bluuebunny/crossref_metadata_2025 huggingface dataset:
A Comparison of Epilepsy Patients in a Traditional Ambulatory Clinic and a Telemedicine Clinic
Authors: Rasmusson Karen A. , Hartshorn Jeanette C.
Published: April 2005

Summary:  Purpose: This article compares a traditional ambulatory clinic in an academic medical center with a telemedicine clinic. The telemedicine clinic is a joint project of the UTMB Telehealth Center and the Epilepsy Foundation of Southeast Texas, with partial funding for clinical operations provided by the Texas Department of Health.

Methods: Data were collected on all the patients (n = 155) in both clinics for 3 months in 2004. In addition to demographic information, outcome data (number of seizures, hospitalizations, and emergency room visits) were gathered. Medication compliance also was collected by using self‐report and medication levels.

Results: Outcome variables were subjected to t test and χ2 analysis. No significant differences were found in any of the demographic data or outcome measures between the two groups.

Conclusions: Telemedicine is an acceptable alternative to in‐person clinics for the provision of care to adults with epilepsy. Because telemedicine programs are designed to bring medical care closer to where patients live, these clinics provide an excellent alternative to provide consistent care in rural and geographically isolated areas. Additional studies are needed to investigate the potential costs associated with telemedicine as well as the potential for cost savings over time as patients are more able to access care and therefore may be more likely to seek the needed routine care and follow‐up.
Lack of Association between the C3435T Polymorphism in the Human Multidrug Resistance (MDR1) Gene and Response to Antiepileptic Drug Treatment
Authors: Sills Graeme J. , Mohanraj Rajiv , Butler Elaine , McCrindle Sheila , Collier Lindsay , Wilson Elaine A. , Brodie Martin J.
Published: April 2005

Summary:  Purpose: P‐glycoprotein (P‐gp) has been implicated in the causation of refractory epilepsy. The expression and efflux efficiency of P‐gp is influenced by a polymorphism (C3435T) in the encoding gene (MDR1). Recent evidence suggests that the homozygous C‐variant, which is associated with higher expression and increased activity of P‐gp, is more common in patients with pharmacoresistant epilepsy. We have investigated the prevalence of this polymorphism in a series of patients attending a specialist epilepsy clinic.

Methods: DNA samples were obtained from 400 patients, irrespective of seizure type or drug treatment. Genotype of the C3435T polymorphism was determined by traditional polymerase chain reaction (PCR) followed by restriction digest. Classification of response to treatment was determined in a blinded fashion by an independent physician. Results were expressed as genotype and allele frequencies per response group and compared by logistic regression analysis.

Results: In total, 170 patients were classified as responders, with ≥12 months seizure freedom on current treatment. The remaining 230 patients were classified as nonresponders. Comparison of responders and nonresponders revealed no significant difference in allele frequency (C vs. T; odds ratio, 1.03; 95% CI, 0.78–1.37; p = 0.83) or genotype frequency (CC vs TT; odds ratio, 1.07; 95% CI, 0.60–1.91; p = 0.81). Subanalyses of individual seizure types were similarly unremarkable.

Conclusions: This study failed to corroborate a previously reported association between the C3435T polymorphism in the human MDR1 gene and pharmacoresistant epilepsy. Whether the C3435T polymorphism can act as a marker for the natural history of treated epilepsy remains to be determined.
Is Variation in the GABA(B) Receptor 1 Gene Associated with Temporal Lobe Epilepsy?
Authors: Tan Nigel C.K. , Heron Sarah E. , Scheffer Ingrid E. , Berkovic Samuel F. , Mulley John C.
Published: April 2005

Summary:  Purpose:γ‐Aminobutyric acid (GABA)‐receptor genes are prime candidates for a role in seizure susceptibility. An association between the c.1465G→A variant in the GABA(B) receptor 1 gene (GABBR1) and susceptibility to temporal lobe epilepsy (TLE) has been reported in an Italian cohort. We sought to replicate this association in an independent Australian cohort.

Methods: The 234 patients with TLE and 164 healthy controls were genotyped for the c.1465G→A variant, by using inclusion criteria identical to those of the first study.

Results: The c.1465G→A variant was found in one TLE patient and one control subject. Genotype and allele frequencies did not differ between groups.

Conclusions: We did not replicate the reported associations between the c.1465G→A variant and susceptibility to TLE. We suggest that the initial positive association may be due to undetected population stratification; the importance of genomic control is emphasized. Population‐specific effects also may play a role, and we highlight the need to demonstrate an in vitro functional effect to give biologic meaning to any proposed association.
Adding Video Recording Increases the Diagnostic Yield of Routine Electroencephalograms in Children with Frequent Paroxysmal Events
Authors: Watemberg Nathan , Tziperman Barak , Dabby Ron , Hasan Mariana , Zehavi Liora , Lerman‐Sagie Tally
Published: April 2005

Summary: Purpose:To report on the usefulness of adding video recording to routine EEG studies of infants and children with frequent paroxysmal events.

Methods:We analyzed the efficacy of this diagnostic means during a 4‐year period. The decision whether to add video recording was made by the pediatric EEG interpreter at the time of the study. Studies were planned to last between 20 and 30 min, and, if needed, were extended by the EEG interpreter. For most studies, video recording was added from the beginning of EEG recording. In a minority of cases, the addition of video was implemented during the first part of the EEG test, as clinical events became obvious. In these cases, a new study (file) was begun. The success rate was analyzed according to the indications for the EEG study: paroxysmal eye movements, tremor, suspected seizures, myoclonus, staring episodes, suspected stereotypias and tics, absence epilepsy follow‐up, cyanotic episodes, and suspected psychogenic nonepileptic events.

