bluuebunny/crossref_metadata_2025 huggingface dataset:
Papillomavirus-Mediated Neoplastic Progression Is Associated with Reciprocal Changes in Jagged1 and Manic Fringe Expression Linked to Notch Activation
Authors: Veeraraghavalu Karthikeyan , Pett Mark , Kumar Rekha V. , Nair Pradip , Rangarajan Annapoorni , Stanley Margaret A. , Krishna Sudhir
Published: July 2004

Infection by high-risk human papillomaviruses (HPV) and persistent expression of viral oncogenes E6 and E7 are causally linked to the development of cervical cancer. These oncogenes are necessary but insufficient for complete transformation of human epithelial cells in vivo. Intracellular Notch1 protein is detected in invasive cervical carcinomas (ICC), and truncated Notch1 alleles complement the function of E6/E7 in the transformation of human epithelial cells. Here we investigate potential mechanisms of Notch activation in a human cervical neoplasia. We have analyzed human cervical lesions and serial passages of an HPV type 16-positive human cervical low-grade lesion-derived cell line, W12, that shows abnormalities resembling those seen in cervical neoplastic progression in vivo. Late-passage, but not early-passage, W12 and progression of the majority of human high-grade cervical lesions to ICC showed upregulation of Notch ligand and Jagged1 and downregulation of Manic Fringe, a negative regulator of Jagged1-Notch1 signaling. Concomitantly, an increase in Notch/CSL (CBF1, Suppressor of Hairless, Lag1)-driven reporter activity and a decrease in Manic Fringe upstream regulatory region (MFng-URR)-driven reporter activity was observed in late-passage versus early passage W12. Analysis of the MFng-URR revealed that Notch signaling represses this gene through Hairy Enhancer of Split 1, a transcriptional target of the Notch pathway. Expression of Manic Fringe by a recombinant adenovirus, dominant-negative Jagged1, or small interfering RNA against Jagged1 inhibits the tumorigenicity of CaSki, an ICC-derived cell line that was previously shown to be susceptible to growth inhibition induced by antisense Notch1. We suggest that activation of Notch in cervical neoplasia is Jagged1 dependent and that its susceptibility to the influence of Manic Fringe is of therapeutic value.
Human Coronavirus 229E Binds to CD13 in Rafts and Enters the Cell through Caveolae
Authors: Nomura Ryuji , Kiyota Asuka , Suzaki Etsuko , Kataoka Katsuko , Ohe Yoshihide , Miyamoto Kaoru , Senda Takao , Fujimoto Toyoshi
Published: July 2004

CD13, a receptor for human coronavirus 229E (HCoV-229E), was identified as a major component of the Triton X-100-resistant membrane microdomain in human fibroblasts. The incubation of living fibroblasts with an anti-CD13 antibody on ice gave punctate labeling that was evenly distributed on the cell surface, but raising the temperature to 37°C before fixation caused aggregation of the labeling. The aggregated labeling of CD13 colocalized with caveolin-1 in most cells. The HCoV-229E virus particle showed a binding and redistribution pattern that was similar to that caused by the anti-CD13 antibody: the virus bound to the cell evenly when incubated on ice but became colocalized with caveolin-1 at 37°C; importantly, the virus also caused sequestration of CD13 to the caveolin-1-positive area. Electron microscopy confirmed that HCoV-229E was localized near or at the orifice of caveolae after incubation at 37°C. The depletion of plasmalemmal cholesterol with methyl β-cyclodextrin significantly reduced the HCoV-229E redistribution and subsequent infection. A caveolin-1 knockdown by RNA interference also reduced the HCoV-229E infection considerably. The results indicate that HCoV-229E first binds to CD13 in the Triton X-100-resistant microdomain, then clusters CD13 by cross-linking, and thereby reaches the caveolar region before entering cells.
Nitric Oxide as an Endogenous Mutagen for Sendai Virus without Antiviral Activity
Authors: Yoshitake Jun , Akaike Takaaki , Akuta Teruo , Tamura Fumio , Ogura Tsutomu , Esumi Hiroyasu , Maeda Hiroshi
Published: July 2004

Nitric oxide (NO) may affect the genomes of various pathogens, and this mutagenesis is of particular interest for viral pathogenesis and evolution. Here, we investigated the effect of NO on viral replication and mutation. Exogenous or endogenous NO had no apparent antiviral effect on influenza A virus and Sendai virus. The mutagenic potential of NO was analyzed with Sendai virus fused to a green fluorescent protein (GFP) gene (GFP-SeV). GFP-SeV was cultured in SW480 cells transfected with a vector expressing inducible NO synthase (iNOS). The mutation frequency of GFP-SeV was examined by measuring loss of GFP fluorescence of the viral plaques. GFP-SeV mutation frequency in iNOS-SW480 cells was much higher than that in parent SW480 cells and was reduced to the level of mutation frequency in the parent cells by treatment with an NO synthase (NOS) inhibitor. Immunocytochemistry showed generation of more 8-nitroguanosine in iNOS-SW480 cells than in SW480 cells without iNOS transfection. Authentic 8-nitroguanosine added exogenously to GFP-SeV-infected CV-1 cells increased the viral mutation frequency. Profiles of the GFP gene mutations induced by 8-nitroguanosine appeared to resemble those of mutations occurring in mouse lungs in vivo. A base substitution that was characteristic of both mutants (those induced by 8-nitroguanosine and those occurring in vivo) was a C-to-U transition. NO-dependent oxidative stress in iNOS-SW480 cells was also evident. Together, the results indicate unambiguously that NO has mutagenic potential for RNA viruses such as Sendai virus without affecting viral replication, possibly via 8-nitroguanosine formation and cellular oxidative stress.
Putative Autocleavage of Outer Capsid Protein μ1, Allowing Release of Myristoylated Peptide μ1N during Particle Uncoating, Is Critical for Cell Entry by Reovirus
Authors: Odegard Amy L. , Chandran Kartik , Zhang Xing , Parker John S. L. , Baker Timothy S. , Nibert Max L.
Published: July 2004

Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein μ1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments μ1N (4 kDa; myristoylated) and μ1C (72 kDa). In this study, we found that μ1 cleavage to yield μ1N and μ1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective μ1 (asparagine → alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including μ1 conformational change and σ1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin α-sarcin). In addition, after these particles were allowed to enter cells, the δ region of μ1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in
trans
by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective μ1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of μ1N/μ1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal μ1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.
Evidence that Rabies Virus Forms Different Kinds of Fusion Machines with Different pH Thresholds for Fusion
Authors: Roche Stéphane , Gaudin Yves
Published: July 2004

Fusion of rabies virus with membranes is triggered at a low pH and is mediated by a viral glycoprotein (G). Fusion of rabies virus with liposomes was monitored by using a lipid mixing assay based on fluorescence resonance energy transfer. Fusion was detected below pH 6.4, and its extent increased with H
+
concentrations to be maximal around pH 6.15. The origin of the partial fusion activity of rabies virus under suboptimal pH conditions (i.e., between pH 6.15 and 6.4) was investigated. We demonstrate unambiguously that fusion at a suboptimal pH is distinct from the phenomenon of low-pH-induced inactivation and that it is not due to heterogeneity of the virus population. We also show that viruses that do not fuse under suboptimal pH conditions are indeed bound to the target liposomes and that the fusion complexes they have formed are blocked at an early stage of the fusion pathway. Our conclusion is that along the fusion reaction, different kinds of fusion machines with different pH thresholds for fusion can be formed. Possible explanations of this difference of pH sensitivity are discussed.
Vβ14+T Cells Mediate the Vaccine-Enhanced Disease Induced by Immunization with Respiratory Syncytial Virus (RSV) G Glycoprotein but Not with Formalin-Inactivated RSV
Authors: Johnson Teresa R. , Varga Steven M. , Braciale Thomas J. , Graham Barney S.
Published: July 2004

Mice immunized with respiratory syncytial virus (RSV) G glycoprotein or with formalin-inactivated RSV (FI-RSV) exhibit severe disease following RSV challenge. This results in type 2 cytokine production and pulmonary eosinophilia, both hallmarks of vaccine-enhanced disease. RSV G-induced T-cell responses were shown to be restricted to CD4+T cells expressing Vβ14 in the T-cell receptor (TCR), and the deletion of these T cells resulted in less severe disease. We therefore examined the role of Vβ14+T cells in FI-RSV-induced disease. BALB/c mice were immunized with vaccinia virus expressing secreted RSV G (vvGs) or with FI-RSV. At the time of challenge with live RSV, mice were injected with antibody to the Vβ14 component of the TCR. vvGs-immunized mice treated with anti-Vβ14 had reduced cytokine levels in the lung. Eosinophil recruitment to the lung was also significantly reduced. In contrast, depletion of Vβ14+T cells in FI-RSV-immunized mice had little impact on cytokine production or pulmonary eosinophilia. An analysis of TCR Vβ chain usage confirmed a bias toward Vβ14 expression on CD4+T cells from vvGs-immunized mice, whereas the CD4+T cells in FI-RSV-immunized mice expressed a diverse array of Vβ chains. These data show that although FI-RSV and vvGs induce responses resulting in similar immunopathology, the T-cell repertoire mediating the response is different for each immunogen and suggest that the immune responses elicited by RSV G are not the basis for FI-RSV vaccine-enhanced disease.
Antiretroviral Drug Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Increase Template-Switching Frequency
Authors: Nikolenko Galina N. , Svarovskaia Evguenia S. , Delviks Krista A. , Pathak Vinay K.
Published: July 2004

