Oncogenic activation of FOXR1 by 11q23 intrachromosomal deletion-fusions in neuroblastoma.

Neuroblastoma tumors frequently show loss of heterozygosity of chromosome 11q with a shortest region of overlap in the 11q23 region. These deletions are thought to cause inactivation of tumor suppressor genes leading to haploinsufficiency. Alternatively, micro-deletions could lead to gene fusion products that are tumor driving. To identify such events we analyzed a series of neuroblastomas by comparative genomic hybridization and single-nucleotide polymorphism arrays and integrated these data with Affymetrix mRNA profiling data with the bioinformatic tool R2 (http://r2.amc.nl). We identified three neuroblastoma samples with small interstitial deletions at 11q23, upstream of the forkhead-box R1 transcription factor (FOXR1). Genes at the proximal side of the deletion were fused to FOXR1, resulting in fusion transcripts of MLL-FOXR1 and PAFAH1B2-FOXR1. FOXR1 expression has only been detected in early embryogenesis. Affymetrix microarray analysis showed high FOXR1 mRNA expression exclusively in the neuroblastomas with micro-deletions and rare cases of other tumor types, including osteosarcoma cell line HOS. RNAi silencing of FOXR1 strongly inhibited proliferation of HOS cells and triggered apoptosis. Expression profiling of these cells and reporter assays suggested that FOXR1 is a negative regulator of fork-head box factor-mediated transcription. The neural crest stem cell line JoMa1 proliferates in culture conditional to activity of a MYC-ER transgene. Over-expression of the wild-type FOXR1 could functionally replace MYC and drive proliferation of JoMa1. We conclude that FOXR1 is recurrently activated in neuroblastoma by intrachromosomal deletion/fusion events, resulting in overexpression of fusion transcripts. Forkhead-box transcription factors have not been previously implicated in neuroblastoma pathogenesis. Furthermore, this is the first identification of intrachromosomal fusion genes in neuroblastoma.

Administration of oral acyclovir suppressive therapy after neonatal herpes simplex virus disease limited to the skin, eyes and mouth: results of a phase I/II trial.

BACKGROUND: Neonatal herpes simplex virus (HSV) infections limited to the skin, eyes and mouth (SEM) can result in neurologic impairment. A direct correlation exists between the development of neurologic deficits and the frequency of cutaneous HSV recurrences. Thus, the National Institutes of Allergy and Infectious Diseases Collaborative Antiviral Study Group conducted a Phase I/II trial of oral acyclovir therapy for the suppression of cutaneous recurrences after SEM disease in 26 neonates. METHODS: Infants < or = 1 month of age with virologically confirmed HSV-2 SEM disease were eligible for enrollment. Suppressive oral acyclovir therapy (300 mg/m2/dose given either twice daily or three times per day) was administered for 6 months. RESULTS: Twelve (46%) of the 26 infants developed neutropenia (< 1000 cells/mm3) while receiving acyclovir. Thirteen (81%) of the 16 infants who received drug 3 times per day experienced no recurrences of skin lesions while receiving therapy. In comparison, a previous Collaborative Antiviral Study Group study found that only 54% of infants have no cutaneous recurrences in the 6 months after resolution of neonatal HSV disease if oral acyclovir suppressive therapy is not initiated. In one infant, HSV DNA was detected in the cerebrospinal fluid during a cutaneous recurrence, and an acyclovir-resistant HSV mutant was isolated from another patient during the course of the study. CONCLUSIONS: Administration of oral acyclovir can prevent cutaneous recurrences of HSV after neonatal SEM disease. The effect of such therapy on neurologic outcome must be assessed in a larger, Phase III study. As such, additional investigation is necessary before routine use of suppressive therapy in this population can be recommended.

Follicular dendritic cells inhibit apoptosis in human B lymphocytes by a rapid and irreversible blockade of preexisting endonuclease.

During germinal center reactions, a minority of B lymphocytes are selected after successful binding to follicular dendritic cells (FDCs). The majority of the B cells, however, die by apoptosis. One of the characteristics of apoptosis is rapid fragmentation of DNA by an endogenous endonuclease. The regulation of apoptosis and endonuclease activity in germinal center (GC) B cells is largely unknown. In this study we have investigated the induction and inhibition of endonuclease activity in GC B cells. We also investigated the role of FDCs, surface Ig (sIg), sIgM, CD21, CD22 CD40, and intracellular Zn2+ in the regulation of endonuclease activity. We have found that DNA fragmentation in GC B cells is caused by a preexisting endonuclease very similar to NUC-18 (an 18-kD endonuclease identified in rat thymocytes). Endonuclease activity in GC B cells appears to be rapidly and irreversibly blocked after interaction with FDCs, but not after cross-linkage of sIg, sIgM, CD21, CD22, or CD40. Addition of soluble CD40-human IgM fusion protein (sCD40) to FDC-B cell cultures also did not interfere with FDC-mediated B cell rescue. Chelation of intracellular Zn2+ during FDC-B cell cultures resulted in abrogated B cell rescue. These data suggest that FDCs inhibit apoptosis in GC B cells by a rapid inactivation of preexisting endonuclease using a mechanism distinct from CD40 ligation.

