Correlation between presence of lactate dehydrogenase-elevating virus RNA and antigens in motor neurons and paralysis in infected C58 mice

Lactate dehydrogenase-elevating virus (LDV) induces poliomyelitis in immunosuppressed C58 mice resulting in fatal paralysis. We have synthesized and cloned cDNA complementary to the LDV genome, and used the cDNA clones as in situ hybridization probes for the detection of LDV RNA in tissue sections. Direct fluorescent antibody staining using IgG from chronically infected mice was used for the detection of LDV antigens. Using these methods, we have detected LDV RNA and antigens in anterior horn neurons of paralyzed mice. The appearance of LDV RNA and antigen positive motor neurons and their location in the spinal cord correlated with the development of paralytic symptoms. No positive neurons were detected in LDV-infected, susceptible mice without signs of paralysis, but some glial cells of the white and gray matter in the spinal cords of these mice were found to contain LDV RNA. These analyses broaden the host cell range of LDV to include neuronal and other cells in the CNS and support the hypothesis of LDV replication in neurons as the cause of poliomyelitis and paralysis.     

Detection of hepatitis C virus (HCV) proteins by immunofluorescence and HCV RNA genomic sequences by non-isotopic in situ hybridization in bone marrow and peripheral blood mononuclear cells of chronically HCV-infected patients

Immunofluorescence (IF) to detect HCV antigens and non-isotopic in situ hybridization (NISH) to detect HCV RNA genome were carried out on bone marrow (BM) and peripheral blood (PB) mononuclear cells (MC) of 11 chronically HCV-infected patients. In four patients (36·4%) HCV antigens were detected in monocytes/macrophages as well as in B lymphocytes in both BMMC and PBMC. Positive T lymphocytes in BMMC were found in three of them, but only one patient showed positive T cells in PBMC. NISH invariably demonstrated minus and plus HCV RNA genomic strands either in monocytes/macrophages or B and T lymphocytes in BMMC and PBMC in the four HCV antigen-positive patients and in two further patients not expressing viral proteins in blood MC. IF signals appeared diffusely distributed within the cytoplasm, or as brilliant granules in distinct submembrane areas or else in cytoplasm membrane. Nuclei never stained. Similarly, NISH displayed HCV RNA accumulation restricted to MC cytoplasm only, nuclei being persistently negative. NISH, however, was unable to detect cell membrane signal. Infection of blood MC is a common event in naturally acquired HCV infection, since none of these patients was conditioned by immunomodulating or immunosuppressive therapies. No difference was found in terms of mean age, length of disease, anti-HCV immune response, type and severity of chronic liver damage between patients with HCV-infected MC and patients without cell infection. These results demonstrate that HCV can infect BMMC and PBMC that represent important extrahepatic sites of virus replication, and may help to explain the immunological abnormalities observed in chronic HCV carriers.     

Refined characterisation of chromosome aberrations in tumours by multicolour banding and electronic mapping resources

Acquired chromosome abnormalities in tumours often reflect pathogenetic events at the gene level. Multicolour fluorescence in situ hybridisation (FISH) with single-copy probes offers extensive possibilities to characterise chromosome breakpoints in relation to the physical map of the human genome. This approach is based on the construction of comprehensive EST-based maps, combinatorial labelling of probes, and tumour cell preparations optimised for metaphase FISH. Information from several electronically available databases is combined into an integrated physical map, to which clones carrying yeast and bacterial artificial chromosomes are anchored. Extracted DNA or PCR products from these clones are then fluorescently labelled by one or several fluors, allowing simultaneous FISH detection of multiple loci. To improve hybridisation efficiency and reduce background fluorescence, standard methods for chromosome preparation from cultured tumour cells are complemented with a prolonged trypsin treatment to obtain complete disaggregation of cells, and exposure of the metaphase spreads to detergent and saline at high temperature, followed by pepsin digestion to remove extracellular matrix and cytoplasmic debris. The resulting colour-banding allows the characterisation of chromosome abnormalities in relation to expressed sequences, even in tumours exhibiting highly complex rearrangements.     

