Centrifugal enhancement of retroviral mediated gene transfer.

Centrifugation has been used for many years to enhance infection of cultured cells with a variety of different types of viruses, but it has only recently been demonstrated to be effective for retroviruses (Ho et al. (1993) J. Leukocyte Biol. 53, 208-212; Kotani et al. (1994) Hum. Gene Ther. 5, 19-28). Centrifugation was investigated as a means of increasing the transduction of a retroviral vector for gene transfer into cells with the potential for transplantation and engraftment in human patients suffering from genetic disease, i.e., gene therapy. It was found that centrifugation significantly increased the rate of transduction into adherent murine fibroblasts and into non-adherent human hematopoietic cells, including primary CD34+ enriched cells. The latter samples include cells capable of reconstitution of hematopoiesis in myeloablated patients. As a step toward optimization of this method, it was shown that effective transduction is: (1) achieved at room temperature; (2) directly related to time of centrifugation and to relative centrifugal force up to 10,000 g; (3) independent of volume of supernatant for volumes > or = 0.5 ml using non-adherent cell targets in test tubes, but dependent upon volume for coverage of adherent cell targets in flat bottom plates; and (4) inversely related to cell numbers per tube using non-adherent cells. The results support the proposal that centrifugation increases the reversible binding of virus to the cells, and together with results reported by Hodgkin et al. (Hodgkin et al. (1988) J. Virol. Methods 22, 215-230), these data support a model in which the centrifugal field counteracts forces of diffusion which lead to dissociation during the reversible phase of binding.

Reactivation of Epstein-Barr virus during early infection with human immunodeficiency virus.

Reactivation of Epstein-Barr virus (EBV) in early human immunodeficiency virus (HIV) infection was investigated in 49 homosexual men who seroconverted to HIV (cases) as compared with 49 matched controls who remained seronegative to HIV during a longitudinal study. EBV infection was reactivated in cases 6 months, but not 12 months, prior to HIV seroconversion as compared with controls and remained reactivated during 18 months of follow-up after HIV seroconversion, as shown by increases in immunoglobulin (Ig) G antibody titers to EBV early antigen. Antibody titers to EBV viral capsid antigen did not differ between cases and controls prior to the time of seroconversion to HIV but were significantly increased among cases by the first seropositive study visit and remained elevated during the 18 months after HIV seroconversion. Total serum IgG levels were increased in cases at the visit of seroconversion, and during 18 months of follow-up, but did not correlate with enhanced IgG production specific for EBV antigens. Significant decreases in numbers of CD4+ cells and increases in numbers of CD8+ cells during this early phase of HIV infection were not associated with changes in patterns of EBV antibody responses. Reactivation of EBV beginning 6 months before HIV seroconversion may have implications regarding the role of this herpesvirus in the pathogenesis of HIV.

Epstein-Barr virus transmission via the donor organs in solid organ transplantation: polymerase chain reaction and restriction fragment length polymorphism analysis of IR2, IR3, and IR4.

Two organ transplant recipients who received organs from a common donor and were diagnosed as having an Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorder were studied to determine the mode of EBV transmission. The results of restriction fragment length polymorphism, polymerase chain reaction, and minisatellite DNA analyses demonstrate that both patients had a common strain of EBV and that this strain was transmitted from the donor's organs to both recipients. Posttransplant lymphoproliferative disorder resulted from the proliferation of EBV-immortalized B lymphocytes of the recipient, not those of the donor.

Enhanced antibody responses to Epstein-Barr virus in HIV-infected homosexual men.

We investigated the association between human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) infections in 593 homosexual men. The status of EBV infection in this group was evaluated based on serological evidence of EBV-specific antibody responses. The geometric mean titers (GMT) of antibody to EBV capsid antigen (EBV-VCA) (1:154) and EBV early antigen (EA) (1:16) in 141 HIV-seropositive men were significantly higher than respective titers in 452 HIV seronegative men (1:95 and 1:12). Antibody titers to EBV were higher in HIV-infected men with lymphadenopathy than in asymptomatic HIV-seropositive men. However, these correlation were less evident in patients with AIDS-related complex. Elevated antibody titers to EBV were found to be independent of levels of total serum IgG. Cytomegalovirus (CMV) antibody titers were also found to be significantly increased among HIV-seropositive men, independent of total IgG. Antibody titers to EBV were not correlated with those to CMV in either HIV-seronegative or HIV-seropositive men. Subjects without evidence of HIV infection, but who had high antibody titers to EBV-VCA and EBV-EA, had elevated mean numbers of CD3+, CD4+, and CD8+ cells, and lower levels of CD4+/CD8+ cell ratios compared to subjects with low EBV-antibody titers. This study suggests that the elevated levels of circulating antibodies against EBV in homosexual men are associated with loss of control of latent EBV due to HIV infection.

