Liver injury and viremia in mice infected with dengue-2 virus

The goal of this study was to test the feasibility of BALB/c mice as an experimental model in the study of dengue disease. BALB/c mice were intraperitoneal infected with DENV-2 obtained from a human patient. Histopathological analysis of infected animals revealed liver injury with viral antigens detection. In initial stages, the most prominent lesions were vacuolization and diffuse steatosis in hepatocytes. Serum levels of ALT and AST increased progressively, reaching the highest values 7 days p.i. and decreasing at the 14th day. Since levels of circulating virus were very low, viremia was analyzed in C6/36 cells. Virus presence was detected by ultrastructural analysis, confirmed by RT-PCR assays. Period of viremia was analyzed by flow cytometry with cells incubated with mouse-infected sera collected in different days, revealing peak virus levels at the 7th day p.i. All such data correlate to the development of the disease described in humans.     

Detection of some dengue-2 virus antigens in infected cells using immuno-microscopy

Summary Immunoelectron microscopy was used to detect the distribution of some dengue-2 virus proteins in infected Vero andAedes albopictus (C 6/36) cells. It was found that the envelope protein (GP 60) was located in clumps on the surface of plasma membrane, and accumulated very little in the infected cytoplasm. However no envelopment of dengue-2 virus nucleocapsids through the plasma membranes was observed. In contrast, the NS 3 (P 67) protein was distributed throughout the whole cytoplasm. No specific association of this protein with the proliferated virus-induced structures was seen. The NS 1 (GP 46) protein showed almost similar distribution as the NS 3 but its quantity appeared to be lower. The NS 3 and NS 1 distribution patterns observed supported the results of immunofluorescent staining but results on E protein were not consistent in both methods.     

Interleukin-1beta modulates proinflammatory cytokine production in human epithelial cells

Periodontitis is a chronic human inflammatory disease initiated and sustained by dental plaque microorganisms. A major contributing pathogen is Porphyromonas gingivalis, a gram-negative bacterium recognized by Toll-like receptor 2 (TLR2) and TLR4, which are expressed by human gingival epithelial cells (HGECs). However, it is still unclear how these cells respond to P. gingivalis and initiate inflammatory and immune responses. We have reported previously that HGECs produce a wide range of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-alpha), and IL-1beta. In this study, we show that IL-1beta has a special role in the modulation of other inflammatory cytokines in HGECs challenged with P. gingivalis. Our results show that the increased production of IL-1beta correlates with the cell surface expression of TLR4, and more specifically, TLR4-normal HGECs produce fourfold more IL-1beta than do TLR4-deficient HGECs after challenge. Moreover, blocking the IL-1beta receptor greatly reduces the production of "secondary" proinflammatory cytokines such as IL-8 or IL-6. Our data indicate that the induction of IL-1beta plays an important role in mediating the release of other proinflammatory cytokines from primary human epithelial cells following challenge with P. gingivalis, and this process may be an inflammatory enhancement mechanism adopted by epithelial cells.     

Reduced virus replication, proinflammatory cytokine production, and delayed macrophage cell death in human PBMCs infected with the newly discovered Bundibugyo ebolavirus relative to Zaire ebolavirus

Bundibugyo ebolavirus is a newly identified Ebolavirus species. The virus was responsible for a recent hemorrhagic fever outbreak in Uganda with an approximate 30% case fatality rate. In this study, we compared the pathogenesis of Bundibugyo with highly lethal Zaire Ebolavirus by using in vitro human PBMCs. We found that PBMCs infected with Bundibugyo ebolaviruses resulted in 1 to 2 log lower virus yields compared to Zaire ebolavirus and produced 2- to 10-fold lower levels of TNF-α, MCP-1, IL-1β, MIP1-α and IL-10 than PBMCs infected with Zaire ebolavirus. In addition, flow cytometric studies have shown lower levels and delay of the macrophage cell death in Bundibugyo ebolavirus compared to Zaire ebolavirus infection. The findings of slower Bundibugyo ebolavirus replication, lower production of proinflammatory cytokines and delay in macrophage cell death provide insight into the basis of the lower case fatality observed with Bundibugyo ebolavirus.     

TLR3-dependent upregulation of RIG-I leads to enhanced cytokine production from cells infected with the parainfluenza virus SV5

Here we address the role of RIG-I and TLR3 in differential cytokine responses against Simian Virus 5 (SV5) and two distinct cytokine inducing SV5 mutants. IFN-beta and IL-6 secretion was induced by infection with P/V-CPI-, an SV5 mutant with P/V substitutions, and were reduced by either siRNA-mediated knockdown of RIG-I expression or by expression of a dsRNA-binding protein. TLR3 overexpression did not alter cytokine secretion induced by P/V-CPI- or by Le-(U5C, A14G), an SV5 promoter mutant. TLR3 signaling by addition of exogenously added dsRNA was not blocked by WT SV5 or either SV5 mutant. Unexpectedly, TLR3 activation in infected cells led to enhanced IL-8 secretion, which correlated with increased RIG-I expression. Dominant negative RIG-I and TRIF supported a model whereby TLR3 activation upregulates RIG-I expression and in turn hypersensitizes cells to RIG-I-mediated cytokine secretion. Implications for crosstalk between different innate immunity pathways in mounting antiviral responses to paramyxoviruses are discussed.     