Decreased expression of ErbB4 and tyrosine hydroxylase mRNA and protein in the ventral midbrain of aged rats

Decreased availability or efficacy of neurotrophic factors may underlie an increased susceptibility of mesencephalic dopaminergic cells to age-related degeneration. Neuregulins (NRGs) are pleotrophic growth factors for many cell types, including mesencephalic dopamine cells in culture and in vivo. The functional NRG receptor ErbB4 is expressed by virtually all midbrain dopamine neurons. To determine if levels of the NRG receptor are maintained during aging in the dopaminergic ventral mesencephalon, expression of ErbB4 mRNA and protein was examined in young (3 months), middle-aged (18 months), and old (24–25 months) Brown Norway/Fischer 344 F1 rats. ErbB4 mRNA levels in the substantia nigra pars compacta (SNpc), but not the adjacent ventral tegmental area (VTA) or subtantia nigra pars lateralis (SNl), were significantly reduced in the middle-aged and old animals when compared to young rats. Protein expression of ErbB4 in the ventral midbrain was significantly decreased in the old rats when compared to the young rats. Expression of tyrosine hydroxylase (TH) mRNA levels was significantly reduced in the old rats when compared to young animals in the SNpc, but not in the VTA or SNI. TH protein levels in the ventral midbrain were also decreased in the old animals when compared to the young animals. These data demonstrate a progressive decline of ErbB4 expression, coinciding with a loss of the dopamine-synthesizing enzyme TH, in the ventral midbrain of aged rats, particularly in the SNpc. These findings may implicate a role for diminished NRG/ErbB4 trophic support in dopamine-related neurodegenerative disorders of aging such as Parkinson's disease.     

Electron Microscopic and Confocal Laser Scanning Microscopic Observation of Subcellular Organelles and Pituitary Hormone mRNA: Application of Ultrastructural In Situ Hybridization and Immunohistochemistry to the Pathophysiological Studies of Pituitary Cells

Nonradioisotopic electron microscopic (EM)in situ hybridization (ISH) (EM-ISH) with biotinylated oligonucleotide probes is utilized for the ultrastructural visualization of pituitary hormone mRNA in rat pituitary cells. EM-ISH is an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. The simultaneous visualization of mRNA and encoded protein in the same cell using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex can provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein. Another focus of this review is the utilization of a recently developed imaging system of confocal laser scanning microscopy (CLSM). The combination of CLSM and image analysis system (IAS) enables us to visualize an individual dimensional image of the intracellular distribution of mRNA and subcellular organelles successfully at any optional cross sections of light microscopic ISH studies, and can be another useful tool for the ultrastructural ISH study of mRNA.     

The nucleoprotein and the viral RNA of infectious salmon anemia virus (ISAV) are localized in the nucleolus of infected cells

The infectious salmon anemia virus (ISAV), which belongs to the new genus Isavirus of the Orthomyxoviridae family, is an important pathogen of the salmon farming industry. Indirect immunofluorescence assays carried out with monoclonal antibodies specific for the nucleoprotein (NP) reveal differential staining of sub-cellular compartments in infected cells. Particularly interesting was the staining of the nucleolus, which showed co-localization with nucleolin in CHSE-214, EPC and SHK-1 cells infected with ISAV. These results were confirmed by co-immunoprecipitation studies showing an interaction between NP and nucleolin. In addition, in situ hybridization carried out with probes specific for each of the 8 RNA segments of ISAV showed that the genomic as well as the anti-genomic strands were also localized in the nucleolus. These results suggest a role of the nucleolus in the replication and/or in the packaging of the ISAV genome.     

Characterization of the translocated chromosome using fluorescencein situ hybridization and random amplified polymorphic DNA on twoTriticum aestivum—Thinopyrum intermedium translocation lines resistant to wheat streak mosaic or barley yellow dwarf virus

