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Biomedical subjects

D H Gilden

Publications and source records attributed to D H Gilden.

At least 55 records · Page 3Linked to original sources
PubMed1998

Profound cerebrospinal fluid pleocytosis and Froin's Syndrome secondary to widespread necrotizing vasculitis in an HIV-positive patient with varicella zoster virus encephalomyelitis.

Demonstration of the direct involvement of cranial blood vessels by varicella zoster virus (VZV) is facilitated by immunohistochemistry (IHC), in situ hybridization (ISH) and polymerase chain reaction (PCR) techniques. The extent to which an inflammatory vasculitis serves as the pathogenic mechanism for VZV encephalomyelitis (VZVE) is still, however, debated. Most VZVE patients are immunocompromised and show little inflammation, either pre-mortem in cerebrospinal fluid (CSF) or at autopsy. We describe an HIV-positive patient with a moderately depressed CD4 count (304) who presented with massively elevated CSF protein (1800 mg/dl), bloody CSF and pleocytosis (1300 white blood cells (WBC)/mm3). His CSF was positive for VZV DNA by PCR. He was treated with acyclovir and foscarnet, but died. At autopsy, an unusually widespread, inflammatory, transmural vasculitis caused by VZV affected meningeal vessels at virtually all brain stem and spinal cord levels, causing multiple subpial hemorrhages and necrosis. Virus DNA in multiple areas of brain, brainstem and spinal cord was readily revealed by PCR, but not by the presence of viral inclusions, IHC or ISH. This case, with a clinically confusing presentation for VZVE, illustrates the extensive, albeit infrequent, degree of necrotizing vasculitis and CSF abnormalities that VZV is capable of producing. Antiviral therapy may have inhibited VZV genome replication and subsequent antigen production, resulting in negative ISH and IHC studies, but generated increased VZV genomic fragments that were detectable by the more sensitive PCR technique.

AIDS-Related Opportunistic Infections↗
PubMed1998

Restricted use of VH4 germline segments in an acute multiple sclerosis brain.

Multiple sclerosis (MS) cerebrospinal fluid and brain contain increased IgG and oligoclonal bands. Whether this oligoclonal and polyclonal IgG is directed against a disease-relevant antigen remains unknown. To distinguish between random activation versus a targeted B-cell response, we analyzed the IgG heavy chain variable region (VH) repertoire expressed in different lesions of an acute MS brain. To obtain a representative sample of the VH repertoire, we constructed directional complementary DNA libraries from plaque-periplaque messenger RNA and amplified VH regions from the library by nested polymerase chain reaction. When MS VH sequences were aligned to germline segments, about 60% of different VH sequences in the acute MS brain were VH4 germline segments, significantly greater than the known approximately 20% VH4 germline prevalence. Specific VH sequences were overrepresented and expressed at multiple plaque sites. Within some overexpressed populations, there were distinct sequence differences (clonal variants) indicative of clonal expansion. Alignment of VH sequences to their closest germline counterparts revealed extensive somatic mutation and the preferential accumulation of amino acid replacement mutations in complementarity determining regions. These observations suggest the limited B-cell response found in this acute MS brain was antigen driven.

Amino Acid Sequence↗
PubMed1998

Varicella-zoster virus gene 21: transcriptional start site and promoter region.

Varicella-zoster virus (VZV) causes chicken pox (varicella), becomes latent in dorsal root ganglia, and reactivates decades later to cause shingles (zoster). During latency, the entire VZV genome is present in a circular form, from which genes 21, 29, 62, and 63 are transcribed. Immediate-early (IE) VZV genes 62 and 63 encode regulators of virus gene transcription, and VZV gene 29 encodes a major DNA-binding protein. However, little is known about the function of VZV gene 21 or the control of its transcription. Using primer extensions, we mapped the start of VZV gene 21 transcription in VZV-infected cells to a single site located at -79 nucleotides (nt) with respect to the initiation codon. To identify the VZV gene 21 promoter, the 284-bp region of VZV DNA separating open reading frames (ORFs) 20 and 21 was cloned upstream from the chloramphenicol acetyltransferase gene. In transient-transfection assays, the VZV gene 21 promoter was transactivated in VZV-infected, but not uninfected, cells. Further, the protein encoded by ORF 62 (IE62), but not those encoded by VZV ORFs 4, 10, 61, and 63, transactivates the VZV gene 21 promoter. By use of transient-cotransfection assays in conjunction with 5' deletions of the VZV gene 21 promoter, a 40-bp segment was shown to be responsible for the transactivation of the VZV gene 21 promoter by IE62. This region was located at -96 to -56 nt with respect to the 5' start of gene 21 transcription.