Template-switching events during reverse transcription are necessary for completion of retroviral replication and recombination. Structural determinants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that influence its template-switching frequency are not known. To identify determinants of HIV-1 RT that affect the frequency of template switching, we developed an in vivo assay in which RT template-switching events during viral replication resulted in functional reconstitution of the green fluorescent protein gene. A survey of single amino acid substitutions near the polymerase active site or deoxynucleoside triphosphate-binding site of HIV-1 RT indicated that several substitutions increased the rate of RT template switching. Several mutations associated with resistance to antiviral nucleoside analogs (K65R, L74V, E89G, Q151N, and M184I) dramatically increased RT template-switching frequencies by two- to sixfold in a single replication cycle. In contrast, substitutions in the RNase H domain (H539N, D549N) decreased the frequency of RT template switching by twofold. Depletion of intracellular nucleotide pools by hydroxyurea treatment of cells used as targets for infection resulted in a 1.8-fold increase in the frequency of RT template switching. These results indicate that the dynamic steady state between polymerase and RNase H activities is an important determinant of HIV-1 RT template switching and establish that HIV-1 recombination occurs by the previously described dynamic copy choice mechanism. These results also indicate that mutations conferring resistance to antiviral drugs can increase the frequency of RT template switching and may influence the rate of retroviral recombination and viral evolution.
Matrix Gene of Influenza A Viruses Isolated from Wild Aquatic Birds: Ecology and Emergence of Influenza A Viruses
Authors: Widjaja Linda , Krauss Scott L. , Webby Richard J. , Xie Tao , Webster Robert G.
Published: July 2004

Wild aquatic birds are the primary reservoir of influenza A viruses, but little is known about the viruses' gene pool in wild birds. Therefore, we investigated the ecology and emergence of influenza viruses by conducting phylogenetic analysis of 70 matrix (M) genes of influenza viruses isolated from shorebirds and gulls in the Delaware Bay region and from ducks in Alberta, Canada, during >18 years of surveillance. In our analysis, we included 61 published M genes of isolates from various hosts. We showed that M genes of Canadian duck viruses and those of shorebird and gull viruses in the Delaware Bay shared ancestors with the M genes of North American poultry viruses. We found that North American and Eurasian avian-like lineages are divided into sublineages, indicating that multiple branches of virus evolution may be maintained in wild aquatic birds. The presence of non-H13 gull viruses in the gull-like lineage and of H13 gull viruses in other avian lineages suggested that gulls' M genes do not preferentially associate with the H13 subtype or segregate into a distinct lineage. Some North American avian influenza viruses contained M genes closely related to those of Eurasian avian viruses. Therefore, there may be interregional mixing of the two clades. Reassortment of shorebird M and HA genes was evident, but there was no correlation among the HA or NA subtype, M gene sequence, and isolation time. Overall, these results support the hypothesis that influenza viruses in wild waterfowl contain distinguishable lineages of M genes.
Human Endogenous Retrovirus Family HERV-K(HML-5): Status, Evolution, and Reconstruction of an Ancient Betaretrovirus in the Human Genome
Authors: Lavie Laurence , Medstrand Patrik , Schempp Werner , Meese Eckart , Mayer Jens
Published: July 2004

The human genome harbors numerous distinct families of so-called human endogenous retroviruses (HERV) which are remnants of exogenous retroviruses that entered the germ line millions of years ago. We describe here the hitherto little-characterized betaretrovirus HERV-K(HML-5) family (named HERVK22 in Repbase) in greater detail. Out of 139 proviruses, only a few loci represent full-length proviruses, and many lack
gag
protease and/or
env
gene regions. We generated a consensus sequence from multiple alignment of 62 HML-5 loci that displays open reading frames for the four major retroviral proteins. Four HML-5 long terminal repeat (LTR) subfamilies were identified that are associated with monophyletic proviral bodies, implying different evolution of HML-5 LTRs and genes. Sequence analysis indicated that the proviruses formed approximately 55 million years ago. Accordingly, HML-5 proviral sequences were detected in Old World and New World primates but not in prosimians. No recent activity is associated with this HERV family. We also conclude that the HML-5 consensus sequence primer binding site is identical to methionine tRNA. Therefore, the family should be designated HERV-M. Our study provides important insights into the structure and evolution of the oldest betaretrovirus in the primate genome known to date.
Determinants of Virulence of Classical Swine Fever Virus Strain Brescia
Authors: van Gennip H. G. P. , Vlot A. C. , Hulst M. M. , de Smit A. J. , Moormann R. J. M.
Published: July 2004

Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB
476
R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB
476
R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB
476
S variant into a His residue did not affect virulence. However, the
710
His in the C1.1.1/CoBrB virus, together with adaptive mutations
276
R,
476
R, and
477
I in E
rns
, resulted in reduced virulence in pigs. These results indicated that mutations in E
rns
and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 E
rns
may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the
710
L→H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.
Internalization of Pseudorabies Virus Glycoprotein B Is Mediated by an Interaction between the YQRL Motif in Its Cytoplasmic Domain and the Clathrin-Associated AP-2 Adaptor Complex
Authors: Van Minnebruggen Geert , Favoreel Herman W. , Nauwynck Hans J.
Published: July 2004

