First screening of Aedes albopictus immunogenic salivary proteins.

Study of the human antibody (Ab) response to Aedes salivary proteins can provide new biomarkers to evaluate human exposure to vector bites. The identification of genus- and/or species-specific proteins is necessary to improve the accuracy of biomarkers. We analysed Aedes albopictus immunogenic salivary proteins by 2D immunoproteomic technology and compared the profiles according to human individual exposure to Ae. albopictus or Ae. aegypti bites. Strong antigenicity to Ae. albopictus salivary proteins was detected in all individuals whatever the nature of Aedes exposure. Amongst these antigenic proteins, 68% are involved in blood feeding, including D7 protein family, adenosine deaminase, serpin and apyrase. This study provides an insight into the repertoire of Ae. albopictus immunogenic salivary proteins for the first time.

On the origin and development of glioblastoma: multifaceted role of perivascular mesenchymal stromal cells.

Glioblastoma, IDH wild-type is the most common and aggressive form of glial tumors. The exact mechanisms of glioblastoma oncogenesis, including the identification of the glioma-initiating cell, are yet to be discovered. Recent studies have led to the hypothesis that glioblastoma arises from neural stem cells and glial precursor cells and that cell lineage constitutes a key determinant of the glioblastoma molecular subtype. These findings brought significant advancement to the comprehension of gliomagenesis. However, the cellular origin of glioblastoma with mesenchymal molecular features remains elusive. Mesenchymal stromal cells emerge as potential glioblastoma-initiating cells, especially with regard to the mesenchymal molecular subtype. These fibroblast-like cells, which derive from the neural crest and reside in the perivascular niche, may underlie gliomagenesis and exert pro-tumoral effects within the tumor microenvironment. This review synthesizes the potential roles of mesenchymal stromal cells in the context of glioblastoma and provides novel research avenues to better understand this lethal disease.

[Physiopathology of chronic arthritis following chikungunya infection in man]. / Physiopathologie de l'arthrite chronique post-chikungunya chez l'homme.

Chronic arthritis following chikungunya infection has no specific treatment. Studies on mice have confirmed involvement of fibroblasts and myoblasts as target cells replicating the virus and shown that macrophages play a key role in the innate immune response involving multiple cytokines and chimiokines. Paradoxically, TH1 and TH2 cytokine levels do not increase significantly during the acute and chronic phases, with the exception of interferon-gamma and IL12 that rise dramatically during the acute phase. The level of IL12 returns to normal in patients who recover. In contrast, patients who develop chronic arthritis show persistently high IL12 levels along with IFN-alpha within PBMC. Histologic examination of synovia reveals joint inflammation due to macrophages containing viral material. Metallo-protease (MMP2) also contributes to tissue damage. Chikungunya virus leads to apoptosis by both the intrinsic and extrinsic pathways.

Identification of an astrocyte cell population from human brain that expresses perforin, a cytotoxic protein implicated in immune defense.

The brain is an immunoprivileged organ isolated from the peripheral immune system. However, it has been shown that resident cells, notably astrocytes and microglia, can express numerous innate immune molecules, providing the capacity to generate a local antipathogen system. Perforin is a cytolytic protein present in the granules of cytotoxic T lymphocytes and natural killer cells. Expression in cells other than those of the hemopoetic lineage has not been described. We report here that fetal astrocytes in culture (passages 2 to 15), astrocytoma, and adult astrocytes expressed perforin. Reverse transcriptase polymerase chain reaction followed by Southern blot was carried out using multiple specific primers and all cDNAs were cloned and sequenced. Human fetal astrocyte perforin cDNA sequence was approximately 100% identical to the reported perforin cDNA cloned from T cells. Western blot analysis using monoclonal and polyclonal antiperforin peptide antibodies revealed a protein of 65 kD in both human fetal astrocyte and rat natural killer cell lysates (n = 4). Immunostaining followed by FACS(R) and confocal and electron microscopy analysis revealed that perforin was expressed by 40-50% of glial fibrillary acidic protein positive cells present in the fetal brain culture (n = 11). Perforin was not localized to granules in astrocytes but was present throughout the cytoplasm, probably in association with the endoplasmic reticulum. Perforin was not detected in normal adult brain tissue but was present in and around areas of inflammation (white and grey matter) in multiple sclerosis and neurodegenerative brains. Perforin-positive cells were identified as reactive astrocytes. These findings demonstrate that perforin expression is not unique to lymphoid cells and suggest that perforin produced by a subpopulation of astrocytes plays a role in inflammation in the brain.