Animals↗
PubMed1998

Localization of varicella-zoster virus gene 21 protein in virus-infected cells in culture.

Although four varicella-zoster virus (VZV) genes have been shown to be transcribed in latently infected human ganglia, their role in the development and maintenance of latency is unknown. To study these VZV transcripts, we decided first to localize their expression products in productively infected cells. We began with VZV gene 21, whose open reading frame (ORF) is 3,113 bp. We cloned the 5' and 3' ends and the predicted antigenic segments of the ORF as 1292-, 1280-, and 880-bp DNA fragments, respectively, into the prokaryotic expression vector pGEX-2T. The three VZV 21 ORFs were expressed as approximately 75-, 73-, and 59-kDa glutathione S-transferase fusion proteins in Escherichia coli. To prepare polyclonal antibodies that would recognize all potential epitopes on the VZV gene 21 protein, rabbits were inoculated with a mixture of the three fusion proteins, and antisera were obtained and affinity purified. Immunohistochemical and immunoelectron microscopic analyses using these antibodies revealed VZV ORF 21 protein in both the nucleus and cytoplasm of VZV-infected cells. When these antibodies were applied to purified VZV nucleocapsids, intense staining was seen in their central cores.

Animals↗
PubMed1998

Infectious simian varicella virus expressing the green fluorescent protein.

Clinical, pathologic, immunologic and virologic features of simian varicella virus (SVV) infection in primates closely resemble varicella-zoster virus (VZV) infection in humans. Such similarities provide a rationale to analyze SVV infection in primates as a model of varicella pathogenesis and latency. Thus, we constructed an SVV-expressing green fluorescent protein (SVV-GFP) by inserting the GFP gene into the unique short segment of the virus genome by homologous recombination. Analysis of recombinant viral DNA and the expressed proteins of plaque-purified SVV-GFP confirmed the location of the GFP insert and that the recombinant SVV expressed the 27 kDa GFP. Infection of monkey kidney cells in tissue culture with SVV-GFP revealed bright green fluorescence associated with the characteristic focal cytopathic effect produced by SVV infection. Microscopic examination of lung from a 3-month-old African green monkey 10 days after infection with SVV-GFP revealed bright green fluorescence in areas of acute necrotizing pneumonitis. SVV-GFP allows ready identification of cells infected with SVV both in vitro and in vivo, and will be useful for further analysis of varicella pathogenesis and latency in experimentally infected animals--studies not possible in humans.

Animals↗
PubMed1997

Extraction and purification of active IgG from SSPE and MS brain.

Immunoglobulin (Ig) G was purified from soluble and membrane fractions of postmortem subacute sclerosing panencephalitis (SSPE) brain, multiple sclerosis (MS) brain plaque-periplaque white matter, and normal human brain (NHB) white matter. After homogenization in 0.32 M sucrose and removal of cell debris and nuclei by low-speed centrifugation, soluble and crude membrane fractions were separated by ultracentrifugation. After removal of sucrose by dialysis, IgG was isolated from the soluble fraction by protein A affinity chromatography. IgG was obtained from the membrane fraction by elution at low pH and purification from the eluate by protein A chromatography. Whereas very little IgG was in NHB white matter, significant levels of IgG were recovered from both SSPE and MS brain. Both immunocytochemical staining of measles virus-infected cells in tissue culture and protein immunoblotting of virus-infected cell lysates showed that the IgG from SSPE brain contained activity specific for measles virus protein. The abundance, purity and functional activity of IgG extracted from SSPE and MS brain indicate that IgG extracted from the brain of humans with an inflammatory disease of unknown etiology can be used to identify its corresponding antigen.

