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.

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.

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.

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.

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.

Down-regulation of telomerase activity in malignant lymphomas by radiation and chemotherapeutic agents.

The effects of radiation and cytotoxic agents on telomerase activity in lymphoma cells were analyzed by a polymerase chain reaction-based telomeric repeat amplification protocol coupled with an enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction for the expression of the catalytic subunit of telomerase (hTERT), and by Western blot analysis in three lymphoma cell lines (Jurkat, Raji, CEM-6). Telomeric repeat amplification protocol-enzyme-linked immunosorbent assay demonstrated high basal levels of telomerase activity in all cell lines compared to normal and activated peripheral blood lymphocytes. A significant decrease in telomerase activity was observed in all cell lines after exposure to vincristine for 24 hours. The decrease in telomerase activity paralleled the decrease in cell viability in Jurkat and CEM-6 cells but not in Raji cells. Radiation exposure inhibited the telomerase activity of Jurkat and CEM-6 cells whereas Raji cells were unaffected. Cell cycle analysis demonstrated a significant G(2)/M arrest by cisplatin, VP-16, and vincristine. In contrast to the decline in telomerase activity, the level of hTERT RNA and protein increased. Furthermore, the induction of hTERT was preceded by increased expression of the cyclin-dependent kinase inhibitor, p27/Kip1 protein, and p53. These results indicate that telomerase activity is down-regulated by anti-neoplastic agents in lymphoma cells, however expression of hTERT may not be correlated with telomerase activity. We also show that p27/Kip1 may be involved in the G(2)/M growth arrest induced by anti-neoplastic agents.

DNA-induced structural changes in the papillomavirus capsid.

Human papillomavirus capsid assembly requires intercapsomeric disulfide bonds between molecules of the major capsid protein L1. Virions isolated from naturally occurring lesions have a higher degree of cross-linking than virus-like particles (VLPs), which have been generated in eukaryotic expression systems. Here we show that DNA encapsidation into VLPs leads to increased cross-linking between L1 molecules comparable to that seen in virions. A higher trypsin resistance, indicating a tighter association of capsomeres through DNA interaction, accompanies this structural change.

EBV-associated perianal Hodgkin's disease in an HIV-positive individual.

Hodgkin's disease (HD) has a higher incidence in HIV-positive individuals. It tends to occur at extranodal sites, frequently exhibits an unfavorable histological type with large numbers of neoplastic cells, and almost invariably harbors Epstein-Barr Virus (EBV). We describe a case of a 33-year-old HIV-positive man who presented with anal pain from a 4-cm mass in the anorectal canal. He had no B symptoms or peripheral lymphadenopathy. A chest X-ray was within normal limits. A biopsy showed an ulcerated mass composed of a mixed infiltrate of lymphocytes, plasma cells, eosinophils, and Reed-Sternberg (RS) cells positive for CD15 and strongly positive for CD30. They were negative for CD45 and CD20. Numerous RS cells and lymphocytes were positive for EBV RNA using the EBER-1 probe. This highly unusual presentation of HD may reflect the greater incidence of anorectal lymphoma and of extranodal HD in the HIV-positive population.

Induction of type-specific neutralizing antibodies by capsomeres of human papillomavirus type 33.

The immunogenicity of capsomeres of human papillomavirus type 33 was evaluated in a dose-response analysis. Capsomeres were obtained free of capsids by expression of L1 carrying the single point mutation C427S. Neutralizing antibodies were detected using an in vitro pseudoinfection assay. Capsomeres induced type-specific, neutralizing antibodies in mice even in the absence of adjuvant. The neutralization titers of immune sera raised without adjuvant were 10- to 20-fold lower than those of antisera to virus-like particles, but virtually identical using Freund's adjuvant. These data indicate that capsomeres may substitute for virus-like particles in future vaccines when used with an adjuvant appropriate for human vaccination.

Immunological analyses of human papillomavirus capsids.

