Maedi-visna virus and caprine arthritis-encephalitis virus: distinct species or quasispecies and its implications for laboratory diagnosis

OBJECTIVE: The authors identify a rare case of Wernicke-Korsakoff amnestic syndrome and highlight its occurrence in the absence of alcohol dependence. METHOD: A longitudinal case history of a patient with schizoaffective disorder who developed Wernicke-Korsakoff syndrome secondary to malnutrition. RESULTS: Refusal to eat based on persecutory delusions in chronic psychotic patients can cause malnutrition and result in Wernicke-Korsakoff syndrome. CONCLUSIONS: With increasing managed-care demands for outpatient treatment of chronic psychotic patients, physicians may see more cases of Wernicke-Korsakoff syndrome in psychiatric populations.     

Dopaminergic sensitization: implications for the pathogenesis of schizophrenia

1. Which transmitters are primarily or secondarily involved in the pathogenesis of schizophrenia has been extensively studied during the last years. This review concentrates on the two systems, that most constantly have been found dysfunctioning in patients; that are the dopaminergic and glutamatergic systems. 2. Numerous neuropathological defects have been found in schizophrenia, but it is as yet unknown which changes are causative and which reflect maladaptive reactions. 3. All findings, however, involve the cortico-striato-thalamo-cortical circuits, which are central for attention and information processing. 4. The article focuses on the consequence of transmitter dysfunction for perception and for the ability of the individual to adapt to a constantly changing environment. Both clinical and experimental studies point to a primary/early cortical defect involving the glutamatergic system, and to a later developed intermittent hyperactivity of the dopaminergic system superimposed on a basal hypodopaminergic state. 5. The authors have previously demonstrated, how it is possible to potentiate mesolimbic dopaminergic activity by intermittent electrical stimulations of the cells in the ventral tegmental area, and that influence on the central mesolimbic dopamine cells is essential for the strengthened neuroplastic response. A changed neuroplastic response to environmental stimulation due to dopaminergic sensitization can explain how an episodic, subcortical hyperactivity can act on a basic glutamatergic and dopaminergic hypofunction to produce psychotic symptoms. Based on our own and others clinical and experimental findings, the "filter" hypothesis for schizophrenia and the state-dependence of schizophrenic symptoms, the authors present a hypothesis for spontaneous mesolimbic dopaminergic sensitization and progressive evolution of psychosis.     

African swine fever virus infection induces tumor necrosis factor alpha production: implications in pathogenesis

We have analyzed the production of tumor necrosis factor alpha (TNF-alpha) induced by in vitro infection with African swine fever (ASF) virus (ASFV) and the systemic and local release of this inflammatory cytokine upon in vivo infection. An early increase in TNF-alpha mRNA expression was detected in ASFV-infected alveolar macrophages, and high levels of TNF-alpha protein were detected by ELISA in culture supernatants from these cells. When animals were experimentally infected with a virulent isolate (E-75), enhanced TNF-alpha expression in mainly affected organs correlated with viral protein expression. Finally, elevated levels of TNF-alpha were detected in serum, corresponding to the onset of clinical signs. TNF-alpha has been reported to be critically involved in the pathogenesis of major clinical events in ASF, such as intravascular coagulation, tissue injury, apoptosis, and shock. In the present study, TNF-alpha containing supernatants from ASFV-infected cultures induced apoptosis in uninfected lymphocytes; this effect was partially abrogated by preincubation with an anti-TNF-alpha specific antibody. These results suggest a relevant role for TNF-alpha in the pathogenesis of ASF.     

Differential sensitivity of hepatitis C virus quasispecies to interferon-alpha therapy

The quasispecies nature of hepatitis C virus was investigated in a patient with chronic hepatitis C virus infection who underwent interferon-alpha therapy. The hepatitis C virus E2/NS1 region was amplified and cloned, and multiple clones were sequenced before and after interferon-alpha therapy. The hepatitis C virus quasispecies can be grouped into three groups by phylogenetic tree analysis. Quasispecies from all the groups were present before interferon-alpha therapy. However, only group 3 remained after interferon-alpha therapy. In addition, only group 3 hepatitis C virus quasispecies were present during the early biochemical relapse. These data indicate that various groups of hepatitis C virus quasispecies may have different sensitivity to interferon-alpha.     

