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Objective: Epstein-Barr virus (EBV) is a causative agent of nasopharyngeal carcinoma (NPC), and EBV gene expression is considered to be closely associated with the pathogenesis of NPC. Among EBV genes expressed in NPC, EBV-encoded non-polyadenylated RNAs, termed EBERs, are the most abundant transcripts of EBV in NPC. However, the role of EBERs still remains unclear. This study was designed to investigate the relevance of EBERs to the oncogenesis of NPC., Methods: Two types of EBERs expression vectors (EBERs-high-expression vector and EBERs-low-expression vector) were constructed and transfected into EBV-negative cells, MDCK or EBV-negative clones of NPC-KT cells. Then, malignant transformation, represented by anchor independent growth, was evaluated between the EBERs-transfected cells and EBERs-negative cells using a soft agar colony formation assay. Apoptosis was induced by serum deprivation (0.1% concentration of fetal bovine serum) and interferon-alpha (IFN-alpha) (500 U/ml) treatment. Cell viability was evaluated with a trypan blue exclusion test. The activation of cellular transcriptional factor NF-kappaB was studied with the IL-8 promoter sequence using a luciferase reporter assay., Results: EBERs-high-expression vector-transfected MDCK cells showed enhanced growth ability in soft agar compared with either EBERs-low-expression vector-transfected MDCK cells or EBERs-untransfected MDCK cells. However, they did not show the acquisition of any anti-apoptotic potential against either IFN-alpha or serum deprivation. Introduction of EBERs-low-expression vector into MDCK cells did not show anchor independent growth characteristics. Neither EBV-negative NPC-KT cells nor MDCK cells transfected with EBERs-high-expression vector showed any difference from EBERs-untransfected EBV-negative NPC-KT cells. Introduction of EBERs into MDCK cells did not transactivate the IL-8 promoter, indicating that neither NF-kappaB nor AP-1 was activated by EBERs., Conclusion: EBERs are believed to induce the initial transformation of epithelial cells, thus contributing to the oncogenesis of NPC. Expression of abundant EBERs is considered to be critical for this transforming property of EBERs.

Antiviral drugs with specific activity against polyomavirus replication have not been developed in the past. This deficiency has become fully apparent with the emergence of polyomavirus-associated nephropathy in kidney-transplant recipients, with a prevalence rate of up to 10%. In most cases, high BK virus replication in tubular epithelial cells causes significant cytopathology, leading to permanently impaired renal allograft function and return to hemodialysis within 6-60 months. In 5-10% of allogenic bone marrow/hematopoietic stem cell transplant recipients, high-level BK virus replication in the ureter/bladder mucosa has been associated with postengraftment hemorrhagic cystitis, which appears to involve significant immunopathology. Thus, in view of the increasing clinical need, a number of drugs have been studied in small case series. We review the antiviral strategies explored to date and specifically discuss available in vivo and in vitro data on cidofovir, leflunomide, fluoroquinolones and intravenous immunoglobulins, regarding mechanism, administration, dosing and outcome and provide a perspective on future therapy options.

Objectives: To discover new potent and selective anti-human immunodeficiency virus (HIV) acyclic nucleoside phosphonate (ANP) drugs with in vivo antiretroviral activity., Methods: New acyclic pyrimidine nucleoside phosphonate derivatives that mimic the structure of the anti-HIV purine nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir) and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA, tenofovir) were designed by linking the acyclic side chain of the ANPs through an ether bond to the C-6 position instead of the N-1 position of the pyrimidine ring. The compounds were evaluated against HIV and Moloney murine sarcoma virus (MSV) in cell culture, including a broad variety of HIV-1 clade clinical isolates and relevant mutant (drug-resistant) HIV-1 isolates. Their antiviral activities were correlated and investigated in an in vivo model consisting of MSV-infected newborn mice. MSV-induced tumour formation and associated death were recorded in drug-treated animals., Results: Several 5-substituted 6-[2-(phosphonomethoxy)ethoxy]-2,4-diaminopyrimidine (PMEO-DAPy) analogues were found to inhibit a broad variety of HIV-1 clinical isolates. They showed a more favourable cross-resistance profile to mutant virus isolates than adefovir and tenofovir. There was a close correlation between inhibition of MSV in C3H/3T3 cells and inhibition of HIV-1 in CEM cells. The PMEO-DAPy derivatives potently inhibited MSV-induced tumour cell formation in newborn mice. The 5-methyl analogue PMEO-5-Me-DAPy proved markedly more inhibitory to MSV-induced tumour cell formation and associated animal death than its unsubstituted parent PMEO-DAPy derivative. When compared with adefovir, PMEO-5-Me-DAPy was less toxic and more antivirally active in MSV-infected mice., Conclusions: PMEO-5-Me-DAPy deserves further (pre)clinical investigations as a candidate anti-HIV drug.

BK virus (BKV) nephropathy has a poor prognosis for renal allograft survival with 30% to 60% risk of allograft loss over 1 year. In the past decade, BKV nephropathy has occurred in 1% to 10% of renal transplant patients, with higher rates observed in patients with increased immunosuppression exposure and renal allograft injury. Vero cells (Green monkey kidney cell origin) were optimized for BKV primary and chronic infection inclusive of culture requirements for 60-day growth and monolayer confluence. Quantification of BKV replication in the culture supernatant (SN) and cells was by real-time polymerase-chain reaction (PCR) using the Roche Lightcycler 2.0. Primary BKV infection of Vero cells is achieved by 2 hour incubation with 6.5 x 10(5) BKV copies with subsequent washing of cells leading to steady-state cellular infection of 10(2) to 10(3) BKV copies. Primary infection is demonstrated within 7 to 10 days by a >10-fold increase of BKV copies in SN. Thereafter, a BKV viral load reduction in SN to a chronic/latent level (<10(2) BKV copies in SN) is observed by 14 days. Vero cells with chronic low-level BKV infection (10(2)-10(3) BKV copies in cells) exhibited reactivation (>10(5) BKV copies in SN) in >72% of late culture wells after 40 days. Vero cells can accommodate primary and chronic BKV infection followed by viral reactivation in late culture. The performance characteristics of 3 different pathogenic BKV strains obtained from patients with BKV nephropathy had infectivity profiles that correlated well the relative clinical profile in this Vero cell culture system.