Role of complement and complement regulators in the removal of apoptotic cells.

Apoptosis, followed by rapid phagocytic clearance, is the primary mechanism by which organisms dispose of unwanted cells. The intracellular and extracellular composition of an apoptotic cell changes to decrease immunogenicity and enhance its uptake. By changing their extracellular composition, apoptotic cells acquire the capacity to bind complement initiation molecules such as C1q and MBL. Binding of these molecules can lead to complement activation. Membrane bound complement inhibitors are down-regulated during apoptosis, which would leave the cell less protected against complement activation; however, recent data show that fluid-phase complement inhibitors may compensate for this loss of regulation. Importantly, binding of complement is a process that mainly takes place during the late stages of apoptosis. Most cells will be cleared before that stage under steady state conditions, but during overwhelming apoptosis or impaired phagocytosis, apoptotic cells may remain in tissues for a longer time and acquire complement proteins. Based on the data from deficiencies of early complement components and the development of systemic lupus erythematosus with accumulation of dead cells, it is clear that, under certain conditions, apoptotic cells persist, becoming necrotic and overloading the scavenging capacities of the complement system. Although the complement system is also involved in inducing apoptosis in target cells, this review will focus on the role of complement in the clearance of apoptotic cells.

The role of primary infection of Schwann cells in the aetiology of infective inflammatory neuropathies.

Numerous different pathogens are responsible for infective peripheral neuropathies and this is generally the result of the indirect effects of pathogen infection, namely anti pathogen antibodies cross reacting with epitopes on peripheral nerve, auto reactive T cells attacking myelin, circulating immune complexes and complement fixation. Primary infection of Schwann cells (SC) associated with peripheral nerve inflammation is rare requiring pathogens to cross the Blood Peripheral Nerve Barrier (BPNB) evade anti-pathogen innate immune pathways and invade the SC. Spirochetes Borrelia bourgdorferi and Trepomema pallidum are highly invasive, express surface lipo proteins, but despite this SC are rarely infected. However, Trypanosoma cruzi (Chaga's disease) and Mycobacterium leprae. Leprosy are two important causes of peripheral nerve infection and both demonstrate primary infection of SC. This is due to two novel strategies; T. cruzi express a trans-silalidase that mimics host neurotrophic factors and infects SC via tyrosine kinase receptors. M. leprae demonstrates multi receptor SC tropism and subsequent infection promotes nuclear reprogramming and dedifferentiation of host SC into progenitor stem like cells (pSLC) that are vulnerable to M. leprae infection. These two novel pathogen evasion strategies, involving stem cells and receptor mimicry, provide potential therapeutic targets relevant to the prevention of peripheral nerve inflammation by inhibiting primary SC infection.

Complement classical pathway expression by human skeletal myoblasts in vitro.

Human myoblasts express immunological properties in vitro and we have previously reported that they produce Complement (C) components of the alternative pathway. Myoblasts activate the classical pathway but are fully protected against C attack by the expression of major C regulators. In order to fully understand the relationship between myoblasts and C, we here report the biosynthesis of C components of the classical pathway by skeletal muscle cells. Human myoblasts in vitro produced C1q, C1r, C1s, C2 and C4 constitutively and all syntheses were upregulated after stimulation with IFN-gamma. We suggest that human myoblasts may constitute a local source of C and therefore C could be implicated in inflammatory or physiopathological processes developed in skeletal muscle.

Role of complement in the aetiology of Pick's disease?