Results:Video recording was added to 137 of 666 routine studies. Mean patient age was 4.8 years. The nature of the event was determined in 61 (45%) of the EEG studies. Twenty‐eight percent were hospitalized patients. The average study duration was 26 min. This diagnostic means was particularly useful for paroxysmal eye movements, staring spells, myoclonic jerks, stereotypias, and psychogenic nonepileptic events. About 46% of 116 patients for whom cognitive data were available were mentally retarded. EEG with added video recording was successfully performed in all 116 cases and provided useful information in 29 (55%) of these 53 patients.

Conclusions:Adding video recording to routine EEG was helpful in 45% of cases referred for frequent paroxysmal events. This technique proved useful for hospitalized children as well as for outpatients. Moreover, it was successfully applied in cognitively impaired patients. Infants and children with paroxysmal eye movements, staring spells, myoclonic jerks, stereotypias, and pseudoseizures especially benefited from this diagnostic means. Because of its low cost and the little discomfort imposed on the patient and his or her family, this technique should be considered as a first diagnostic step in children with frequent paroxysmal events.
Visually Self‐induced Seizures Sensitive to Round Objects
Authors: Brockmann Knut , Huppke Peter , Karenfort Michael , Gärtner Jutta , Höger Christoph
Published: April 2005

Summary: Purpose:To describe a previously unreported type of self‐induced pattern‐sensitive seizures in a child.

Methods:Evaluation of clinical and EEG features.

Results:An 18‐month‐old boy was initially seen with series of short focal tonic seizures self‐induced by gazing intermittently at round objects. Self‐induction of these seizures had an obvious relieving effect on the child. Covering the round object foiled further seizures but resulted in a tantrum. Later in the course, an increasing variety of patterns proved to be capable of inducing seizures, which occurred with increasing frequency and severity, including secondarily generalized tonic–clonic seizures. Interictal EEG revealed multifocal sharp‐and‐slow‐waves. Ictal EEG showed no abnormalities during short focal seizures and rapidly generalizing epileptic discharges during a secondarily generalized seizure. No photosensitivity was noted. Motor and mental development of the boy stagnated over a period of 2 years. Behavioral therapy as well as medical treatment, consisting of various combinations of antiepileptic drugs (AEDs) together with a selective serotonin‐reuptake inhibitor, were of merely transient benefit. Only combined pharmacotherapy, including valproate, sulthiame, and clobazam, resulted in seizure control at age 3 years 9 months.

Conclusions:Visual capture of geometric patterns other than stripes or gratings may trigger focal seizures with secondary generalization. Synchronization of cortical neurons responsible for pattern recognition may account for epileptogenesis in this child.
Cataracts in a laboratory colony of African green monkeys (Chlorocebus aethiops)*
Authors: Plesker Roland , Hetzel Udo , Schmidt Werner
Published: April 2005

Abstract:  Three cases of spontaneous cataracts were investigated in a colony of African green monkeys (Chlorocebus aethiops). Clinical, pathological and microbiological investigations were conducted in two cases of juvenile cataracts and in one case of a mature cataract. These investigations revealed no indication of an infection as the cause of cataract development. A genetic correlation existed between the affected individuals. Clinical chemistry gave a hint that calcium might be a ‘key factor’ in the development of these cataracts: in both cases of the juvenile cataracts, the calcium content in the serum and in the aqueous humor was clearly decreased in the affected babies. The calcium uptake in the affected baby itself was not altered but the calcium content in the mother's milk was low.
Mitogen-Activated Protein Kinases Regulate LSF Occupancy at the Human Immunodeficiency Virus Type 1 Promoter
Authors: Ylisastigui Loyda , Kaur Rupinderjeet , Johnson Holly , Volker Janet , He Guocheng , Hansen Ulla , Margolis David
Published: April 2005

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent, nonproductive state within a small population of memory CD4
+
cells. The transcription factor LSF binds to sequences within the HIV-1 long terminal repeat (LTR) initiation region and recruits a second factor, YY1, to the LTR. These factors then cooperatively recruit histone deacetylase 1 to the LTR, resulting in inhibition of transcription. This appears to be one mechanism contributing to HIV persistence within resting CD4
+
T cells. We sought to further detail LSF binding to the HIV-1 LTR and factors that regulate LSF occupancy. We find that LSF binds the LTR as a tetramer and that binding is regulated by phosphorylation mediated by mitogen-activated protein kinases (MAPKs). In vitro, phosphorylation of LSF by Erk decreases binding to the LTR, while binding is increased by p38 phosphorylation. LSF occupancy at LTR chromatin is increased by the p38 agonist anisomycin and decreased by specific p38 inhibition. p38 inhibition also results in increased acetylation of histone H4 at the LTR nucleosome adjacent to the LSF binding site. p38 inhibition also blocked the ability of YY1 to inhibit activation of the integrated HIV promoter. Finally, HIV was recovered from the resting CD4
+
T cells of aviremic, HIV-infected donors upon treatment of these cells with specific inhibitor of p38. These data suggest that the MAPK pathway regulates LSF binding to the LTR and thereby one aspect of the regulation of HIV expression. This mechanism could be exploited as a novel therapeutic target to disrupt latent HIV infection.
Virus-Specific CD4+and CD8+Cytotoxic T-Cell Responses and Long-Term T-Cell Memory in Individuals Vaccinated against Polio
Authors: Wahid Rahnuma , Cannon Martin J. , Chow Marie
Published: April 2005