Differential effects of the bicyclic imidazoles on cytokine biosynthesis in human monocytes and endothelial cells

The effects of bicyclic imidazoles on human monocyte and endothelial cell cytokine production were examined. These compounds constitute the CSAID^TM class of anti-inflammatories and are inhibitors of cytokine biosynthesis. The bicyclic imidazoles differ from glucocorticoids and phosphodiesterase inhibitors in their chemical structure as well as pharmacological profile. At optimal concentrations of LPS (50 ng/ml), SK&F 86002, a prototypic compound, inhibited IL-1 and TNF but not g-CSF or IRAP production in human monocytes. At suboptimal concentrations of LPS (50 pg/ml), IL-6 and IL-8 production were also inhibited. Inhibition of cytokine biosynthesis was stimulus independent. For example, induction of IL-1 or TNF expression by phosphatase 1 and 2A inhibitors (Okadaic acid or Calyculin A) and Vitamin D3-dependent induction of IL-1 or TNF was also inhibited. In addition, IL-8 production, but not ICAM/E-Selectin expression in IL-1-stimulated HUVEC, was inhibited at similar IC₅₀s. Taken together, the bicyclic imidazoles inhibit cytokine production selectively in a stimulus and cell type independent manner.     

Accumulation of the proinflammatory cytokine endothelial-monocyte-activating polypeptide II in ramified microglial cells in brains of Borna virus infected Lewis rats

Borna disease virus (BDV) infection of adult Lewis rats induces a severe and often fatal neurologic disease characterized by a massive mononuclear meningo-encephalitis, and activation of microglial cells. Therefore, we analyzed expression of endothelial monocyte activating polypeptide II (EMAP II) by immunohistology as a marker for activation of microglial cells in BDV infected rat brains. EMAP II is a chemotactic peptide, inducing activation of macrophages and endothelial cells, and is considered a proinflammatory mediator of the innate immune system. An up to 30-fold increase in numbers of EMAP II+ microglial cells and a massive expression by infiltrating macrophages at perivascular inflammatory foci was observed in infected brains, with a maximum on day 25 after infection. These results provide evidence that EMAP II contributes to immune responses in inflammatory processes caused by viral infections.     

Hemozoin differentially regulates proinflammatory cytokine production in human immunodeficiency virus-seropositive and -seronegative women with placental malaria

Pregnant women are at an increased risk for malarial infection. Plasmodium falciparum accumulates in the placenta and is associated with dysregulated immune function and poor birth outcomes. Malarial pigment (hemozoin) also accumulates in the placenta and may modulate local immune function. In this study, the impact of hemozoin on cytokine production by intervillous blood mononuclear cells from malaria-infected placentas was investigated. There was a dose-dependent, suppressive effect of hemozoin on production of gamma interferon (IFN-gamma), with less of an effect on tumor necrosis factor alpha (TNF-alpha) and interleukin-10, in human immunodeficiency virus-seronegative (HIV(-)) women. In contrast, IFN-gamma and TNF-alpha production tended to increase in HIV-seropositive women with increasing hemozoin levels. Production patterns of cytokines, especially IFN-gamma in HIV(-) women, followed different trends as a function of parasite density and hemozoin level. The findings suggest that the influences of hemozoin accumulation and high-density parasitemia on placental cytokine production are not equivalent and may involve different mechanisms, all of which may operate differently in the context of HIV infection. Cytokine production dysregulated by accumulation of hemozoin or high-density parasitemia may induce pathology and impair protective immunity in HIV-infected and -uninfected women.     

