Pyroptosis,and its Role in Central Nervous System Disease

Pyroptosis is an inflammatory form of cell death executed by transmembrane pore-forming proteins known as gasdermins and can be activated in an inflammasome-dependent or -independent manner. Inflammasome-dependent pyroptosis is triggered in response to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and has emerged as an important player in the pathogenesis of multiple inflammatory diseases, mainly by releasing inflammatory contents. More recently, numerous studies have revealed the intricate mechanisms of pyroptosis and its role in the development of neuroinflammation in central nervous system (CNS) diseases. In this review, we summarize current understandings of the molecular and regulatory mechanisms of pyroptosis. In addition, we discuss how pyroptosis can drive different forms of neurological diseases and new promising therapeutic strategies targeting pyroptosis that can be leveraged to treat neuroinflammation.     

Pathogenesis of Borna Disease Virus: Granulocyte Fractions of Psychiatric Patients Harbor Infectious Virus in the Absence of Antiviral Antibodies

Borna disease virus (BDV) causes acute and persistent infections in various vertebrates. During recent years, BDV-specific serum antibodies, BDV antigen, and BDV-specific nucleic acid were found in humans suffering from psychiatric disorders. Furthermore, viral antigen was detected in human autopsy brain tissue by immunohistochemical staining. Whether BDV infection can be associated with psychiatric disorders is still a matter of debate; no direct evidence has ever been presented. In the present study we report on (i) the detection of BDV-specific nucleic acid in human granulocyte cell fraction from three different psychiatric patients and (ii) the isolation of infectious BDV from these cells obtained from a patient with multiple psychiatric disorders. In leukocyte preparations other than granulocytes, either no BDV RNA was detected or positive PCR results were obtained only if there was at least 20% contamination with granulocytes. Parts of the antigenome of the isolated virus were sequenced, demonstrating the close relationship to the prototype BDV strains (He/80 and strain V) as well as to other human virus sequences. Our data provide strong evidence that cells in the granulocyte fraction represent the major if not the sole cell type harboring BDV-specific nucleic acid in human blood and contain infectious virus. In contrast to most other reports of putative human isolates, where sequences are virtually identical to those of the established laboratory strains, this isolate shows divergence in the region previously defined as variable in BDV from naturally infected animals.     

A New Method for the Detection of Neutralizing Antibodies against Mumps Virus

Neutralization test is the most reliable method of evaluating immunity against viral diseases but there is no standard procedure for mumps virus, with tests differing in the infectivity of the challenge virus, 50% plaque reduction or complete inhibition of cytopathic effects (CPE), and usage of complement. A reliable, easy, and simple neutralization test for mumps virus was developed in this study. A recombinant mumps virus expressing GFP was generated as a challenge virus. Complement was added to the neutralizing mixture at 1∶200 when stocked serum samples were used. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution that did not exceed two-fold of FU values (GFP expression) of the cell control wells. A total of 1,452 serum samples were assayed by inhibition of GFP expression in comparison with those examined by conventional 100% inhibition of CPE. 1,367 (94.1%) showed similar neutralizing antibody titers when examined by both methods. The GFP expression inhibition assay, using a recombinant mumps virus expressing GFP, is a simple and time- saving method.     

腺苷的中枢作用

腺苷是包括中枢神经系统（ＣＮＳ）细胞外液在内的体液的正常组成成分,其正常水平为０．０３～０．３μｍｏｌ／Ｌ。ＡＴＰ合成与分解失衡的条件下明显升高,如缺血时可升高１０００倍之多。腺苷通过腺苷受体（ａｄｅｎｏｄｉｎｅｒｅｃｅｐｔｏｒ,ＡＲ）对ＣＮＳ具有多方面的生理与病理作用,被认为是ＣＮＳ的抑制性神经调质,具有神经保护作用。     

Elemental Analysis in Murine Central Nervous System: Elevation of Rubidium Subsequent to Newcastle Disease Virus Encephalopathy

The Cg strain of Newcastle disease virus (NDV) produces neurologic signs and death in mice. This illness is unusual because of the lack of typical features of a viral encephalitis. Specifically, there is a paucity of infectious virus, detectable cellular inflammatory reaction, cytopathic effect, and viral antigen by immunofluorescence. We previously showed an elevation of alpha-aminoisobutyric acid in the CNS of moribund NDV-infected mice, indicating cellular membrane dysfunction. In an attempt to further our understanding of the pathogenesis of the illness, we evaluated CNS concentrations of sodium, potassium, iron, copper, zinc, magnesium, selenium, and rubidium. Elemental analysis revealed no difference between infected and control mice for all elements except for rubidium, which was significantly elevated in infected mice. Elevation in rubidium was detected in infected mice by X-ray fluorescence and atomic absorption spectrophotometry, whereas rubidium concentrations for control mice were similar by both methods. Neurologic symptoms correlated directly with rising rubidium concentrations. Our data suggest that abnormal trace element levels during viral infection may be one mechanism responsible for the clinical symptoms.     