Complement in the postmortem brains of 15 cases of Pick's disease has been widely analyzed immunohistochemically and, in 2 cases, by immunoelectron microscopy. Astrocytes and the Pick bodies and cytoplasm of ballooned neurons were immunoreactive with antibodies to classical pathway components C1, C1q, C4, C2 and C3 and the terminal complex components C5, C6 and C8. In almost all cases, no immunostaining was obtained with antibodies against C9 and neoepitopes in the membrane attack complex (MAC), the complement complex responsible for cytotoxicity. However, unequivocal staining with antibodies to two soluble complement regulatory proteins, S-protein and clusterin, and to the membrane complement inhibitor CD59 was found, although three other membrane inhibitors, CR1(CD35), DAF (CD55), and MCP (CD46), were not detected. The complement immunoreactivity of astrocytes and neurons could be the result of complement biosynthesis or attack. Complement attack will be restricted by the expressed regulatory proteins. However, neurons may be the victims of attack since they show pathological change. The internalization of complement-attacked membrane, perhaps involving the genesis of Pick bodies and ballooning, may explain the intracellular immunolocalization of complement in damaged neurons. Immunoglobulins, as a possible source of complement activation, were observed in only two cases, leaving unresolved the trigger for complement activation in the other cases.

C3A binds to the seven transmembrane anaphylatoxin receptor expressed by epithelial cells and triggers the production of IL-8.

The complement (C) plays an important role in many acute inflammatory processes. C3a is an inflammatory polypeptide named anaphylatoxin, generated during C activation and which acts through a specific receptor C3aR. In this study, we demonstrated that the epithelial cell line ECV 304 constitutively expressed C3aR (by flow cytometry and immunofluorescence) and that binding of purified C3a to epithelial cells resulted in a time- and dose-dependent upregulation of interleukin-8 (IL-8). Pre-treatment of ECV 304 with pertussis toxin inhibited IL-8 response induced by C3a, indicating that the action of C3a was mediated by a G protein coupled pathway.

Complement activation on human neuroblastoma cell lines in vitro: route of activation and expression of functional complement regulatory proteins.

Two human neuroblastoma cell lines activated the classical pathway of complement in serum. Activation caused the opsonisation of these cells with complement fragments but with moderate cell killing. Neuroblastoma expressed regulators MCP and CD59 but did not express DAF or CR1. Neutralisation of CD59 rendered the cells susceptible to killing. Neuroblastoma also expressed C1-inhibitor, factor H, clusterin and S-protein. Expression of several regulators was enhanced by incubation with cytokines. Complement inhibition using soluble CRI markedly reduced opsonisation and killing of neuroblastoma. Our results suggest that complement might play a role in neuronal loss and that treatment with complement inhibitors might be of therapeutic value.

The endogenous benzodiazepine receptor ligand ODN increases cytosolic calcium in cultured rat astrocytes.

We have investigated the production of diazepam-binding inhibitor (DBI)-related peptides by astrocytes in primary culture and we have determined the effect of the octadecaneuropeptide DBI[33-50] (ODN) on the intracellular calcium concentration ([Ca2+]i) in astrocytes. Immunocytochemical labeling with antibodies against ODN showed that cultured astrocytes retain their ability to synthesize DBI in vitro. Cultured astrocytes were also found to release substantial amounts of ODN-immunoreactive material, and a brief exposure of astrocytes to a depolarizing potassium concentration resulted in a 5-fold increase in the rate of release of the ODN-like peptide. Microfluorimetric measurement of [Ca2+]i with the fluorescent probe indo-1 showed that nanomolar concentrations of ODN induced a marked increase in [Ca2+]i. The stimulatory effect of ODN on [Ca2+]i was not affected by calcium channel blockers or by incubation in Ca(2+)-free medium. In contrast, thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase activity, totally abolished the ODN-induced increase in [Ca2+]i. Repeated pulses of ODN caused attenuation of the response, indicating the existence of a desensitization phenomenon. Preincubation of astrocytes with pertussis toxin totally blocked the effect of ODN on [Ca2+]i. The present study indicates that ODN-related peptides are synthesized and released by glial cells. Our results also show that synthetic ODN induces calcium mobilization from an intracellular store through stimulation of pertussis toxin-sensitive G protein. Taken together, these data suggest that endozepines act as paracrine and/or autocrine factors controlling the activity of astroglial cells.