The presence of poliovirus (PV)-specific CD4+T cells in individuals vaccinated against polio has been shown, but CD8+T-cell responses have not been described. Here, we functionally characterize the CD4+T-cell response and show for the first time that dendritic cells and macrophages can stimulate PV-specific CD8+T-cell responses in vitro from vaccinees. Both CD4+T and CD8+T cells secrete gamma interferon in response to PV antigens and are cytotoxic via the perforin/granzyme B-mediated pathway. Furthermore, the T cells also recognize and kill Sabin 1 vaccine-infected targets. The macrophage-stimulated CD4+T and CD8+T cells most likely represent memory T cells that persist for long periods in vaccinated individuals. Thus, immunity to PV vaccination involves not only an effective neutralizing antibody titer but also long-term CD4+and CD8+cytotoxic T-cell responses.
Constitutive Activation of the Transcription Factor NF-κB Results in Impaired Borna Disease Virus Replication
Authors: Bourteele Soizic , Oesterle Katja , Pleschka Stephan , Unterstab Gunhild , Ehrhardt Christina , Wolff Thorsten , Ludwig Stephan , Planz Oliver
Published: April 2005

The inducible transcription factor NF-κB is commonly activated upon RNA virus infection and is a key player in the induction and regulation of the innate immune response. Borna disease virus (BDV) is a neurotropic negative-strand RNA virus, which replicates in the nucleus of the infected cell and causes a persistent infection that can lead to severe neurological disorders. To investigate the activation and function of NF-κB in BDV-infected cells, we stably transfected the highly susceptible neuronal guinea pig cell line CRL with a constitutively active (IKK EE) or dominant-negative (IKK KD) regulator of the IKK/NF-κB signaling pathway. While BDV titers were not affected in cells with impaired NF-κB signaling, the expression of an activated mutant of IκB kinase (IKK) resulted in a strong reduction in the intracellular viral titer in CRL cells. Electrophoretic mobility shift assays and luciferase reporter gene assays revealed that neither NF-κB nor interferon regulatory factors (IRFs) were activated upon acute BDV infection of wild-type or vector-transfected CRL cells. However, when IKK EE-transfected cells were used as target cells for BDV infection, DNA binding to an IRF3/7-responsive DNA element was detectable. Since IRF3/7 is a key player in the antiviral interferon response, our data indicate that enhanced NF-κB activity in the presence of BDV leads to the induction of antiviral pathways resulting in reduced virus titers. Consistent with this observation, the anti-BDV activity of NF-κB preferentially spread to areas of the brains of infected rats where activated NF-κB was not detectable.
Myxoma Virus M141R Expresses a Viral CD200 (vOX-2) That Is Responsible for Down-Regulation of Macrophage and T-Cell Activation In Vivo
Authors: Cameron Cheryl M. , Barrett John W. , Liu Liying , Lucas Alexandra R. , McFadden Grant
Published: April 2005

M141R is a myxoma virus gene that encodes a cell surface protein with significant amino acid similarity to the family of cellular CD200 (OX-2) proteins implicated in the regulation of myeloid lineage cell activation. The creation of an M141R deletion mutant myxoma virus strain (vMyx141KO) and its subsequent infection of European rabbits demonstrated that M141R is required for the full development of a lethal infection in vivo but is not required for efficient virus replication in susceptible cell lines in vitro. Minor secondary sites of infection were detected in the majority of rabbits infected with the M141R deletion mutant, demonstrating that the M141R protein is not required for the dissemination of virus within the host. When compared to wild-type myxoma virus-infected rabbits, vMyx141KO-infected rabbits showed higher activation levels of both monocytes/macrophages and lymphocytes in situ through assessments of inducible nitric oxide synthase-positive and CD25+infiltrating cells in infected and lymphoid tissues. Purified peripheral blood mononuclear cells from vMyx141KO-infected rabbits demonstrated an increased ability to express gamma interferon upon activation by phorbol myristate acetate plus ionomycin compared to cells purified from wild-type myxoma virus-infected rabbits. We concluded that the M141R protein is a bona fide CD200-like immunomodulator protein which is required for the full pathogenesis of myxoma virus in the European rabbit and that its loss from the virus results in increased activation levels of macrophages in infected lesions and draining lymph nodes as well as an increased activation level of circulating T lymphocytes during infection. We propose a model whereby M141R transmits inhibitory signals to tissue macrophages, and possibly resident CD200R+dendritic cells, that reduce their ability to antigenically prime lymphocytes and possibly provides anergic signals to T cells directly.
Receptor-Independent Spread of a Highly Neurotropic Murine Coronavirus JHMV Strain from Initially Infected Microglial Cells in Mixed Neural Cultures
Authors: Nakagaki Keiko , Nakagaki Kazuhide , Taguchi Fumihiro
Published: April 2005