Animals↗
PubMed1997

Identification of simian varicella virus homologues of varicella zoster virus genes.

Clinical, pathologic, immunologic and virologic features of simian varicella virus (SVV) infection in primates resemble human varicella-zoster virus (VZV) infection. Further, the SVV and VZV genomes are similar in size and structure, show striking homology in their configuration and DNA sequences, and encode antigenically related polypeptides. Although the entire VZV genome is present during latency in human ganglia, transcription is limited. VZV genes 21, 29, 62 and 63 are transcribed during latency, while genes 4, 10, 40, 51 and 61 are not transcribed. The entire VZV genome has been sequenced, but the SVV genome has not. Thus, to analyze SVV genes transcribed during latency, we have begun to identify SVV homologues of the above VZV genes. We used nick-translated [32p]-labeled-VZV open reading frame (ORF)-specific probes to screen Southern blots containing EcoRI-digested SVV genomic DNA and recombinant clones of SVV EcoRI and BamHI DNA fragments spanning approximately 97% of the virus genome. We showed that SVV homologues of VZV ORFs 4, 10, 29, 40, 51 and 61 mapped to SVV DNA fragments EcoRI I, A, N, BamHI E, EcoRI D and E, respectively. We also confirmed earlier reports that SVV homologues of VZV genes 21 and 63 mapped to SVV EcoRI DNA fragments H and C, respectively. Viral genes on the SVV and VZV genomes seem to be collinear.

Animals↗
PubMed1996

Expression of protein encoded by varicella-zoster virus open reading frame 63 in latently infected human ganglionic neurons.

The ganglionic cell type in which varicella-zoster virus (VZV) is latent in humans was analyzed by using antibodies raised against in vitro-expressed VZV open reading frame 63 protein. VZV open reading frame 63 protein was detected exclusively in the cytoplasm of neurons of latently infected human trigeminal and thoracic ganglia. This is, to our knowledge, the first identification of a herpesvirus protein expressed during latency in the human nervous system.

Adult↗
PubMed1996

Epstein-Barr virus--associated acute autonomic neuropathy.

A 44-year-old man developed blurry vision, photosensitivity, orthostasis, constipation, and acrodysesthesias after a febrile illness. The neurologic examination and ancillary studies were consistent with a dysautonomic small fiber neuropathy. The cerebrospinal fluid (CSF) contained both Epstein-Barr virus (EBV) DNA and antibody to EBV. This is the first report of an acute autonomic neuropathy with documented EBV infection in CSF.

Adult↗
PubMed1996

The patterns of varicella zoster virus encephalitis.

Varicella zoster virus (VZV) encephalitis has become increasingly prevalent in the era of acquired immunodeficiency syndrome (AIDS), and a widening spectrum of pathological lesions has defined the disease in these and other severely immunosuppressed patients. VZV produces three distinct morphological patterns of brain damage. VZV can cause bland or hemorrhagic infarctions secondary to a large or medium vessel vasculopathy. Deep white matter, ovoid mixed necrotic, and demyelinative lesions occur as a consequence of small vessel vasculopathy, with demyelination dependent on the degree of additional oligodendrocyte infection. Distinctive Cowdry A intra-nuclear viral inclusions are rare in either large or small blood vessels or near infarctions, but are commonly found in glial cells at the edge of the smaller ovoid, demyelinative lesions. Ependymal and periventricular necrosis occurs as a result of vasculopathy of subependymal vessels and secondary infection of ependymal and other glial cells in the periventricular region. To clarify these patterns of VZV encephalitis and shed light on their pathogenesis, the authors have examined all cases of VZV encephalitis seen at our institution since 1984. Additionally, the authors review the extensive literature in an attempt to classify the patterns of VZV encephalitis into (1) large/ medium vessel vasculopathy with bland or hemorrhagic infarctions, (2) small vessel vasculopathy with mixed ischemic/demyelinative lesions, and (3) ventriculitis/periventriculitis. Although one of these three patterns often predominates clinically and radiographically, careful histological examination at autopsy shows mixed features in many cases.