Regulation of proinflammatory cytokines in human lung epithelial cells infected with Mycoplasma pneumoniae

Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans. It has also been associated with chronic conditions, such as arthritis, and extrapulmonary complications, such as encephalitis. Although the interaction of mycoplasmas with respiratory epithelial cells is a critical early phase of pathogenesis, little is known about the cascade of events initiated by infection of respiratory epithelial cells by mycoplasmas. Previous studies have shown that M. pneumoniae can induce proinflammatory cytokines in several different study systems including cultured murine and human monocytes. In this study, we demonstrate that M. pneumoniae infection also induces proinflammatory cytokine expression in A549 human lung carcinoma cells. Infection of A549 cells resulted in increased levels of interleukin-8 (IL-8) and tumor necrosis factor alpha mRNA, and both proteins were secreted into culture medium. IL-1 beta mRNA also increased after infection and IL-1 beta protein was synthesized, but it remained intracellular. In contrast, levels of IL-6 and gamma interferon mRNA and protein remained unchanged or undetectable. Using protease digestion and antibody blocking methods, we found that M. pneumoniae cytoadherence is important for the induction of cytokines. On the other hand, while M. pneumoniae protein synthesis and DNA synthesis do not appear to be prerequisites for the induction of cytokine gene expression, A549 cellular de novo protein synthesis is responsible for the increased cytokine protein levels. These results suggest a novel role for lung epithelial cells in the pathogenesis of M. pneumoniae infection and provide a better understanding of M. pneumoniae pathology at the cellular level.     

Chemokine production predominates in human monocytes infected with Tula virus

Many reports suggest the hypothesis of a complex immune response accompanying hantaviral infections. However, little is known about the immunopathogenesis of nonpathogenic hantaviruses, especially Tula virus (TULV). The aim of our study was to determine the cytokine/chemokine profile induced after the infection of human macrophages with TULV and the role of viral replication in this process. Also, we wanted to establish how the study of TULV is relevant to our previous study of pathogenic hantaviruses. We showed that TULV-infected macrophages produced chemokines (interleukin-8, macrophage chemoattractant protein-1, and macrophage inflammatory protein-1beta) important for recruiting inflammatory cells, whereas no significant changes were recorded in the tested cytokine levels. This property was not influenced by ultraviolet inactivation. There were some differences in chemokine production compared with our previous study with pathogenic hantaviruses. A possible explanation could be a different way of entering host cells found in the pathogenic and nonpathogenic hantaviruses and activation of different intracellular signaling pathways.     

Innate response of human endothelial cells infected with mycobacteria

Endothelial cells are susceptible to infection by several pathogens, but little is known about mycobacterial infection. We analyzed some features of mycobacteria-endothelial cell interactions and the innate response to the infection. Intracellular growth in human umbilical vein endothelial cells (HUVECs) of three Mycobacterium species: M. tuberculosis (MTB), M. abscessus (MAB) and M. smegmatis (MSM) was analyzed. M. smegmatis was eliminated; M. abscessus had an accelerate intracellular replication and M. tuberculosis did not replicate or was eliminated. M. abscessus infection induced profound cytoskeleton rearrangements, with M. tuberculosis infection changes were less marked, and with MSM were slight. Nitric oxide (NO) production was induced differentially: M. abscessus induced the highest levels followed by M. tuberculosis and M. smegmatis; the contrary was true for reactive oxygen species (ROS) production. Only M. tuberculosis infection caused beta-1 defensin over-expression. As a whole, our results describe some aspects of the innate response of HUVEC infected by mycobacteria with different virulence and suggest that a strong cytoskeleton mobilization triggers a high NO production in these cells.     

Interferon production and virus replication in lymphoblastoid cells infected with different viruses

Summary A semicontinuous infection system was used to test viral replication and interferon induction in lymphoblastoid cells: measles virus, Newcastle disease virus (NDV), Sendai virus, human parainfluenza virus (type II and III), Semliki forest virus (SFV) and Vesicular stomatitis virus (VSV). With the exception of Sendai virus, all viruses replicated in the Namalva cell line. Only measles virus was able to induce high levels of interferon. Three other cell lines, NC37, Raji (TK⁺-variant) and Raji (TK^–-variant) were tested using measles virus as inducer. The interferon yields from these cells were inferior to those obtained from Namalva cells.     

Increased proinflammatory cytokine and chemokine responses and microglial infection following inoculation with neural stem cells infected with polytropic murine retroviruses

Proinflammatory cytokines and chemokines are often detected in brain tissue of patients with neurological diseases such as multiple sclerosis (MS), HIV-associated dementia (HAD) and Alzheimer's disease (AD). We have utilized a mouse model of retrovirus-induced neurological disease to examine how these proinflammatory responses contribute to neuropathogenesis. In previous studies with this model, a correlation was found between neurovirulence and cytokine and chemokine expression. However, it was unclear whether the induction of these cytokines and chemokines was in response to specific virus envelope determinants or was regulated by the level of virus infection in the brain. In the current study, we demonstrated that multiple polytropic retroviruses induced cytokine and chemokine mRNA expression following increased virus levels in the brain. Increased virus levels of polytropic viruses also correlated with increased neuropathogenesis. In contrast, the ecotropic retrovirus, FB29, did not induce cytokine or chemokine mRNA expression or neurological disease, despite virus levels either similar to or higher than the polytropic retroviruses. As polytropic and ecotropic viruses utilize different receptors for entry, these receptors may play a critical role in the induction of these innate immune responses in the brain.     

