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Biomedical subjects

E O Major

Publications and source records attributed to E O Major.

At least 73 records · Page 4Linked to original sources
PubMed1992

Detection of JC virus DNA in peripheral lymphocytes from patients with and without progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) results from lytic infection of oligodendrocytes by JC virus (JCV). Although JCV has been identified in mononuclear cells in bone marrow and hematogenous dissemination of the virus to the central nervous system has been suspected, JCV has never been clearly demonstrated in the peripheral circulation. Using polymerase chain reaction technology, we examined peripheral lymphocytes of 19 patients with brain biopsy-proven PML for the JCV genome. Two non-PML control groups, consisting of 26 patients seopositive for human immunodeficiency virus type 1 (HIV-1) and 30 immunocompetent patients with Parkinson's disease, were also examined for the presence of the JCV genome in lymphocytes. Cerebrospinal fluid from 10 patients with PML was examined for the presence of the JCV genome as well. The JCV genome was detected in the lymphocytes of 89% (17) of the patients with PML, 38% (10) of the HIV-1-seropositive patients without PML, and none of the patients with Parkinson's disease. Sequencing of the JCV regulatory region from the lymphocytes of three patients revealed the prototype MAD-1 strain of JCV in one patient with PML, a MAD-4 strain in a second patient with PML, and a slightly modified MAD-4 strain in an HIV-1-positive patient without PML. Only 3 of 10 patients with PML who had JCV detected in lymphocytes had the JCV genome in their cerebrospinal fluid. These results demonstrate that the JCV genome can be found in circulating lymphocytes from patients with PML and suggest that lymphocytes are an important vector for hematogenous dissemination of JCV to the central nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Lack of JC viral genomic sequences in multiple sclerosis brain tissue by polymerase chain reaction.

With DNA extracted from brain specimens from 19 multiple sclerosis, 5 progressive multifocal leukoencephalopathy, 1 Alzheimer's disease, and 8 nonneurological control subjects, polymerase chain reaction was performed using nested sets of primer pairs amplifying segments of the large T and VP1 antigen-encoding sequences of JC virus. Both sequences were detected in each of the 5 brain specimens of progressive multifocal leukoencephalopathy but in none of the 19 multiple sclerosis, 1 Alzheimer's disease, or the 8 control brain specimens.

Base Sequence↗
PubMed1992

Interaction of the human polyomavirus, JCV, with human B-lymphocytes.

The human polyomavirus, JCV, is the causative agent of the central nervous system demyelinating disease progressive multifocal leukoencephalopathy (PML). The principal target of JCV infection in the central nervous system (CNS) is the myelinating oligodendrocyte. However, the site of JCV multiplication outside of the CNS and the mechanism by which virus gains access to the brain are not known. Recently, JCV infected B-lymphocytes have been demonstrated in PML patients in several lymphoid organs, in circulating peripheral lymphocytes, and in brain, suggesting a possible role of B-lymphocytes in the dissemination of virus to the brain. The experiments reported here were undertaken to understand more about the interactions of JCV with human B-lymphocytes. The data show that JCV is able to multiply in either Epstein-Barr virus transformed (EBV) or EBV negative human B cell lines resulting in production of infectious, progeny virions. In addition, nuclear proteins extracted from these B cells bind to similar nucleotides within the JCV regulatory region that are bound by nuclear proteins extracted from human fetal glial cells, the most susceptible host and principal target cell for JCV infection in vitro. It is not known, however, whether these DNA binding proteins from susceptible B cells and glial cells are similar.

B-Lymphocytes↗
PubMed1992

Progressive multifocal leukoencephalopathy in HIV-1-infected children.

