Platelets: much more than bricks in a breached wall.

Platelets have various roles in vascular biology and homeostasis. They are the first actor in primary haemostasis and play important roles in thrombosis pathogenesis, but they are also part of innate immunity, which initiates and accelerate many inflammatory conditions. In some contexts, their immune functions are protective, while in others they contribute to adverse inflammatory outcomes. Platelets express numerous receptors and contain hundreds of secretory molecules that are crucial for platelet functional responses. The capacity of platelets to produce and secrete cytokines, chemokines and related molecules, under the control of specific intracellular pathways, is intimately related to their key role in inflammation. They are also able to intervene in tissue regeneration and repair because they produce pro-angiogenic mediators. Due to this characteristic platelets are involved in cancer progression and spreading. In this review we discuss the complex role of platelets, which bridges haemostasis, inflammation and immune response both in physiological and pathological conditions.

Is the TAM receptor Axl a receptor for lymphocytic choriomeningitis virus?

A recent publication indicated that overexpression of Axl, a cellular receptor that negatively regulates Toll-like receptor signaling, enhanced the entry of viruses pseudotyped with the glycoprotein of lymphocytic choriomeningitis virus (LCMV) in vitro. In testing the biological relevance of these observations, we found differences in neither viral kinetics between LCMV infections of Axl(-/-) and wild-type mice nor T-cell responses prior to spontaneous viral clearance. Thus, Axl is not required for productive LCMV infection of mice.

Pathogenesis of arenavirus hemorrhagic fevers.

Viral hemorrhagic fevers (VHFs) caused by arenaviruses belong to the most devastating emerging human diseases and represent serious public health problems. Arenavirus VHFs in humans are acute diseases characterized by fever and, in severe cases, different degrees of hemorrhages associated with a shock syndrome in the terminal stage. Over the past years, much has been learned about the pathogenesis of arenaviruses at the cellular level, in particular their ability to subvert the host cell's innate antiviral defenses. Clinical studies and novel animal models have provided important new information about the interaction of hemorrhagic arenaviruses with the host's adaptive immune system, in particular virus-induced immunosuppression, and have provided the first hints towards an understanding of the terminal hemorrhagic shock syndrome. The scope of this article is to review our current knowledge on arenavirus VHF pathogenesis with an emphasis on recent developments.

Expression level of a pancreatic neo-antigen in beta cells determines degree of diabetes pathogenesis.

It is not fully understood how the expression level of autoantigens in beta cells impacts autoimmune diabetes (T1D) development. Earlier studies using ovalbumin and also insulin had shown that secreted antigens could enhance diabetes development through facilitated presentation by antigen presenting cells. Here we sought to determine how the expression level of a membrane bound, non-secreted or cross-presented neo-antigen, the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), would influence T1D. We found that an RIP-LCMV transgenic mouse line exhibiting higher levels of beta cell GP expression developed more severe diabetes after LCMV infection or transfer of high numbers of activated autoreactive T cells. Importantly, all beta cells were lost and a significant increase in morbidity and mortality from T1D was noted. Insulitis and accumulation of autoaggressive CD8 cells was more profound in the RIP-LCMV-GP high-expressor line. Interestingly, the additional introduction of neo-antigen-specific CD4(+) helper or regulatory T cells was able to influence diabetogenesis positively or negatively. We conclude that a higher degree of autoantigen expression results in increased diabetes susceptibility. Therefore, autoantigens such as insulin that are expressed at higher levels in beta cells might have a more profound impact on diabetes pathogenesis.

Molecular determinants of Pichinde virus infection of guinea pigs--a small animal model system for arenaviral hemorrhagic fevers.

Arenaviruses are enveloped single-strand RNA viruses that mostly have natural hosts in rodents. Upon infection of humans, several arenaviruses can cause severe hemorrhagic fever diseases, including Lassa fever that is endemic in West Africa. The virulence mechanism of these deadly arenaviruses can be studied in a safe and economical small animal model-guinea pigs infected by a nonpathogenic arenavirus Pichinde virus (PICV), a virulent strain of which can cause similar disease syndromes in guinea pigs as arenaviral hemorrhagic fevers in humans. We have recently developed molecular clones for both the virulent and avirulent strains of PICV. Using the available reverse genetics tools, we are characterizing the molecular determinants of virulent arenavirus infections in vivo.

Unknown disease in South Africa identified as arenavirus infection.

On 12 September 2008, a tourist guide organising safari trips, residing in Lusaka, Zambia, was evacuated in a critical condition to Johannesburg, South Africa. She was admitted to a clinic where she died on 14 September about 10 days after the onset of symptoms. The symptoms included a prodromal phase with fever, myalgia, vomiting, diarrhoea, followed by rash, liver dysfunction and convulsions [1]. Cerebral oedema was detected on scan examination. No laboratory specimen was available for investigation.

Cytokine patterns in a comparative model of arenavirus haemorrhagic fever in guinea pigs.