The influence of BK virus nephropathy (BKVN) in pancreas after kidney (PAK) transplantation is unclear. A retrospective analysis of PAK transplants performed at our center was conducted to determine the impact of BKVN. Among 40 PAK transplants performed using sequential immunosuppression, four patients developed BKVN, as defined by a >20% rise in serum creatinine and BK viremia (BK plasma load >4 log copies/mL), at a median of 19 months following PAK. In all four patients, treatment of BKVN consisted of reduction in tacrolimus, cessation of mycophenolate mofetil, and introduction of leflunomide. With this approach, two patients experienced improvement or stabilization of renal function. The remaining two patients progressed to dialysis dependence despite treatment. Plasma BK load < or =5 log copies/mL was associated with graft preservation. Gender, age, delay between transplants, cumulative Thymoglobulin dose, and type of kidney donor were not associated with BK virus infection. Pancreas graft rejection or dysfunction was not observed with the above immunosuppression modification. Mean amylase and lipase > or =6 months following BKVN treatment remained normal. BKVN is an important cause of kidney allograft loss in PAK patients. Screening and early treatment of BKVN may enable preservation of kidney and pancreas grafts.

We have isolated a substance with anti-retroviral activity from the freshly prepared crude extract of Chelidonium majus L. (greater celandine) by 9-aminoacridine precipitation method and ion exchange chromatography using Dowex-50W/H+ resin followed by the gel filtration on Sephadex-75 column. Elemental and phenol/sulfuric acid method analyses as well as the mass spectrometry of the purified substance indicated that it may represent a low-sulfated poly-glycosaminoglycan moiety with molecular weight of approximately 3800 Da. The substance prevented infection of human CD4+ T-cell lines AA2 and H9 with HIV-1 at concentration of 25 microg/mL as well as the cell-to-cell virus spread in H9 cells continuously infected with HIV-1, as determined by the measurement of reverse transcriptase activity and p24 content in cell cultures. Furthermore, we have shown in a murine AIDS model that the treatment with purified substance significantly prevented splenomegaly and the enlargement of cervical lymph nodes in C57Bl/6 mice chronically infected with the pool of murine leukemia retroviruses. The mechanism(s) of anti-retroviral activity of this substance have to be elucidated.

Background: Polyomavirus-associated nephropathy (PVAN) is an important cause of kidney dysfunction and graft loss. Different treatment regimens have been used, including low-dose intravenous cidofovir. Anterior uveitis, a complication of this treatment, has been reported after its use in patients with cytomegalovirus-associated retinitis. We analyzed the incidence and associated risk factors for this disorder in patients with PVAN., Patients and Methods: The study included 14 kidney-transplant patients who had received low doses of cidofovir after being diagnosed with PVAN., Results: Five (35%) patients developed an episode of anterior uveitis. The mean number of cidofovir doses given was 6.8 +/- 1.6 as compared with 9.1 +/- 2.1 in patients who did not develop the disease. Creatinine clearance at diagnosis of the nephropathy and after terminating treatment was lower in the uveitis patients, who had a graft survival of 40% versus 100% in the patients who did not develop eye involvement. Treatment was suspended in the affected patients, with complete resolution in 80% after the administration of topical corticoids and cycloplegics., Conclusions: Anterior uveitis secondary to low-dose treatment with cidofovir is a common complication in patients with PVAN and is associated with the degree of renal involvement. In the absence of larger studies, cidofovir should be used with caution in patients with creatinine clearance below 30 mL/min.

Background: BK virus renal allograft nephropathy (BKVAN) in the setting of simultaneous kidney-pancreas transplantation (SKPT) has been inadequately studied and reported. We analyzed our data on the incidence of BKVAN and its outcome in SKPT recipients at University of Pittsburgh Medical Center (UPMC) and affiliated centers and report significant differences compared to previous studies., Methods: This study used retrospective review and case studies., Results: A review of 243 consecutive SKPT recipients from January 1, 1996 to December 31, 2004 identified seven cases (three females; ages = 23-54 yrs) of BKVAN following SKPT (incidence = 2.9%). The immunosuppressive protocols during this period were divided into: Period I (pre-August 2001) with no antibody induction and Period II (post-August 2001) with alemtuzumab or antithymocyte globulin induction with steroid avoidance. One BKVAN case was diagnosed in Period II (incidence = 1.4%). Six of seven patients were treated with intravenous cidofovir (0.20-0.50 mg/kg) every two to four weeks over one to six months. Three patients lost the renal allograft 8-22 months following diagnosis of BKVAN, whereas four patients had prolonged allograft survival. Pancreatic function was well preserved in five; one patient lost the pancreatic function due to surgical complications and one has had partial preservation., Conclusions: There was a relatively lower incidence of BKVAN among SKPT patients at our center. Although overall graft loss rate was comparable to other series, BKVAN patients had a slightly prolonged graft life. The BKVAN incidence was further reduced in patients receiving modified immunosuppression with antibody preconditioning. The underlying reasons may include less toxic immunosuppressive protocols, earlier diagnosis and the use of antiviral therapy.

BK virus is ubiquitously present in the latent state in humans, and awareness of the importance of BK polyomavirus is emerging among the kidney transplant community. First discovered in 1971 in the urine of a renal transplant recipient, BK virus nephropathy (BKVN) has come to be recognized as a significant cause of genitourinary disease and potential graft loss in the kidney transplant patient. In this review, we discuss the risk factors, available methods of diagnosis and therapeutic monitoring, and current approaches to therapy of BKVN.

