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.

HLA heterogeneity of insulin-dependent diabetes mellitus at diagnosis. The Pittsburgh IDDM study.

Although some previous studies have suggested that insulin-dependent diabetes mellitus (IDDM) is a heterogeneous condition with variant forms being associated with HLA-DR types, the evidence, thus far, is conflicting. To address this issue, we have examined the presenting characteristics of a consecutive admission series of 200 newly diagnosed cases of IDDM from the Children's Hospital of Pittsburgh. Because HLA-DR frequencies vary by race, data are presented only for the 172 white cases with complete HLA-DR typing. HLA-DR3 was found more frequently among male cases and DR4 among female cases (P less than 0.005). Generally, patients with DR4 presented with a severer clinical picture, being more likely to have impaired consciousness and significant dehydration. In addition, patients with DR4 were more likely to be acidotic, ketotic, and to more frequently report a recent viral infection. This latter finding was supported by a greater frequency of antibodies to Coxsackie-B viruses in the DR4 cases at presentation. These results therefore suggest that there is considerable heterogeneity in IDDM, at least in presenting characteristics, according to HLA-DR type.

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.

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.

Analyses on possible heterogeneity of IDDM based on presence of islet cell cytoplasmic antibody at diagnosis.

In a large, representative sample of newly-diagnosed IDDM patients, using a highly sensitive assay to detect islet cell cytoplasmic antibodies (ICA), no marked differences were found between ICA+ and ICA- patients on various clinical, genetic, immunologic, and epidemiologic characteristics. In particular, there was no evidence for associations between ICA status at diagnosis and either sex, race, family history of IDDM, HLA-DR phenotype, antibody titers to Coxsackie B viruses, immunoglobulin levels, C-peptide and glycosylated hemoglobin concentrations, or insulin requirements. The most significant relationship was between the presence of ICA and a young age at diagnosis; however, the large overlap between the distributions of the ages at onset for ICA+ and ICA- groups on this variable suggests that this association is of limited importance. These data suggest that the presence or absence of ICA at diagnosis may not be useful in defining possible subtypes of IDDM.

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.

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.

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.

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).

Epidemiological and immunological studies of Cryptococcus neoformans.

Walter, Jinks E. (University of Pittsburgh, Pittsburgh, Pa.), and Robert W. Atchison. Epidemiological and immunological studies of Cryptococcus neoformans. J. Bacteriol. 92:82-87. 1966.-The complement-fixation fluorescent-antibody test provided a means of differentiating between antibodies of Cryptococcus neoformans and Candida albicans. The test was applied to the sera of 134 pigeon fanciers for detection of antibodies to C. neoformans only. About 22% were positive as compared with 3% of a control group composed of 36 non-pigeon breeders. Positive reactions were observed only with C. neoformans types A and B cells. It was concluded that the pigeon fanciers had presumably been infected previously with C. neoformans type A or type B. Moreover, 48 of 49 isolates of C. neoformans cultured from the pigeon habitats of 72 fanciers studied were serotype A. These findings would seem to substantiate the hypothesis that pigeon habitats serve as reservoirs for human infections, and also that subclinical cryptococcosis is more prevalent than is realized.

Echovirus hepatic failure in infancy: report of four cases with speculation on the pathogenesis.

Disseminated echovirus infection with fulminant hepatic failure occurs almost exclusively in newborns. Although a relatively uncommon condition, it is on occasion associated with neonatal death accompanied by diffuse and extensive hemorrhagic necrosis of the liver and adrenals as the defining finding. We report four cases of severe systemic neonatal echovirus infection and present histologic and clinical evidence to demonstrate the two histologic patterns of liver involvement; intravascular coagulation in the early clinical course and a veno-occlusive component in later stages of the disease. Viral damage to vascular endothelium and hepatic venous endothelium by a "hit-and-run" process in the early viremic phase rather than direct hepatocyte injury is postulated to be a mechanism.

Slide method for detection of antibody-coated bacteria in urine sediments.

