Oral cefixime is similar to continued intravenous antibiotics in the empirical treatment of febrile neutropenic children with cancer.

Empiric oral antibiotic therapy for febrile neutropenic cancer patients has been suggested as a means to decrease hospitalization, but the safety of this approach has not been adequately studied in children. We compared continued iv antibiotic therapy with switching treatment to orally administered cefixime in a group of selected febrile neutropenic children for whom blood cultures were sterile after 48 h of incubation. Two hundred episodes of febrile neutropenia were studied (156 patients), and 100 episodes were randomized to receive each treatment. Failure to respond to therapy was defined by documented or suspected bacterial infection, recurrent fever, or discontinuation of assigned therapy for any reason before neutropenia resolved. Rates of treatment failure were similar in the oral cefixime group (28%) and in the iv antibiotic group (27%; P=1.0). Results support the safety of oral cefixime therapy for low-risk febrile neutropenic children, a therapeutic approach that would facilitate earlier outpatient management and decrease the costs of treatment.

Epstein-Barr virus-targeted therapy for AIDS-related primary lymphoma of the central nervous system.

Epstein-Barr virus (EBV) targeted therapeutic strategies for viral associated malignant diseases have received only perfunctory consideration, first, because latent herpesviruses have been intractable to antiviral chemotherapy and, second, because the role EBV has in maintenance of the malignant cell phenotype has been uncertain. Two patients with EBV related primary central nervous system lymphoma (PCNSL) in the setting of advanced AIDS, were treated with low dose hydroxyurea based on in vitro anti-EBV activity. The responses obtained here suggest the promise of antiviral approaches in select cancers.

Epstein-Barr virus infection in salivary gland tumors in children and young adults.

BACKGROUND: Epstein-Barr virus (EBV) infection has been implicated in the pathogenesis of certain subtypes of salivary gland tumors in the adult population. However, to the authors' knowledge its role in pediatric salivary gland tumors, a rare disease, has not been explored previously. METHODS: Thirteen cases of primary tumors of the salivary gland occurring in children were retrieved from the tumor registry at the St. Jude Children's Research Hospital in Memphis, Tennessee. Clinical data were analyzed from the medical records and formalin fixed, paraffin embedded tumor tissues were examined by the in situ hybridization (ISH) technique for the presence of latent EBV infection. RESULTS: Twelve of 13 tumors originated from the parotid gland and 1 originated from the submandibular gland. Mucoepidermoid carcinoma was the predominant tumor type; it was observed in seven patients, rhabdomyosarcoma was the diagnosis in three patients, acinic cell carcinoma was noted in two patients, and malignant fibrous histiocytoma was diagnosed in one patient. The ages of the patients ranged from 4.1-29.2 years, with a median age of 11 years. The outcome was excellent with all patients alive and free of disease at the time of last follow-up. The ISH tested negative in all tumor samples. CONCLUSIONS: Based on the results of the current study, EBV infection does not appear to play a major role in the pathogenesis of pediatric salivary gland tumors.

Molecular evidence of ocular Epstein-Barr virus infection.

Ocular manifestations have been attributed to the Epstein-Barr virus (EBV), largely on the basis of seroepidemiologic data. Two patients who developed conjunctival disease as the presenting feature of EBV infection are reported, each confirmed by in situ hybridization of EBV genome in affected tissue biopsy specimens. Recognition of EBV-induced ocular disease as an initial presentation of clinical EBV infection is important to the practitioner because of the ubiquitous nature of this herpesvirus.

Patterned entry and egress by Epstein-Barr virus in polarized CR2-positive epithelial cells.

In polarized epithelium direction of viral entry and release correlates with proclivity of a virus to establish local versus systemic infection. The Epstein-Barr virus (EBV), whose principal tissue reservoir is B lymphocytes, also has disease manifestations in epithelium, suggesting intertissue spread potentially influenced by epithelial cell polarity. We stably transfected the B lymphocyte EBV receptor (CR2/CD21) into Madin-Darby canine kidney (MDCK) epithelial cells used extensively to study effects of cell polarity on infection by both DNA and RNA viruses. CR2/CD21 was detected on both apical and basolateral surfaces of polarized MDCK cells, with predominant expression basolaterally. However, infectivity was up to four-fold greater apically, suggesting that endogenous cell surface molecules, sorted asymmetrically onto polarized plasma membranes, may be involved in EBV entry into MDCK cells. EBV gp350/220, a replicative cycle glycoprotein added to the virus envelope on egress through the cell membrane, was immunolocalized by confocal microscopy to basolateral cell surfaces only. Apical entry of EBV with subsequent basolateral release of newly replicated virus favors systemic infection by viral dissemination to underlying lymphocytic aggregations. Under conditions of long-term culture, latent EBV was not stably maintained in these cells, suggesting that the epithelial phase of acute EBV infection may be transient.

Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients.

Epstein-Barr virus (EBV) causes potentially lethal immunoblastic lymphoma in up to 25% of children receiving bone marrow transplants from unrelated or HLA-mismatched donors. Because this complication appears to stem from a deficiency of EBV-specific cytotoxic T cells, we assessed the safety and efficacy of donor-derived polyclonal (CD4(+) and CD8(+)) T-cell lines as immunoprophylaxis and treatment for EBV-related lymphoma. Thirty-nine patients considered to be at high risk for EBV-induced lymphoma each received 2 to 4 intravenous infusions of donor-derived EBV-specific T lymphocytes, after they had received T-cell-depleted bone marrow from HLA-matched unrelated donors (n = 33) or mismatched family members (n = 6). The immunologic effects of this therapy were monitored during and after the infusions. Infused cells were identified by detection of the neo marker gene. EBV-specific T cells bearing the neo marker were identified in all but 1 of the patients. Serial analysis of DNA detected the marker gene for as long as 18 weeks in unmanipulated peripheral blood mononuclear cells and for as long as 38 months in regenerated lines of EBV-specific cytotoxic T cells. Six patients (15.5%) had greatly increased amounts of EBV-DNA on study entry (>2, 000 genome copies/10(6) mononuclear cells), indicating uncontrolled EBV replication, a complication that has had a high correlation with subsequent development of overt lymphoma. All of these patients showed 2 to 4 log decreases in viral DNA levels within 2 to 3 weeks after infusion and none developed lymphoma, confirming the antiviral activity of the donor-derived cells. There were no toxic effects that could be attributed to prophylactic T-cell therapy. Two additional patients who did not receive prophylaxis and developed overt immunoblastic lymphoma responded fully to T-cell infusion. Polyclonal donor-derived T-cell lines specific for EBV proteins can thus be used safely to prevent EBV-related immunoblastic lymphoma after allogeneic marrow transplantation and may also be effective in the treatment of established disease.

Primary central nervous system lymphoma in a child with acute B-cell lymphoblastic leukaemia: consecutive Epstein-Barr virus-related malignancies.

Primary central nervous system lymphoma (PCNSL), observed among immunocompromised AIDS patients, has not been reported during chemotherapy for acute lymphoblastic leukaemia (ALL). We report a case of PCNSL occurring in a child receiving intensive multiagent chemotherapy for B-cell ALL. In situ hybridization studies demonstrated Epstein-Barr virus genome in both tumours, suggesting a possible link between the two diseases. The clinical response of the PCNSL to conservative therapy highlights the importance of accurately diagnosing such EBV-related disorders, especially in patients where immune compromise can be reversed.

Spontaneous loss of viral episomes accompanying Epstein-Barr virus reactivation in a Burkitt's lymphoma cell line.

Life-long viral persistence is a hallmark of human herpesvirus infection. In the Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell line, Mutu, spontaneous loss of all viral episomes accompanied productive viral DNA replication. The molecular configuration of intracellular EBV DNA evolved from monoclonal episomes in cells retaining the original tumor phenotype to predominantly replicating linear DNA and, subsequently, only integrated forms in BL cells that had acquired the lymphoblastoid cell phenotype. Transient appearance of deleted, rearranged WZhet EBV DNA capable of disrupting viral latency, along with the integration of viral DNA into human chromosomes, indicates a genetic instability in the host cell which, if duplicated in vivo, may affect configuration and persistence of the viral genome in expanding malignant cell clones.

Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for the treatment of patients with EBV-positive relapsed Hodgkin's disease.

Adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) is effective prophylaxis and treatment of EBV-positive immunoblastic lymphoma in immunocompromised patients. In 50% of patients with Hodgkin's disease, the tumor cells are EBV antigen-positive and may therefore also be suitable targets for treatment with virus-specific CTLs. However, Hodgkin's disease may produce several inhibitory effects on immune induction and effector function in vivo, which may preclude the generation or effector function of CTLs reactive against EBV viral proteins, including those expressed by the tumor cells. We have investigated whether EBV-specific CTLs could be generated ex vivo from 13 patients with Hodgkin's disease: nine with active relapsed disease and four who were in clinical remission after a first or subsequent relapse. CTL lines were successfully generated from nine of 13 patients (five active disease, four remission). Although these lines had an abnormal pattern of expansion comparable to EBV-specific CTLs generated from normal donors, their phenotype was normal except for reduced expression of the zeta chain of the T-cell receptor (TCR). Their cytotoxicity was also compared to EBV-specific lines generated from normal donors and included activity against LMP2a, one of the three weakly immunogenic viral antigens expressed by Hodgkin's tumor cells. To assess the activity of the CTLs in vivo, they were gene-marked and infused into three patients with multiply relapsed disease. The CTLs persisted for more than 13 weeks postinfusion and retained their potent antiviral effects in vivo, thereby enhancing the patient immune response to EBV. This approach may therefore have value in the treatment of EBV-positive Hodgkin's disease.

Eradication of latent Epstein-Barr virus by hydroxyurea alters the growth-transformed cell phenotype.

The hallmark of infection by human herpesviruses, life-long persistence in the host, is unaffected by current antiviral therapies effective against replication of virus. In vitro studies indicated that low concentrations of the ribonucleotide reductase inhibitor, hydroxyurea, completely eliminated Epstein-Barr virus (EBV) episomes from latently infected Burkitt's lymphoma (BL) cell subsets, providing the first example of chemotherapeutic eradication of a latent herpesvirus from any cell population. Unlike parental EBV-positive BL cells, virus-free cell progeny from one treated cell line no longer exhibited the malignant phenotype in tumorigenicity assays. Hydroxyurea-treated primary B lymphocytes immortalized by EBV ceased to proliferate as episomes were lost. The altered growth phenotype of both BL cells and immortalized primary B cells suggests that latent EBV is an appropriate and accessible therapeutic target for treatment of some EBV-induced lymphoproliferations.

Acute Epstein-Barr virus infection complicated by severe thrombocytopenia.

We describe one patient with acute Epstein-Barr virus (EBV) infection associated with severe thrombocytopenia and review 36 additional cases reported in the literature. Complications of EBV infection due to severe thrombocytopenia occurred in 10 (27.0%) of 37 patients, and 2 (5.4%) of 37 patients died. Although acute EBV infections are generally benign and self-limiting, thrombocytopenia, a potentially serious complication, should not be overlooked.

Epstein-Barr virus in pediatric Hodgkin disease: age and histiotype are more predictive than geographic region.

Epidemiologic studies have implicated Epstein-Barr virus (EBV) in the great majority (80%-100%) of Hodgkin disease (HD) cases in South American countries, versus only 30%-40% in the United States and other industrialized countries. Other EBV-related malignancies are known to be geographically localized, including nasopharyngeal carcinoma in south China and Burkitt lymphoma in equatorial Africa. Some studies, however, have suggested that age and histiotype, rather than geographic region, are the major determinants of the association between EBV and HD. To further characterize this relationship in children, we matched 26 cases of pediatric Hodgkin disease from south Brazil and 26 cases from the U.S.-forhistiotype and age. The Brazilian children (22 males, 4 females) had a median age of 9 years, while the median age of the U.S. group (11 males, 15 females) was 7.5 years. Formalin-fixed, paraffin-embedded biopsy material was examined for EBV early RNA1 (EBER1) expression by in situ hybridization. This antigen was detected solely in Reed-Sternberg cells or their variants in positive samples. The same proportion of cases was positive (15/26 or 58%) in both groups of children. After adjustment for histiotype and age, the association between EBV and HD remained independent of geographic location, but was more frequent in children aged < or = 10 years at diagnosis. These findings support the multiple-etiology hypothesis for Hodgkin disease.

Epithelial cell polarization is a determinant in the infectious outcome of immunoglobulin A-mediated entry by Epstein-Barr virus.