BK virus allograft nephropathy (BKVAN) is a rising complication in kidney transplant recipients. Reducing immunosuppression has been the initial form of therapy in most cases, but is not always associated with improvement in graft function. Anti-viral therapy with low-dose cidofovir (0.25-0.42 mg/kg/dose) has been used successfully in some patients, but dose-related nephrotoxicity has limited its use. We present our experience with 3 kidney transplant recipients diagnosed with BKVAN who received intermediate-dose cidofovir (0.75-1.0 mg/kg/dose) without probenecid, and without concomitant nephrotoxicity. Three female patients, ages 8, 19 and 20 yr, presented with elevated serum creatinine (SCr) values, BK virus stain positive on renal biopsy and high plasma BK viral loads. As a result of viral loads being >2 million copies/ml in two patients and a lack of response to reduction in immunosuppression in the third, we initiated therapy with low-dose cidofovir. Because of persistent positive BK stain and positive plasma viral load, we then administered intermediate-dose cidofovir, without probenecid, for several subsequent doses (seven to 15 infusions till date). All patients tolerated the intermediate-dose cidofovir with no significant rise in SCr during the course of the infusions. The most recent SCr values in all three patients were improved from those at the initial diagnosis of BKVAN. All three patients showed a marked drop in BK viral loads when on intermediate-dose cidofovir, with complete clearing of viremia in two patients. In our experience, intermediate-dose cidofovir without probenecid, used judiciously, is not associated with additional nephrotoxicity and may provide an additional alternative for treatment.

BK viremia and nephritis are increasing problems in renal transplant recipients. The exact cause of the increasing prevalence of this condition remains poorly understood. Increasing prevalence has been correlated with newer immunosuppressive agents and the decline in acute rejection rates in recent years. The clinical manifestation varies from the asymptomatic state of viremia and nephritis to clinical renal dysfunction. The diagnosis of this infection is based on the combination of the presence of urinary decoy cells, virus in the urine/blood, and typical renal histological findings of interstitial nephritis. Routine post-transplant screening for BK viremia and viruria prior to the occurrence of nephritis and the reduction in immunosuppressive therapy for subjects with viremia appear to be attractive approaches. The treatment of BKV nephritis (BKVN) consists of reduction in immunosuppressive therapy and antiviral therapy with cidofovir or leflunomide or a combination of both. Approximately 30-60% of subjects with BKVN experienced irreversible graft failure. However, in recent years, the combinations of early detection, prompt diagnosis, and appropriate reduction in immunosuppressive therapy have been associated with better outcome. The pathogenesis of BK virus infection in renal transplant recipients needs to be explored. The source of BKV infection (donor as opposed to recipient), the role of host humoral, and cellular immunity to BKV, and the role of alloimmune activation in renal graft to the occurrence of nephritis are discussed in this review.

BK virus associated nephropathy (BKVAN) has emerged as an important cause of renal allograft dysfunction and graft loss. Although several treatment strategies have been proposed, the rate of graft loss remains high. We studied the outcome of renal transplant patients with BKVAN treated with IVIG. After 11.4 +/- 3.9 months (mean +/- SEM) from the time of transplantation, 8 renal allograft recipients were diagnosed with BKVAN. In addition to a reduction of immunosuppressive therapy, patients received 2 g/kg IVIG. After a mean follow-up of 15 months, all except one patient are currently off dialysis. In summary, after IVIG therapy, 88% of patients still have functioning grafts, although renal function continues to be impaired. The benefit of concomitant IVIG and reduction of immunosuppressive therapy in BKVAN needs to be further addressed in randomized, multicentered trials.

Human papillomaviruses (HPVs) are the causative agents of several disease states, including genital warts and cervical cancer. There are around 500 million cases of genital warts per annum worldwide and around 450,000 cases of cervical cancer. Although HPV vaccines should eventually reduce the incidence of these diseases, new and effective treatments are still urgently required. The E2 (early) proteins from some HPV types induce growth arrest and apoptosis, and these proteins could be used as therapeutics for HPV-induced disease. A major obstacle to this approach concerns the delivery of the protein to HPV-transformed cells and/or HPV-infected cells in vivo. One possible solution is to use recombinant viruses to deliver E2. Another possible solution is to use purified E2 proteins or E2 fusion proteins. The herpes simplex virus VP22 protein is one of a small number of proteins that have been shown to cross the cell membrane with high efficiency. VP22-E2 fusion proteins produced in bacterial cells are able to enter mammalian cells and induce apoptosis. This suggests that VP22-E2 fusion proteins could be topically applied as a treatment for HPV-induced diseases, most probably post-surgery. In this review, we discuss this and other approaches to the topical delivery of selective therapeutic agents against HPV-associated conditions.

Haemorrhagic cystitis (HC) is a distinct clinical disorder of multiple aetiologies. It is characterized by painful haematuria due to haemorrhagic inflammation of the urinary bladder mucosa. In allogeneic haematopoietic stem cell transplantation (HSCT), HC occurring before engraftment is mostly transient and self-limiting, whereas that after engraftment is severe and sometimes life-threatening. Pre- and post-engraftment HC represent distinct disorders with different aetiologies and treatment implications. Recent data suggest that reactivation of the polyoma BK virus (BKV) plays a pivotal role in post-engraftment HC. Urotoxicity of the conditioning regimen and alloimmune reaction accompanying graft-versus-host disease (GVHD) upon engraftment are also important pathogenetic factors. Based on data from BKV studies, we propose that HC may be divided into three phases. In the first phase, the conditioning regimen damages uroepithelial cells, providing a milieu for BKV replication. In the second phase, unchecked uroepithelial BKV replication leads to BK viruria. In the last phase after engraftment, alloimmunity against BKV-infected uroepithelial cells leads to HC. The quinolone antibiotics suppress BKV replication in vivo and in vitro, suggesting that their prophylactic use may prevent the occurrence of HC.

In a previous study, it was found that even though more male cats were infected by feline leukaemia virus (FeLV), females seemed to progress easier to overt disease. To study the effect of female hormones, 17beta-estradiol and progesterone were added in different concentrations (10(-3) M to 10(-12) M) to a culture of persistently FeLV-infected cells. The effect of both hormones was very similar. After 24 h the cell viability was very low at 10(-3) M and 10(-4) M but similar to controls at the remaining concentrations. Liberation of viral particles was estimated by the reverse transcriptase activity (RT), which was the lowest also at 10(-3) M and 10(-4) M. However, low viability could not account for this low RT, as when cells were lysed with lysis buffer RT was high. Thus, cells were dying without freeing viral particles, suggestive of apoptosis. This possibility was confirmed by staining hormone-treated cells with annexin V and propidium iodide. The FeLV antigen p27 measured in the cultures had a maximum at 10(-3) M and 10(-4) M, higher than controls and lysed cells, so the presence of p27 in the supernatant was not only due to cell lysis but a consequence of hormone effect. In conclusion, 17beta-estradiol and progesterone induce death of FeLV-infected cells at high concentrations, probably through a process of apoptosis, which might limit the spread of the infection, as infective viral particles would be hampered from budding.