Interferon induction as a quasispecies marker of vesicular stomatitis virus populations

The interferon (IFN)-inducing capacity of different isolates of vesicular stomatitis virus (VSV) of the Indiana (IN) and New Jersey (NJ) serotypes were measured to assess the extent of variability of this phenotype. Over 200 preparations of wild-type field isolates, laboratory strains, and plaque-derived subpopulations were examined. Marked heterogeneity was found in the ability of these viruses to induce IFN, covering a 10,000-fold range. A good fit to a normal distribution for the log of the IFN yields suggests a continuum of incremental changes in the viral genome may govern the IFN-inducing capacity of consensus populations derived from independently arising infections. A broad range in the magnitude of these changes, skewed towards inducers of high IFN yields, is consistent with a comparable series of ribonucleotide changes in the VSV genome, a sine qua non of a quasispecies population. Plaque- or vesicle-derived populations displayed standard deviations less than the mean IFN yields, though skewed to higher yielders, whereas populations from field and laboratory samples which differed widely in time and origin of isolation gave standard deviations greater than the means. The plaque isolation of IFN-inducing particles of VSV-IN, normally masked in populations by the predominance of non-IFN-inducing particles that suppress IFN induction, and the isolation of potent wild-type IFN-inducing VSV-IN from cows during an outbreak of vesicular stomatitis in a region that had yielded only virus expressing the non-IFN-inducing phenotype in prior and subsequent years, supports the view that genetic bottlenecks are operative in the natural transmission of this disease.     

Apoptosis in restenosis versus stable-angina atherosclerosis: implications for the pathogenesis of restenosis

Decreases in programmed cell death (apoptosis) may contribute to restenotic hyperplasia by prolonging the life span of intimal cells. Apoptotic events were compared in restenotic versus primary lesions, by using atherectomy samples from 16 restenotic and 30 primary human peripheral and coronary lesions from patients presenting with stable angina. We used transmission electron microscopy to identify apoptosis, quantify its frequency, distinguish apoptosis from necrosis, and relate these events to cellular composition. Smooth muscle cell (SMC) density was higher in restenotic versus primary lesions (P<0.0001), whereas the number of macrophages was significantly reduced (P<0.01) and the number of lymphocytes was lower, but not significantly (P=0.06). As the main finding, restenotic lesions contained fewer apoptotic cells compared with primary lesions (3% versus 13%, P=0.002), whereas no differences were found for cellular necrosis. With regard to cell type, the lower frequency of apoptotic cells observed in restenotic tissue was attributable to both SMCs and macrophages. The key finding of less apoptosis in restenotic versus primary lesions was in agreement with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis (2% versus 9%, P<0.001). For all lesions analyzed, significant inverse correlations were observed between the density of SMCs and the frequency of apoptotic cell death (r=-0.60, P<0.001) as well as the density of SMCs and that of macrophages (r=-0.74, P<0.001). No relationship was seen between the frequency of apoptosis and the density of macrophages. In conclusion, the data of the present study indicate that a low level of apoptosis may be an important mechanism leading to restenotic intimal lesion development after interventional procedures.     

Heat-shock proteins and molecular chaperones: implications for pathogenesis, diagnostics, and therapeutics

Cells react to physical (e.g., heat) or chemical (e.g., anoxia, low pH) stressors, mounting a stress (heat-shock) response. Most genes are turned down or off, while a few are activated. The latter encode the stress or heat-shock proteins (Hsps), whose levels increase in stressed cells. Various Hsps are molecular chaperones. These, and other molecular chaperones that are not Hsps, help the other cellular proteins to achieve their native state (correct folding or functional conformation), reach their final destination (e.g., the endoplasmic reticulum or the mitochondria), resist denaturing by stressors, and regain the native state after partial denaturation. Thus the Hsps and molecular chaperones occupy the stage's center whenever and wherever there is cellular and tissue injury caused by local or systemic stressors via protein damage. This feature, their participation in protein folding and transport, and their evolutionary conservation within the three phylogenetic domains, strongly suggest a vital role for Hsps and molecular chaperones. Their importance in pathogenesis, and as diagnostic markers and prognostic indicators, is beginning to be appreciated. The role of Hsps and molecular chaperones in cell recovery from injury by a variety of noxae of clinical and surgical relevance is also being assessed. Consequently, the potential of these molecules (and corresponding genes) as targets for treatment or as therapeutic tools is emerging and is being explored. Stroke, myocardial infarction, inflammatory syndromes, infectious and parasitic diseases, autoimmune disorders, cancer, and aging are but some examples of conditions in which Hsps and molecular chaperones are being scrutinized. The era of Hsp and molecular chaperone pathology has dawned. It is likely that genetic and acquired defects of Hsp and molecular chaperone structure and function will be identified, and will play a primary, or auxiliary but determinant, role in disease.     

