JC virus multiplication in human hematopoietic progenitor cells requires the NF-1 class D transcription factor.

JCV, a small DNA virus of the polyomavirus family, has been shown to infect glial cells of the central nervous system, hematopoietic progenitor cells, and immune system lymphocytes. A family of DNA binding proteins called nuclear factor-1 (NF-1) has been linked with site-coding specific transcription of cellular and viral genes and replication of some viruses, including JC virus (JCV). It is unclear which NF-1 gene product must be expressed by cells to promote JCV multiplication. Previously, it was shown that elevated levels of NF-1 class D mRNA were expressed by human brain cells that are highly susceptible to JCV infection but not by JCV nonpermissive HeLa cells. Recently, we reported that CD34(+) precursor cells of the KG-1 line, when treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA), differentiated to cells with macrophage-like characteristics and lost susceptibility to JCV infection. These studies have now been extended by asking whether loss of JCV susceptibility by PMA-treated KG-1 cells is linked with alterations in levels of NF-1 class D expression. Using reverse transcription-PCR, we have found that PMA-treated KG-1 cells express mRNA that codes for all four classes of NF-1 proteins, although different levels of RNA expression were observed in the hematopoietic cells differentiated into macrophages. Northern hybridization confirms that the expression of NF-1 class D gene is lower in JCV nonpermissive PMA-treated KG-1 cells compared with non-PMA-treated cells. Further, using gel mobility shift assays, we were able to show the induction of specific NF-1-DNA complexes in KG-1 cells undergoing PMA treatment. The binding increases in direct relation to the duration of PMA treatment. These results suggest that the binding pattern of NF-1 class members may change in hematopoietic precursor cells, such as KG-1, as they undergo differentiation to macrophage-like cells. Transfection of PMA-treated KG-1 cells with an NF-1 class D expression vector restored the susceptibility of these cells to JCV infection, while the transfection of PMA-treated KG-1 cells with NF-1 class A, B, and C vectors was not able to restore JCV susceptibility. These data collectively suggest that selective expression of NF-1 class D has a regulatory role in JCV multiplication.

Detection of JC virus DNA in human tonsil tissue: evidence for site of initial viral infection.

Progressive multifocal leukoencephalopathy is a demyelinating disease of the human central nervous system that results from lytic infection of oligodendrocytes by the polyomavirus JC (JCV). Originally, JCV was thought to replicate exclusively in human glial cells, specifically oligodendrocytes. However, we have recently shown that JCV can replicate in cells of lymphoid origin such as hematopoietic precursor cells, B lymphocytes, and tonsillar stromal cells. To determine whether tonsils harbor JCV, we tested a total of 54 tonsils, 38 from children and 16 from adult donors. Nested PCRs with primer sets specific for the viral T protein and regulatory regions were used for the detection of JCV DNA. JCV DNA was detected in 21 of 54 tonsil tissues, or 39% (15 of 38 children and 6 of 16 adults) by using regulatory-region primers and in 19 of 54 tonsil tissues, or 35% (13 of 38 children and 6 of 16 adults) by using the T-protein primers. The DNA extracted from children's nondissected tonsil tissue, isolated tonsillar lymphocytes, and isolated stromal cells that demonstrated PCR amplification of the JCV regulatory region underwent cloning and nucleotide sequencing. Of the regulatory-region sequences obtained, nearly all contained tandem repeat arrangements. Clones originating from nondissected tonsil tissue and tonsillar lymphocytes were found to have sequences predominantly of the Mad-1 prototype strain, whereas the majority of clones from the DNA of tonsillar stromal cells had sequences characteristic of the Mad-8br strain of JCV. A few clones demonstrated structures other than tandem repeats but were isolated only from tonsillar lymphocytes. These data provide the first evidence of the JCV genome in tonsil tissue and suggest that tonsils may serve as an initial site of viral infection.

Evaluation of the role of cytokine activation in the multiplication of JC virus (JCV) in human fetal glial cells.

The human polyomavirus, JCV, is the etiologic agent of the fatal central nervous system demyelinating disease, progressive multifocal leukoencephalopathy. Progressive multifocal leukoencephalopathy occurs most frequently in patients with underlying immunosuppressive disorders and is the direct result of virus multiplication in oligodendrocytes, the myelin producing cell in the central nervous system. In this report we test the ability of cellular activation signals to modulate expression of the JCV genome in either transfected or infected human fetal glial cells. In addition, we analyze the binding of nuclear proteins isolated from untreated and cytokine treated human fetal glial cells to transcription factor binding sites in the JCV regulatory region. In contrast to the effects of cellular activation on the expression of the HIV-1 promoter in these cells, none of the cellular activators tested increased expression of JCV. The cytokine, TNF-alpha, increased binding of NF kappa B (p50/p65) to a JC NF kappa B site but did not modulate the binding of nuclear proteins to the overlapping NF-1/AP1 region of the JCV enhancer. When taken together these results suggest that the response of JCV to cellular activation signals may be fundamentally different from the response of HIV-1 to these signals in human fetal glial cells and that the JC NF kappa B site may not be required for JCV gene expression or multiplication in vivo.