OBJECTIVE: To describe the clinical and pathologic features of two HIV-1-infected children with progressive multifocal leukoencephalopathy (PML). DESIGN: Case report. SETTING: University-affiliated, public-health trust hospital. METHODS: Two HIV-1-infected children with PML are described. A 13-year-old girl, presumed to be congenitally infected with HIV-1, presented with dysarthria and paresthesias of the tongue and chin that evolved rapidly to dementia, muteness and severe spastic quadriparesis. The other patient, a 10-year-old boy who developed HIV-1 infection from a blood transfusion at the age of 3 years, presented with a facial palsy with subsequent development of right hemiparesis and aphasia. RESULTS: Brain biopsy in the first child and autopsy in the second confirmed the diagnosis of PML. In both patients, the CD4 T-lymphocyte count was less than 100 x 10(6)l at the time of neurological presentation. CONCLUSION: Despite seroepidemiological studies suggesting that the majority of individuals are infected with JC virus during childhood, PML is rare in children with impaired cell-mediated immunity. Our patients illustrate that PML is among the neurological complications of HIV-1 infection in children.

Adolescent↗
PubMed1992

Pathogenesis and molecular biology of progressive multifocal leukoencephalopathy, the JC virus-induced demyelinating disease of the human brain.

Studies of the pathogenesis and molecular biology of JC virus infection over the last two decades have significantly changed our understanding of progressive multifocal leukoencephalopathy, which can be described as a subacute viral infection of neuroglial cells that probably follows reactivation of latent infection rather than being the consequence of prolonged JC virus replication in the brain. There is now sufficient evidence to suggest that JC virus latency occurs in kidney and B cells. However, JC virus isolates from brain or kidney differ in the regulatory regions of their viral genomes which are controlled by host cell factors for viral gene expression and replication. DNA sequences of noncoding regions of the viral genome display a certain heterogeneity among isolates from brain and kidney. These data suggest that an archetypal strain of JC virus exists whose sequence is altered during replication in different cell types. The JC virus regulatory region likely plays a significant role in establishing viral latency and must be acted upon for reactivation of the virus. A developing hypothesis is that reactivation takes place from latently infected B lymphocytes that are activated as a result of immune suppression. JC virus enters the brain in the activated B cell. Evidence for this mechanism is the detection of JC virus DNA in peripheral blood lymphocytes and infected B cells in the brains of patients with progressive multifocal leukoencephalopathy. Once virus enters the brain, astrocytes as well as oligodendrocytes support JC virus multiplication. Therefore, JC virus infection of neuroglial cells may impair other neuroglial functions besides the production and maintenance of myelin. Consequently our increased understanding of the pathogenesis of progressive multifocal leukoencephalopathy suggests new ways to intervene in JC virus infection with immunomodulation therapies. Perhaps along with trials of nucleoside analogs or interferon administration, this fatal disease, for which no consensus of antiviral therapy exists, may yield to innovative treatment protocols.

Animals↗
PubMed1992

Improved electrochemiluminescent label for DNA probe assays: rapid quantitative assays of HIV-1 polymerase chain reaction products.

We describe the characterization and utility of a new electrochemiluminescent (ECL) label for oligonucleotides, utilizing phosphoramidite chemistry. This phosphoramidite of the tris(2,2-bipyridine)ruthenium(II) complex, bis(2,2-bipyridine)(4-[4-(2-cyanoethoxy-N,N-diisopropyl-amino) phosphinoxybutyl]4'-methyl)2,2-bipyridine ruthenium(II) dihexafluorophosphate or Origen phosphoramidite, enables the direct incorporation of the label during automated DNA synthesis. Efficiency of this automated synthesis allows the direct utilization of probes without further purification. Introduction of this labeling group is reproducible, and the ECL signal recovered is not influenced by hybridization. Furthermore, neither hybridization kinetics nor hybrid stability was affected by our label. We also demonstrate the utility of these labels for the development of rapid assays with oligonucleotides direct from automated synthesis. The clinical utility of these labeled oligonucleotides is shown with assays of total nucleic acid, extracted from peripheral blood lymphocytes of patients with acquired immunodeficiency syndrome (AIDS), to detect the human immunodeficiency virus (HIV-1). The results demonstrate the ability of the assay to quantify 30-2000 copies of HIV1 gag genes and to rapidly detect (less than 45 min) HIV-1 gag genes in a nonseparation assay. The application of this assay to clinical samples demonstrates the utility of these assays for rapid and quantitative analysis.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Human astrocytes stimulate HIV-1 expression in a chronically infected promonocyte clone via interleukin-6.