A new in vivo anti-viral assay using microencapsulated infected cell cultures.

Microencapsulation technology makes it possible to encapsulate virus infected human or animal cells in microcapsules with semipermeable membranes. These may be implanted intraperitoneally into mice which may then be treated with antiviral drugs. The implanted microcapsules may be recovered at various intervals following in vivo treatment and the effect of the drug is evaluated by assaying the virus titers inside the microcapsules. In this paper, the feasibility of this model was tested using microencapsulated human or non-human cells infected with herpes simplex virus type 1. The microcapsules were implanted in the peritoneal cavity of mice, and the effect of systematically administered acyclovir on HSV-1 replication was ascertained. We found that (a) HSV-1 can replicate in both human (A549 and FEMx) and non-human (Vero) cells after they are infected and encapsulated. (b) HSV-1 replication was inhibited by 0.005 microgram/ml to 0.08 mg/ml of acyclovir in the medium when virus producing A549 cells were encapsulated or when they were in monolayers. (c) Acyclovir (20-80 mg/kg), injected twice daily by intraperitoneal, subcutaneous or intravenous routes in mice, significantly inhibited HSV-1 production in encapsulated Vero cells implanted in the peritoneal cavity. The major advantage of this in vivo model is that it can be used to study antivirals in experimental animals in which viruses do not replicate in non-permissive animals. Toxicity, pharmacokinetic and efficacy data may be obtained. It can also be used to test drugs which require activation in vivo to be effective.

The frequency of Epstein-Barr virus infection and associated lymphoproliferative syndrome after transplantation and its manifestations in children.

Twenty cases of Epstein-Barr virus (EBV)-associated lymphoproliferative syndrome (LPS), defined by the presence of EBV nuclear antigen and/or EBV DNA in tissues, were diagnosed in 1467 transplant recipients in Pittsburgh from 1981-1985. The frequency of occurrence in pediatric transplant recipients was 4% (10/253), while in adults it was 0.8% (10/1214) (P less than .0005). The frequency of LPS in adults declined after 1983 coincidental with the introduction of cyclosporine monitoring. However there was no apparent decline of LPS in children. We describe these ten pediatric cases and one additional case of LPS in a child who received her transplant before 1981. The frequency of EBV infection in 92 pediatric liver recipients was 63%. Of these subjects, 49% were seronegative and 77% of those acquired primary infection. Of 11 cases of pediatric EBV-associated LPS, 10 were in children who had primary infection shortly before or after transplantation. These results reinforce the importance of primary EBV infection in producing LPS, which was previously shown in adults. Children are at greater risk because they are more likely to be seronegative for EBV and to acquire primary infection. Three clinical types of LPS were recognized in children. The first (5 cases) was a self-limited mononucleosislike syndrome. The second syndrome (4 cases) began similarly, but then progressed over the next two months to widespread lymphoproliferation in internal organs and death. The third type (2 cases) was an extranodal intestinal monoclonal B cell lymphoma, occurring late after primary infection.

Association of HTLV-III with Epstein-Barr virus infection and abnormalities of T lymphocytes in homosexual men.

Homosexual men were studied for associations among human T-lymphotropic virus type III (HTLV-III) infection, Epstein-Barr virus (EBV) infection, and T cell abnormalities. The presence of IgG antibody to EBV capsid antigen and antibody to EBV early antigen was significantly associated with augmented counts of suppressor T cells in healthy HTLV-III-seronegative men. HTLV-III-seropositive asymptomatic subjects had significantly enhanced titers of antibody to EBV and lower ratios of helper to suppressor T cells compared with HTLV-III-seronegative homosexual men. Of three men who seroconverted to HTLV-III, two had a greater than fourfold increase in titer of IgG antibody to EBV capsid antigen after seroconversion. These results suggest that the interaction of HTLV-III and EBV and their immunologic perturbations are significant in the natural history of this retrovirus infection in homosexual men.

Epstein-Barr virus infections and DNA hybridization studies in posttransplantation lymphoma and lymphoproliferative lesions: the role of primary infection.

Fourteen patients who developed B cell lymphomas or lymphoproliferative lesions after kidney, liver, heart, or heart-lung transplantation in Pittsburgh during 1981-1983 had active infection with Epstein-Barr virus (EBV) of the primary (six patients), reactivated (seven patients), or chronic (one patient) type. In transplant patients without tumors, the incidence of EBV infection was 30% (39 of 128). Only three of these patients had primary infections. Thus the frequency of active infection was significantly higher in patients with tumors, and patients with primary infections were at greater risk of developing tumors. Five of 13 tumors tested contained EBV nuclear antigen (EBNA) and nine of 11 contained EBV genomes detected by DNA-DNA hybridization with BamHI K, BamHI W, or EcoRI B cloned probes. All EBNA-positive tumors, except one, were also positive by hybridization. Only one tumor was negative for both EBNA and EBV DNA. These data suggest that EBV plays an etiologic role in the development of these lesions.