Microculture System for Detection of Newcastle Disease Virus Antibodies

A microculture system utilizing cytopathic effect (CPE) and hemadsorption (HAd) end points was effective in determining the level of Newcastle disease virus (NDV) antibodies. The microculture system was of comparable sensitivity to the plaque reduction test for the detection of NDV antibodies. The standards by which the CPE and HAd microculture tests would be considered reproducible were defined. The results indicate that the CPE and HAd microculture tests are reproducible within one twofold dilution.     

Role of Interleukin-2 and Herpes Simplex Virus 1 in Central Nervous System Demyelination in Mice

We have reported previously that ocular infection of different strains of mice with recombinant herpes simplex virus 1 (HSV-1) constitutively expressing interleukin-2 (IL-2) provokes central nervous system (CNS) demyelination and optic neuropathy, as determined by changes in visual evoked cortical potentials and pathological changes in the optic nerve and CNS, whereas recombinant viruses expressing IL-4, gamma interferon, IL-12p35, IL-12p40, or IL-12p70 do not induce this neuropathy. The goal of this study was to dissect the mechanism underlying the interplay between the immune system (elevation of IL-2) and an environmental factor (infection with HSV-1) that elicits this pathology. Similar results were obtained upon delivery of IL-2 into the mouse brain using osmotic minipumps or injection of mice with recombinant IL-2 protein, IL-2 DNA, or IL-2 synthetic peptides prior to infection with wild-type (wt) HSV-1 strains McKrae and KOS. The critical role of IL-2 is further supported by our data, indicating that a single mutation at position T27A in IL-2 completely blocks the HSV-1-induced pathology. This study shows a novel model of autoimmunity in which viral infection and enhanced IL-2 cause CNS demyelination.     

Identification of Shared Populations of Human Immunodeficiency Virus Type 1 Infecting Microglia and Tissue Macrophages outside the Central Nervous System

Infection of microglia and other cells of the macrophage/monocyte lineage in the central nervous system (CNS) by human immunodeficiency virus type I (HIV-1) underlies the development of giant cell encephalitis (GCE). It is currently unknown whether GCE depends on the emergence of virus populations specifically adapted to replicate in cells of the monocyte/macrophage lineage and whether this also leads to the specific targeting of macrophages in other nonlymphoid tissues. Autopsy samples from lymph node, brain (frontal region), lung, and full-thickness colon sections were obtained from nine study subjects with GCE and from nine without. The two groups showed no significant differences in CD4 counts, disease progression, or treatment history before death. Genetic relatedness between variants recovered from lymph node and nonlymphoid tissues was assessed by sequence comparison of V3 and p17(gag) regions using a newly developed method that scores the sample composition at successive nodes in a neighbor-joining tree. The association index enabled objective, numerical comparisons on the degree of tissue compartmentalization to be made. High proviral loads and p24 antigen expression in the brain were confined to the nine individuals with GCE. GCE was also associated with significantly higher proviral loads in colon samples (median of the GCE(+) group: 1,010 copies/10(6) cells; median of GCE(-) group, 10/10(6) cells; P = 0.006). In contrast, there were no significant differences in proviral load between the GCE(+) and GCE(-) groups in lymph node or lung samples, where HIV infection was manifested predominantly by infiltrates of lymphoid cells. V3 sequences from brain samples of individuals with GCE showed the greatest compartmentalization from those of lymph node, although samples from other tissues, particularly the colon, frequently contained variants phylogenetically related to those found in brain. The existence of shared, distinct populations of HIV specifically distributed in cells of the monocyte/macrophage lineage was further indicated by immunocytochemical detection of CD68(+), multinucleated giant cells expressing p24 antigen in samples of lung and colon in two individuals with GCE. This study provides the basis for future investigation of possible phenotypic similarities that underline the shared distributions of HIV variants infecting microglia and tissue macrophages outside the CNS.     

Cadherins in the Developing Central Nervous System: An Adhesive Code for Segmental and Functional Subdivisions

In this review, we describe general features of the expression of cadherins in the developing central nervous system (CNS) of vertebrates. In the early neuroepithelium, the expression of several cadherins is restricted to specific regions corresponding to segmental domains. Segmental boundaries often coincide with changes in cadherin expression, subdividing the primordial CNS into different adhesive domains. In the different neuromeric domains, early neurons are generated which differentially express cadherins. In the mantle layer, these early neurons seem to sort out according to which cadherin they express, and they aggregate into various gray matter regions (brain nuclei and cortical lamina and regions). The gray matter structures expressing a given cadherin become connected to one another to form parts of particular functional systems or neuronal circuits. Together, these findings show that cadherins provide a molecular system reflecting both early embryonic and mature nervous system architecture. The possible roles of cadherins in the formation and maintenance of segmental and functional nervous system structures are discussed.     