Human promonocyte cells chronically infected with human immunodeficiency virus type (HIV-1) (clone U1.1.5) were grown in the presence of media conditioned by human astrocytes and glioma cell lines U251 and 253. HIV-1 expression was assessed by measuring reverse transcriptase activity. All media conditioned by unstimulated and lipopolysaccharide (LPS) stimulated glial cells induced HIV-1 expression and contained detectable levels of interleukin-6 (IL-6) but not tumor necrosis factor-alpha (TNF-alpha). An antibody against IL-6, but not against TNF-alpha, reduced the induction of HIV-1 by the conditioned media in a concentration-dependent manner. The magnitude of HIV-1 induction by the conditioned media was proportional to the concentration of IL-6 in them. The data indicate that normal and transformed human astrocytes are capable of stimulating HIV-1 expression in chronically infected promonocytic cells by secreting IL-6. The results demonstrate that cytokines secreted by neural cells could play an important role in regulating HIV-1 expression in the brain.

Astrocytes↗
PubMed1991

Persistent human immunodeficiency virus type 1 infection in human fetal glial cells reactivated by T-cell factor(s) or by the cytokines tumor necrosis factor alpha and interleukin-1 beta.

Human immunodeficiency virus type 1 (HIV-1) infection of the brain has been associated with a severe dementing illness in children and adults. However, HIV-1 antigens are most frequently found in macrophages and microglial cells. To determine the extent of susceptibility of neuroglial cells to infection, the HIV-1 genome was introduced into cells cultured from human fetal brain tissue. Astroglial cells rapidly transcribed the viral genome producing high levels of p24 protein and infectious virions which peaked two to three days posttransfection. Thereafter HIV-1 genome expression progressively diminished and a persistent phase of infection developed during which neither virus nor viral proteins could be demonstrated by immunodetection methods. Cocultivation with CD4+ T cells at any time during the persistent infection resulted in resumption of p24 synthesis and virus multiplication. The release of persistence did not require direct cell-cell contact between the glial and T cells, since separation of the two cell types across a permeable membrane resulted in a delayed but similar resumption of p24 synthesis and virus multiplication. The persistently infected glial cells could also be stimulated to produce viral p24 protein if either tumor necrosis factor alpha or interleukin-1 beta was added to the medium without T cells present. These results suggest that astrocytes may serve as an undetected reservoir for HIV-1 and disseminate the virus to other susceptible cells in the brain upon triggering by some cellular or biochemical signal.

Astrocytes↗
PubMed1991

Human fetal Schwann cells support JC virus multiplication.

The human papovavirus JC virus (JCV), the etiologic agent of progressive multifocal leukoencephalopathy, displays a narrow host range for growth, preferentially infecting oligodendrocytes, the myelin-producing cells of the central nervous system. In tissue culture, human fetal brain cells have been used for JCV propagation because of their ability to support JCV virion production. In this study, we evidence that a human fetal cell type derived from the peripheral nervous system can be productively infected with JCV. Schwann cells, the cell type responsible for myelination in the peripheral nervous system, support the expression of JCV T antigen and JCV DNA replication. However, viral proteins and DNA replication were not detected either in dorsal root ganglion neurons or fibroblasts. These results extend the host range of JCV to include another cell of the glial lineage whose function is myelin formation.

Antigens, Viral↗
PubMed1990

Glial cells of the human developing brain and B cells of the immune system share a common DNA binding factor for recognition of the regulatory sequences of the human polyomavirus, JCV.