AIDS-Related Opportunistic Infections↗
PubMed1996

Varicella-zoster virus (VZV) transcription during latency in human ganglia: detection of transcripts mapping to genes 21, 29, 62, and 63 in a cDNA library enriched for VZV RNA.

Information on the extent of virus DNA transcription and translation in infected tissue is crucial to an understanding of herpesvirus latency. To detect low-abundance latent varicella-zoster virus (VZV) transcripts, poly(A)+ RNA extracted from latently infected human trigeminal ganglia was enriched for VZV transcripts by hybridization to biotinylated VZV DNA. After hybridization, the RNA-DNA hybrid was isolated by binding to avidin-coated beads and extensively washed, and the RNA was released by heat denaturation. A lambda-based cDNA library was then constructed from the enriched RNA. PCR and DNA sequencing of DNA extracted from the cDNA library revealed the presence of VZV genes 21, 29, 62, and 63, but not VZV genes 4, 10, 40, 51, and 61, in the enriched cDNA library. These findings confirm the detection of VZV gene 29 and 62 transcripts on Northern (RNA) blots prepared from latently infected human ganglia (J.L. Meier, R.P. Holman, K.D. Croen, J.E. Smialek, and S.E. Straus, Virology 193:193-200, 1993) and the presence of VZV gene 21 transcripts in a cDNA library from mRNA of latently infected ganglia (R.J. Cohrs, K. Srock, M.B. Barbour, G. Owens, R. Mahalingam, M.E. Devlin, M. Wellish and D.H. Gilden, J. Virol. 68:7900-7908,1994) and also reveal, for the first time, the presence of VZV gene 63 RNA in latently infected human ganglia.

Base Sequence↗
PubMed1996

A simian varicella virus (SVV) homolog to varicella-zoster virus gene 21 is expressed in monkey ganglia latently infected with SVV.

We have sequenced a simian varicella virus (SVV) open reading frame (ORF), 3,123 bp in length, whose product has 51% amino acid homology with the sequence encoded by the ORF of varicella-zoster virus gene 21. Several regions are highly conserved between the two ORFs, with homologies of approximately 80%. The SVV gene is transcribed in tissue culture cells productively infected with SVV and in monkey ganglia latently infected with SVV.

Amino Acid Sequence↗
PubMed1996

The search for virus in multiple sclerosis brain.

Plaque-periplaque areas from MS brain tissue were explanted and propagated in tissue culture. The same in vitro techniques that successfully rescued measles virus from SSPE brain, papovavirus from PML brain, and HSV from normal human trigeminal ganglia, were applied. MS brain cells were also inoculated into chimpanzees, multiple rodent species, and embryonated hens eggs. No neurologic disease developed in experimentally infected animals, and no cytopathic effect was observed in explanted cells, or after cocultivation or fusion of MS brain cells with indicator cells. Further analysis of explanted and cocultivated cells by indirect immunofluorescence with various antiviral antisera prepared against viruses associated with post-infectious encephalomyelitis, as well as antisera to other ubiquitous viruses, failed to detect viral antigen. Finally, attempts to detect a latent enveloped virus in MS brain cells by 'superinfecting' MS brain cells in culture with vesicular stomatitis virus (VSV) did not reveal a VSV non-neutralizable fraction. Nevertheless, since oligoclonal bands (OGBs) in the CSF of patients with chronic infectious diseases of the CNS are directed against the causative agent, it is likely that OGBs in MS CSF are antibody directed against the agent or antigen that triggered disease. Although the relevant antibody may be scarce relative to irrelevant antibody in MS CSF, and only small amounts of an MS-specific antigen may be present in brain, this report provides a rationale for strategies proposed in our companion report by Owens et al which will allow detection of an MS-specific antigen or its cognate RNA in brain.