Fluorescencein situ hybridization (FISH) was used to determine the breakpoint of the translocation chromosome in two bread wheat (Triticum aestivum) germplasm lines withThinopyrum intermedium chromatin carrying resistance to either wheat streak mosaic virus (WSMV) or barley yellow dwarf virus (BYDV). In addition, genome-specific random amplified polymorphic DNA (RAPD) markers were used to ascertain the genomic sources of theTh. intermedium chromosomes carrying the WSMV or BYDV resistance. CI17766, a WSMV-resistant wheat germplasm line derived from induced homoeologous pairing by using theph1b mutant, had a translocation chromosome composed of the complete 4AL and about 45% of proximal 4AS from wheat, and the entire 4ES ofTh. intermedium. The BYDV-resistant translocation line, TC14, derived from tissue culture, had a very short distal segment of 7StL fromTh. intermedium terminally attached to 56% of the proximal 7DL. These observations indicate that translocations in these wheat germplasm lines did not involve centromeric breaks and fusion but were a result of homoeologous chromosome recombination.accepted for publication by J. S. (Pat) Heslop-Harrison     

Easy flat embedding of oriented samples in hydrophilic resin (LR White) under controlled atmosphere: application allowing both nucleic acid hybridizations (CARD-FISH) and ultrastructural observations

Hydrophilic resins present the advantage of making possible both hybridization experiments involving either antibodies or oligonucleotide probes and ultrastructural observations. Whereas various embedding protocols are available, only very few concern flat-embedded preparations. In this study we describe an easy protocol for flat embedding of small-oriented biological samples in hydrophilic resins (LR White). The most important constraints are (i) to polymerize the samples under argon-saturated atmosphere (avoiding oxygen which is an inhibitor of LR White polymerization) and (ii) to use transparent flat embedding molds. Two kinds of samples were analyzed: small pieces of large tissue that need to be accurately oriented for a valuable analysis and very small organisms such as free-living nematodes, which are very hard to investigate with conventional paraffin wax embedding techniques. Semi-thin sections strongly reinforce the quality of the observations from oligonucleotidic in situ hybridization experiments by reducing the background usually encountered in oligonucleotide probe hybridization experiments from sections. Such protocols could also permit a cheap alternative to the use of laser scanning confocal microscopes for oligonucleotidic in situ hybridization as in FISH and CARD-FISH experiments from histological sections. The interest of this embedding protocol is reinforced by the fact that molecular in situ hybridization experiments and ultrastructural observations from thin sections can be carried out from a single-small individual (<1mm in length) sample.     

Compartmentalization of cellular and viral DNAs in adenovirus type 5 infection as revealed by ultrastructuralin situ hybridization

We describe the respective distributions of cellular DNA and viral genomes in adenovirus type 5 (Ad5)-infected HeLa cells by means of electron microscopein situ hybridization using biotinylated Alu DNA and Ad5 DNA probes in a post-embedding technique. When hybridization was performed on sections of formaldehyde-fixed material, Alu elements were restricted to the cellular chromatin, irrespective of the stage of the infectious cycle, without any interpenetration within the virus-induced regions. Viral DNA was confined entirely to the latter. Similar topological distributions of cellular and viral DNA were obtained whenin situ hybridizations were performed at the optical level on methanol—acetone fixed cells. Such separation between cellular and viral DNA persisted under experimental conditions which partially loosened the nucleoproteins. In addition, the viral DNA of the different viral structures spread without interpenetration. On the other hand, the procedure did not dissociate the clusters of viruses which remained, as usual, closely surrounded by innumerable viral genomes. It also preserved the shapes, content (viral ssDNA + viral 72 kD protein) and the topological localization of the pleomorphic viral ssDNA accumulation sites. These data, which suggest that all viral DNAs are spatially linked in the infected nuclei, suggest a prominent role for the internal nuclear matrix in virus replication, assembly and maturation.     

Brain content of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid and immune response in aggressive C57Bl/6J mice

Catabolism of 5-hydroxytryptamine in the midbrain raphe nuclei of aggressive C57Bl/6J mice increased after 10 and 20 days of confrontations. Both catabolism and concentration of 5-hydroxytryptamine increased in the dopaminergic nuclei A11, A10, A9, and in the amigdala. The level of 5-hydroxyindoleacetic acid in the A9 and raphe nuclei decreased after 20 days of confrontations, which coincided with manifestation of the immune response. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 10, pp. 399–401, October, 2000     

Influence of N-linked glycans on intracellular transport of hepatitis C virus E1 chimeric glycoprotein and its role in pseudotype virus infectivity