Cells of the nervous system and the immune system perform highly specialized functions which reflect the tissue-specific regulation of their genes. However there are some functions between these two cell systems such as antigen presentation, cytokine release, and expression of MHC molecules which suggest a common mechanism for regulation of certain genes. We present data that extend this observation to include the recognition of specific neurotropic viral DNA sequences by glial cells and B cells. The experiments here provide evidence that both human glial cells and B cells possess nuclear DNA binding proteins that interact with nucleotide sequences on the regulatory region of the JC viral genome. These DNA binding proteins are present in human lymphoma B cell lines and fetal glial cultures. The fetal glial cultures are characterized as astrocytes by unique cDNA expression and the presence of GFAP. Data are also presented that demonstrate the presence of JCV infected B cells in brain tissue derived from progressive multifocal leucoencephalopathy, the demyelinating disease caused by JCV infection. The possibility that the glial and B cell protein factor(s) responsible for recognition of the JCV genome belong to a family of proteins similar to known transcriptional control elements such as the Octamer binding proteins or Nuclear Factor-1 is discussed.

Astrocytes↗
PubMed1990

Varicella-zoster virus infection of human astrocytes, Schwann cells, and neurons.

Human fetal cell cultures enriched for astrocytes, Schwann cells, or dorsal root ganglia neurons were infected with cell-free varicella-zoster virus (VZV), and the course of these infections was compared with that in fetal fibroblasts. Virus replication was detected in each neural cell type as early as 10-16 hr postinfection. Permissiveness of each cell type was confirmed by electron microscopy. However, the kinetics of virus spread varied between the neural cell types. Moreover, the accumulation, progression, and localization of VZV putative immediate early (IE), early (E), and late proteins was neural cell-type specific. VZV replication was slower in Schwann cells and neurons than in astrocytes. In Schwann cells and neurons VZV E proteins could be detected before IE proteins, a reversal of the order of accumulation noted with astrocytes and fibroblasts. There was also relatively more VZV IE protein in the perinuclear cytoplasm of Schwann cells and neurons, suggesting a delay in the transport of this antigen to its nuclear site. The permissiveness of non-neuronal cell types suggests that they could play a role in the pathogenesis of herpes zoster.

Antigens, Viral↗
PubMed1989

Interaction of a nuclear factor-1-like protein with the regulatory region of the human polyomavirus JC virus.

We have initiated a study to identify host proteins which interact with the regulatory region of the human polyomavirus JC (JCV), which is associated with the demyelinating disease, progressive multifocal leukoencephalopathy. We examined the interaction of nuclear proteins prepared from different cell lines with the JCV regulatory region by DNA binding gel retardation assays. Binding was detected with nuclear extracts prepared from human fetal glial cells, glioma cells, and HeLa cells. Little or no binding was detected with nuclear extracts prepared from human embryonic kidney cells. Competitive binding assays suggest that the nuclear factor(s) which interacted with the JCV regulatory region was different from those which interacted with the regulatory region of the closely related polyomavirus SV40. We found three areas in the JCV regulatory region protected from DNase I digestion: site A, located just upstream from the TATA sequence in the first 98-base pair (bp) repeat; site B, located upstream from the TATA sequence in the second 98-bp repeat; and site C, located just following the second 98-bp repeat. There were some differences in the ability of the nuclear factor(s) from the two brain cell lines and HeLa cells to completely protect the nucleotides within the footprint region. The results from the DNase I protective studies and competitive DNA binding studies with specific oligonucleotides, suggest that nuclear factor-1 or a nuclear factor-1-like factor is interacting with all three sites in the JCV regulatory region. In addition, the results suggest that the nuclear factor which interacts with the JCV regulatory region from human brain cell lines is different from the factor found in HeLa cells.

Base Sequence↗
PubMed1989

Extension of JC virus host range to monkey cells by insertion of a simian virus 40 enhancer into the JC virus regulatory region.