Role of Matricellular Proteins in Disorders of the Central Nervous System

Matricellular proteins (MCPs) are actively expressed non-structural proteins present in the extracellular matrix, which rapidly turnover and possess regulatory roles, as well as mediate cell–cell interactions. MCPs characteristically contain binding sites for other extracellular proteins, cell surface receptors, growth factors, cytokines and proteases, that provide structural support for surrounding cells. MCPs are present in most organs, including brain, and play a major role in cell–cell interactions and tissue repair. Among the MCPs found in brain include thrombospondin-1/2, secreted protein acidic and rich in cysteine family (SPARC), including Hevin/SC1, Tenascin C and CYR61/Connective Tissue Growth Factor/Nov family of proteins, glypicans, galectins, plasminogen activator inhibitor (PAI-1), autotaxin, fibulin and perisostin. This review summarizes the potential role of MCPs in the pathogenesis of major neurological disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, ischemia, trauma, hepatic encephalopathy, Down’s syndrome, autism, multiple sclerosis, brain neoplasms, Parkinson’s disease and epilepsy. Potential therapeutic opportunities of MCP’s for these disorders are also considered in this review.     

Mutations in Hepatitis C Virus E2 Located outside the CD81 Binding Sites Lead to Escape from Broadly Neutralizing Antibodies but Compromise Virus Infectivity