Functional diversity of the CD8(+) T-cell response to Epstein-Barr virus (EBV): implications for the pathogenesis of EBV-associated lymphoproliferative disorders

Epstein-Barr virus (EBV)-specific CD8(+) cytotoxic T cells are thought to be critical for the control of EBV, which persists in healthy individuals as a latent infection of B cells. However, recent observations have indicated that CD8(+) T-cell responses are not uniformly cytotoxic and that CD8(+) T cells may be subdivided into type 1 and type 2 subsets that parallel the classically described Th1 and Th2 subsets of CD4(+) T cells. Using two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, we have identified two distinct but overlapping subsets of EBV-specific CD8(+) T cells, the first of which expressed high levels of interferon gamma (IFNgamma), but little or no interleukin-4 (IL-4), whereas the second subset was IFNgamma+/IL-4(+) double-positive. A significant proportion of EBV-specific CD8(+) T cells also expressed IL-13. Subsequent analysis of a panel of 27 EBV-specific CD8(+) T-cell clones showed inverse relationships between EBV-specific cytotoxicity and secretion of IL-4, IL-10, and IFNgamma, respectively. IL-10 was not secreted by the 11 most strongly cytotoxic clones, suggesting that IL-10 secretion may provide a functional definition of an EBV-specific type 2 CD8(+) T-cell subset with reduced EBV-specific cytotoxicity. Finally, we have demonstrated that EBV-specific CD8(+) T cells that express type 2 cytokines possess the ability to activate resting B cells. EBV-specific CD8(+) T cells thus have the potential to reactivate latent EBV infection in vivo and may contribute to the development of EBV-associated lymphoproliferative disorders and lymphoma.     

Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS

The evolution of human immunodeficiency virus type 1 (HIV-1) quasispecies at the envelope gene was studied from the time of infection in 11 men who experienced different rates of CD4+ cell count decline and 6 men with unknown dates of infection by using DNA heteroduplex mobility assays. Quasispecies were genetically homogeneous near the time of seroconversion. Subsequently, slower proviral genetic diversification and higher plasma viremia correlated with rapid CD4+ cell count decline. Except for the fastest progressors to AIDS, highly diverse quasispecies developed in all subjects within 3 to 4 years. High quasispecies diversity was then maintained for years until again becoming more homogeneous in a subset of late-stage AIDS patients. Individuals who maintained high CD4+ cell counts showed continuous genetic turnover of their complex proviral quasispecies, while more closely related sets of variants were found in longitudinal samples of severely immunocompromised patients. The limited number of variants that grew out in short-term PBMC cocultures were rare in the uncultured proviral quasispecies of healthy, long-term infected individuals but more common in vivo in patients with low CD4+ cell counts. The slower evolution of HIV-1 observed during rapid progression to AIDS and in advanced patients may reflect ineffective host-mediated selection pressures on replicating quasispecies.     

Hepatitis B virus genomic heterogeneity: variation between quasispecies may confound molecular epidemiological analyses of transmission incidents

Nucleotide sequence variability studies were conducted on a 263-base pair fragment of the core-coding genomic region of hepatitis B virus (HBV), amplified by the polymerase chain reaction (PCR) from three surgeons with varying circulating levels of HBV, all of whom were thought to have transmitted HBV to their patients post-surgically. DNA sequencing was applied to amplicons obtained directly from serum and those cloned into plasmid vectors, and from single HBV molecules in serum separated by a limiting dilution procedure. In one surgeon, who had a titre of approximately 3 x 10(5) genome equivalents ml-1, the direct sequence was identical to none of 29 other sequences and differed by one base substitution from the sequence amplified from the single patient he infected. In another surgeon, who had a titre of approximately 2 x 10(6) genome equivalents ml-1, the direct sequence was identical to 17 of 36 (47%) sequences; however, the sequence common to all three infected patients was identical to a unique sequence in the surgeon that differed by three base substitutions from the direct sequence. By contrast, the direct sequence in the third surgeon, who had a titre of approximately 4 x 10(7) genome equivalents ml-1, was identical to 25 of 38 (66%) sequences, and to the sequence common to all 11 infected patients. Assessment of HBV DNA sequences directly amplified from clinical specimens may not be appropriate to studies of transmission in which the source of infection harbours a relatively dilute, heterogeneous mix of viral variants.     