Characterization of rat C5a anaphylatoxin receptor (C5aR): cloning of rat C5aR cDNA and study of C5aR expression by rat astrocytes.

Complement system activation within the central nervous system (CNS) is involved in demyelinating and neurodegenerative disorders, but the role of complement in the pathogenic process or in the repair remains unclear. Besides the direct lytic effects of complement on target cells (oligodendrocytes or neurons), complement can exert other functions through interaction of complement fragments with specific receptors. The C5a anaphylatoxin, an inflammatory peptide which is formed during complement activation, might play a role in the CNS pathogenesis, and activation and recruitment of glial cells by binding to its receptor (C5aR) on CNS cells. Using degenerate primers corresponding to homologous regions between human and mouse C5aR cDNAs, we have cloned a rat C5aR cDNA probe from rat monocytes RNAs after RT-PCR experiment. The rat C5aR probe isolated by this procedure allowed us to clone the rat C5aR cDNA-coding sequence using a library screening cloning strategy. This probe was also used to study the expression of the C5aR mRNA in the rat CNS. Northern blotting and RT-PCR experiments demonstrated the constitutive expression of C5aR mRNA in brain, spleen, kidney and lung. This transcript was also observed in primary culture of rat astrocytes. Microfluorimetry experiments demonstrated that C5aR expressed by astrocytes in culture is functional since the addition of C5a induced a dose-dependent increase of intracellular calcium concentration. The expression of the C5aR by astrocytes suggests new roles for the C5a anaphylatoxin in reactive astrogliosis to CNS injuries.

Culture supernatants of patient-derived Aspergillus isolates have toxic and lytic activity towards neurons and glial cells.

Infection of the central nervous system by the ubiquitous fungi Aspergillus spp. is a life-threatening disease. Therefore we investigated the mechanism of brain damage by fungal infection. To examine whether secretory factors of Aspergillus isolates derived from patients can induce death of different brain cells, culture supernatants of Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger were added to different astrocytes as well as to the neuroblastoma cell line SK-N-SH, and to the microglial cell line CHME. All four fungal species were shown to secrete toxic factors with neurons being most sensitive against these factors. Very low amounts and short incubation times are sufficient to induce irreversible cell damage, indicating that secreted factors might also affect distant brain regions. Further characterization of the toxic factors revealed that A. fumigatus and A. terreus produced small, heat-stable components whereas the toxic activity of A. niger filtrates was triggered by a high molecular mass factor which could be inactivated by heat. The active component of A. flavus had a molecular mass similar to that of A. niger but was heat-stable and had a significantly lower activity. Taken together these results indicate that secretion of different necrotizing factors might contribute to brain lesions in patients with cerebral aspergillosis.

Encephalitis due to emerging viruses: CNS innate immunity and potential therapeutic targets.

The emerging viruses represent a group of pathogens that are intimately connected to a diverse range of animal vectors. The recent escalation of air travel climate change and urbanization has meant humans will have increased risk of contacting these pathogens resulting in serious CNS infections. Many RNA viruses enter the CNS by evading the BBB due to axonal transport from the periphery. The systemic adaptive and CNS innate immune systems express pattern recognition receptors PRR (TLRs, RiG-1 and MDA-5) that detect viral nucleic acids and initiate host antiviral response. However, several emerging viruses (West Nile Fever, Influenza A, Enterovirus 71 Ebola) are recognized and internalized by host cell receptors (TLR, MMR, DC-SIGN, CD162 and Scavenger receptor B) and escape immuno surveillance by the host systemic and innate immune systems. Many RNA viruses express viral proteins WNF (E protein), Influenza A (NS1), EV71 (protein 3C), Rabies (Glycoprotein), Ebola proteins (VP24 and VP 35) that inhibit the host cell anti-virus Interferon type I response promoting virus replication and encephalitis. The therapeutic use of RNA interference methodologies to silence gene expression of viral peptides and treat emerging virus infection of the CNS is discussed.