An immunofluorescent slide method incorporating 0.1% (wt/vol) Evans blue as a counterstain was developed and compared with a recently described tube method. Seventy-one urine specimens were tested concurrently by both methods. Results of the two methods agreed in 70 specimens and disagreed in only 1. We found the slide method to be less costly and time consuming than the tube method.

Containment of hepatitis B virus infection in a hemodialysis unit.

The unsuspected introduction of a carrier of hepatitis B virus into a hepatitis-free hemodialysis unit coincided with a routine serologic survey in early April 1976. Thus the prevalence of hepatitis B surface antigen and its antibody was known for the 42 patients and 23 personnel at risk. Control consisted of isolating the patient identified as a carrier seven days after admission. Immune globulin was not given to potentially exposed persons. During a 15-month period, only one case of icteric hepatitis B occurred, in a nurse who was probably infected through defective gloves while attending the carrier in isolation. Monthy serologic tests showed that none of the other personnel and patients became infected with hepatitis B virus. This limited spread of hepatitis B indicated that isolation of the carrier was an effective preventive measure in given setting.

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.

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.

Effect of urea on the hemagglutinating and complement-fixing antigens of type 2 Dengue virus.

Sephadex G-200 filtration was used for fractionating dengue type 2 (DEN-2)-infected suckling mouse brain (SMB) supernatant fluids. The high-molecular-weight fraction, which was eluted in the void volume, showed no increase in type specificity when tested against immune ascitic fluid to the four prototype dengue viruses. A void-volume fraction obtained after the infected SMB supernatant fluids were treated with urea displayed significant increases in complement fixation (CF) type specificity. Immune ascitic fluid prepared against the more typespecific DEN-2 antigen demonstrated neutralizing ability and greater CF type specificity. When DEN-2 sucrose-acetone-extracted hemagglutinating (HA) antigens were treated with 6 m urea at 37 C for various time intervals, all HA antigen was destroyed in 15 min. Urea treatment of infected SMB supernatant fluids indicated that the CF antigens were more stable to the effect of urea than were the HA antigens. After urea treatment of the SMB supernatant fluids, the CF type specificity increased as the hemagglutination inhibition titer decreased.

Evaluation of anti-complement immunofluorescence test in cytomegalovirus infection.

The anti-complement immunofluorescence (ACIF) technique was evaluated for the diagnosis of human cytomegalovirus (CMV) infection in a group of sera derived from renal transplant recipients and donors by comparing it with the indirect immunofluorescence (FA) and complement fixation (CF) TESTS. The ACIF and FA tests yielded similar results. However, the ACIF test had a distinct advantage over the indirect FA test, since it eliminated the nonspecific cytoplasmic staining that may result in false positive readings in inexperienced hands. Both the indirect FA and ACIF tests were more sensitive than the CF test. In primary CMV infection, the FA and ACIF antibodies appeared earlier and had significantly higher titer than corresponding CF titers. This difference in titers was not seen in seropositive individuals who lacked overt infection. Our previously reported correlation between the seropositivity of the donor and CMV infection in seronegative recipients has been confirmed.

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.

The development of Type 1 (insulin-dependent) diabetes mellitus: two contrasting presentations.

Genetic, immunological and viral factors have been implicated in pathogenesis of Type 1 diabetes mellitus. The development of Type 1 diabetes in two siblings of patients with Type 1 diabetes studied as part of a large epidemiological study, is described. One case, a 13-year-old male not sharing either HLA haplotype with his diabetic sister, had virtually normal glucose tolerance 80 days before symptomatic presentation. He showed serological evidence of infection by Coxsackie CB4 (at diagnosis) and influenza A virus (soon after diagnosis). The other case, a 15-year-old male, had impaired glucose tolerance for over 500 days (i.e., since the diagnosis of diabetes in his HLA-identical brother) before symptomatic presentation which was not associated with serological evidence of acute viral infection. The former case was negative for islet cell antibody (cytoplasmic) when first seen though positive at diagnosis, while the latter was positive throughout. These two cases suggest contrasting interactions of the main pathogenetic factors associated with Type 1 diabetes.