Progressive multifocal leukoencephalopathy is a fatal demyelinating disease caused by infection of oligodendrocytes by the human polyomavirus known as JC virus. Over the past 10 years, the disease has been documented almost exclusively in AIDS patients, who constitute a rapidly growing population of immunosuppressed individuals. More recently, progressive multifocal leukoencephalopathy has also been described in patients undergoing solid organ or cell transplant, as a result of immunosuppressive therapy to avoid graft rejection. Although there are several reports of successful treatment of progressive multifocal leukoencephalopathy, large-scale prospective trials have been few, and with mixed results. As more is discovered about the biology of JC virus infection and advances are made in targeted parenchymal delivery of therapeutic agents, there is hope for the development of an effective therapy for progressive multifocal leukoencephalopathy.

BK virus interstitial nephritis (BKVIN) is a serious complication after kidney grafting, necessitating drastic reduction of immunosuppressive therapy in order to enable viral clearance. Despite these measures, progressive graft dysfunction and graft loss occur in the majority of recipients. We diagnosed BKVIN in 21 recipients grafted between 1998 and 2004. Eight of 21 patients were treated with weekly, adjuvant low-dose cidofovir in addition to reduction of immunosuppressive therapy. BKVIN caused irreversible deterioration of graft function in all patients but renal function stabilized after antiviral treatment (creatinine clearance: 51.8-32 mL/min; p=0.001) and no graft loss occurred in cidofovir-treated recipients during 24.8 (8-41) months follow-up. Peak serum cidofovir concentrations were dose-dependent and attained approximately one-tenth of thein vitroEC50 for cidofovir against BK-virus, while pre-treatment with probenecid did not alter peak serum concentrations nor affected the incidence of nephrotoxicity. In fact, no cidofovir-related renal toxicity occurred; few patients had minor transient side effects (nausea, skin rash). In contrast, 9 of 13 patient who received no adjuvant cidofovir therapy lost their graft after median 8 (4-40) months. In this selected group of recipients with BKVIN, the use of adjuvant low-dose cidofovir therapy resulted in prolonged graft survival and stabilized graft function.

The aim of our investigation was to examine the mechanisms of protective features of plaferon LB and fenovit in mice infected with Rausche virus. It was found that in erythroblasts from peripheral blood and spleen, which appeared 12-14 days after infection, the highest percent of determined virus antigens was approximately 47% (range 35,5 to 58,4) which was followed by decrease of the concentration of virus antigens (between 5-12%) in following days and increase of uninfected cells as a result of division of non-contaminated erythroblasts. Mechanism of action of plaferon LB and fenovit was manifested in the reconstitution of mechanisms of apoptosis among erythroblasts, which did not contain synthesized Rausche virus. Impairment of functional activity and antivirus resistance of macrophagal phagocytes are playing an important role not only in the pathogenesis of murine viral leukemia caused by Rausche virus, but also during human leukemia. Correction of functional status of above -- mentioned diseases using plaferon LB and fenovit is presenting as a new and prospective way.

Adult T-cell leukemia/lymphoma (ATLL) is a malignant lymphoproliferation of mature activated T-cells, mostly CD4, which develops after a long period of latency following Human T cell Lymphotropic virus Type 1 infection. It is characterized by the clonal integration of one or more HTLV-1 proviruses in the tumor cells. There are 4 major subtypes of ATLL: a smoldering type, a chronic type, a lymphoma type and a leukemic/acute type. The survival rate of ATLL patients, especially those who develop the acute leukemic or lymphomas forms, is very poor and such a tumor remains one of the most severe lymphoproliferations. Treatment of ATLL patients using conventional chemotherapy has very limited benefit, since HTLV-1 transformed cells are resistant to most apoptosis-inducing agents. Recently, antiretroviral therapy using the combination of zidovudine (AZT) and interferon alpha (IFN-alpha) has been shown to induce a high complete remission rate and to prolong the survival of ATLL patients. Based on the current physiopathology, other drugs such as arsenic trioxide combined to IFN-alpha have also been demonstrated to synergize in vitro for inducing apoptosis in HTLV-1 infected T cells. Such drugs have now been used in vivo for treating ATLL patients. Both in vitro and in vivo data will be discussed.

Introduction: BK virus has been described previously in renal transplant patients, but has also been reported in cases of progressive multifocal leukoencephalopathy., Method: A case of progressive BK virus multifocal leukoencephalopathy induced by long-term corticosteroid therapy is reported., Results: In our case, BK virus was detected in cerebrospinal fluid by polymerase chain reaction (PCR). A detailed review of similar cases in the literature showed most of them occurred in AIDS patients, but four involved immunocompetent patients., Conclusion: Though BK virus infection usually leads to urinary tract symptoms in immunologically suppressed patients, it is important to be aware of its neurological manifestations and to recognize BK infections that can occur in immunocompetent patients.

The ribonucleotide reductase inhibitor hydroxyurea (HU) has demonstrated some benefit as a component of drug cocktails for the treatment of HIV-1 infection. However, HU is notoriously myelosuppressive and often administered only as salvage therapy to patients with late-stage disease, potentially exacerbating the bone marrow toxicity of HU. In this report we have compared the antiviral effects of HU and two novel RR inhibitors trimidox (3,4,5-trihydroxybenzamidoxime) and didox (3,4-dihydroxybenzohydroxamic acid) in combination with didanosine (2,3-didoxyinosine; ddI) in the LPBM5 MuLV retrovirus model (murine AIDS). We also evaluated the effects of these drug combinations on the hematopoietic tissues of LPBM5 MuLV-infected animals. The combination of RR inhibitors and ddI was extremely effective (DX>TX>HU) in inhibiting development of retrovirus-induced disease (splenomegaly, hypergammaglobulinemia, activated B-splenocytes and loss of splenic architecture). In addition, relative levels of proviral DNA were significantly lower in combination drug-treated animals compared to infected controls. Evaluation of femur cellularity, numbers of marrow-derived myeloid progenitor cells (CFU-GM and BFU-E) and peripheral blood indices revealed that TX and DX in combination with ddI were well-tolerated. However, treatment with HU and ddI induced moderate myelosuppression. These data demonstrate that RR inhibitors in combination with ddI provide significant protection against retroviral disease in murine AIDS. Moreover, the novel RR inhibitors TX and DX appear to be more effective and less myelosuppressive than HU when administered with ddI in this model.