Although neurovirulent mouse hepatitis virus (MHV) strain JHMV multiplies in a variety of brain cells, expression of its receptor carcinoembryonic antigen cell adhesion molecule 1 (CEACAM 1) (MHVR) is restricted only in microglia. The present study was undertaken to clarify the mechanism of an extensive JHMV infection in the brain by using neural cells isolated from mouse brain. In contrast to wild-type (wt) JHMV, a soluble-receptor-resistant mutant (srr7) infects and spreads solely in an MHVR-dependent fashion (F. Taguchi and S. Matsuyama, J. Virol. 76:950-958, 2002). In mixed neural cell cultures, srr7 infected a limited number of cells and infection did not spread, although wt JHMV induced syncytia in most of the cells. srr7-infected cells were positive for GS-lectin, a microglia marker. Fluorescence-activated cell sorter analysis showed that about 80% of the brain cells stained with anti-MHVR antibody (CC1) were also positive for GS-lectin. Pretreatment of those cells with CC1 prevented virus attachment to the cell surface and also blocked virus infection. These results show that microglia express functional MHVR that mediates JHMV infection. As expected, in microglial cell-enriched cultures, both srr7and wt JHMV produced syncytia in a majority of cells. Treatment with CC1 of mixed neural cell cultures and microglia cultures previously infected with wt virus failed to block the spread of infection, indicating that wt infection spreads in an MHVR-independent fashion. Thus, the present study indicates that microglial cells are the major population of the initial target for MHV infection and that the wt spreads from initially infected microglia to a variety of cells in an MHVR-independent fashion.
The B30.2(SPRY) Domain of the Retroviral Restriction Factor TRIM5α Exhibits Lineage-Specific Length and Sequence Variation in Primates
Authors: Song Byeongwoon , Gold Bert , O'hUigin Colm , Javanbakht Hassan , Li Xing , Stremlau Matthew , Winkler Cheryl , Dean Michael , Sodroski Joseph
Published: April 2005

Tripartite motif (TRIM) proteins are composed of RING, B-box 2, and coiled coil domains. Some TRIM proteins, such as TRIM5α, also possess a carboxy-terminal B30.2(SPRY) domain and localize to cytoplasmic bodies. TRIM5α has recently been shown to mediate innate intracellular resistance to retroviruses, an activity dependent on the integrity of the B30.2 domain, in particular primate species. An examination of the sequences of several TRIM proteins related to TRIM5 revealed the existence of four variable regions (v1, v2, v3, and v4) in the B30.2 domain. Species-specific variation in TRIM5α was analyzed by amplifying, cloning, and sequencing nonhuman primate
TRIM5
orthologs. Lineage-specific expansion and sequential duplication occurred in the TRIM5α B30.2 v1 region in Old World primates and in v3 in New World monkeys. We observed substitution patterns indicative of selection bordering these particular B30.2 domain variable elements. These results suggest that occasional, complex changes were incorporated into the TRIM5α B30.2 domain at discrete time points during the evolution of primates. Some of these time points correspond to periods during which primates were exposed to retroviral infections, based on the appearance of particular endogenous retroviruses in primate genomes. The results are consistent with a role for TRIM5α in innate immunity against retroviruses.
Infectious Bronchitis Virus 3a Protein Localizes to a Novel Domain of the Smooth Endoplasmic Reticulum
Authors: Pendleton Amanda R. , Machamer Carolyn E.
Published: April 2005

All coronaviruses possess small open reading frames (ORFs) between structural genes that have been hypothesized to play important roles in pathogenesis. Infectious bronchitis virus (IBV) ORF 3a is one such gene. It is highly conserved among group 3 coronaviruses, suggesting that it has an important function in infection. IBV 3a protein is expressed in infected cells but is not detected in virions. Sequence analysis predicted that IBV 3a was a membrane protein; however, only a fraction behaved like an integral membrane protein. Microscopy and immunoprecipitation studies demonstrated that IBV 3a localized to the cytoplasm in a diffuse pattern as well as in sharp puncta in both infected and transfected cells. These puncta did not overlap cellular organelles or other punctate structures. Confocal microscopy demonstrated that IBV 3a puncta lined up along smooth endoplasmic reticulum (ER) tubules and, in a significant number of instances, were partially surrounded by these tubules. Our results suggest that IBV 3a is partially targeted to a novel domain of the smooth ER.
Relocalization of the Mre11-Rad50-Nbs1 Complex by the Adenovirus E4 ORF3 Protein Is Required for Viral Replication
Authors: Evans Jared D. , Hearing Patrick
Published: April 2005

Adenovirus replication is controlled by the relocalization or modification of nuclear protein complexes, including promyelocytic leukemia protein (PML) nuclear domains and the Mre11-Rad50-Nbs1 (MRN) DNA damage machinery. In this study, we demonstrated that the E4 ORF3 protein effects the relocalization of both PML and MRN proteins to similar structures within the nucleus at early times after infection. These proteins colocalize with E4 ORF3. Through the analysis of specific viral mutants, we found a direct correlation between MRN reorganization at early times after infection and the establishment of viral DNA replication domains. Further, the reorganization of MRN components may be uncoupled from the ability of E4 ORF3 to rearrange PML. At later stages of infection, components of the MRN complex disperse within the nucleus, Nbs1 is found within viral replication centers, Rad50 remains localized with E4 ORF3, and Mre11 is degraded. The importance of viral regulation of the MRN complex is underscored by the complementation of E4 mutant viruses in cells that lack Mre11 or Nbs1 activity. These results illustrate the importance of nuclear organization in virus growth and suggest that E4 ORF3 regulates activities in both PML nuclear bodies and the MRN complex to stimulate the viral replication program.
Epitope-Mapping Studies Define Two Major Neutralization Sites on the Vaccinia Virus Extracellular Enveloped Virus Glycoprotein B5R
Authors: Aldaz-Carroll Lydia , Whitbeck J. Charles , Ponce de Leon Manuel , Lou Huan , Hirao Lauren , Isaacs Stuart N. , Moss Bernard , Eisenberg Roselyn J. , Cohen Gary H.
Published: April 2005