Diseases of the nasopharyngeal epithelium due to Epstein-Barr virus (EBV) infection typically occur in chronic virus carriers with preexisting virus-specific antibodies. In vitro studies have shown that EBV-specific immunoglobulin A (IgA) promotes infection of human epithelial cells, otherwise refractory to EBV, via the polymeric immunoglobulin receptor (pIgR). To determine if EBV similarly exploits IgA transport mechanisms in vivo, we examined the fate of IgA-EBV complexes in the blood of mice, where pIgR-mediated transcytosis of IgA immune complexes through hepatocytes eliminates exogenous antigens from the circulation. By PCR analysis we showed hepatobiliary transport of IgA-EBV in viremic mice, but without detectable hepatocellular infection by immunostaining. Because efficient transport of EBV immune complexes might avert an infectious outcome, we modulated the transcytotic pathway in polarized Madin-Darby canine kidney (MDCK) cells transfected with pIgR to determine the effect on viral antigen expression. Like hepatocytes in vivo, MDCK cells in polarized monolayers translocated IgA-EBV from the basal cell face into apical medium without evidence for infection. However, when exposed to IgA-EBV as unpolarized single-cell suspensions, MDCK cells expressed EBV immediate-early and early antigens. These results suggest that pIgR-mediated transcytosis of pIgA-EBV through epithelium facilitates endogenous spread of EBV in long-term virus carriers, with infection being confined to cells with altered polarity from prior cytopathology.

Detection of Epstein-Barr virus genome in ocular tissues.

BACKGROUND: Epstein-Barr virus (EBV) is a ubiquitous mucosal pathogen with a propensity for lifelong, asymptomatic persistence. Because of reported association between EBV and ocular inflammatory disorders, we tested ocular tissues from normal eyes for presence of the EBV genome. METHODS: Ten freshly harvested cadaveric human eyes were dissected into limbal cornea, central cornea, aqueous humor, iris, vitreous humor, and optic nerve. Total cellular DNA preparations were screened for DNA sequences specific to EBV's large internal repeat region. After Southern transfer, polymerase chain reaction products were detected by a 32P-labeled oligonucleotide probe specific to amplified sequences internal to the polymerase chain reaction primers. RESULTS: Seven of ten eyes from deceased donors yielded a polymerase chain reaction product, indicating presence of EBV genome. In all, 12 (20%) of 60 cadaveric ocular samples contained EBV DNA. Only the optic nerve was consistently negative for EBV DNA. CONCLUSIONS: Detection of EBV DNA in cadaveric ocular tissues indicates a broad anatomic distribution of this persistent mucosal pathogen. The frequency with which EBV was found at apparently normal ocular sites raises the possibility for viral involvement in disease states, but emphasizes the need for specific criteria to implicate EBV in ocular pathology.

Epstein-Barr Virus DNA recombination and loss in sporadic Burkitt's lymphoma.

Epstein-Barr virus (EBV) antigens in tumor tissue define associations of virus with human malignancies and provide clues as to mechanisms of viral oncogenesis. In Burkitt's lymphoma, EBV markers are absent from 85% of sporadic cases and 4% of endemic (African) cases, raising questions as to the exact role EBV in the disease. Standard screening criteria may be insufficient to determine the EBV status of all tumors. One of 9 tumors from American patients expressed EBV nuclear antigen 1 (EBNA1) and contained standard episomal EBV DNA, making this series consistent with the 15% EBV association traditionally ascribed to sporadic Burkitt's lymphoma. Surprisingly, 3 tumors without detectable EBNA1 contained partial EBV genomes. Identification of defective, integrated viral DNA in some tumors indicates greater involvement of virus in sporadic Burkitt's lymphoma than previously documented and suggests a process of viral DNA rearrangement and loss during malignant progression most consistent with an initiating role for EBV in tumorigenesis.

Epstein-Barr virus induction of recombinase-activating genes RAG1 and RAG2.

In experimental B-cell infections, Epstein-Barr virus induced sustained expression of V(D)J recombinase-activating genes RAG1 and RAG2, whose aberrant activity has been implicated in chromosomal translocations in B-cell neoplasms. In cell lines in which RAG1 and RAG2 were detected, virus integrated into cellular DNA rather than assumed the configuration of extrachromosomal episomes. Expression of the Epstein-Barr virus nuclear antigen 1 in transient transfection assays was sufficient to induce both recombinase-activating genes.

Detection of cell-free Epstein-Barr virus DNA in serum during acute infectious mononucleosis.