Reactivation of polyoma virus (BK virus) is a significant cause of morbidity in kidney transplant patients. This seemingly insignificant viral infection that affects the majority of population at a young age, once reactivated by immunosuppression, is a major factor contributing to graft loss. Screening techniques have been developed for early prediction of BK virus reactivation. These include plasma and urine assays for detection of BK virus DNA by PCR, urine cytology for detection of "decoy cells" and electron microscopy. Combining urine cytology and serology screening can be more effective for early detection of BK virus reactivation. Immunohistochemistry can be utilized as an additional tool to support the diagnosis. Once screening tests reveal a suspicious BK virus reactivation, tissue biopsy should be performed to confirm the diagnosis, rule out acute cellular rejection and plan treatment approaches. Treatment normally includes decreasing immunosuppression and the use of antiviral drug therapy. Unfortunately, disease outcome is often unfavorable and can culminate with eventual graft loss. Renal retransplantation has been performed with mixed results. As new data emerges, we will gain a better understanding of the disease caused by BK virus and respond with improved early diagnosis and treatment to preserve graft function.

Background: Topical immunomodulators include both immunostimulatory and immunosuppressive agents. If successful, topical immunotherapy may represent an important improvement in the therapy of inflammatory dermatoses, viral infections, and cancers of the skin and genital mucosa. Topical immunotherapy using obligate contact sensitizers such as diphencyprone or dinitrochlorobenzene has been used against viral (e.g., common warts) and autoimmune diseases (e.g., alopecia areata)., Results: Newer agents such imidazoquinolines (imiquimod and resiquimod) act by cytokine secretion from monocytes/macrophages (interferon-alpha, interleukin-12, tumor-necrosis factor-alpha). The locally generated immune milieu leads to a Th1-dominance and cell-mediated immunity that have been clinically used to treat viral infections such as human papillomavirus, herpes simplex virus, and mollusca. Although these agents improve antigen presentation by dendritic cells, they also act on B cells leading to the synthesis of antibodies such as IgG2a. We have also introduced this treatment against cancerous lesions including initial squamous cell and basal cell carcinoma in immunocompetent and immunosuppressed patients. We provide examples of successful treatment of squamous cell cancer using topical imiquimod., Conclusion: The available and additional Toll-like receptor agonists will help to improve the specific dermatologic therapy. Topical immunotherapy with both immunostimulatory and immunosuppressive agents bears potential for effective and patient friendly treatment of inflammatory, infectious, and cancerous skin diseases. Long-term evaluation will define the tolerability and safety profile of these novel topical agents.

Background. Smoking may increase the risk of cervical cancer, a disease that is related to human papillomavirus (HPV) infection. However, the effects of smoking on the natural history of HPV are poorly understood, especially in women coinfected with human immunodeficiency virus (HIV).Methods. HIV-infected (n=1797) and HIV-uninfected (n=496) women were assessed every 6 months for type-specific HPV DNA. Smoking status was self-reported. Covariates included age, parity, sexual behavior, HIV load, CD4(+) T cell count, and antiretroviral therapy.Results. Smoking was positively associated with HPV prevalence at baseline in HIV-infected women (P=.002) and was significantly associated with type-specific HPV detection (e.g., type 18, odds ratio [OR], 2.45; 95% confidence interval [CI], 1.86-3.22). In Cox models, detection of HPV was significantly associated with smoking in HIV-infected women (relative hazard [RH], 1.33; 95% CI, 1.10-1.60; P=.003), but HPV persistence was not (RH, 0.97; 95% CI, 80-1.16; P=.72). The overall likelihood of acquiring persistent HPV was higher in smokers (OR, 1.39; 95% CI, 1.05-1.86; P=.023) because of greater incidence.Conclusions. Among HIV-infected women, smoking is associated with a significantly higher prevalence and incidence of HPV infection. Smoking during HIV infection may alter the natural history of HPV infection and increase the risk of cervical disease.

Nasopharyngeal carcinomas (NPC) challenge clinicians and biologists in various fields including epidemiology, genetics, virology and immunology. These tumours have a striking geographical distribution. They are constantly associated with the Epstein-Barr virus (EBV) and contain a massive lymphocytic infiltrate. Their study has major implications especially at this moment while a pathological role of EBV is suspected in several other human epithelial malignancies (for example gastric, mammary and thyroid carcinomas). The North-South Workshop on Nasopharyngeal Carcinoma was held at the Institut Gustave-Roussy in early December 2003. Its main goal was to support the exchanges between clinical research on NPC in the South and basic research in the North. With regard to epidemiology and genetics, the main information was the possible existence of several susceptibility genes (including two of them on the 4p and 5p chromosomes). In virology, participants have emphasized the selection of peculiar EBV variants within the malignant cells and the expression of novel oncogenic viral proteins : LMP2 and BARF1. Cellular gene alterations also contribute to NPC development, especially inactivation of tumor suppressor genes located on the 3p chromosome. Therapeutic research was not forgotten. Hope of higher rate of cure relies on improved ballistic processes in radiotherapy (IMRT) and on the development of targeted therapeutics : induction of the lytic/productive viral cycle, gene therapy with conditional replicative adenoviruses, antitumor vaccination directed against the viral protein LMP2.

Photodynamic diagnosis (PDD) is a new method based on the detection of different forms of fluorescence of tissues after previous administration of photosensitizers. The photosensitizer is gathered in the pathological tissue at much higher concentration than in the healthy tissue, thus the fluorescence differs. Localizing wrong fluorescence allows precise choosing of the spot to collect tissue for histopathological or cytological study. Photodynamic therapy (PDT) is a technique in which tissue is irradiated with light after the use of a photosensitizing drug that produces singlet oxygen, which has a cytotoxic effect. The authors describe new trends in photodynamic diagnosis and treatment of some vulvar epithelial diseases (VIN, lichen sclerosus, condylomata acuminata) and cervical intraepithelial neoplasia. They describe photodynamic method in their own studies: diagnosis and treatment of lichen sclerosus and diagnosis of uterine cervix cancer.