The cytoplasmic domain of pseudorabies virus (PRV) glycoprotein B (gB) contains three putative internalization motifs. Previously, we demonstrated that the tyrosine-based YQRL motif at positions 902 to 905, but not the YMSI motif at positions 864 to 867 or the LL doublet at positions 887 and 888, is required for correct functioning of gB during antibody-mediated internalization of PRV cell surface-bound glycoproteins. In the present study, we demonstrate that the YQRL motif is also crucial to allow spontaneous internalization of PRV gB, and thus, that spontaneous and antibody-mediated internalizations of PRV gB occur through closely related mechanisms. Furthermore, we found that PRV gB colocalizes with the cellular clathrin-associated AP-2 adaptor complex and that this colocalization depends on the YQRL motif. In addition, by coimmunoprecipitation assays, we found that during both spontaneous and antibody-dependent internalization, PRV gB physically interacts with AP-2, and that efficient interaction between gB and AP-2 required an intact YQRL motif. Collectively, these findings demonstrate for the first time that during internalization of an alphaherpesvirus envelope protein, i.e., PRV gB, a specific amino acid sequence in the cytoplasmic tail of the protein interacts with AP-2 and may constitute a common AP-2-mediated mechanism of internalization of alphaherpesvirus envelope proteins.
Virus Persistence in an Animal Model of Multiple Sclerosis Requires Virion Attachment to Sialic Acid Coreceptors
Authors: Kumar A. S. Manoj , Reddi Honey V. , Kung Aisha Y. , Dal Canto Mauro , Lipton Howard L.
Published: July 2004

Persistent Theiler's virus infection in the central nervous system (CNS) of mice provides a highly relevant animal model for multiple sclerosis. The low-neurovirulence DA strain uses sialic acid as a coreceptor for cell binding before establishing infection. During adaptation of DA virus to growth in sialic acid-deficient cells, three amino acid substitutions (G1100D, T1081I, and T3182A) in the capsid arose, and the virus no longer used sialic acid as a coreceptor. The adapted virus retained acute CNS virulence, but its persistence in the CNS, white matter inflammation, and demyelination were largely abrogated. Infection of murine macrophage but not oligodendrocyte cultures with the adapted virus was also significantly reduced. Substitution of G1100D in an infectious DA virus cDNA clone demonstrated a major role for this mutation in loss of sialic acid binding and CNS persistence. These data indicate a direct role for sialic acid binding in Theiler's murine encephalomyelitis virus persistence and chronic demyelinating disease.
Expression in a Recombinant Murid Herpesvirus 4 Reveals the In Vivo Transforming Potential of the K1 Open Reading Frame of Kaposi's Sarcoma-Associated Herpesvirus
Authors: Douglas Jill , Dutia Bernadette , Rhind Susan , Stewart James P. , Talbot Simon J.
Published: July 2004

Murid herpesvirus 4 (commonly called MHV-68) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV) and provides an excellent model system for investigating gammaherpesvirus-associated pathogenesis. MHV-76 is a naturally occurring deletion mutant of MHV-68 that lacks 9,538 bp of the left end of the unique portion of the genome encoding nonessential pathogenesis-related genes. The KSHV K1 protein has been shown to transform rodent fibroblasts in vitro and common marmoset T lymphocytes in vivo. Using homologous recombination techniques, we successfully generated recombinants of MHV-76 that encode green fluorescent protein (MHV76-GFP) and KSHV K1 (MHV76-K1). The replication of MHV76-GFP and MHV76-K1 in cell culture was identical to that of MHV-76. However, infection of BALB/c mice via the intranasal route revealed that MHV76-K1 replicated to a 10-fold higher titer than MHV76-GFP in the lungs at day 5 postinfection (p.i.). We observed type 2 pneumocyte proliferation in areas of consolidation and interstitial inflammation of mice infected with MHV76-K1 at day 10 p.i. MHV76-K1 established a 2- to 3-fold higher latent viral load than MHV76-GFP in the spleens of infected mice on days 10 and 14 p.i., although this was 10-fold lower than that established by wild-type MHV-76. A salivary gland tumor was present in one of four mice infected with MHV76-K1, as well as an increased inflammatory response in the lungs at day 120 p.i. compared with that of mice infected with MHV-76 and MHV76-GFP.
Classic AIDS in a Sooty Mangabey after an 18-Year Natural Infection
Authors: Ling Binhua , Apetrei Cristian , Pandrea Ivona , Veazey Ronald S. , Lackner Andrew A. , Gormus Bobby , Marx Preston A.
Published: July 2004

Prevailing theory holds that simian immunodeficiency virus (SIV) infections are nonpathogenic in their natural simian hosts and that lifelong infections persist without disease. Numerous studies have reported that SIV-infected sooty mangabeys (SMs;Cercocebus atys) remain disease free for up to 24 years despite relatively high levels of viral replication. Here, we report that classic AIDS developed after an 18-year incubation in an SM (E041) with a natural SIVsm infection. Unlike that described in previous reports of SIV-related disease in SMs, the SIVsm infecting E041 was not first passaged through macaques; moreover, SM E041 was simian T-cell leukemia virus antibody negative. SM E041 was euthanized in 2002 after being diagnosed with severe disseminated B-cell lymphoma. The plasma virus load had been approximately the same for 16 years when a 100-fold increase in virus load occurred in years 17 and 18. Additional findings associated with AIDS were CD4+-cell decline, loss of p27 core antibody, and loss of control of SIVsm replication with disseminated giant cell disease. These findings suggest that the time to development of AIDS exceeds the average lifetime of SMs in the wild and that the principal adaptation of SIV to its natural African hosts does not include complete resistance to disease. Instead, AIDS may develop slowly, even in the presence of high virus loads. However, a long-term relatively high virus load, such as that in SM E041, is consistent with AIDS development in less than 18 years in humans and macaques. Therefore, the results also suggest that SMs have a special mechanism for resisting AIDS development.
Heparan Sulfate-Independent Infection Attenuates High-Neurovirulence GDVII Virus-Induced Encephalitis
Authors: Reddi Honey V. , Kumar A. S. Manoj , Kung Aisha Y. , Kallio Patricia D. , Schlitt Brian P. , Lipton Howard L.
Published: July 2004