Radiographic recommendations for the primary dentition: comparison of general dentists and pediatric dentists.

A survey which included brief case histories and intraoral photos of four primary dentitions ranging from healthy to severely carious was mailed to a random sample of 2000 general dentists and 1000 pediatric dentists. Radiographic options were listed, from which the dentist was to indicate all films needed for each child's examination. Surveys were received from 1273 (43%) dentists, including 713 (36%) general dentists and 560 (56%) pediatric dentists. The pediatric dentists recommended significantly more diagnostic radiographs than did the general dentists across all four primary dentition cases. This trend was apparent in the absence of clinically visible caries. When radiographs were recommended, bite-wing radiographs were the most frequently ordered films. The most frequently ordered combination among all respondents was bite-wing radiographs plus anterior periapical films. The results suggest that, frequently, neither general dentists nor pediatric dentists prescribe radiographs for the primary dentition patient that conform to the USDHHS guidelines for radiographic examination (1987).

Radiographic recommendations for the transitional dentition: comparison of general dentists and pediatric dentists.

A survey which included brief case histories and intraoral photos of four transitional dentitions, including examples of ectopic and delayed eruption, as well as carious lesions, was mailed to a random sample of 2000 general dentists and 1000 pediatric dentists. Radiographic options were listed, from which each dentist was to indicate all films needed for each child's examination. Surveys were returned by 1273 (43%) dentists, including 713 (36%) general dentists and 560 (56%) pediatric dentists. The pediatric dentists took significantly more diagnostic radiographs than did the general dentists for each of the four transitional dentition cases. Pediatric dentists were more likely than the general dentists to take panoramic films and combinations which included panoramic films, bite-wing radiographs and periapical films. The most frequently ordered combinations were bite-wing radiographs plus panoramic films and bite-wing radiographs plus anterior periapical films. General dentists recommended bite-wing radiographs films only more frequently than did pediatric dentists. In view of the results of this study and the USDHHS guidelines for radiographic examinations, (1987) education must be provided for both general dentists and pediatric dentists regarding appropriate radiographic examinations for transitional dentition patients.

Antibody response to teichoic acid and peptidoglycan in Staphylococcus aureus osteomyelitis.

An enzyme-linked immunosorbent assay was used to evaluate the immunoglobulin G (IgG) response to Staphylococcus aureus crude teichoic acid (TA) and peptidoglycan (PG) in both rabbits and patients with osteomyelitis. In rabbits with experimental S. aureus osteomyelitis, elevated levels of IgG to TA were present in 13/18 (72%) of the serum samples obtained at 4 and 10 weeks postinfection. In contrast, only 5/18 (28%) of these sera were found to be positive for antibodies to PG. Of a total of 39 patients with confirmed S. aureus osteomyelitis (11 acute, 28 chronic), IgG to TA was elevated in 17 (44%), whereas antibodies to PG were found to be increased in only 1 (3%). Cross-reacting antibodies to S. aureus TA were detected in only 1/18 (6%) of the patients with osteomyelitis caused by organisms other than S. aureus. These studies indicate that IgG to TA is more prevalent than IgG to PG in patients with staphylococcal osteomyelitis. Although these results are encouraging, a larger number of patients is required for an adequate evaluation of the TA enzyme-linked immunosorbent assay for the diagnosis and management of suspected S. aureus osteomyelitis.

Enzyme-linked immunosorbent assay for detection of antibodies to Staphylococcus aureus cell walls in experimental osteomyelitis.

An enzyme-linked immunosorbent assay was used to evaluate and compare the immunoglobulin G antibody response to Staphylococcus aureus cell walls of rabbits with either chronic staphylococcal osteomyelitis or subcutaneous abscesses. Osteomyelitis of the femur was produced by the intramedullary application of a sclerosing agent (3% sodium tetradecyl sulfate) and S. aureus. Radiographic evidence of osteomyelitis was observed in 10 of the 13 animals that survived the 10-week experimental period, and the diagnosis was confirmed by histopathology in 8 of the 10 instances. Abscess formation was initiated in a separate group of rabbits by the subcutaneous injection of S. aureus cells. All 10 of these rabbits subsequently developed abscesses, which usually resolved spontaneously within 3 to 5 weeks. Elevated levels of immunoglobulin G antibodies to the cell wall antigen were detected in 7 of 10 rabbits with osteomyelitis at 21 days postinfection, and these animals continued to display high antibody levels even at 59 days postinfection. In contrast, elevated levels of anti-cell-wall antibodies were only detected in 1 of 10 rabbits with subcutaneous abscesses. The enzyme-linked immunosorbent assay was found to be a rapid and sensitive serological technique for the detection of cell wall antibodies in this experimental osteomyelitis model and may be useful for the diagnosis of staphylococcal bone infections in humans.