The authors established a protocol for the use of 5-fluorouracil (5FU) adjuvant in lasertherapy for clinical and subclinical HPV infection in immunosuppressed patients, persistent lesions and as reinforcement treatment in cases of poor progress. Sixty-four patients were evaluated, of whom 26 were immunosuppressed, 34 presented persistent lesions and four received intravaginal reinforcement treatment with 2.5 g 5% 5FU every two weeks, or biweekly vulvar reinforcement after lasertherapy. On average, five 5FU courses were used, but in the immunossuppressed patients its use was maintained indefinitely. The rate of complete response was 66%, but the immunossuppressed patients showed less response (46.2%) when compared with the persistent lesion/reinforcement treatment group (78.9%). The responses were positive in the two groups when compared to that with no response. We deem the use of low-dose 5FU an excellent alternative in cases of difficult HPV progress, presenting a low cost and minimal side-effects.

Polyomavirus BK viral allograft nephropathy is a potentially reversible cause of deteriorating function of kidney allografts. Initial treatment involves reducing immunosuppressive medications, with low-dose cidofovir an effective alternative in refractory cases. We describe two cases of BK viral allograft nephropathy responding to low-dose cidofovir after a reduction in immunosuppressive medications failed to clear the virus or stabilize the deterioration in renal function. There were no significant side-effects from this treatment in either patient.

The effects of fig tree latex in treating teat papillomatosis in cow in comparison with salicylic acid were evaluated. For this purpose, 12 cows of 1-3 years of age (average 2.25) affected by teat papillomatosis were divided into three groups. In group A, four cows were treated by fig tree (Ficus carica) latex; in group B, four cows were treated with 10% salicylic acid solution and in group C, four cows were kept as control animals receiving no treatment. Animals in each treatment group received their treatment once every 5 days. In groups A and B, de-epithelialization and shrinking of the warts began from the fifth day of treatment and all the warts disappeared within 30 days. However, in the control group no changes in the number of warts were observed until day 15 but thereafter a number of warts disappeared spontaneously in some of the animals. Both salicylic acid and fig tree latex were evaluated as having similar therapeutic effects in treating teat papillomatosis in cow.

Purpose of Review: Viral nephropathies, particularly those caused by polyomaviruses of the BK-virus strain, are serious complications following renal transplantation. The review will highlight the morphological, pathophysiological and clinical aspects of BK-virus nephropathy. New patient management strategies are discussed., Recent Findings: Immunosuppression with tacrolimus and mycophenolate-mofetil promotes the activation of latent BK-virus in the urinary tract and increases the odds ratio for developing BK-virus nephropathy significantly. A productive infection with BK-viruses shows viral replication in tubular epithelial cells and acute tubular injury. BK-virus nephropathy can be further complicated by concurrent acute rejection episodes contributing to graft demise. Risk assessment after transplantation and patient management during ongoing viral nephropathy have undergone revision by the introduction of real time quantitative polymerase chain reaction techniques measuring BK-virus genome load fluctuations in the serum. Treatment strategies for BK-virus nephropathy include not only low-dose immunosuppression but also drugs with antiviral effects: cidofovir and leflunomide. Transient anti-rejection therapy, including anti-lymphocytic preparations, is a therapeutic option in cases of BK-virus nephropathy and concurrent acute rejection. Recent advances in patient management strategies have resulted in markedly improved graft survival. In cases of graft loss due to BK-virus nephropathy, re-transplantation should be considered., Summary: BK-virus nephropathy is a significant complication following renal transplantation. Recent advances have improved our understanding of the morphological changes, potential risk factors and patient management strategies would be optimized. The availability of quantitative viral load measurements now offers the opportunity for a more accurate and timely clinical intervention.

Topical immunomodulators (TIMs) include both immunostimulatory and immunosuppressive agents. Newer immunostimulatory compounds such as imidazoquinolines (e.g. imiquimod) act by cytokine secretion from monocytes/macrophages (interferon-alpha, interleukin-12, tumour-necrosis factor-alpha), leading to a Th1-dominance and cell-mediated immunity. This immune milieu has been clinically used to treat viral infections such as human papillomavirus (condyloma and common warts), herpes simplex virus and mollusca in immunocompetent and immunosuppressed patients.

SV40 has been linked to some human malignancies, and the evidence that this virus plays a causative role in mesothelioma and brain tumors is mounting. The major SV40 oncoprotein is the Large tumor antigen (Tag). A key Tag transforming activity is connected to its capability to bind and inactivate cellular p53. In this study we developed an effective, high throughput, ELISA-based method to study Tag-p53 interaction in vitro. This assay allowed us to screen a chemical library and to identify a chemical inhibitor of the Tag binding to p53. We propose that our in vitro assay is a useful method to identify molecules that may be used as therapeutic agents for the treatment of SV40-related human cancers.

Objective: The paper presents the role of immunomodulatory treatment with Iscador QuS and Intron A of women with CIN1 and CIN2 with concurrent HPV infection., Material and Methods: Clinical material consisted of 96 women aged 18-52 years of life. The women were divided into three groups. Group A (35 women) treated with Iscador QuS administered s.c. twice a week for 3 months, group B (30 women) treated with Intron A, administered twice a week in the cervical injections for 3 months and control group K (31 women) without treatment followed up with cytology and colposcopy., Results: In the group A (Iscador QuS) CIN remission was observed in slightly higher percentage (non significant) comparing to the control group. In the group B (Intron A) remission CIN was observed in 24 (80%) cases which was statistically significant comparing to the control and A groups. There were no progression of CIN in the group B and the stationery process was observed statistically more frequent comparing to the control and A groups. There was observed statistically higher percentage of cases without HPV infection in all groups during the experiment. The remission concerned both high and low oncogenic potency viruses. In the highest percentage CIN with concurrent HPV infection remission was observed in the B (Intron A) group., Conclusions: 1/Iscador QuS and specially Intron A increases the CIN1 and CIN2 remission rate. 2/These two agents may also affect the HPV remission.