Vaccinia extracellular enveloped virus (EEV) is critical for cell-to-cell and long-range virus spread both in vitro and in vivo. The B5R gene encodes an EEV-specific type I membrane protein that is essential for efficient EEV formation. The majority of the B5R ectodomain consists of four domains with homology to short consensus repeat domains followed by a stalk. Previous studies have shown that polyclonal antibodies raised against the B5R ectodomain inhibit EEV infection. In this study, our goal was to elucidate the antigenic structure of B5R and relate this to its function. To do this, we produced multimilligram quantities of vaccinia virus B5R as a soluble protein [B5R(275t)] using a baculovirus expression system. We then selected and characterized a panel of 26 monoclonal antibodies (MAbs) that recognize B5R(275t). Five of these MAbs neutralized EEV and inhibited comet formation. Two other MAbs were able only to neutralize EEV, while five others were able only to inhibit comet formation. This suggests that the EEV neutralization and comet inhibition assays measure different viral functions and that at least two different antigenic sites on B5R are important for these activities. We further characterized the MAbs and the antigenic structure of B5R(275t) by peptide mapping and by reciprocal MAb blocking studies using biosensor analysis. The epitopes recognized by neutralizing MAbs were localized to SCR1-SCR2 and/or the stalk of B5R(275t). Furthermore, the peptide and blocking data support the concept that SCR1 and the stalk may be in juxtaposition and may be part of the same functional domain.
Discontinuous Subgenomic RNA Synthesis in Arteriviruses Is Guided by an RNA Hairpin Structure Located in the Genomic Leader Region
Authors: van den Born Erwin , Posthuma Clara C. , Gultyaev Alexander P. , Snijder Eric J.
Published: April 2005

Nidoviruses produce an extensive 3′-coterminal nested set of subgenomic (sg) mRNAs, which are used to express structural proteins and sometimes accessory proteins. In arteriviruses and coronaviruses, these mRNAs contain a common 5′ leader sequence, derived from the genomic 5′ end. The joining of the leader sequence to different segments derived from the 3′-proximal part of the genome (mRNA bodies) presumably involves a unique mechanism of discontinuous minus-strand RNA synthesis in which base pairing between sense and antisense transcription-regulating sequences (TRSs) plays an essential role. The leader TRS is present in the loop of a hairpin structure that functions in sg mRNA synthesis. In this study, the minimal sequences in the 5′-proximal region of the
Equine arteritis virus
genome that are required for sg RNA synthesis were delimited through mutagenesis. A full-length cDNA clone was engineered in which this domain was duplicated, allowing us to make mutations and monitor their effects on sg RNA synthesis without seriously affecting genome replication and translation. The leader TRS present in the duplicated sequence was used and yielded novel sg mRNAs with significantly extended leaders. Our combined findings suggest that the leader TRS hairpin (LTH) and its immediate flanking sequences are essential for efficient sg RNA synthesis and form an independent functional entity that could be moved 300 nucleotides downstream of its original position in the genome. We hypothesize that a conformational switch in the LTH region regulates the role of the 5′-proximal region of the arterivirus genome in subgenomic RNA synthesis.
Sequence Variability, Gene Structure, and Expression of Full-Length Human Endogenous Retrovirus H
Authors: Jern Patric , Sperber Göran O. , Ahlsén Göran , Blomberg Jonas
Published: April 2005

Recently, we identified and classified 926 human endogenous retrovirus H (HERV-H)-like proviruses in the human genome. In this paper, we used the information to, in silico, reconstruct a putative ancestral HERV-H. A calculated consensus sequence was nearly open in all genes. A few manual adjustments resulted in a putative 9-kb HERV-H provirus with open reading frames (ORFs) in
gag
,
pro
,
pol
, and
env
. Long terminal repeats (LTRs) differed by 1.1%, indicating proximity to an integration event. The
gag
ORF was extended upstream of the normal myristylation start site. There was a long leader (including a “pre-
gag
” ORF) region positioned like the N terminus of murine leukemia virus (MLV) “glyco-Gag,” potentially encoding a proline- and serine-rich domain remotely similar to MLV pp12. Another ORF, starting inside the 5′ LTR, had no obvious similarity to known protein domains. Unlike other hitherto described gammaretroviruses, the reconstructed Gag had two zinc finger motifs. Alternative splicing of sequences related to the HERV-H consensus was confirmed using dbEST data.
env
transcripts were most prevalent in colon tumors, but also in normal testis. We found no evidence for full length
env
transcripts in the dbEST. HERV-H had a markedly skewed nucleotide composition, disfavoring guanine and favoring cytidine. We conclude that the HERV-H consensus shared a gene arrangement common to gammaretroviruses with
gag
separated by stop codon from
pro-pol
in the same reading frame, while
env
resides in another reading frame. There was also alternative splicing. HERV-H consensus yielded new insights in gammaretroviral evolution and will be useful as a model in studies on expression and function.
Use of Confocal Immunofluorescence Microscopy To Localize Viral Nonstructural Proteins and Potential Sites of Replication in Pigs Experimentally Infected with Foot-and-Mouth Disease Virus
Authors: Monaghan P. , Simpson J. , Murphy C. , Durand S. , Quan M. , Alexandersen S.
Published: April 2005