Recombinant human papillomavirus (HPV) virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease. Questions remain, however, concerning the extent of capsid antigenic similarity between closely related virus genotypes. To investigate this issue, we produced VLPs and corresponding polyclonal immune sera from several anogenital HPV types, and examined these reagents in enzyme-linked immunosorbent assays (ELISAs) and in cross-neutralization studies. Despite varying degrees of L1 genetic sequence relatedness, VLPs of each type examined induced high-titer serum polyclonal antibody responses that were entirely genotype-specific. In an in vitro infectivity assay, only cognate VLP antisera were able to neutralize pseudovirions of HPV-16, HPV-18 and HPV-33, with two exceptions: HPV-31 and HPV-45 VLP post-immune sera demonstrated low levels of neutralizing activity against pseudovirions of HPV-33 and HPV-18, respectively. In other experiments, epitopes shared between closely related types were found to be less immunogenic than, and antigenically distinct from, primary type-specific B-cell determinants of the viral capsid. In addition, results from epitope blocking experiments suggested a close correlation between primary type-specific capsid antigenic sites and virion neutralization. These findings support the view that papillomavirus genotypes denote unique viral serotypes, and suggest that a successful vaccine for these viruses will likely require the inclusion of VLPs of each serotype for which protection is desired.

Human papillomavirus infection requires cell surface heparan sulfate.

Using pseudoinfection of cell lines, we demonstrate that cell surface heparan sulfate is required for infection by human papillomavirus type 16 (HPV-16) and HPV-33 pseudovirions. Pseudoinfection was inhibited by heparin but not dermatan or chondroitin sulfate, reduced by reducing the level of surface sulfation, and abolished by heparinase treatment. Carboxy-terminally deleted HPV-33 virus-like particles still bound efficiently to heparin. The kinetics of postattachment neutralization by antiserum or heparin indicated that pseudovirions were shifted on the cell surface from a heparin-sensitive into a heparin-resistant mode of binding, possibly involving a secondary receptor. Alpha-6 integrin is not a receptor for HPV-33 pseudoinfection.

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.

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.

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.

Sexual behaviour and papillomavirus exposure in cervical intraepithelial neoplasia: a population-based case-control study.

Sexual history is an established risk determinant for cervical neoplasia. It is not clear if human papillomavirus (HPV) exposure entirely explains the sexual behaviour-related risk or if other sexually transmitted agents may act as cofactors for HPV in carcinogenesis. The aim of this study was to elucidate whether HPV exposure or HPV persistence explains the sexual history-related risk of high-grade cervical intraepithelial neoplasia (CIN) using a population-based case-control study of most of the 254 women referred to colposcopy in the Vasterbotten county in Sweden because of an abnormal cervical smear during October 1993 to December 1995 and 320 age-matched women from the general population. The women were interviewed for sexual history and tested for presence of serum antibodies to HPV-16, -18 and -33 as well as for presence of HPV DNA in cervical brush samples. HPV-16, -18 and -33 seropositivity was specific for the corresponding type of HPV DNA, dependent on the lifetime sexual history and associated with a two- to threefold increased risk of CIN 3. There was no sexual history-related risk of CIN among HPV-seropositive women and adjustment for HPV DNA presence explained the sexual history-related risk of CIN. In conclusion, HPV exposure appeared to explain the sexual history-related risk of high-grade CIN.

A survey of seroprevalence of human papillomavirus types 16, 18 and 33 among children.

The importance and natural history of HPV infections in childhood is incompletely understood. We performed a survey for presence of serum antibodies to HPV capsids among 1031 children aged 0 to 13 years, resident in Stockholm, Sweden. The HPV seroprevalence among these children was 3.0% for HPV16, 0.6% for HPV18 and 2.7% for HPV33. By comparison, among simultaneously analyzed positive control panels comprising women with CIN or healthy women with type-specific cervical HPV DNA, seroprevalence of HPV 16, 18 and 33 was 69%, 58% and 63% respectively. The results suggest that HPV infection in childhood is not common.

Prospective study on cervical neoplasia IV. Presence of HPV antibodies.

Sera collected in the course of a prospective study carried out in Prague in 1975-1983 were assayed for the presence of human papillomavirus (HPV) antibodies. Women with cervical neoplasia proven by biopsy at enrollment possessed antibodies to peptides derived from E2, E4 and E7 proteins of HPV16 and to virus-like particles (VLPs) of HPV16, -18 and -33 significantly more frequently than matched controls. Women without cervical neoplasia at enrollment who developed the disease in the course of the study differed from matched controls by a higher prevalence of antibodies against VLPs of HPV16 and -18 but not against early antigens of HPV16. In 19 of the latter subjects, paired serum specimens were tested, the first samples having been taken at enrollment and the second at diagnosis. Development of the disease was associated with seroconversion from negativity to positivity to at least one HPV antigen in 11 (57.9%) women.

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.