The regulation of the CNS innate immune response is vital for the restoration of tissue homeostasis (repair) after acute brain injury: a brief review.

Neurons and glia respond to acute injury by participating in the CNS innate immune response. This involves the recognition and clearance of "not self " pathogens and "altered self " apoptotic cells. Phagocytic receptors (CD14, CD36, TLR-4) clear "not self" pathogens; neurons and glia express "death signals" to initiate apoptosis in T cells.The complement opsonins C1q, C3, and iC3b facilitate the clearance of apoptotic cells by interacting with CR3 and CR4 receptors. Apoptotic cells are also cleared by the scavenger receptors CD14, Prs-R, TREM expressed by glia. Serpins also expressed by glia counter the neurotoxic effects of thrombin and other systemic proteins that gain entry to the CNS following injury. Complement pathway and T cell activation are both regulated by complement regulatory proteins expressed by glia and neurons. CD200 and CD47 are NIRegs expressed by neurons as "don't eat me" signals and they inhibit microglial activity preventing host cell attack. Neural stem cells regulate T cell activation, increase the Treg population, and suppress proinflammatory cytokine expression. Stem cells also interact with the chemoattractants C3a, C5a, SDF-1, and thrombin to promote stem cell migration into damaged tissue to support tissue homeostasis.

Innate immunity and protective neuroinflammation: new emphasis on the role of neuroimmune regulatory proteins.

Brain inflammation due to infection, hemorrhage, and aging is associated with activation of the local innate immune system as expressed by infiltrating cells, resident glial cells, and neurons. The innate immune response relies on the detection of "nonself" and "danger-self" ligands behaving as "eat me signals" by a plethora of pattern recognition receptors (PRRs) expressed by professional and amateur phagocytes to promote the clearance of pathogens, toxic cell debris (amyloid fibrils, aggregated synucleins, prions), and apoptotic cells accumulating within the brain parenchyma and the cerebrospinal fluid (CSF). These PRRs (e.g., complement, TLR, CD14, scavenger receptors) are highly conserved between vertebrates and invertebrates and may represent the most ancestral innate scavenging system involved in tissue homeostasis. However, in some diseases, these protective mechanisms lead to neurodegeneration on the ground that several innate immune molecules have neurocytotoxic activities. The response is a "double-edged sword" representing a fine balance between protective and detrimental effects. Several key regulatory mechanisms have now been evidenced in the control of CNS innate immunity, and these could be harnessed to explore novel therapeutic avenues. We will herein provide new emphasis on the role of neuroimmune regulatory proteins (NIRegs), such as CD95L, TNF, CD200, CD47, sialic acids, CD55, CD46, fH, C3a, HMGB1, which are involved in silencing innate immunity at the cellular and molecular levels and suppression of inflammation. For instance, NIRegs may play an important role in controlling lymphocyte/macrophage/microglia hyperinflammatory responses, while sparing host defense and repair mechanisms. Moreover, NIRegs have direct beneficial effects on neurogenesis and contributing to brain tissue remodeling.

Structure-function studies of the receptors for complement C1q.