Stability and utility of the unique human small cell carcinoma line SHP-77.

The human small cell (oat cell) carcinoma line, SHP-77, established by Fisher and Paulson in 1977 and originally described as a "large cell variant of oat cell cancer" has been evaluated by several different parameters and shown even after more than 200 passages to retain properties described for the original cell line. Karyotypic, histological, and biochemical features are retained, as well as tumorigenicity in nude mice. The original authors' suggestion that this is a propitious cell line for both in vitro and in vivo studies is supported by this report. Modulation of growth characteristics in vivo (in xenografts) emphasizes the plasticity of this unique line which serves as a valuable model for basic as well as therapeutic studies. SHP-77 can serve as an in vitro target in 51Cr and 111In release cytotoxicity assays as well as in in vivo nude mouse assays for evaluating immune reactivity of cells and serum from lung cancer patients. The potential histological variability of SHP-77, despite its biochemical stability, calls attention to the inadequacy of histological criteria for lung tumor classification.

Epstein-Barr virus-induced lymphoma in a cardiac transplant recipient.

A monoclonal diffuse histiocytic lymphoma developed during the course of a serologically documented primary Epstein-Barr virus infection in a 22-year-old cardiac transplant recipient taking cyclosporine and prednisone. Throat washings revealed the virus at tumor presentation, and the tumor was shown to contain Epstein-Barr nuclear antigen-positive cells and the viral genome. Prolonged inversion of the T cell helper/suppressor ratio was demonstrated. A brief course of acyclovir appeared to halt viral shedding in the throat but had no apparent effect on the tumor.

Fatal Epstein-Barr virus infection in a 63-year-old man. An autopsy report.

A 63-year-old man with acute, heterophil-negative Epstein-Barr (EBV) viral infection displayed neurologic impairment that progressed to coma and death. Fever, pharyngitis, and lymphadenopathy were notably absent. There was no lymphocytosis, and multiple peripheral smears revealed few atypical lymphocytes. Results of specific EBV serology were diagnostic of acute infection. At the time of autopsy, there was massive intravascular and perivascular infiltration of all organs by lymphocytes and atypical mononuclear cells. There was depletion of the paracortical T-lymphocyte areas of lymph nodes. The atypical mononuclear cells did not contain intracytoplasmic immunoglobulin, as shown by the immunoperoxidase technique, nor did they take up esterase stains, but their electron-microscopic features were characteristic of lymphoid cells. These morphologic findings suggest a T-cell defect, with unrestricted proliferation of B lymphocytes. The lack of characteristic clinical and hematologic features in this case underscores the value of specific EBV serology in the diagnosis of acute heterophil-negative EBV infection.

Adenovirus and ileocecal intussusception.

Intranuclear inclusion bodies were found by light microscopy in epithelial cells in more than one-third of the specimens from children operated on for ileocecal intussusception. Electron microscopic examination done on hematoxylin and eosin-stained slides showed the intranuclear inclusion bodies to be composed of viral particles in large and small crystalline arrays. Adenovirus of serotypes 2, 3, and 5 were isolated from the five cases with inclusions in which isolation was attempted. These findings strongly suggest a pathogenetic role for adenovirus in those cases of intussusception in which intranuclear inclusion bodies are found in the epithelial cells of the appendix or the terminal ileum.

Paracrystal formation in cell cultures infected with adenovirus type 2.

Large rod-shaped structures corresponding to paracrystals were seen in the nucleus, cytoplasm, or both of adenovirus type 2 (Ad2)-infected cells by immunofluorescence staining with antibody prepared against purified Ad2. In exception to this, Ad2-induced crystals did not stain with either hexon or fiber antibody. The crystalline structures were first observed in Ad2-infected Vero cells at 28 hr with a maximum number at 70 hr postinoculation. The kinetics of paracrystalline formation closely paralleled the experimental synthesis of infectious progeny virus. Acridine-orange staining revealed the lack of nucleic acids associated with the crystal. Also, the paracrystals stained intensely with phenanthrenequinone, suggesting that they are composed of basic proteins. Interferon induced by Newcastle disease virus from African green monkey kidney cell cultures was used to pretreat Vero cells prior to Ad2 infection. This resulted in inhibiting the formation of viral-induced paracrystals in 97% of the cells and reduced virus yields by 95%. The African green monkey kidney cell culture interferon did not reduce Ad2 yields in HeLa cell cultures or display any virus inhibitory activity in rabbit kidney cell cultures. Staining procedures, fluorescent-antibody tests with whole virus, hexon or fiber antibody, and interferon studies suggested that the paracrystals were viral-directed and composed of basic proteins (possibly core proteins).