The high-neurovirulence Theiler's murine encephalomyelitis virus (TMEV) strain GDVII uses heparan sulfate (HS) as a coreceptor to enter target cells. We report here that GDVII virus adapted to growth in HS-deficient cells exhibited two amino acid substitutions (R3126L and N1051S) in the capsid and no longer used HS as a coreceptor. Infectious-virus yields in CHO cells were 25-fold higher for the adapted virus than for the parental GDVII virus, and the neurovirulence of the adapted virus in intracerebrally inoculated mice was substantially attenuated. The adapted virus showed altered cell tropism in the central nervous systems of mice, shifting from cerebral and brainstem neurons to spinal cord anterior horn cells; thus, severe poliomyelitis, but not acute encephalitis, was observed in infected mice. These data indicate that the use of HS as a coreceptor by GDVII virus facilitates cell entry and plays an important role in cell tropism and neurovirulence in vivo.
Immune Escape Precedes Breakthrough Human Immunodeficiency Virus Type 1 Viremia and Broadening of the Cytotoxic T-Lymphocyte Response in an HLA-B27-Positive Long-Term-Nonprogressing Child
Authors: Feeney M. E. , Tang Y. , Roosevelt K. A. , Leslie A. J. , McIntosh K. , Karthas N. , Walker B. D. , Goulder P. J. R.
Published: July 2004

The emergence of cytotoxic T-lymphocyte (CTL) escape mutations in human immunodeficiency virus type 1 (HIV-1) proteins has been anecdotally associated with progression to AIDS, but it has been difficult to determine whether viral mutation is the cause or the result of increased viral replication. Here we describe a perinatally HIV-infected child who maintained a plasma viral load of <400 copies/ml for almost a decade until a nonbinding escape mutation emerged within the immunodominant CTL epitope. The child subsequently experienced a reemergence of HIV-1 viremia accompanied by a marked increase in the number of CTL epitopes targeted. This temporal pattern suggests that CD8 escape can play a causal role in the loss of immune control.
RNA Interference Directed against Poly(ADP-Ribose) Polymerase 1 Efficiently Suppresses Human Immunodeficiency Virus Type 1 Replication in Human Cells
Authors: Kameoka Masanori , Nukuzuma Souichi , Itaya Asako , Tanaka Yasuharu , Ota Katsuya , Ikuta Kazuyoshi , Yoshihara Koichiro
Published: July 2004

We established small interfering RNA (siRNA) directed against poly(ADP-ribose) polymerase 1 (PARP-1) that effectively reduces the expression of PARP-1 in two human cell lines. Established siRNA against PARP-1 significantly suppressed human immunodeficiency virus type 1 (HIV-1) replication, as well as the activation of the integrated HIV-1 long terminal repeat promoter. These results indicate that PARP-1 is required for efficient HIV-1 replication in human cells. We propose that PARP-1 may serve as a cellular target for RNA interference-mediated gene silencing to inhibit HIV-1 replication.
The Gene Encoding the Nucleocapsid Protein of Gill-Associated Nidovirus of
Penaeus monodon
Prawns Is Located Upstream of the Glycoprotein Gene
Authors: Cowley Jeff A. , Cadogan Lee C. , Spann Kirsten M. , Sittidilokratna Nusra , Walker Peter J.
Published: July 2004

The ORF2 gene of
Gill-associated virus
(GAV) of
Penaeus monodon
prawns resides 93 nucleotides downstream of the ORF1a-ORF1b gene and encodes a 144-amino-acid hydrophilic polypeptide (15,998 Da; pI, 9.75) containing 20 basic (14%) and 13 acidic (9%) residues and 19 prolines (13%). Antiserum to a synthetic ORF2 peptide or an
Escherichia coli
-expressed glutathione
S
-transferase-ORF2 fusion protein detected a 20-kDa protein in infected lymphoid organ and gill tissues in Western blots. The GAV ORF2 fusion protein antiserum also cross-reacted with the p20 nucleoprotein in virions of the closely related
Yellow head virus
. By immuno-gold electron microscopy, it was observed that the ORF2 peptide antibody localized to tubular GAV nucleocapsids, often at the ends or at lateral cross sections. As GAV appears to contain only two structural protein genes (ORF2 and ORF3), these data indicate that GAV differs from vertebrate nidoviruses in that the gene encoding the nucleocapsid protein is located upstream of the gene encoding the virion glycoproteins.
Multiplicity of Human Immunodeficiency Virus Infections in Lymphoid Tissue
Authors: Dixit Narendra M. , Perelson Alan S.
Published: July 2004