A chimeric polyomavirus genome was constructed by inserting the 72- and the 21-bp repeats of simian virus 40 (SV40) into the JC virus (JCV) regulatory region on the late side of the JCV 98-bp repeats. Although the chimeric polyomavirus DNA was able to replicate very well in human fetal glial cells, deletions were found in sequences of the regulatory region. DNA sequence analysis of a selected clone indicated a 294-bp deletion from the original construction that retained the sequences for the JCV replication origin, 78 bp of one 98-bp repeat, 33 bp of one SV40 72-bp repeat, and one intact 72-bp repeat. Of significant interest was that this genome demonstrated an extended species and cell-type host range, producing infectious virus in human fetal brain and embryonic kidney as well as in rhesus monkey fetal and adult glial cells. However, the species host range was not extended beyond primate cells since the chimeric polyomavirus was unable to multiply in rodent glial cells. Analysis of the viral RNA transcripts from either kidney or glial cells indicated that the major start sites for early RNA mapped within the JCV sequences of the regulatory region and major start sites for late RNA mapped within the JCV and SV40 sequences. This extension of the JC virus host range was most likely attributable to changes in the regulatory region and not the viral T protein since a recombinant DNA clone which placed the coding sequences for the wild-type JCV T protein subject to regulation by the deleted chimeric regulatory region showed a similar extension of host range.

Animals↗
PubMed1989

A rapid method for in situ hybridization for viral DNA in brain biopsies from patients with AIDS.

Brain biopsy is often necessary in the diagnosis of neurological complications found in AIDS patients. We describe here a rapid method of tissue preparation and in situ DNA hybridization for detecting JC virus DNA in frozen brain biopsy sections which allows the diagnosis of progressive multifocal leukoencephalopathy to be established on the day of surgery. Once the diagnosis is established, therapeutic and management decisions can be made more easily. The commercial availability of biotinylated probes for several of the DNA viruses most frequently encountered in brain infections of AIDS patients will provide wide application of these techniques to patient management.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Human fetal astrocytes in culture support the growth of the neurotropic human polyomavirus, JCV.

JC virus (JCV) has frequently been described as a tissue specific agent in the human population targeting the myelin producing oligodendrocyte whose destruction results in the demyelinating disease, progressive multifocal leukoencephalopathy (PML). JC virus growth in cell culture systems has generally reflected that observation because its multiplication is most efficient in cultures derived from human fetal brain. These cultures, however, are composed of a mixed population of cells phenotypically categorized as either astrocytes or precursors to oligodendrocytes. We have been able to separate mixed populations of glial cells from human fetal brain in culture and produce a pure population of astrocytes. We then analyzed the astrocyte cell cultures for their ability to support JCV transcription, replication, and virion production. Our results demonstrate that cultures of astrocytic cell types support JCV gene expression leading to virus multiplication. These astrocytic cell cultures also can be passaged several times in culture without loss of their astrocytic phenotypes or susceptibility to JCV infection. These data are consistent with our studies of JCV gene expression in fixed brain tissues of PML patients and virus immortalized astrocytic cell lines. These results strongly suggest that astrocytes as well as oligodendrocytes play a role in the pathogenesis of PML and should focus additional experiments on this particular cell type.

Astrocytes↗
PubMed1988

Early appearance of type II astrocytes in developing human fetal brain.

To compare rat and human glial development, we examined the cellular composition of human fetal brain in short-term cultures and fresh cell suspensions from fetal ages ranging from 7 to 16 weeks, utilizing the cell type-specific markers which define glial subsets in rats. Here we report that unlike the early rat central nervous system (CNS), 7-10 week human fetal brain contains mostly astrocytes that can be characterized as type II rather than type I. Type I astrocytes become more prevalent in 16-week gestational age human brain. Although cells morphologically and immunocytochemically similar to the rat 02-A cell are found in human fetal brain and spinal cord, these cells were not induced to express galactocerebroside in serum-free media and did not have vimentin-containing intermediate filaments as do rat 02-A cells. These results suggest that functional differences may exist between rat type I and type II astrocytes and phenotypically similar cells found in humans.

Astrocytes↗