Functionally active Epstein-Barr virus-transformed follicular dendritic cell-like cell lines.

Follicular dendritic cells (FDC) are unique nonlymphoid cells found only in germinal centers. FDC can be distinguished from other accessory cells based on a characteristic set of cell surface markers. It is known that FDC are able to rescue germinal center B cells from apoptosis. To investigate the role of FDC in the process of selection and maturation of B cells during germinal center reactions, we tried to establish factor-independent immortalized FDC-like cell lines. Because freshly isolated FDC express the Epstein-Barr Virus (EBV) receptor CD21, we attempted EBV transformation on isolated FDC. After incubation of FDC-enriched cell populations with EBV, cell lines were obtained consisting of slowly duplicating very large cells. These cell lines have a fibroblast-like morphology but could be clearly distinguished from several human fibroblast cell lines by displaying a different phenotype including intercellular adhesion molecule 1, CD40, and CD75 expression. Detection of the EBV-encoded proteins latent membrane protein 1 and Epstein-Barr virus nuclear antigen 2 in our FDC-like cell lines implicated successful EBV transformation. FDC-like cells are able to bind nonautologous B cells and preserve the latter from apoptosis. The binding of B cells to FDC-like cells is dependent on adhesion via lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 and closely resembles the pattern of emperipolesis as described by others. These data demonstrate that FDC can be successfully infected by EBV, and that the cell lines obtained share phenotypic and functional characteristics with freshly isolated FDC.

Neurovirulence in an experimental focal herpes encephalitis: relationship to observed seizures.

An animal model of focal herpes simplex encephalitis was used to study several strains of type-1 herpes simplex virus. Rabbits were inoculated in the olfactory bulb by a standardized technique. Virus strains resulting in mortality of greater than 70% produced seizures of 3 types, and all animals that seized became moribund or died. In contrast, a virus strain resulting in a 20% mortality produced no seizures. Administration of 60 mg phenobarbital intramuscularly daily reduced mortality significantly in animals given the epileptogenic viruses. Cultures from temporal and frontal lobes showed viral growth more frequently than did cultures of other brain areas. Microscopic examination of routine and immunoperoxidase-stained brain sections confirmed the focal nature of the infection. Clinical syndromes such as seizures arising from viral brain disease may influence mortality in animal model systems.

Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells.

Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of 51Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a+ Leu-4- granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of 51Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.

A rabbit model of focal herpes simplex encephalitis.

Results from studies designed to create a model of focal encephalitis caused by herpes simplex virus (HSV) are reported. Anesthetized rabbits underwent exposure and inoculation of the olfactory bulb with three different doses of a wild-type HSV. Lethal infection resulted in 69% of the animals, without evidence for a dose-response relationship. Necropsy specimens obtained on or before day 10 after inoculation routinely yielded HSV in culture. In 76% of the animals with positive cultures for virus, these cultures originated exclusively or primarily from the pyriform (or temporal) cortex and frontal lobes. Virus could not be cultured from animals killed more than two weeks after inoculation. Histological examination of brains obtained three or more days after inoculation demonstrated evidence of viral infection, with more severe involvement of temporal cortex than of the surrounding brain in 80%. Immunohistochemical demonstration of viral antigens persisted for up to three weeks after inoculation.

Analysis of DNA from recurrent genital herpes simplex virus isolates by restriction endonuclease digestion.

In a blind study, DNA from 40 clinical isolates of herpes simplex viruses was analyzed by restriction endonucleases to determine whether serial isolates from an individual patient could be identified and whether exogenous reinfection occurred within this population. Five of the 40 isolates served as controls. Based on restriction patterns obtained following Bam H1 cleavage of DNA, 35 isolates were assigned to 15 patients. Isolates from two patients displayed variation in the electrophoretic mobility of certain Bam H1 fragments. However, the isolates from one patient were identical when digested with four additional enzymes. One of three sequential isolates from a patient, when cleaved with Eco R1 and Hind III, showed variable fragments in the terminal and joint regions of the S segment of the genome. The appearance of the fragments was not due to the addition of a known restriction site. We conclude that exogenous reinfection with a genetically distinct strain of herpes simplex virus type 2 (HSV-2) did not occur among these 15 patients with recurrent genital HSV infections.

Herpes simplex burn wound infections: epidemiology of a case cluster and responses to acyclovir therapy.

Nosocomial transmission of herpes simplex virus (HSV) has been described in intensive care units. A cluster of three patients with HSV wound infections within a 6-week period prompted temporary closure of a burn unit and suggested nosocomial cross infection. However, restriction endonuclease "fingerprint" analysis of the HSV isolates showed them to be genetically and therefore epidemiologically unrelated. This report describes these cases and the use of intravenous acyclovir in the treatment of HSV burn wound infections.

Effect of cloned human interferons on the replication of and cell fusion induced by herpes simplex virus.