Arenaviruses such as Lassa virus cause a spectrum of disease in humans ranging from mild febrile illness to lethal haemorrhagic fever. The contributions of innate immunity to protection or pathogenicity are unknown. We compared patterns of expression of cytokines of innate immunity in mild versus severe arenavirus disease using an established guinea pig model based on the macrophage-tropic arenavirus Pichinde virus (PICV). Cytokine transcripts were measured by using real-time RT-PCR in target organs and blood during mild infection (caused by PICV, P2 variant) and lethal haemorrhagic fever (caused by PICV, P18 variant). In the initial peritoneal target cells, virulent P18 infection was associated with significantly increased gamma interferon (IFN-gamma) and monocyte chemoattractant protein-1 (MCP-1, CCL2) mRNA levels relative to P2 infection. Peritoneal cells from P18-infected animals had decreased tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-8 (CXCL-8) and IL-12p40 transcripts relative to mock-infected animals. Late in infection, P18-infected peripheral blood leukocytes (PBL) had decreased TNF-alpha, IFN-gamma, and regulated upon activation, normal T cell expressed and secreted (RANTES, CCL-5) cytokine transcripts relative to P2-infected PBL. We conclude that, in severe arenavirus disease, patterns of cytokine expression in the initially infected cells favour recruitment of additional target monocytes, while inhibiting some of their pro-inflammatory responses. Suppression rather than overexpression of pro-inflammatory cytokines accompanied the terminal shock in this model of arenavirus haemorrhagic fever.

Animal models of highly pathogenic RNA viral infections: hemorrhagic fever viruses.

A diverse group of highly pathogenic RNA viruses cause a severe multisystemic illness in humans commonly referred to as viral hemorrhagic fever (VHF). Although they can vary widely in clinical presentation, all VHFs share certain features that include intense fever, malaise, bleeding and shock. Effective antiviral therapies for most of the VHFs are lacking. Complicating development of intervention strategies is the relative infrequency and unpredictability of VHF outbreaks making human clinical trials extremely challenging or unfeasible. Therefore, animal models that can recapitulate human disease are essential to the development of effective antivirals and vaccines. In general, a good animal model of VHF will demonstrate systemic dispersion of the virus through infection of mononuclear phagocytes and dendritic cells, which induces the release of inflammatory mediators that increase vascular permeability and facilitate coagulation. The culmination of this process leads to significant loss of plasma volume and terminal hypovolemic shock. Although it is clear that nonhuman primate models are the most faithful to human disease, the more accessible and less costly rodent models, including those based on infection with related surrogate viruses, can reproduce certain components of VHF and can serve as suitable preclinical models for initial development of effective countermeasures. Such models are sufficient for testing of drugs that directly block viral replication, but may be inadequate for evaluating therapies that depend for their success on the activation or inhibition of host responses.

Superinfection exclusion in BHK-21 cells persistently infected with Junín virus.

We characterized a persistently Junín virus (JUNV)-infected BHK-21 cell line obtained by experimental infection with the XJCl3 strain. This cell line, named K3, produced low levels of virus in supernatants which were not influenced by the presence of defective interfering (DI) particles after the first year of infection. K3 cells were able to exclude superinfection of the homologous JUNV and the antigenically related Tacaribe virus (TCRV), whereas the non-related arenaviruses lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV) could replicate normally. Although superinfecting virus binding and internalization to persistently infected cells were slightly reduced, earlier biosynthesis of antigenomic RNA was observed in comparison with BHK-21 cells. Despite the fact that superinfection did not increase the number of cells expressing viral antigens, de novo synthesis of superinfecting virus proteins was detected. The virus produced by JUNV-superinfected K3 cells remained mostly cell-associated in the form of particles tethered to the plasma membrane and aberrant tubular structures. JUNV restriction was correlated with an overexpression of cellular protein TSG101 in K3 cells, which has been pointed out as involved in the budding of several RNA viruses. This correlation was also observed in a cell clone isolated from K3. Reduction of TSG101 expression favoured the release of infectious virus to the supernatant of JUNV-superinfected K3 cells. Our data suggest that overexpression of TSG101 in K3 cells is a novel mechanism that may contribute, along with a diminished synthesis of superinfecting virus proteins, to explain superinfection exclusion in persistently arenavirus-infected cells.

LCMV-mediated hepatitis in rhesus macaques: WE but not ARM strain activates hepatocytes and induces liver regeneration.

Lymphocytic chorimeningitis virus (LCMV), the prototype arenavirus, and Lassa virus (LASV), causative agent of Lassa hemorrhagic fever (LHF), belong to the Old World group of the family Arenaviridae. Both viruses have extensive strain diversity and significant variations in lethality and pathogenicity for man and experimental animals. We have shown that the LHF-like infection of rhesus macaques with the WE strain of LCMV affects liver functions, induces hepatocyte proliferation, and causes a rise in IL-6 and soluble TNF receptors (sTNFR) concomitant with a rise in viremia. The levels of IL-6 and sTNFR can serve as an additional diagnostic tool for liver involvement in pathogenesis of arenavirus infection. Mucosal inoculation of rhesus macaques with LCMV-WE can result in attenuated infection with a transient viremia and liver enzyme abnormalities. The ARM strain of LCMV shares 88% amino acid homology with WE. In contrast to LCMV-WE, ARM strain does not induce manifested disease in monkeys, does not affect liver functions, and does not induce hepatocyte proliferation. Previously we demonstrated that LCMV-ARM infection protected rhesus macaques challenged with LCMV-WE. Here we have shown that the protected animals have no signs of hepatitis and hepatocyte proliferation.