Replication of foot-and-mouth disease virus in infected pig epithelium has been studied by immunofluorescence labeling of the viral nonstructural protein 3ABC and confocal microscopy. The results were correlated with viral RNA copy numbers in tissue samples from adjacent sites determined by reverse transcription-PCR (RT-PCR). Lesion formation was seen in the tongues and coronary band epithelia of infected pigs 2 days after infection. Viral replication was observed in cells of the epithelium of the tongue and coronary band but not in the associated stromal cells. Infected epithelial cells were present in the stratum spinosum, away from the lesion, with small lesions formed above the basement membrane. Viral replication was markedly reduced in tongue epithelium by day 3 postinfection but remained apparent in the coronary band tissue up to 5 days postinfection. These results were confirmed by the RNA copy number determined by RT-PCR.
Interleukin-1 Is Responsible for Acute Lung Immunopathology but Increases Survival of Respiratory Influenza Virus Infection
Authors: Schmitz Nicole , Kurrer Michael , Bachmann Martin F. , Kopf Manfred
Published: April 2005

Interleukin-1α (IL-1α) and IL-1β are proinflammatory cytokines, which induce a plethora of genes and activities by binding to the type 1 IL-1 receptor (IL-1R1). We have investigated the role of IL-1 during pulmonary antiviral immune responses in IL-1R1−/−mice infected with influenza virus. IL-1R1−/−mice showed markedly reduced inflammatory pathology in the lung, primarily due to impaired neutrophil recruitment. Activation of CD4+T cells in secondary lymphoid organs and subsequent migration to the lung were impaired in the absence of IL-1R1. In contrast, activation of virus-specific cytotoxic T lymphocytes and killing of virus-infected cells in the lung were intact. Influenza virus-specific immunoglobulin G (IgG) and IgA antibody responses were intact, while the IgM response was markedly reduced in both serum and mucosal sites in IL-1R1−/−mice. We found significantly increased mortality in the absence of IL-1R1; however, lung viral titers were only moderately increased. Our results demonstrate that IL-1α/β mediate acute pulmonary inflammatory pathology while enhancing survival during influenza virus infection. IL-1α/β appear not to influence killing of virus-infected cells but to enhance IgM antibody responses and recruitment of CD4+T cells to the site of infection.
Acylation-Mediated Membrane Anchoring of Avian Influenza Virus Hemagglutinin Is Essential for Fusion Pore Formation and Virus Infectivity
Authors: Wagner Ralf , Herwig Astrid , Azzouz Nahid , Klenk Hans Dieter
Published: April 2005

Attachment of palmitic acid to cysteine residues is a common modification of viral glycoproteins. The influenza virus hemagglutinin (HA) has three conserved cysteine residues at its C terminus serving as acylation sites. To analyze the structural and functional roles of acylation, we have generated by reverse genetics a series of mutants (Ac1, Ac2, and Ac3) of fowl plague virus (FPV) containing HA in which the acylation sites at positions 551, 559, and 562, respectively, have been abolished. When virus growth in CV1 and MDCK cells was analyzed, similar amounts of virus particles were observed with the mutants and the wild type. Protein patterns and lipid compositions, characterized by high cholesterol and glycolipid contents, were also indistinguishable. However, compared to wild-type virus, Ac2 and Ac3 virions were 10 and almost 1,000 times less infectious, respectively. Fluorescence transfer experiments revealed that loss of acyl chains impeded formation of fusion pores, whereas hemifusion was not affected. When the affinity to detergent-insoluble glycolipid (DIG) domains was analyzed by Triton X-100 treatment of infected cells and virions, solubilization of Ac2 and Ac3 HAs was markedly facilitated. These observations show that acylation of the cytoplasmic tail, while not necessary for targeting to DIG domains, promotes the firm anchoring and retention of FPV HA in these domains. They also indicate that tight DIG association of FPV HA is essential for formation of fusion pores and thus probably for infectivity.
Differences in the Postfusion Conformations of Full-Length and Truncated Class II Fusion Protein E of Tick-Borne Encephalitis Virus
Authors: Stiasny Karin , Kössl Christian , Heinz Franz X.
Published: April 2005

The trimeric postfusion structure of the C-terminally truncated fusion protein E of the flavivirus tick-borne encephalitis virus, a class II viral fusion protein, was previously determined (S. Bressanelli, K. Stiasny, S. L. Allison, E. A. Stura, S. Duquerroy, J. Lescar, F. X. Heinz, and F. A. Rey, EMBO J.
23:
728-738, 2004). In this study we compared the properties of this truncated form with the full-length trimer and found that the so-called stem-anchor region not only confers additional stability to the full-length molecule but also structurally modifies the protein domain carrying the fusion peptide loop. These data provide experimental evidence to support the model of a fusion process that leads to the interaction of the stem-anchor region with the fusion peptide loop in the postfusion trimer.
Characterization of Human Immunodeficiency Virus Type 1 (HIV-1) Envelope Variation and Neutralizing Antibody Responses during Transmission of HIV-1 Subtype B
Authors: Frost Simon D. W. , Liu Yang , Pond Sergei L. Kosakovsky , Chappey Colombe , Wrin Terri , Petropoulos Christos J. , Little Susan J. , Richman Douglas D.
Published: April 2005