This report describes two cases of ureteral stricture in renal graft recipients related to cytomegalovirus (CMV) and human polyoma BK virus (BKV) ureteritis with the same onset characterized by acute graft failure with no clinical signs of systemic viral infections. The histological analysis did not show other causes of graft impairment (i.e. drug toxicity and acute rejection). Ultrasound scan (US) revealed absent or mild hydronephrosis. The diuretic-MAG3 renal scan showed a urinary flow obstruction. The viral genomes were isolated from urine, peripheral blood and graft or ureteral tissues samples. A percutaneous nephrostomy confirmed the stricture, but it restored urine flow only in the graft affected by CMV ureteritis, the association with a specific antiviral therapy probably produced a stable restoration of graft function. In BKV ureteritis,the graft prognosis was poor; graft loss could be due to the progress of BKV nephropathy. A correct differential diagnosis of the etiologic agent responsible for the ureteritis is mandatory, because treatment and outcome of the infection are different.

Various herpes- and poxviruses contain DNA sequences encoding proteins with homology to cellular chemokine receptors, which belong to the family of G protein-coupled receptors (GPCRs). Since GPCRs play a crucial role in cellular communication and chemokine receptors play a prominent role in the immune system, the virally encoded GPCRs may be crucial determinants of viral action. The Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8), implicated in the pathogenesis of Kaposi's sarcoma (KS), a highly vascularized tumor, encodes a GPCR, referred to as ORF74. This virally encoded receptor was found to induce tumorigenesis and transgenic expression of ORF74 induces an angioproliferative disease resembling KS. Cytomegalovirus (CMV), suggested to play a role in atherosclerosis, encodes four GPCRs, among which US28. This virally encoded GPCR is able to induce migration of smooth muscle cells, a feature essential for the development of atherosclerosis. Remarkably, the KSHV and some CMV-encoded GPCRs display constitutive activity, while their cellular homologs do not. It remains to be determined whether this phenomenon contributes to the pathogenesis of viral action. Also, the family of poxviruses encodes GPCRs of which the function is not clear yet. In this review we will give an overview of the different virally encoded GPCRs, and discuss their putative role in viral action and potential as drug target.

Cervical cancer chemoprevention agents under study include diet and micronutrients (particularly beta-carotene, folate, and vitamins A, C, and E); medications such as retinoids (retinyl acetate gel, all-trans-retinoic acid, and 4-hydroxyphenylretinamide) that are chemically related to micronutrients; and other chemopreventives meant to affect the carcinogenic process at the cellular level, including such polyamine synthesis inhibitors as alpha-difluoromethylornithine. Agents become reasonable candidates for study when they have a biologic rationale, they are of low toxicity, and they can be taken for a long period of time. Since the human papillomavirus (HPV) is the major etiologic agent, the medication should show activity against HPV-positive preinvasive and invasive cell lines. The medication needs to be of low toxicity because it may be taken for long periods of time and less toxicity is tolerated in the precancerous setting. Until 1995, none of the studies used surrogate end point biomarkers (SEBs), relying instead on histologic and colposcopic regression as end points. All studies typically included subjects with cervical intraepithelial neoplasia. Conclusions to be drawn from these studies include the following: Though micronutrients are logical candidates for chemoprevention, they haven't worked consistently, and the reasons remain unclear. Furthermore, SEBs need to be validated in phase I trials. Finally, a better understanding of the role of HPV needs elucidation, including an understanding of the relationship of the medication to HPV status and of the immunobiology of HPV throughout the trial.

Objective: We sought to determine the efficacy of intralesional injection of cidofovir in improving resolution of recurrent respiratory papillomatosis (RRP). Study design and setting We conducted a prospective, observational trial at an academic tertiary children's hospital., Results: Four children with RRP requiring more than 6 surgical excisions per year were treated with intralesional cidofovir. Cidofovir (5 mg/mL) was injected into airway sites where papillomas had just been excised using sharp technique. Each patient had 6 treatments performed 6 to 8 weeks apart. Biopsies confirmed benign papilloma lesions in all cases. During treatment with intralesional cidofovir there was diminished growth of the papillomas in each patient. Once cidofovir treatment was stopped, the rate of regrowth and frequency of surgical excision returned to pretreatment levels in 3 of the 4 patients., Conclusions: Intralesional cidofovir may provide benefit in reducing the rate of RRP growth while under treatment, but RRP severity returned to pretreatment levels once cidofovir treatment was stopped using this treatment program.

It is known that cell-free DNA circulates in plasma/serum of patients with cancer and that part of this DNA circulates as nucleosomes that can be quantified by ELISA. We analyzed the effect of tumor and chemotherapy upon the levels of nucleosomes in vitro, in vivo and in cervical cancer patients. The levels of nucleosomes pre- and post-treatment were correlated with response in 11 patients receiving chemotherapy. Nucleosomes were determined in nude mice treated with or without cisplatin and carrying tumors generated with HeLa cells, and in the cell lysate and supernatant of HeLa cells exposed to cisplatin in culture. In addition, nucleosomes were determined at different time points in patients and in rats receiving chemotherapy. Nucleosomes were higher in patients that controls (1,760 vs. 601, p = 0.0001). After 24 hr of treatment with oxaliplatin and gemcitabine, the levels decreased in 6 patients of whom 5 had response. Nucleosome levels differed between mice xenografted and not xenografted (765 vs. 378, p = 0.001) and between xenografted treated with or without cisplatin (650 vs. 765, p = 0.010), but not in tumor-free animals treated and untreated with cisplatin (378 vs. 379, p = 0.99). In vitro, nucleosomes reached at peak 8 hr in cell lysates to decrease thereafter, whereas in supernatant, levels continued to increase up to 24 hr. Serial determination of nucleosomes in patients showed a rise within 6-12 hr and then a reduction to below the basal at 24 hr. In rats, nucleosomes had no major changes in those receiving oxaliplatin or the triple combination of cisplatin, gemcitabine and paclitaxel as compared to untreated controls. An overdose of this triple combination produced a transient elevation of almost 1,000 AU over the basal. Our results demonstrate that most of circulating nucleosomes originate from the tumor and that chemotherapy produces an early rise most likely due to tumor apoptosis and that nucleosomes are rapidly cleared from circulation. On the contrary, chemotherapy within the therapeutic range of doses has no effect on nucleosome levels in healthy mice and rats. This data suggests that the determination of circulating nucleosomes pre- and post-treatment could be a useful test to predict response to chemotherapy in cancer patients., (Copyright 2003 Wiley-Liss, Inc.)