Human immunodeficiency virus type 1 (HIV-1)-infected splenocytes in humans were recently shown to harbor three to four proviruses per cell on average (A. Jung et al., Nature 418:144, 2002). However, the mechanisms that lead to such extensive multiple infections are not understood. Here, we find by using mathematical analysis that two mechanisms quantitatively capture the distribution of proviral genomes in HIV-1-infected splenocytes, one where multiple genomes are acquired one at a time in a series of sequential infectious contacts of a target cell with free virions and infected cells, and the other where cell-to-cell transmission of multiple virions or genomes results from a single infectious contact of a target cell with an infected cell. The two mechanisms imply different genetic diversities of proviruses within an infected cell and therefore different rates of emergence of drug resistance via recombination.
Functional Compensation of a Detrimental Amino Acid Substitution in a Cytotoxic-T-Lymphocyte Epitope of Influenza A Viruses by Comutations
Authors: Rimmelzwaan G. F. , Berkhoff E. G. M. , Nieuwkoop N. J. , Fouchier R. A. M. , Osterhaus A. D. M. E.
Published: July 2004

Influenza A viruses accumulate amino acid substitutions in cytotoxic-T-lymphocyte (CTL) epitopes, allowing these viruses to escape from CTL immunity. The arginine-to-glycine substitution at position 384 of the viral nucleoprotein is associated with escape from CTLs. Introduction of the R384G substitution in the nucleoprotein gene segment of influenza virus A/Hong Kong/2/68 by site-directed mutagenesis was detrimental to viral fitness. Introduction of one of the comutations associated with R384G, E375G, partially restored viral fitness and nucleoprotein functionality. We hypothesized that influenza A viruses need to overcome functional constraints to accumulate mutations in CTL epitopes and escape from CTLs.
Mucin expression in pleomorphic adenoma of salivary gland: a potential role for MUC1 as a marker to predict recurrence
Authors: Hamada T , Matsukita S , Goto M , Kitajima S , Batra S K , Irimura T , Sueyoshi K , Sugihara K , Yonezawa S
Published: July 2004

Background:Pleomorphic adenoma of the salivary gland (PA) is essentially a benign neoplasm. However, patients with recurrent PA are difficult to manage. There are rare reports on useful immunohistochemical markers to detect a high risk of recurrence when the primary lesions are resected.

Aims:To find a new marker to predict the recurrence of PA.

Methods:Primary lesions of PA were collected from nine patients showing subsequent recurrence and from 40 patients without recurrence during at least 10 years of follow up of the disease. Paraffin wax embedded tumour samples of the two groups were examined for the expression profiles of MUC1 (differentially glycosylated forms), MUC2, MUC4, MUC5AC, and MUC6 using immunohistochemistry. Several clinicopathological factors were also examined.

Results:In univariate analysis of the factors examined, MUC1/DF3 high expression (more than 30% of the neoplastic cells stained) in the primary lesions was seen more frequently in patients with recurrence (four of nine) than in those without recurrence (three of 40; p  =  0.011). Larger tumour size (more than 3.0 cm) of the primary PA was also a significant (p  =  0.035) risk factor for the recurrence of PA. In multivariate analysis, only high expression of MUC1/DF3 was found to be a significant independent risk factor for the recurrence of PA (p  =  0.021).

Conclusions:Expression of MUC1/DF3 in PA is a useful marker to predict its recurrence. Those patients with PA showing positive MUC1/DF3 expression should be followed up carefully.
BEST PRACTICE NO 178
Authors: Hargitai B , Marton T , Cox P M
Published: July 2004

The human placenta is an underexamined organ. The clinical indications for placental examination have no gold standards. There is also inconsistency in the histological reports and the quality is variable. There is great interobserver variability concerning the different entities. Although there are still grey areas in clinicopathological associations, a few mainstream observations have now been clarified. The histopathological examination and diagnosis of the placenta may provide crucial information. It is possible to highlight treatable maternal conditions and identify placental or fetal conditions that can be recurrent or inherited. To achieve optimal benefit from placental reports, it is essential to standardise the method of placenta examination. This article summarises the clinical indications for placenta referral and the most common acknowledged clinicopathological correlations.
Gene expression profiles of giant hairy naevi
Authors: Dasu M R K , Barrow R E , Hawkins H K , McCauley R L
Published: July 2004

Background:Congenital neomelanocytic naevi appear in nearly 1% of newborns. Giant hairy naevi (GHN) are uncommon lesions covering large areas of the body. They are of concern because they have the potential to transform into malignant melanomas.

Aims:To describe gene expression profiles of GHN and nearby normal skin from patients with GHN and normal control skin (from patients with cleft lip/palate).

Methods:Tissues from three patients with GHN and two normal controls were studied for differences in gene expression profiles. Total RNA was isolated from normal skin near the hairy naevus, GHN, and skin from normal controls. The RNA samples were subjected to probe labelling, hybridisation to chips, and image acquisition according to the standard Affymetrix protocol.

Results:There were 227 genes affected across all samples, as determined by DNA microarray analysis. There was increased expression of 22 genes in GHN compared with nearby normal skin. Decreased expression was noted in 73 genes. In addition, there was increased expression of 36 genes in normal skin near GHN compared with normal control skin, and decreased expression of five genes. Categories of genes affected were those encoding structural proteins, proteins related to developmental processes, cell death associated proteins, transcription factors, growth factors, stress response modulators, and collagen associated proteins. Changes in mRNA expression were checked by reverse transcription polymerase chain reaction.

