18, and 33. SUBJECTS: 81 cases and 240 controls matched for sex, age, and storage time of serum samples. MAIN OUTCOME MEASURES: Odds ratios of developing non-cervical anogenital cancers in presence of IgG antibodies to specific micro-organisms. RESULTS: Subjects seropositive for human papillomavirus type 16 had an increased risk of developing non-cervical anogenital cancers (odds ratio 3.1 (95% confidence interval 1.4 to 6.9)). Subjects seropositive for type 33 also had an increased risk (odds ratio 2.8 (1.0 to 8.3)) but not significantly after adjustment for infection with type 16. Seropositivity for human papillomavirus type 16 was associated with an increased risk of developing vulvar and vaginal cancers (odds ratio 4.5 (1.1 to 22)) and a strongly increased risk of developing preinvasive vulvar and vaginal lesions (odds ratio infinity (3.8 to infinity)). Seropositivity for human papillomavirus type 18 increased the risk of developing preinvasive lesions (odds ratio 12 (1.2 to 590)). High, but non-significant odds ratios for types 16 and 33 were seen for penile cancers. CONCLUSIONS: This study provides prospective seroepidemiological evidence that infection with human papillomavirus type 16 confers an increased risk of developing non-cervical genital cancers, particularly vulvar and vaginal cancers.

Differential cytokine responses in human and mouse lymphatic endothelial cells to cytokines in vitro.

BACKGROUND: Inflammatory cytokines dysregulate microvascular function, yet how cytokines affect lymphatic endothelial cells (LEC) are unclear. METHODS AND RESULTS: We examined effects of TNF-α, IL-1 beta, and IFN-gamma on LEC proliferation, endothelial cell adhesion molecule (ECAM) expression, capillary formation, and barrier changes in murine (SV-LEC) and human LECs (HMEC-1a). RESULTS: All cytokines induced ICAM-1, VCAM-1, MAdCAM-1, and E-selectin in SV-LECs; TNF-α, IL-1 beta; and IFN-gamma induced ECAMs (but not MAdCAM-1) in HMEC-1a. IL-1 beta increased, while IFN-gamma and TNF-α reduced SV-LEC proliferation. While TNF-α induced, IFN-gamma decreased, and IL-1 beta did not show any effect on HMEC-1a proliferation. TNF-α, IL-1 beta, and IFN-gamma each reduced capillary formation in SV-LEC and in HMEC-1a. TNF-α and IL-1 beta reduced barrier in SV-LEC and HMEC-1a; IFN-gamma did not affect SV-LEC barrier, but enhanced HMEC-1a barrier. Inflammatory cytokines alter LEC growth, activation and barrier function in vitro and may disturb lymphatic clearance increasing tissue edema in vivo. CONCLUSION: Therapies that maintain or restore lymphatic function (including cytokines blockade), may represent important strategies for limiting inflammation.