We analyzed neutralization sensitivity and genetic variation of transmitted subtype B human immunodeficiency virus type 1 (HIV-1) in eight recently infected men who have sex with men and the virus from the six subjects who infected them. In contrast to reports of heterosexual transmission of subtype C HIV-1, in which the transmitted virus appears to be more neutralization sensitive, we demonstrate that in our study population, relatively few phenotypic changes in neutralization sensitivity or genotypic changes in envelope occurred during transmission of subtype B HIV-1. We suggest that limited genetic variation within the infecting host reduces the likelihood of selective transmission of neutralization-sensitive HIV.
Expression of secreted Wnt antagonists in gastrointestinal tissues: potential role in stem cell homeostasis
Authors: Byun T , Karimi M , Marsh J L , Milovanovic T , Lin F , Holcombe R F
Published: April 2005

Background:Wnt signalling dysregulation has been implicated in cancer, including colon and gastric cancer. Initiation of Wnt signalling is modulated by soluble Wnt antagonists (sWAs), including soluble frizzled related proteins, dickkopf (Dkk) proteins, and Wnt inhibitory factor-1 (Wif1).

Aims:To evaluate the role of sWAs in upper (gastric) and lower (colon) gastrointestinal tract tumorigenesis.

Methods:Dkk1–3, Wif1, and FrzB expression was evaluated by in situ RNA hybridisation on normal and malignant human gastric and colon tissues. Expression was graded semiquantitatively.

Results:Wif1, Dkk1, and Dkk2 were not expressed in normal gastric tissue. Dkk3 was expressed in some samples, with stronger expression in deep gastric glands. FrzB was expressed in several normal gastric samples, but not in matched tumour specimens. In contrast, Dkk1 and FrzB were not expressed in normal colon. Wif1 was expressed in most colon samples, with stronger expression at crypt bases. Dkk3 and Dkk2 expression was also concentrated at crypt bases. There were no differences between sWA expression in malignant colon and matched normal tissue.

Conclusions:sWA expression differed between upper and lower gastrointestinal tract. The loss of FrzB in gastric cancer suggests that it acts as a tumour suppressor. The graded expression of Dkk3 in gastric tissue, and Dkk2, Dkk3, and Wif1 in colon tissue, with increased expression in the deep gastric glands/colonic crypt bases, where gastrointestinal stem cells reside, suggests that sWAs may be crucial Wnt signalling regulators in these tissues, and may contribute to stem cell pool maintenance. sWAs are important components of the gastrointestinal proliferative regulatory network.
p16INK4A, CDC6, and MCM5: predictive biomarkers in cervical preinvasive neoplasia and cervical cancer
Authors: Murphy N , Ring M , Heffron C C B B , King B , Killalea A G , Hughes C , Martin C M , McGuinness E , Sheils O , O’Leary J J
Published: April 2005

Aim:To analyse and compare expression patterns of three potential biomarkers—p16INK4A, CDC6, and MCM5—and evaluate their use as predictive biomarkers in squamous and glandular cervical preinvasive neoplasia.

Methods:Immunocytochemical analysis of p16INK4A, MCM5, and CDC6 expression was performed on 20 normal, 38 cervical intraepithelial neoplasia 1 (CIN1), 33 CIN2, 46 CIN3, 10 squamous cell carcinoma, 19 cervical glandular intraepithelial neoplasia (cGIN), and 10 adenocarcinoma samples. Staining intensity was assessed using a 0–3 scoring system. p16INK4A, MCM5, and CDC6 expression was also examined in ThinPrep slides exhibiting mild, moderate, and severe dyskaryosis. Human papillomavirus (HPV) was detected using a modified SYBR green assay. Fluorogenic polymerase chain reaction (PCR) and solution phase PCR were used for specific HPV typing.

Results:All three markers showed a linear correlation between expression and grade of dysplasia. p16INK4Aand MCM5 protein expression was upregulated in all grades of squamous and glandular dysplasia. CDC6 protein was preferentially expressed in high grade lesions and in invasive squamous cell carcinoma.

Conclusion:p16INK4Aexpression was closely associated with high risk HPV infection—all grades of squamous and glandular cervical lesions were immunohistochemically positive. MCM5 staining intensity was independent of high risk HPV infection, highlighting its potential as a biomarker in both HPV dependent and independent cervical dysplasia. CDC6 may be a biomarker of high grade and invasive lesions of the cervix, with limited use in low grade dysplasia. p16INK4Awas the most reliable marker of cervical dysplasia. Combinations of dysplastic biomarkers may be useful in difficult diagnostic cases.
Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease
Authors: Yu B , Sawyer N A , Caramins M , Yuan Z G , Saunderson R B , Pamphlett R , Richmond D R , Jeremy R W , Trent R J
Published: April 2005

Aims:To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND).

Methods:The coding sequence and intron–exon boundaries of the cardiac β myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods.

Results:Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening.