Conclusions:Genetic profiles of GHN may provide insight into their pathogenesis, including their potential for malignant transformation. Such information may be useful in improving the understanding and management of these lesions.
Aberrant cellular retinol binding protein 1 (CRBP1) gene expression and promoter methylation in prostate cancer
Authors: Jerónimo C , Henrique R , Oliveira J , Lobo F , Pais I , Teixeira M R , Lopes C
Published: July 2004

Aims:Retinoids are involved in cell growth, differentiation, and carcinogenesis. Their effects depend on cytosolic transport and binding to nuclear receptors. CRBP1 encodes a protein involved in this process. Because altered CRBP1 expression and promoter hypermethylation occur in several tumours, these changes were investigated in prostate tumorigenesis.

Methods:The CRBP1 promoter was assessed by methylation specific polymerase chain reaction on tissue samples from 36 radical prostatectomy specimens (paired normal tissue, adenocarcinoma, and high grade prostatic intraepithelial neoplasia (HGPIN)), 32 benign prostatic hyperplasias (BPHs), and 13 normal prostate tissue samples from cystoprostatectomies. Methylation of DNA extracted from microdissected tissue was examined blindly. CRBP1 expression was assessed by immunohistochemistry on formalin fixed, paraffin wax embedded tissue.

Results:Loss of CRBP1 expression was seen in 15 of 36 adenocarcinomas and 18 of 36 HGPINs. Fifteen adenocarcinomas and nine HGPINs showed overexpression, whereas the remainder showed normal expression. BPH displayed normal expression. No significant associations were found between CRBP1 expression and Gleason score or stage. CRBP1 promoter hypermethylation was found in 17 of 36 adenocarcinomas, three of 35 HGPINs, one of 36 normal prostate tissues from the same patients, none of 32 BPHs, and none of 13 normal prostate tissues from cystoprostatectomies. Loss of expression and hypermethylation of CRBP1 were not significantly associated.

Conclusions:Altered CRBP1 expression and hypermethylation are common in prostate carcinoma, although CRBP1 hypermethylation is not an early event in tumorigenesis. Moreover, both adenocarcinoma and HGPIN show frequent CRBP1 overexpression. The molecular mechanisms underlying altered CRBP1 expression in prostate cancer deserve further study.
Aberrant epithelial expression of trefoil family factor 2 and mucin 6 inHelicobacter pyloriinfected gastric antrum, incisura, and body and its association with antralisation
Authors: Xia H H-X , Yang Y , Lam S K , Wong W M , Leung S Y , Yuen S T , Elia G , Wright N A , Wong B C-Y
Published: July 2004

Aims:To determine gastric expression of trefoil family factor 2 (TFF2) and MUC6 inHelicobacter pyloripositive and negative subjects, and its association with antralisation at the gastric incisura.

Methods:Gastric biopsies from the antrum, incisura, and body of 76 dyspeptic patients without ulcers were used for the determination ofH pyloriinfection, histological changes, and epithelial TFF2 and MUC6 expression.

Results:In the foveola, the rates of TFF2 and MUC6 immunostaining were greater inH pyloriinfected (n  =  27) than in uninfected patients (n  =  49) at the antrum (59.3%v4.1% for TFF2 and 63.0%v4.1% for MUC6; both p < 0.001) and incisura (44.4%v2.0% for TFF2 and 48.1%v0% for MUC6; both p < 0.001). In the deeper glands, the rates were also greater inH pyloriinfected than in uninfected patients at the incisura (85.2%v22.4% for both TFF2 and MUC6; p < 0.001). Antral-type mucosa was present at the incisura in 28 of the 76 patients. TFF2 and MUC6 expression in the foveola and deeper glands was significantly associated with antral-type mucosa, independent ofH pyloristatus.

Conclusions:Helicobacter pyloriinfection increases the expression of TFF2 and MUC6 in the gastric epithelium. Aberrant TFF2 and MUC6 expression is associated with antralisation of gastric incisura.
Increased neutrophil apoptosis in chronic fatigue syndrome: Table 1
Authors: Kennedy G , Spence V , Underwood C , Belch J J F
Published: July 2004

Background/Aims:Many patients with chronic fatigue syndrome (CFS) have symptoms that are consistent with an underlying viral or toxic illness. Because increased neutrophil apoptosis occurs in patients with infection, this study examined whether this phenomenon also occurs in patients with CFS.

Methods:Apoptosis was assessed in patients with CFS in conjunction with concentrations of the anti-inflammatory cytokine, transforming growth factor β1 (TGFβ1).

Results:The 47 patients with CFS had higher numbers of apoptotic neutrophils, lower numbers of viable neutrophils, increased annexin V binding, and increased expression of the death receptor, tumour necrosis factor receptor-I, on their neutrophils than did the 34 healthy controls. Patients with CFS also had raised concentrations of active TGFβ1 (p < 0.005).

Conclusions:These findings provide new evidence that patients with CFS have an underlying detectable abnormality in their immune cells.
Node retrieval in axillary lymph node dissections: recommendations for minimum numbers to be confident about node negative status
Authors: Somner J E A , Dixon J M J , Thomas J S J
Published: July 2004

Aims:To determine the minimum number of lymph nodes needed in an axillary lymph node dissection (ALND) specimen to be confident that the axilla is free from metastases.