The Epstein-Barr virus (EBV) is involved in the carcinogenesis of several human cancers such as nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). Given the consistent role of EBV in transformation and maintenance of malignant phenotype, antiviral strategies provide an attractive approach to target EBV-expressing cells. In that aim, we have tested the Cidofovir, which is an acyclic nucleoside phosphonate analog known to exert an antiproliferative activity in some human virus-related tumors. Here, we show that Cidofovir induces a downregulation of the EBV oncoprotein LMP1 associated with a decrease of the antiapoptotic Bcl-2 and an increase of the proapoptotic Bax protein in Raji (BL) and C15 (NPC) cells. Using BL cell line BL2 B95-8 (BL2 infected with the B95.8 strain of EBV), we addressed the relation between EBV genome expression and modulation of viral oncoproteins by Cidofovir and/or ionizing radiation (IR). Cidofovir was able to significantly reduce LMP1 and EBNA2 mRNA and protein expression. This effect was associated with inhibition of proliferation, stimulation of apoptosis, and decrease of Bcl-2 expression in BL2 B95.8 cells. In addition, Cidofovir enhanced the radiation-induced apoptosis and the radiosensitivity through the proteolytic cleavage of death effectors caspase-9 and -3, which was specifically induced by combined treatment in EBV-positive cells compared to their negative counterparts. Furthermore, the combined treatment in nude mice led to a complete tumor remission without increasing toxicity in two human EBV-related cancer xenografts (Raji and C15). These results provide the basis for a novel anticancer strategy to enhance the therapeutic ratio of IR in EBV-related cancers.

This article discusses infections of the female genital tract, including vaginitis, sexually transmitted diseases, and endometriosis, and includes methods of diagnosis, treatment, and specific management strategies for these infections.

Human papillomavirus E6 forms a complex with p53 tumor suppressor and E6-associated protein, leading to the degradation of p53 via the ubiquitination pathway, resulting in the oncogenesis of cervical carcinomas. Several viral and cellular gene promoters were shown to be transactivated by E6 oncogene. In this study, to understand the role of transcription activity of E6 related to cervical carcinogenesis, the effect of cervical cancer drugs on E6 induced transcription activity has been elucidated. Several viral promoter (RSV, CMV, SV40, and HIV)-luciferase reporter gene constructs, and eukaryotic E6 expression vector were prepared as an E6 transcription analysis system and an exogenous E6 protein source, respectively. It was shown that the promoters of RSV, SV40, and HIV, but not CMV, were transactivated by HPV 16 E6 in cervical cancer cell line. Several known cervical cancer drugs were investigated for their effects on transcription activity of E6 in SiHa stably transfected with E6-responsive promoters. Cervical cancer drugs consistently reduced luciferase activity, in transfectants with RSV-luc (SiHa/pRSV-luc, KCTC 0427BP) E6 mRNA also. Thus, in this study, we have demonstrated that the promoters of RSV, HIV, and SV40 were transactivated by E6 in cervical cancer cells. Three cervical cancer drugs downregulated RSV-luc transcription and E6 expression by a p53 independent pathway. RSV-luc promoter analysis system could be useful for understanding the role of transcription activity of E6 related to cervical cancer and also for screening drugs against cervical cancers caused by HPV infection.

A constituent of green tea, (-)-epigallocatechin-3-gallate (EGCG) has been known to possess antiproliferative properties. In this study, we investigated the anticancer effects of EGCG in human papillomavirus (HPV)-16 associated cervical cancer cell line, CaSki cells. The growth inhibitory mechanism(s) and regulation of gene expression by EGCG were also evaluated. EGCG showed growth inhibitory effects in CaSki cells in a dose-dependent fashion, with an inhibitory dose (ID)(50) of approximately 35 microM. When CaSki cells were further tested for EGCG-induced apoptosis, apoptotic cells were significantly observed after 24 h at 100 microM EGCG. In contrast, an insignificant induction of apoptotic cells was observed at 35 microM EGCG. However, cell cycles at the G1 phase were arrested at 35 microM EGCG, suggesting that cell cycle arrests might precede apoptosis. When CaSki cells were tested for their gene expression using 384 cDNA microarray, an alteration in the gene expression was observed by EGCG treatment. EGCG downregulated the expression of 16 genes over time more than twofold. In contrast, EGCG upregulated the expression of four genes more than twofold, suggesting a possible gene regulatory role of EGCG. This data supports that EGCG can inhibit cervical cancer cell growth through induction of apoptosis and cell cycle arrest as well as regulation of gene expression in vitro. Furthermore, in vivo antitumor effects of EGCG were also observed. Thus, EGCG likely provides an additional option for a new and potential drug approach for cervical cancer patients.

BK virus-associated nephropathy (BKVN) has become recognized as an important cause of allograft dysfunction in renal transplant recipients and despite reduction in immunosuppression, 30-40% of recipients ultimately progress to allograft loss. Cidofovir is an antiviral agent that demonstrates in vitro activity against murine polyomavirus and has been proposed for treatment of BKVN in renal allograft recipients. We describe the clinical course, renal function, serial renal histology and urine and blood viral load measurements in two consecutive patients with refractory BKVN who were treated with low-dose cidofovir (0.25 mg/kg IV). In each case, renal dysfunction and BK viral load progressed despite reduced immunosuppression, and persistent BK virus infection was documented in serial renal allograft biopsy specimens. Administration of low-dose cidofovir was associated with clearance of BK virus DNA from blood and allograft, and stabilization of renal function in both patients, without significant toxicity. These preliminary data suggest that low-dose cidofovir may be tolerated, even among renal transplant recipients with significant renal dysfunction due to BKVN. Prospective, controlled trials are warranted to further define the optimal dose, toxicity and potential role of cidofovir in renal transplant recipients with BK virus nephropathy.