C1q is an essential component of the phylogenetically ancient innate complement (C) system and is crucial to our natural ability to ward off infection and clear toxic cell debris (e.g. amyloid fibrils, apoptotic cells). Several candidate C1q receptors [C1q receptor for phagocytosis enhancement (C1qRp), complement receptor (CR) 1, calreticulin (CRT), binding protein for the globular head of C1q (gC1qbp)] have been described, and the aim of this review is to shed light on their structure-function relationships. One cell-surface molecule, C1qRp, has emerged as a defence collagen receptor for C1q, as well as mannose-binding lectin (MBL) and surfactant protein A. C1qRp (also known as the AA4 antigen in rodents) is the antigen recognized by a pro-adhesive monoclonal antibody called mNI-11 and antibodies against CD93, but recent results failed to confirm C1q binding activity. CR1 (CD35), a multifunctional receptor both in its ligand specificity and in its C regulation activities, is found on circulating monocytes and neutrophils, but the major site of expression is B-lymphocytes. As a receptor, CR1 binds to C1q, other C opsonins (C4b, C3b, iC3b) and MBL, and as such, has been involved in promoting phagocytosis. Several studies support a role for the cell surface receptor for the collagenous domains of C1q (cC1qR; also known as CRT). CRT belongs to the family of heat-shock proteins, the most abundant and ubiquitous soluble intracellular proteins. Though CRT does not have a transmembrane domain, it seems to mediate phagocytosis of the apoptotic cells through association with CD91. A 33 kDa protein interacts with the globular head of C1q and, logically, has been termed gC1qbp. This protein is located in mitochondria, suggesting that gC1qbp is not a cell-surface receptor itself.

Complement regulatory protein expression by a human oligodendrocyte cell line: cytokine regulation and comparison with astrocytes.

Rat oligodendrocytes spontaneously activate complement (C) and lack the C inhibitor CD59. As a consequence, rat oligodendrocytes are susceptible to lysis by autologous C in vitro. Expression of C inhibitors on human oligodendrocytes in vitro and other human glia has yet to be well characterized. We have previously shown expression at the mRNA level of the membrane inhibitors CD59, decay-accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46) in human astrocytes. We here examine the expression of membrane and secreted C inhibitors by the oligodendrocyte cell line, HOG. HOG cells abundantly expressed CD59, assessed at protein and mRNA level, and expressed DAF and MCP, albeit at a lower level. Expression of all three inhibitors was enhanced by incubation with interferon-gamma or with phorbol ester (PMA). Complement receptor type 1 (CR1; CD35) was neither expressed constitutively nor induced by cytokines. HOG also constitutively secreted C1-inhibitor, S-protein and clusterin. Factor H was secreted only after stimulation with cytokines. C4b binding protein was expressed at a very low level and was detected only at the mRNA level by reverse transcriptase-polymerase chain reaction (RT-PCR). For comparison, astrocyte expression of CD59, DAF, MCP and CR1 was confirmed at the mRNA and protein levels. HOG did not activate C spontaneously, as judged by the lack of deposition of C fragments, and were not lysed by C even after inhibition of CD59 and DAF using specific monoclonal antibodies.

Expression of the complement alternative pathway by human myoblasts in vitro: biosynthesis of C3, factor B, factor H and factor I.

In this study, we demonstrate expression in vitro of complement alternative pathway components C3, factor B, factor H and factor I by normal human myoblasts and human rhabdomyosarcoma cell lines CRL1558 and HTB153. Proteins in culture supernatants were detected by Western (protein) blot analysis and biosynthetic labeling followed by immunoprecipitation experiments, and quantified by ELISA. Newly secreted proteins were structurally and functionally similar to their serum counterparts. An additional polypeptide of 43 kDa with factor H immunoreactivity was detected, which could correspond to the N-terminal truncated form found in plasma. Protein expression was correlated with mRNA expression by reverse transcriptase-polymerase chain reaction analysis. The major proteins of complement alternative pathway C3, factor B and factor H were produced constitutively by skeletal muscle cells at a rate of 50 to 150 ng/10(6) cells/ml and factor I was expressed 20 ng/10(6) cells/ml. These syntheses in vitro were regulated by inflammatory cytokines. Interferon-gamma significantly upregulated C3, factor B and factor H expression, but had no effect on factor I production. Interleukin-1 beta strongly enhanced C3 and factor B production and had a weak enhancing or no effect on factor I and factor H secretion. Human myoblast cell lines constitute an interesting model to analyze complement biosynthesis by human skeletal muscle cells. Local complement expression by skeletal muscle in vivo may be implicated in some muscular inflammatory or pathological processes.