Conclusions:DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.
Unnecessary repeat requesting of tests: an audit in a government hospital immunology laboratory
Authors: Kwok J , Jones B
Published: April 2005

Unnecessary repeat requesting of tests can make up a large proportion of a laboratory’s workload. This audit set out to establish the size of this problem and to identify the circumstances under which these repeat requests were made in a government tertiary hospital immunology laboratory. The numbers of tests for immunoglobulin measurement, common autoantibodies, and tumour markers that were repeated over a 12 month period were analysed by interrogating the Delphic laboratory computer system using a management information system for raw data enquiry protocol. Repeat requests within 12 weeks of a previous request made up 16.78% of the total workload. The total cost of the tests was estimated at US$ 132 151. The waste of technician time and reagents as a result of unnecessary repeat testing is excessive. Many of these tests might be eliminated with the use of interventions such as computerised reminders.
5-Fluorouracil/irinotecan induced lethal toxicity as a result of a combined pharmacogenetic syndrome: report of a case
Authors: Steiner M , Seule M , Steiner B , Bauer I , Freund M , Köhne C H , Schuff-Werner P
Published: April 2005

Combination cancer chemotherapy induced toxicity can be associated with combined pharmacogenetic syndromes. Dihydropyrimidine dehydrogenase (DPD) is the principal enzyme involved in the catabolic detoxification of 5-fluorouracil (5FU). A heterozygous G > A transition at the 5′ splicing donor consensus sequence in intron 14 leading to exon 14 skipping (IVS14+1 G > A, DPYD*2A) with partial loss of enzyme activity may be partly responsible for 5FU induced toxicity, whereas irinotecan associated toxicity may in part be explained by an aberrant UGT1A1 promoter (TA)ngenotype underlying Gilbert’s syndrome with reduced liver glucuronidation activity. This report describes a 44 year old white woman who suffered from severe gastrointestinal and haematological toxicity while undergoing 5FU24h/folinic acid/irinotecan treatment for adenocarcinoma of the sigmoid colon. Despite appropriate supportive treatment, her condition rapidly deteriorated and led to death. Molecular analysis revealed a hitherto undescribed combined pharmacogenetic syndrome, consisting of heterozygosity for the DPD IVS14+1 G > A mutation and UGT1A1 (TA)6/7heterozygosity, which probably contributed to the fatal outcome in this patient.
Angiocentric immunoproliferative lesion and angiocentric lymphoma of lymph node in children. A report of two cases
Authors: Drut R , Drut R M
Published: April 2005

Aim:To report two examples of an angiocentric immunoproliferative lesion (AIL) and angiocentric angiodestructive lymphoma (AL) presenting in lymph nodes in children. Most commonly involving extranodal sites, AIL/AL rarely presents in the spleen and lymph nodes.

Methods/Results:Case 1 presented as a cervical lymphadenopathy in a 3 year old girl being treated for pre-B cell acute lymphoblastic leukaemia. Histological and immunohistochemistry studies revealed an Epstein-Barr virus positive (EBV+), large B cell (CD20 and CD30+) AIL with large areas of necrosis, the whole resembling lymphomatoid granulomatosis. Case 2 presented as a large supraclavicular lymphadenopathy in a 13 year old boy. Histology and immunohistochemistry revealed an EBV–, large T cell (CD45RO, CD56, and CD30+) AL, presenting the features of so called angiocentric T cell/natural killer cell lymphoma, nasal type.

Conclusions:The term AIL/AL refers to a heterogeneous group of conditions not unique to a particular type of lymphoid cell. These lesions are easily recognised by the histopathologist because of their extremely unusual angiocentric pattern. Although rare, AIL/AL may present as nodal lesions in children ab initio.
Analysis of Bcl-2 protein expression in choroidal melanomas
Authors: Hussein M R
Published: April 2005

Background:Bcl-2 protooncogene alterations are involved in tumorigenesis and may have prognostic ramifications.

Aims:To investigate normal ocular structures and choroidal melanoma for: (1) Bcl-2 protein expression (semiquantitative staining values: SI, staining intensity; PP, percentage of positive cells; and IRS, immunoreactivity score) and (2) any associations between the staining values and clinicopathological features in these lesions.

Materials/Methods:Bcl-2 protein expression was analysed in 24 choroidal melanomas using immunoperoxidase staining methods.

Results:Bcl-2 protein expression was seen in corneal epithelium, lens epithelium, the ciliary body, and retinal cells. In these structures, the mean (SEM) values were: 1.1 (0.1), 1.6 (0.3), 1.1 (0.1), and 2.3 (0.3), respectively, for SI; 1.6 (0.2), 1.7 (0.1), 1.7 (0.2), and 1.7 (0.2) for PP, respectively; and 1.9 (0.4), 2.7 (0.5), 1.9 (0.1), and 4.0 (0.8), respectively, for IRS. Based on Bcl-2 immunoreactivity, the lesions were divided into two groups. The first group comprised 12 tumours with Bcl-2 expression. Bcl-2 expression was significantly higher in this group compared with normal ocular structures (1.5 (0.1)v2.8 (0.2), 1.7 (0.1)v3.5 (0.1), and 2.6 (0.3)v9.3 (0.9) for mean (SEM) SI, PP, and IRS scores, respectively; p  =  0.00). The second group comprised 12 tumours lacking Bcl-2 protein expression. There was no significant correlation between Bcl-2 protein expression and most of the clinicopathological features of these lesions.

Conclusions:Bcl-2 protein expression is altered in choroidal melanomas.