Selective quasispecies transmission after systemic or mucosal exposure of macaques to simian immunodeficiency virus

OBJECTIVES: Mutans streptococci (MS) are the primary pathogens involved in the development of early childhood caries. However, factors that may affect their acquisition in the mouths of young children are not well understood, and the period of initial colonization remains controversial. This study investigated the relationship of age, number of teeth, and bottle usage/content with regard to the isolation of MS in 6-24-month-old children. METHODS: A total of 122 children from low-income families attending a nutritional supplement program, and their mothers, participated in this study. Children were examined for dental caries and number of erupted teeth and were sampled for MS. Mothers were administered a questionnaire to obtain details of baby bottle use, including what food items were put in the bottle during the last week. RESULTS: MS was detected in more than one-third of the 6-24-month-olds. Unlike some studies that suggest a later period of infectivity, approximately 20% of children under 14 months of age, including 4 of 22 infants aged 6-9 months, were colonized with MS. When examined separately, age, number of teeth, and bottle usage/content were each found to be related to the presence of MS. Mutans streptococci colonization was more likely with increasing age and number of teeth, and children whose bottles contained sweetened beverages were more likely to be colonized than children whose bottles contained milk. Logistic regression models that controlled for both age and number of teeth indicated that children who consumed sweetened beverages in their baby bottle had a statistically significant, four-fold increase in the odds of colonization by MS relative to children who consumed milk. CONCLUSIONS: The finding that approximately 20% of the children under 14 months of age were infected with MS indicates that colonization in this sample of low-income preschool children may begin earlier than suggested by some investigations. Additionally, the risk of MS colonization appears lower among infants who consume milk rather than sweetened beverages in the bottle.     

Interferon resistance of hepatitis C virus genotype 1b: relationship to nonstructural 5A gene quasispecies mutations

A 40-amino-acid sequence located in the nonstructural 5A (NS5A) protein of hepatitis C virus genotype 1b (HCV-1b) was recently suggested to be the interferon sensitivity-determining region (ISDR), because HCV-1b strains with an ISDR amino acid sequence identical to that of the prototype strain HCV-J were found to be resistant to alpha interferon (IFN-alpha) whereas strains with amino acid substitutions were found to be sensitive (N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, N. Izumi, F. Marumo, and C. Sato, J. Clin. Invest. 96:224-230, 1995; N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, Y. Ogura, N. Izumi, F. Marumo, and C. Sato, N. Engl. J. Med. 334:77-81, 1996). We used single-strand conformation polymorphism (SSCP) analysis, combined with cloning and sequencing strategies, to characterize NS5A quasispecies in HCV-1b-infected patients and determine the relationships between pre- and posttreatment NS5A quasispecies mutations and the IFN-alpha sensitivity of HCV-1b. The serine residues involved in phosphorylation of NS5A protein were highly conserved both in the various patients and in quasispecies in a given patient, suggesting that phosphorylation is important in NS5A protein function. A hot spot for amino acid substitutions was found at positions 2217 to 2218; it could be the result of either strong selection pressure or tolerance to these amino acid replacements. The proportion of synonymous mutations was significantly higher than the proportion of nonsynonymous mutations, suggesting that genetic variability in the region studied was the result of high mutation rates and viral replication kinetics rather than of positive selection. Sustained HCV RNA clearance was associated with low viral load and low nucleotide sequence entropy, suggesting (i) that the replication kinetics when treatment is started plays a critical role in HCV-1b sensitivity to IFN-alpha and (ii) that HCV-1b resistance to IFN-alpha could be conferred by numerous and/or related mutations that could be patient specific and located at different positions throughout the viral genome and could allow escape variants to be selected by IFN-alpha-stimulated immune responses. No NS5A sequence appeared to be intrinsically resistant or sensitive to IFN-alpha, but the HCV-J sequence was significantly more frequent in nonresponder quasispecies than in sustained virological responder quasispecies, suggesting that the balance between NS5A quasispecies sequences in infected patients could have a subtle regulatory influence on HCV replication.     