Methods:The Edinburgh Breast Unit selects patients with large and high grade tumours for ALND; 609 consecutive ALNDs performed between October 1999 and December 2002 were reviewed. Full data about the underlying invasive breast cancer were available for 520 patients. Data were collected regarding number of positive nodes and total number of nodes collected, tumour size and grade, and presence of lymphovascular invasion.

Results:Axillary node metastases were seen in 64% of patients. The mean number of positive nodes found was 3.56, with a mean of 17.9 nodes collected. The highest proportion of patients with lymph node metastases were in the group with 16–20 nodes recovered/specimen (68%); specimens with >20 nodes recovered did not have a higher rate of nodal involvement. There was a significant difference between the proportion of metastasis positive specimens in those with 1–15 nodes recovered (58.5%) and those with 16 or more recovered (69.1%). A linear association test showed a direct correlation between the number of nodes collected and presence of node metastasis (p = 0.0005).

Conclusions:Although there is no minimum number of nodes that should be recovered in an ALND specimen, 16 nodes should be regarded as a target to ensure a high level of confidence that the nodes are negative. Node positivity in an ALND specimen appears to obey the law of diminishing returns.
Genetic pathways to melanoma tumorigenesis
Authors: Hussein M R
Published: July 2004

The incidence of cutaneous malignant melanomas is growing faster than that of any other cancer and therefore posing a major heath threat worldwide. In melanocytic skin tumours, the feasibility of correlating a specific pathological stage with a corresponding genetic alteration provides a remarkable opportunity to study the multistep tumorigenesis model. This multistep melanoma tumorigenesis is best described as a continuum of transformation of the melanocytes, melanocytic dysplasia, and melanoma formation. These steps involve genotypic alterations including loss of tumour suppressor genes, microsatellite instability, and alterations of the mismatch repair system. This review seeks to examine melanoma tumorigenesis based on these genetic changes.
Sertoli cell nodules in the undescended testis: a histochemical, immunohistochemical, and ultrastructural study of hyaline deposits
Authors: Govender D , Sing Y , Chetty R
Published: July 2004

Aims:To document the morphology, immunohistochemical staining properties, and ultrastructural features of hyaline material in Sertoli cell nodules of undescended testis and contrast them with those of sex cord tumour with annular tubules (SCTAT), which is histologically similar. To highlight the need to distinguish these nodules from other Sertoli cell hyperplasias, such as intratubular Sertoli cell proliferations, which occur in specific clinical contexts.

Materials/methods:A retrospective study of 46 orchidectomy specimens from cryptorchid testes, 27 of which contained Sertoli cell nodules. Special histochemical stains, immunohistochemical stains for type IV collagen and fibronectin, and ultrastructural examination of the hyaline material were performed using tissue from paraffin wax embedded tissue blocks.

Results:The hyaline deposits in SCTAT and Sertoli cell nodules had similar staining patterns—periodic acid Schiff (PAS) and PAS-diastase positivity with variable staining of Martius scarlet blue and Masson trichrome. Type IV collagen immunoreactivity was seen in hyaline areas, although fibronectin was negative. Electron microscopy of hyaline areas confirmed a compact matrix identical to components of the basement membrane in the adjacent seminiferous tubules.

Conclusion:This study describes an unusual form of Sertoli cell proliferation in undescended testes, which must be distinguished from Sertoli cell tumours and other forms of proliferation. In addition, the hyaline material within Sertoli cell nodules in the cryptorchid testis is histochemically, immunohistochemically, and ultrastructurally consistent with both matrix and fibrous components of seminiferous tubule basement membranes. Increased production of basement membrane material, with subsequent invagination into tubules, is the most likely origin of this material.
Secular trends of nocardia infection over 15 years in a tertiary care hospital
Authors: Matulionyte R , Rohner P , Uçkay I , Lew D , Garbino J
Published: July 2004

Aims:To assess the incidence of nocardia infection over 15 years in a tertiary care hospital.

Methods:Over a 15 year period,Nocardiaspp were isolated from 20 patients hospitalised at the Geneva University Hospitals, Switzerland.

Results:Sixteen patients had one or more underlying conditions. The median time between symptom onset and diagnosis was 30 days. The most common initial unconfirmed diagnosis was pulmonary tuberculosis (four). The lung was involved in 16 cases, followed by the central nervous system (two) and skin (two); one patient had disseminated infection. The most common species identified wasN asteroides. In vitro susceptibility testing was performed on 14 of 20 strains. All strains were susceptible to imipenem and amikacin. Initial treatment with trimethoprim/sulfamethoxazole (TMP/SMX) was started in 14 patients, although five patients had to be switched to another treatment because of side effects or lack of efficacy. A cure was observed in 15 patients, death in three, and relapse or complications in two.

Conclusions:Nocardiosis can become a severe infection and mainly affects profoundly immunocompromised patients. Differential diagnosis often delays the time to diagnosis, which worsens the outcome. New diagnostic tools, such as the polymerase chain reaction, could provide more rapid and reliable results. TMT/SMX was the most commonly prescribed treatment, but needed to be changed for another treatment because of side effects or lack of efficacy in a considerable proportion of patients. Imipenem should be used as an alternative treatment for severely ill patients, and the sulfa combination for less severe infections.