Human alpha- and beta-interferons, expressed from cloned genes, block the replication of, and cell fusion induced by herpes simplex viruses. This inhibition is neutralized by antiserum to interferon and demonstrates species specificity. The block in replication appears to be late in the replication cycle of herpes simplex virus, since similar levels of viral DNA are synthesized in both interferon-treated and untreated cells.

Monoclonal antibodies for rapid diagnosis and typing of genital herpes infections during pregnancy.

Fluorescein-conjugated, type-specific monoclonal antibodies to herpes simplex virus (HSV-1 and HSV-2) and group-specific antibody which recognizes both HSV-1 and HSV-2 were used to detect HSV-infected cells in clinical specimens. Specimens were collected from 66 pregnant women with a past history of herpes genitalis or with suspected lesions. Fourteen of 18 samples from which virus was isolated were positive by immunofluorescence test; four of 18 specimens had an insufficient number of cells (less than 50 per smear) for analysis. In one specimen, a positive reaction by immunofluorescence was not confirmed by virus isolation. HSV was typed directly on smears of clinical specimens in 14 instances: three samples were identified as HSV-1, and 11 as HSV-2. Moreover, the immunofluorescence test was used to type HSV isolates in cell cultures in these cases and 40 additional strains. Examination of viral deoxyribonucleic acid fragments obtained by restriction enzyme digestion confirmed the typings; complete agreement was found among the three methods. Immunofluorescence with monoclonal antibodies is a reliable test for rapid diagnosis and simultaneous typing of genital HSV infections, even in asymptomatic women. With adequate specimens, the specificity and sensitivity of this method approach 100%.

Use of restriction enzymes to investigate the source of a primary cytomegalovirus infection in a pediatric nurse.

The risk of transmission of cytomegalovirus (CMV) infection from congenitally infected infants to nonimmune medical attendants is unknown. The case of a CMV-seronegative, pregnant nurse who seroconverted after caring for an infant with symptomatic CMV infection is reported. She elected to be aborted and the fetal tissue contained CMV. Isolates from the nurse, the fetal tissue, and the infant to whom the nurse was exposed were examined for genetic relatedness by restriction enzyme analysis. As expected, the isolates from the nurse and the fetal tissue were identical. However, the virus isolated from the symptomatic infant was different from the strain infecting the nurse. These data indicate that the nurse acquired her infection from a source other than the index infant, either within the hospital or within the community.

Size of infectious DNA from human and murine cytomegaloviruses.

Viral DNA was isolated from human and murine cytomegalovirus by equilibrium centrifugation in cesium chloride gradients. The size of the DNA was measured relative to T4 DNA by velocity sedimentation in neutral glycerol gradients, and fractions were assayed for infectious DNA. Infectious murine cytomegalovirus DNA sedimented as a single peak with an estimated molecular weight of 136 X 10(6). Infectious human cytomegalovirus DNA was detected in two peaks with molecular weights of 130 X 10(6) and 150 X 10(6).

Genetic analysis of a cytomegalovirus-like agent isolated from human brain.

An unusual cytomegalovirus (CMV, strain Colburn) isolated from brain biopsy of a boy with clinical encephalopathy was studied for genetic relatedness to human and simian CMV. Cross-examination of the purified viral DNA by DNA-DNA reassociation kinetics analyses showed more than 90% homology between Colburn virus and simian CMV (strain GR2757) and a lack of detectable homology between Colburn virus and human CMV (strains AD-169 and TW-87). Restriction endonuclease analysis of Colburn DNA showed some similarity of the DNA fragment pattern with that of simian CMV DNA, although the DNA fragment patterns were not identical, and showed no similarity to that of human CMV DNA. The molecular size and density of viral DNA were close to those of simian CMV DNA. The antigenic study, as performed by complement fixation and neutralization tests, showed strong cross-reactivity of Colburn virus to simian GR2757 virus. One-way cross-reaction of Colburn virus to several human CMV isolates (AD-169, Davis, and Town) was detected by complement fixation; this one-way cross-reaction was not obvious in a plaque neutralization test. It was concluded that Colburn is a simian CMV-related virus.

Experimental infection of marmosets with a cytomegalovirus of human origin.

Two adult and two neonatal cotton-topped marmosets and two neonatal white-lipped marmosets (Saguinus species) were inoculated with 10(7) plaque-forming units of cytomegalovirus (Colburn strain). No overt clinical disease developed in four marmosets during observation for eight months; one adult and one neonatal cotton-topped marmoset died from nonspecific causes 63 and 259 days after inoculation, respectively. By days 7-16 after inoculation, all marmosets developed plasma antibodies, which were detectable by neutralization and immunofluorescence assays (peak titers, 1:128-1:256 and 1:64-1:256, respectively). Attempts to isolate virus from whole blood, peripheral lymphocytes, oropharyngeal swabs, or vaginal swabs by cocultivation with permissive cell cultures were unsuccessful. Virus was recovered, however, by cocultivation from the kidney tissues of the adult marmoset that died. Immunosuppressive treatment with azathioprine resulted in a fourfold increase in antibody levels in plasma of two of three marmosets.