Hippocampal pyramidal cell disarray correlates negatively to cell number: implications for the pathogenesis of schizophrenia

It has been well documented that the medial parvocellular subnucleus of the hypothalamic paraventricular nucleus (PVN) participates in immune regulation by releasing corticotrophin-releasing hormone (CRH), which triggers the hypothalamus-pituitary-adrenal axis, leading to immunosuppression. Little is known about other possible influences of PVN on immunomodulation. Evidence, however, has been accumulating recently, indicating possible involvement of other subnuclei of this nucleus. By using the c-fos technique, the present study investigated the neuronal groups of the PVN that were activated in response to intracerebroventricularly administered IL-1 beta. In addition to strong Fos expression in the dorsal part of medial parvocellular subnucleus of the PVN, where CRH neurons are located, two more neuronal groups were found to express Fos protein. One of which was the oxytocin-immunoreactive magnocellular neurons, mainly concentrated in the anterior and medial magnocellular subnuclei of the PVN. The magnocellular PVN subnuclei are known to project to, and release their hormones, in the posterior pituitary. Another group of Fos-immunoreactive neurons were found in the brainstem and spinal cord projecting area of the PVN. By combining retrograde tracing technique and Fos immunohistochemistry, it was proved that many of the spinal cord projecting PVN neurons were activated following IL-1 beta administration, through which the spinal cord sympathetic outflow might be regulated. The present study indicates that the hypothalamic PVN may serve as an integrative center for immunomodulation via three channels, i.e., the CRH and oxytocin neuroendocrinological and the PVN-spinal cord sympathetic neural channels.     

X-Linked agammaglobulinemia patients are not infected with Epstein-Barr virus: implications for the biology of the virus

Epstein-Barr virus (EBV) infects both B lymphocytes and squamous epithelial cells in vitro, but the cell type(s) required to establish primary and persistent infection in vivo has not been definitively elucidated. The aim of this study was to investigate a group of individuals who lack mature B lymphocytes due to the rare heritable disorder X-linked agammaglobulinemia in order to determine the role of the B cell in the infection process. The results show that none of these individuals harbored EBV in their blood or throat washings. Furthermore, no EBV-specific memory cytotoxic T lymphocytes were found, suggesting that they had not undergone infection in the past. In contrast, 50% of individuals were found to carry human herpesvirus 6, showing that they are infectible by another lymphotropic herpesvirus. These results add weight to the theory that B lymphocytes, and not oropharyngeal epithelial cells, may be required for primary infection with EBV.     

Differential distribution of ciliated epithelial cells in the trachea of hamsters: implications for studies of pathogenesis

The morphology of the inner aspect of the adult hamster trachea was examined by scanning electron microscopy. Relatively large patches of unciliated cells were observed in the epithelial layer. The patches, which covered several hundreds to thousands of square microns, were most conspicuous on the ventral surface of the trachea, especially in the middle third. The frequency of these areas of unciliated cells, both isolated and in patches, was much greater in hamsters than in mice, rats, or cats. Greatest ciliation in the hamster trachea was observed over the strip of trachealis muscle between the open ends of the cartilaginous rings. Areas with the heaviest ciliation also had the greatest activity of cellular metabolism, as measured by the tetrazolium reduction assay. The attachment of tritium-labeled cells of Mycoplasma pneumoniae was inversely correlated with extensive ciliation, since the greatest numbers of counts were found on the middle third and ventral regions of the tracheal surface. The results of this study suggest that the regional differences in ciliation of respiratory epithelium in hamsters may influence studies of pathogenesis and isolation of M. pneumoniae and that these differences should therefore be considered and controlled in the experimental design.     

Quasispecies and the implications for virus persistence and escape

We have examined whether the interaction of peptide-loaded MHC molecules on the surface of B-cells with antigen-specific T-cell receptors (TCRs) enhances Ig secretion in the presence of other antigen-independent interactions in vitro. B-cells specific for region 25-40 of beta-lactoglobulin (beta-LG) were stimulated in a T-cell dependent manner using plasma membranes (PM) derived from two different T-helper (Th) clones, culture supernatants of activated Th2 cells and beta-LG as a specific antigen. PM were obtained from either the beta-LG-specific T-cell clone H1.1 which can mediate specific TCR/MHC class II interactions as well as antigen-independent ones or from the D10 clone which bears a TCR of an irrelevant specificity and thus, can only mediate antigen-independent interactions. IgG, but not IgM, secretion was specifically enhanced by H1.1 PM, but not D10 PM in the presence of beta-LG. Furthermore, a blockade of TCR/MHC class II interactions using either anti-T-cell receptor, beta or anti-CD4 monoclonal antibodies inhibited this enhanced IgG secretion in response to beta-LG. The results show that while antigen-independent interactions between T- and B-cells can enhance secretion of IgM antibodies, specific interactions between TCRs and peptide:MHC complexes stimulate B-cells to enhance secretion of IgG but not IgM antibodies. This mechanism may contribute to antibody secretion only from B-cells activated through cognate interaction in vivo.