Targeting Schwann cells by nonlytic arenaviral infection selectively inhibits myelination.

Members of the arenavirus family, famous for their hemorrhagic syndromes, cause distinct neurological disorders; however, cellular and molecular targets as well as pathogenesis of peripheral nervous system disorders associated with these viruses are unknown. Using noncytolytic lymphocytic choriomeningitis virus, the prototype arenavirus, and pseudotyped Lassa fever virus, we showed that the Schwann cells, but not the neurons, were preferentially targeted and harbored the virus. This permissiveness was caused by the viral glycoprotein usage of its receptor alpha-dystroglycan, which was highly abundant on Schwann cell membranes. Persistent lymphocytic choriomeningitis virus infection rendered immature Schwann cells defective or incapable of forming compact myelin sheathes when they differentiated to myelinating phenotype in an in vitro differentiation model of Schwann cells. Persistent infection did not cause Schwann cell apoptosis or cytopathic effect. Defects in myelination coincided with the down-regulation of dystroglycan expression and disruption of the laminin-2 organization and basal lamina assembly on Schwann cell-axon units. The data provide evidence for a selective perturbation of laminin-2-laminin-2 receptor communication pathway in the peripheral nervous system by a nonlytic virus and the resulting myelin defects, which may partly contribute to neurological abnormalities associated with arenaviral infection.

Long-lasting astrocyte reaction to persistent Junin virus infection of rat cortical neurons.

Immunoperoxidase labeling was performed in histological sections from rat brain harvested during acute (10-30 days), clinically inapparent (90-270 days) and late (450-540 days) stages of Junin virus-induced neurological disease. In frontoparietal cortex, count of viral antigen (+) neurons peaked during the acute period (27.7+/-6.8), dropped within the intermediate (4.8+/-4.0 to 1.4+/-1.1) and increased (7.6+/-4.3) at the onset of the late neurological syndrome. In infected vs. control rats, the number of GFAP (+) astrocytes maximized during the acute stage (19+/-4 vs. 11+/-5), and from the end of the intermediate (27+/-5 vs. 21+/-5) up to the late (37+/-7 vs. 26+/-6) periods. In turn, surface density of GFAP (+) material in infected samples peaked at 0.196+/-0.066, while it failed to exceed 0.090+/-0.043 in controls. Both astrocyte hypertrophy relapsing into chronicity, as depicted by surface density, and astrocyte hyperplasia preceding the onset of the late neurological syndrome, support their pathogenic contribution to disease expression.

Endothelial cell function alteration after Junin virus infection.

Hematologic involvement is the main feature of Argentine hemorrhagic fever (AHF), an endemo-epidemic disease caused by Junin virus (JV). Since endothelial dysfunction could play a role in AHF-altered hemostasis, we studied human umbilical vein endothelial cell (HUVEC) infection with a virulent (JVv) and a non-virulent (JVa) JV strain. Cells were infected by the two JV variants with no detectable apoptosis or cytopathic effect. Both viral variants up-regulated ICAM-1 and VCAM-1 levels, while von Willebrand factor (VWF) production was decreased. Prostacyclin (PGI2) release and decay accelerating factor (DAF) expression were greater in JVv- than in JVa-infected or control cells. Furthermore, nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression was only raised in JVv-infected supernatants. Significant NO and PGI2 values were also detected in AHF patient sera. These data demonstrate that endothelial cell responses are triggered subsequently by JV infection, suggesting that such alterations play a major role in the pathogenesis of AHF and perhaps in other viral-induced hemorrhagic diseases.

Effects of promyelocytic leukemia protein on virus-host balance.

The cellular promyelocytic leukemia protein (PML) associates with the proteins of several viruses and in some cases reduces viral propagation in cell culture. To examine the role of PML in vivo, we compared immune responses and virus loads of PML-deficient and control mice infected with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV). PML(-/-) mice exhibited accelerated primary footpad swelling reactions to very-low-dose LCMV, higher swelling peaks upon high-dose inoculation, and higher viral loads in the early phase of systemic LCMV infection. T-cell-mediated hepatitis and consequent mortality upon infection with a hepatotropic LCMV strain required 10- to 100-times-lower inocula despite normal cytotoxic T-lymphocyte reactivity in PML(-/-) mice. Furthermore, PML deficiency rendered mice 10 times more susceptible to lethal immunopathology upon intracerebral LCMV inoculation. Accordingly, 10-times-lower VSV inocula elicited specific neutralizing-antibody responses, a replication-based effect not observed with inactivated virus or after immunization with recombinant VSV glycoprotein. These in vivo observations corroborated our results showing more virus production in PML(-/-) fibroblasts. Thus, PML is a contributor to innate immunity, defining host susceptibility to viral infections and to immunopathology.