We have previously reported a functional role associated with hepatitis C virus (HCV) E1 glycoprotein using vesicular stomatitis virus (VSV)/HCV pseudotype. In this study, we have investigated the role of glycosylation upon intracellular transport of chimeric E1-G, and in infectivity of the pseudotyped virus. Interestingly, surface expressed E1-G exhibited sensitivity to Endoglycosidase H (Endo H) treatment, which was similar to full-length E1, suggesting that additional complex oligosaccharides were not added while E1-G was in transit from the endoplasmic reticulum (ER) to the mammalian cell surface. As a next step, each of the four potential N-linked glycosylation sites located at amino acid position 196, 209, 234, or 305 of the E1 ectodomain were mutated separately (asparagine --> glutamine), or in some combination. FACS analysis suggested that mutation(s) of the glycosylation sites affect the translocation of E1-G to the cell surface to different extents, with no single site being particularly essential. VSV pseudotype virus generated from glycosylation mutants exhibited a decrease in titer with an increasing number of mutations at the glycosylation sites on chimeric E1-G. In a separate experiment, N-glycosidase F treatment of pseudotype generated from the already synthesized E1-G or its mutants decreased virus titer by approximately 35%, and the neutralization activity of patient sera was not significantly altered with N-glycosidase F-treated pseudotype virus. Taken together, our results suggested that E1-G does not add complex sugar moieties during transport to the cell surface and retain the glycosylation profile of its parental E1 sequence. Additionally, the removal of glycans from the E1-G reduced, but does not completely impair, virus infectivity.     

Detection and Localization of <Emphasis Type="Italic">Rice stripe virus</Emphasis> Gene Products <Emphasis Type="Italic">In Vivo</Emphasis>

The genome of the Tenuivirus, Rice stripe virus (RSV) comprises four RNAs, the smallest three of which each contain two open reading frames (ORFs) arranged in an ambisense manner. The expression of the ORFs from RNAs 2–4 in plants and the insect vector, Laodelphax striatellus, was studied using antisera raised against the gene products. In Western blotting of the proteins from infected plants, the molecular masses of p2, p3, pc3 (nucleocapsid protein, N) and p4 (major non-structural protein, NCP) were as expected; that of pc4 appeared larger than expected. Antisera to the N- and C-terminal parts of the complementary ORF on RNA 2, analogous to that encoding glycoproteins on genomes of bunyaviruses and tospoviruses, revealed banding patterns suggestive of processing of the product; the possible processing is discussed. Four types of inclusion bodies were identified by immunofluorescent and immunogold microscopy of thin sections of infected leaves. Most electron-dense amorphous semi-electron-opaque inclusion bodies (dASO) contained only p4 while some contained at least p2, pc2-N, p3, pc3 as well as p4. A ring-like structure containing at least pc2-N, p4 and pc4 was also identified in infected plant cells. Fibrillar amorphous semi-electron-opaque inclusion bodies (fASO) contained only p4. Filamentous electron-opaque inclusion bodies (FEO), which consist of pc2-N^.and p4, were found both in infected plant cells and in the mid-gut lumen and mid-gut epithelial cells of L. striatellus. This suggests an interaction between p4 and pc2-N and a function of pc2-N distinct from that of its-homologue in Bunyaviridae. Our results confirm the in vivo ambisense coding strategy of Tenuivirus RNA 2 and provide further evidence that RSV does not produce enveloped virions in infected rice plants.     

Infection of murine precision cut lung slices (PCLS) with respiratory syncytial virus (RSV) and chlamydophila pneumoniae using the Krumdieck technique

The Krumdieck technique allows the investigation of the so-called precision cut lung slices (PCLS) with a special microtome. It is thus possible to evaluate morphologic changes over a longer period of time using only a small group of animals. Chlamydophila pneumoniae (Cp) and respiratory syncytial virus (RSV) proved to be important causes of pneumonia, rhinitis and exacerbations of asthma bronchiale, as well as of lower respiratory tract infections in young children. PCLS should be tested for their suitability as an in vitro model for these infections. The PCLS were infected with Cp and RSV over different periods of time. Investigations were carried out by light and transmission electron microscopy (TEM). Furthermore, immunofluorescence (IF) studies with antibodies against bacterial or viral proteins and cell-specific markers were done using confocal laser scanning microscopy (CLSM). Non-infected and infected PCLS showed a well-preserved morphology up to 72 hours. After short infection intervals, typical inclusions of Cp or RSV were detected in vacuoles of different cell types. Infection and cell types could be verified using IF. Cytopathic effects were not prominent. Ciliary beat was detectable up to 96 hours after infection. This in vitro technique offers the possibility of studying mechanisms and effects of bacterial and viral infections on viable tissue complexes.     