Brain↗
PubMed1996

Strategies to identify sequences or antigens unique to multiple sclerosis.

Chronic inflammatory and infectious diseases of the central nervous system (CNS) are characterized by increased IgG and oligoclonal bands (OGBs) in the brain and cerebrospinal fluid (CSF). The OGBs in CNS infectious diseases of known cause have been shown to be directed against the pathogenic agent. In multiple sclerosis (MS), the antigenic specificity of the OGBs is unknown, but could be directed against an infectious agent, an autoantigen, or both. In a molecular approach to identify antigens specific for MS, we constructed directional cDNA expression libraries with mRNA extracted from chronic and acute MS plaques and periplaque white matter. The libraries were: (1) screened to identify clones whose expression products react with MS CSF, but not with CSF from other infectious and inflammatory diseases of the CNS; (2) subtracted by hybridization to mRNA from normal human brain white matter and differentially screened to detect unique MS transcripts; and (3) used as template in polymerase chain reactions to amplify, clone, and sequence IgG heavy and light chain variable regions (VH and VL, respectively) expressed in MS plaques. Analysis of the VH and VL IgG repertoire in MS brain may identify disease-relevant IgG sequences that can be assembled into functional antibodies using recombinant phage technology. Such recombinant antibodies will be useful to probe brain tissue to identify antigens unique to MS.

DNA, Complementary↗
PubMed1996

Zoster myelitis: improvement with antiviral therapy in two cases.

This report describes two patients with acquired immunodeficiency syndrome (AIDS) and herpes zoster myelopathy. Patient one had a T-8 myelitis that preceded the onset of T-8-distribution zoster and was followed by cervical myelopathy. Antibody to varicella zoster virus (VZV) was present in the CSF. He never received steroids or other immunosuppressive drugs, and his condition improved dramatically after treatment with intravenous acyclovir. The second patient had a rapidly progressive myelitis with paralysis of both legs. Detection of VZV DNA and antibody to VZV in his CSF led to successful treatment with famciclovir despite discontinuation of dexamethasone and earlier treatment failure with acyclovir. These cases support the idea that VZV myelopathy in the immunosuppressed host is caused by virus invasion. CSF analysis for antiviral antibody and for VZV DNA by polymerase chain reaction are helpful in establishing the diagnosis. Aggressive antiviral therapy is advised.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Varicella zoster virus, a cause of waxing and waning vasculitis: the New England Journal of Medicine case 5-1995 revisited.

A 73-year-old man developed an ill-defined fatal vasculitis involving the central nervous system. The case report was published as a clinicopathologic exercise in February 1995 in The New England Journal of Medicine. We restudied the pathologic material and found both varicella zoster virus (VZV) DNA and VZV-specific antigen, but not herpes simplex virus (HSV) or cytomegalovirus (CMV) DNA or HSV- or CMV-specific antigen, in three of the five cerebral arteries examined. The inflammatory response, disruption of the internal elastic lamina, and detection of viral antigen were patchy from one artery to another, as well as within a given artery. A search for VZV should be conducted in cases of vasculitis when both the central and peripheral nervous systems are involved, when focal narrowing is present in large arteries, when brain imaging reveals infarction in gray and white matter, both deep and superficial, and when white matter is disproportionally involved. Zosteriform rash is not required for diagnosis.

Aged↗
PubMed1996

Another case of virologically confirmed zoster sine herpete, with electrophysiologic correlation.

A third virologically-confirmed case of thoracic-distribution zoster sine herpete is reported. Electromyography (EMG) of paraspinal muscles demonstrated frequent fibrillation potentials restricted to chronically painful thoracic root segments. Treatment with intravenous acyclovir and oral famciclovir were ineffective. These findings suggest the usefulness of EMG of muscles corresponding to painful dermatomes, combined with virologic studies, to support the diagnosis of zoster sine herpete.

Aged↗