Broadly neutralizing antibodies are commonly present in the sera of patients with chronic hepatitis C virus (HCV) infection. To elucidate possible mechanisms of virus escape from these antibodies, retrovirus particles pseudotyped with HCV glycoproteins (HCVpp) isolated from sequential samples collected over a 26-year period from a chronically infected patient, H, were used to characterize the neutralization potential and binding affinity of a panel of anti-HCV E2 human monoclonal antibodies (HMAbs). Moreover, AP33, a neutralizing murine monoclonal antibody (MAb) to a linear epitope in E2, was also tested against selected variants. The HMAbs used were previously shown to broadly neutralize HCV and to recognize a cluster of highly immunogenic overlapping epitopes, designated domain B, containing residues that are also critical for binding of viral E2 glycoprotein to CD81, a receptor essential for virus entry. Escape variants were observed at different time points with some of the HMAbs. Other HMAbs neutralized all variants except for the isolate 02.E10, obtained in 2002, which was also resistant to MAb AP33. The 02.E10 HCVpp that have reduced binding affinities for all antibodies and for CD81 also showed reduced infectivity. Comparison of the 02.E10 nucleotide sequence with that of the strain H-derived consensus variant, H77c, revealed the former to have two mutations in E2, S501N and V506A, located outside the known CD81 binding sites. Substitution A506V in 02.E10 HCVpp restored binding to CD81, but its antibody neutralization sensitivity was only partially restored. Double substitutions comprising N501S and A506V synergistically restored 02.E10 HCVpp infectivity. Other mutations that are not part of the antibody binding epitope in the context of N501S and A506V were able to completely restore neutralization sensitivity. These findings showed that some nonlinear overlapping epitopes are more essential than others for viral fitness and consequently are more invariant during earlier years of chronic infection. Further, the ability of the 02.E10 consensus variant to escape neutralization by the tested antibodies could be a new mechanism of virus escape from immune containment. Mutations that are outside receptor binding sites resulted in structural changes leading to complete escape from domain B neutralizing antibodies, while simultaneously compromising viral fitness by reducing binding to CD81.Over 170 million people worldwide are infected with hepatitis C virus (HCV). While acute infection is usually silent, the majority of infected individuals develop persistent infections. Approximately 30% of acute infections are spontaneously resolved. Cellular immunity is clearly necessary, as robust and sustained CD4⁺ and CD8⁺ T-cell responses are temporally associated with virus clearance leading to disease resolution (7). Persistent infection is associated with an inability to sustain a vigorous CD4⁺ response. The role of antibodies in disease resolution is increasingly recognized but less understood. Clinical trials with gamma globulin administration prior to the discovery of HCV achieved prophylactic effects on transfusion-associated non-A, non-B hepatitis cases, most of which were subsequently shown to be HCV related (28, 46). Animal studies showed that gamma globulin therapy delayed the onset of acute HCV infection (29). Preincubation of the infectious inoculum with pooled gamma globulin from HCV-positive donors prevented infection in challenged chimpanzees (55). The protection afforded by gamma globulin preparations correlated with antibody titers blocking infection of target cells with retroviral pseudotype particles expressing HCV E1E2 glycoproteins (HCVpp) (4). In addition, chimpanzees vaccinated with recombinant HCV E2 glycoproteins were protected against infection in a manner that correlated with serum antibody titers inhibiting binding of E2 to CD81 (19, 40, 41), a receptor required for entry by both HCVpp and cell culture infectious HCV (HCVcc) (5, 17, 33, 53, 56). Two recent studies observed that patients with strong and progressive neutralizing antibody responses demonstrated decreasing viremia and control of viral replication (31, 39). A third study, however, reported the lack of neutralizing antibodies to heterologous HCVpp isolates in the sera of patients who eventually controlled their viremia during acute HCV infection (21). Furthermore, 10⁴ to 10⁶ virions per milliliter of serum are usually detected during chronic infection in the presence of high titers of serum neutralizing antibodies.A driver of persistent viremia is a high degree of viral variants, or “quasispecies.” Owing to a high viral replication rate (10¹² copies per day) and an error-prone viral RNA-dependent polymerase, the estimated mutation rate is 2.0 × 10⁻³ base substitutions per genome per year (9, 34). This high rate of quasispecies formation contributes to the emergence of escape viral variants from immune surveillance. Mutations within major histocompatibility complex class I-restricted HCV epitopes lead to escape from cytotoxic T-cell responses (7). Mutations leading to escape from humoral immunity, particularly in E2 hypervariable region 1 (HVR1), known to be the target of host neutralizing antibodies, are also documented (10, 22, 30, 45). Protection in chimpanzees is achieved following challenge with an inoculum that had been preincubated with antibodies to autologous HVR1 (10). Yet over time, these isolate-specific antibodies drive the emergence of new viral variants that the concurrent immune response poorly recognizes. A study of sequential HCV isolates obtained from a patient, H, who was meticulously followed for a 26-year period starting 3 weeks after exposure to the virus, showed that the serial HCV variants were poorly neutralized by the concurrent serum antibodies (52). Escape was associated in part with mutations in HVR1 leading to decreased binding and neutralization by monoclonal antibodies (MAbs) to HVR1 that were produced against the first isolate obtained from this patient.Broadly neutralizing antibodies are usually directed against conformational epitopes within E2 (2, 8, 13, 14, 44). We previously described a panel of neutralizing and nonneutralizing human MAbs (HMAbs) to conformational epitopes on HCV E2 that were derived from peripheral B cells of individuals infected with either genotype 1a or 1b HCV. Cross-competition analyses delineated at least three immunogenic clusters of overlapping epitopes with distinct functions and properties (23-25). All nonneutralizing antibodies fell within one cluster, designated domain A (24). Neutralizing HMAbs segregated into two clusters, designated domains B and C, with domain B HMAbs having greater potency than domain C HMAbs in blocking infection with the strain JFH1 genotype 2a HCVcc (23, 25).The epitopes of increasing numbers of anti-HCV E2 neutralizing antibodies include residues that are also critical for binding of E2 to CD81. All of our domain B HMAbs inhibit binding of E2 to CD81. Alanine scanning mutagenesis of E2 regions implicated in binding to CD81 identified two highly conserved residues, G530 and D535, that are needed for all domain B antibodies, with a subset also requiring W529 (25, 26, 36). Other laboratories have isolated similar neutralizing antibodies to epitopes containing these residues (20, 32, 38). A similar panel of E2 mutants was previously used to identify five amino acid residues, W420, Y527, W529, G530, and D535, that are essential for interaction with CD81 (37, 42). These findings show that domain B antibodies exert their potent neutralization of HCV infectivity by directly competing with CD81 for binding to E2. It also explains the breadth of neutralization against different HCV genotypes and subtypes for many of these antibodies, since any changes in their epitopes could affect CD81 binding and virus entry. The conserved nature of this cluster of overlapping epitopes makes them of interest for vaccine and immunotherapeutic development. A critical question involves the likelihood that immune selection could lead to escape from neutralization by domain B HMAbs. The series of sequential HCVpp variants derived from patient H over a span of 26 years (52) provide a unique resource for studying the extent and mechanisms of virus escape from broadly neutralizing antibodies. This report describes evidence of escape from immune containment of some but not other domain B HMAbs. Interestingly, a single H variant with reduced HCVpp infectivity and diminished CD81 binding was resistant to neutralization by all domain B antibodies as well as MAb AP33, recognizing a highly conserved linear epitope spanning residues 413 to 420 (35, 47). Sequence analysis revealed multiple mutations on E2 at a considerable distance from CD81 binding residues that could account for the immune escape, although it is unlikely that they are part of the domain B HMAb or the AP33 epitopes. Site-directed substitutions at these mutations restored neutralization sensitivity to all antibodies and CD81 dependency.