The West Nile virus mutant spectrum is host-dependant and a determinant of mortality in mice

To define the impact of mosquitoes and birds on intrahost WNV population dynamics, the mutant spectra that arose as a result of 20 serial in vivo passages in Culex pipiens and young chickens were examined. Genetically homogeneous WNV was serially passaged 20 times in each host. Genetic diversity was greater in mosquito-passaged WNV compared to chicken-passaged WNV. Changes in the viral consensus sequence occurred in WNV passaged in mosquitoes earlier and more frequently than in chicken-passaged WNV. Analysis of synonymous and nonsynonymous variation suggested that purifying selection was relaxed during passage in mosquitoes. Mortality in mice was significantly negatively correlated with the size of the WNV mutant spectrum. These studies suggest that mosquitoes serve as sources for WNV genetic diversity, that birds are selective sieves, and that both the consensus sequence and the mutant spectrum contribute to WNV phenotype.     

Detection of inhibin α and βA subunits (inhibin A, B, activin A,AB) in the human pituitary gland and in pituitary adenomas by immunohistochemistry and nonradioisotopicin situ hybridization

Inhibin and activin are gonadal hormones produced in human ovaries. They are known to act on anterior pituitary cells to regulate the synthesis and secretion of follicle-stimulating hormone (FSH). The purpose of the present study was to determine the localization of inhibin and activin subunits α and βA as endocrine markers in the human normal pituitary gland and pituitary adenomas, using immunohistochemistry andin situ hybridization (ISH) methods. Pituitary tissues from surgical and autopsy materials were fixed in 10% formalin and embedded in paraffin. Five normal pituitary glands and 79 pituitary adenomas were immunostained with the avidin-biotin peroxidase complex (ABC) method using polyclonal antibodies against inhibin and activin subunits α and βA. The other antibodies against anterior pituitary hormones used in this study were as follows: antigrowth hormone (anti-GH), antiprolactin (anti-PRL), antiadrenocorticotropic hormone (anti-ACTH), anti-FSHβ, antilutenizing hormone (anti-LH) β, antithyroid-stimulating hormone (anti-TSH) β, and antiglycoprotein α-subunit (anti-α-SU). We analyzed gene expressions of subunits α and βA by nonradioisotopic ISH in pituitary adenomas. In the normal human pituitary glands, inhibin and activin subunits α and βA immunoreactivities were found diffusely in the cytoplasm of anterior pituitary cells. The percentage of subunit α-immunopositive cells was 40% of the anterior pituitary cells. Subunit βA immunoreactivities were observed in about 15% of the anterior pituitary cells. By the double-staining method, subunit α immunoreactivity was detected in all types of anterior pituitary cells, and it was colocalized most frequently with GH and α-SU-positive cells. Subunit βA immunoreactivity was colocalized predominantly with PRL, FSH-β, LH-β, and α-SU. Among the 79 adenomas, 75 cases (94.9%) were positive for subunit α, and 50 cases (63.3%) were positive for subunit βA. Subunit βA was positive in tumor cells with the following incidences: GH adenomas, 3 of 14 (21.4%); PRL adenomas, 5 of 8 (62.5%); ACTH adenomas, 6 of 6 (100%); TSH adenomas, 7 of 7 (100%); nonfunctioning adenomas, 29 of 44 (65.9%), including gonadotropin-positive, 16 of 22 (80.0%). The ISH signals for subunits α and βA were strongly expressed in gonadotropin-positive adenomas among the nonfunctioning adenomas. The mRNA signals were low and infrequent in the GH-producing adenomas. Inhibin and activin subunit α localization did not demonstrate cell-type specificity in pituitary adenomas. In contrast, subunit βA demonstrated predominant positivity in the functioning pituitary adenomas (ACTH- and TSH-secreting) and nonfunctioning adenomas (including gonadotropin-positive adenomas). The present results suggest that the functional role of inhibin and activin in the differentiation of cells in normal human pituitary glands and adenomas is present in subunit βA.     