Infectious Epstein-Barr virus (EBV) is shed from the oropharynx of infected hosts intermittently throughout life, but in the peripheral circulation the viral genome characteristically maintains itself in a noninfectious, cell-associated form. Sera from 125 persons with heterophil-positive acute infectious mononucleosis or EBV-associated nasopharyngeal carcinoma or who were healthy virus carriers were examined for evidence of cell-free viral DNA. EBV DNA suggesting viremia was detected in 11 (27%) of 41 infectious mononucleosis patients by polymerase chain reaction analysis but infrequently in healthy seropositive carriers and patients with nasopharyngeal carcinoma. In serial samples examined from 2 patients, serum EBV DNA was detected over a 3-day interval. Viral DNA was found in concert with one serologic marker of acute infection, EBV-specific polymeric IgA, that could affect patterns of viral spread and clinical symptomatology.

Thymic carcinoma with a defective Epstein-Barr virus encoding the BZLF1 trans-activator.

The diagnosis of Epstein-Barr virus (EBV)-associated carcinomas is often heralded by high antibody titers to the viral replicative antigens, suggesting EBV reactivation may be a factor in tumor evolution. EBV DNA and nuclear antigen was detected in a newly diagnosed thymic carcinoma. Polymerase chain reaction analysis revealed the presence of a rearranged EBV DNA fragment, BamHI WZhet. This rearrangement is found in a defective EBV that up-regulates the BZLF1 (BamHI Z leftward reading frame) gene product in vitro and induces the EBV lytic cycle. Molecular analysis of the EBV termini demonstrated low levels of the lytic (linear) genomic configuration among a predominantly latent (episomal) population at diagnosis. The episomal populations were of uniform molecular weight at diagnosis and relapse, indicating clonal tumor expansion from a single EBV-infected progenitor. The presence within malignant epithelium of defective virus that can disrupt EBV latency, and perhaps cellular gene regulation, suggests a potential mechanism for EBV reactivation and concurrent malignant progression.

Evasion of host immune responses by tumours and viruses.

Viruses and tumours use various mechanisms to avoid immune surveillance. Oncogenic viruses have achieved a balance with the immune system through evolutionary time to ensure long-term persistence. Mutations that promote escape mechanisms favouring tumour growth to the detriment of host survival through reproductive age offer no selective advantage and will not generally be maintained in the viral genome that persists in nature. Conventional (non-oncogenic) and tumour viruses interact with various immune mediators and T cells in different ways. Oncogenic viruses cannot operate solely in the context of a lytic cycle, though this may be characteristic of the initial phase of infection that is limited by the acute immune response. Some oncogenic viruses interact with normal cellular growth control and signalling mechanisms. Synthesis of key viral proteins may be tightly controlled in replicating cells that are subject to T cell surveillance, such as basal epithelia, while productive infection occurs in non-proliferating progeny that are lost under normal physiological conditions, such as desquamating epithelia. Tumorigenesis may be an aberrant consequence of the molecular mechanisms needed to maintain this pattern of viral growth regulation in the context of the cell cycle. Vaccines designed to limit the acute phase of infection with cell-free oncogenic viruses should be as effective as those for conventional viruses.

Human oropharyngeal lesions with a defective Epstein-Barr virus that disrupts viral latency.

The Epstein-Barr virus (EBV), a human herpesvirus that is predominantly latent after infection, can be induced to replicate by deleted, rearranged EBV DNA from cultures of laboratory strain P3HR-1. Because mucosal surfaces are permissive of EBV replication, 101 oral biopsies from 70 Chinese and 5 American patients were examined for natural counterparts to tissue culture defective virus (WZhet), using as marker the abnormal juxtaposition of BamHI W and Z EBV DNA restriction fragments. Of the 49 oral biopsies that contained EBV DNA, 12 (24%) had the rearranged WZ fragment by polymerase chain reaction analysis: 3 (42%) of 7 EBV-positive epithelial dysplasias or carcinomas, 6 (38%) of 16 hairy leukoplakias, and 3 (12%) of 25 nonmalignant salivary gland biopsies. Accompanying viral replication was confirmed by in situ cytohybridization and demonstration of the linear configuration of the genome in select WZhet-positive lesions. These findings indicate that defective EBV with the unusual property of disrupting EBV latency is prevalent in natural infections and may contribute to EBV's pathogenic diversity.