Generation and maintenance of Listeria-specific CD8+ T cell responses in perforin-deficient mice chronically infected with LCMV

Disruption of the perforin gene results in primary immunodeficiency and an increased susceptibility to opportunistic pathogens. Perforin-deficient (PKO) mice fail to clear primary lymphocytic choriomeningitis virus (LCMV) Armstrong, resulting in persistent infection and functional exhaustion of virus-specific CD8+ T cells. CD8+ T cell responses to Listeria monocytogenes (LM) challenge within the first week after LCMV infection were diminished in both WT and PKO mice, and correlated with enhanced bacterial clearance. However, bacterial challenge at later time points generated similar CD8 T cell responses in both groups of mice. The phenotype and function of pre-existing LM-specific memory CD8+ T cells were maintained in persistently infected PKO mice. Thus persistent LCMV infection, as a result of perforin deficiency, results in dysfunction of the virus-specific CD8+ T cell response but does not compromise the host's ability to maintain pre-existing memory CD8+ T cells or to generate new memory CD8+ T cell responses against other pathogens.     

Detection of Epstein–Barr virus nucleic acid sequences and protein in nodal T-cell lymphomas: relation between latent membrane protein-1 positivity and clinical course

Forty-six nodal T-cell lymphomas, classified according to the updated Kiel classification, were investigated for the presence of Epstein–Barr virus (EBV) DNA by polymerase chain reaction (PCR), EBER 1 and 2 (EBER 1/2) and latent membrane protein-1 (LMP-1) expression. A combination of RNA in situ hybridization and immunohistochemistry was used to establish the phenotype of the Epstein–Barr virus harbouring cells. In 21 of 45 cases Epstein–Barr virus DNA sequences could be detected with the polymerase chain reaction. In 15 cases (14 of 21 EBV PCR positive cases), EBER 1/2 positive cells could be demonstrated. As judged by morphology, EBER 1/2 expression was found in non neoplastic and neoplastic lymphoid cells. Double staining revealed that more than 80% of the EBER 1/2 harbouring cells, lacked B-, T- or histiocytic markers, suggesting down regulation of T- and B-cell markers by Epstein–Barr virus. In eight of 15 cases some EBER 1/2 positive T-cells (CD3, CD45RO, CD43) morphologically resembling tumour cells were found. In nine of 14 cases tested EBER 1/2 positive non-neoplastic B-cells (CD20) were seen. Based on in situ hybridization results, four patterns of EBER 1/2 positive cells were found, i.e. single cells (≤1 per medium power field (mpf), n = 3), scattered (1–25/mpf, n = 4), clustered (26–100/mpf, n = 5) and diffuse (≥100/mpf, n = 3). In eight of 15 cases a clustered or diffuse pattern of EBER 1/2 positive cells was found and these lymphomas were therefore considered to be strongly associated with Epstein–Barr virus. In these lymphomas LMP-1 expression was found to be associated with an aggressive clinical course and hepatosplenomegaly.     

Characterization of the CD8+ T cell responses directed against respiratory syncytial virus during primary and secondary infection in C57BL/6 mice

The BALB/c mouse model for human respiratory syncytial virus infection has contributed significantly to our understanding of the relative role for CD4+ and CD8+ T cells to immune protection and pathogenic immune responses. To enable comparison of RSV-specific T cell responses in different mouse strains and allow dissection of immune mechanisms by using transgenic and knockout mice that are mostly available on a C57BL/6 background, we characterized the specificity, level and functional capabilities of CD8+ T cells during primary and secondary responses in lung parenchyma, airways and spleens of C57BL/6 mice. During the primary response, epitopes were recognized originating from the matrix, fusion, nucleo- and attachment proteins, whereas the secondary response focused predominantly on the matrix epitope. C57BL/6 mice are less permissive for hRSV infection than BALB/c mice, yet we found CD8+ T cell responses in the lungs and bronchoalveolar lavage, comparable to the responses described for BALB/c mice.