Long-term survival and virus production in human primary macrophages infected by human immunodeficiency virus

The role of macrophages in the pathogenesis and progression of human immunodeficiency virus (HIV)-related infection is substantiated by in vitro and in vivo evidence. The unique ability to survive HIV infection and produce viral particles for long periods is postulated. Detailed studies of this phenomenon are lacking. The dynamics of HIV-1 replication and cumulative virus production was studied in long-term cultures of macrophages in the presence or in the absence of antiviral drugs. Multiply spliced and unspliced HIV-RNA production was assessed by quantitative PCR, and the number of infected cells was monitored by FACS analysis. Cumulative HIV-1 production was determined by a trapezoidal equation, including such parameters as times of collection and experimental values of genomic-RNA and p24 gag antigen. Unspliced and multiply spliced HIV-RNA increased linearly after macrophage infection; reached levels of 1.5 x 10(8) and 2.8 x 10(5) copies/10(5) cells, respectively, at day 10; and then remained stable throughout the course of the experiment. Cumulative production of genomic-RNA and p24 gag antigen was 10(10) copies/10(6) cells and 10(7) pg/10(6) cells, respectively, with an average of >200 virus particles produced daily by each macrophage. AZT decreased the cumulative production of both genomic-RNA and p24 gag antigen down to 2.5 x 10(9) copies and 1.1 x 10(6) pg/10(6) cells (73.8% and 88.9% inhibition, respectively) up to day 50 without virus breakthrough. Ritonavir had a limited, but consistent, efficacy on the release of mature virus proteins (about 40% inhibition), but not on HIV-RNA production. In conclusion, the long-term dynamics and the high cumulative virus production that characterize HIV-1 infection of macrophages underscore the peculiar role of these cells as a persistently infected reservoir of HIV.     

Protective efficacy in mice of a secreted form of recombinant dengue-2 virus envelope protein produced in baculovirus infected insect cells

Summary We constructed a recombinant baculovirus encoding a dengue (DEN)-2 virus envelope glycoprotein truncated of 102 amino acids (aa) at its C-terminus (D2E102). The production, processing and transportation of the recombinant protein in baculovirus-infectedSpodoptera frugiperda (Sf9) cells and its immunogenic properties in mice were compared to those of a previously characterized recombinant DEN-2 E-protein with a 71aa C-terminal truncation (D2E71). Both proteins were transported through the Golgi complex and their N-oligosaccharides of the high man-nose type were processed to the complex mannose type. D2E102 transited to the plasma membrane and was secreted whereas D2E71 presumably remained associated with the plasma membrane. The reactivities of the recombinant proteins with neutralizing monoclonal antibodies were similar. Both intracellular and extracellular D2E102 induced neutralizing antibodies in mice and were thus immunogenic. The level of protective immunity to DEN-2 virus encephalitis challenge in mice vaccinated with intracellular D2E102 (80%, p<0.01) was lower than that induced with D2E71 (90%, P<0.001). Sixty-eight percent (P<0.001) of mice vaccinated with 5 µg of extracellular D2E102 protein were protected against lethal challenge.     

Enzyme secretion by human endothelial cells infected with Rickettsia conorii

When cultured endothelial cells from the human umbilical vein were infected with rickettsia conorii, a greater increase was noticed in the inhibitor of plasminogen activator than in tissue type plasminogen activator. This could be linked to clinical observation of coagulation abnormalities in Boutonneuse fever (BF). Otherwise, transaminase levels increased significantly in the supernatant of R. conorii infected endothelial cells which could explain the increased levels of these enzyme in BF.     

Aspergillus fumigatus stimulates leukocyte adhesion molecules and cytokine production by endothelial cells in vitro and during invasive pulmonary disease

Invasive aspergillosis is characterized by hyphal invasion of the blood vessels, which contributes to the pathogenesis of this disease. During this angioinvasion, Aspergillus fumigatus interacts with the endothelial cell lining of the blood vessels. We investigated the response of vascular endothelial cells to A. fumigatus infection in vitro and in mouse models of invasive pulmonary aspergillosis. Infection with hyphae, but not with conidia, stimulated endothelial cells to synthesize E-selectin, vascular cell adhesion molecule 1 (VCAM-1), interleukin 8, and tumor necrosis factor alpha (TNF-alpha) in vitro. Killed hyphae induced approximately 40% less stimulation than did live hyphae. Endothelial cell stimulation required contact between the hyphae and endothelial cells but not endocytosis of the organisms. Studies with DeltagliP and DeltastuA null mutants of A. fumigatus indicated that the extent of endothelial cell stimulation was not influenced by gliotoxin or other StuA-dependent factors synthesized by A. fumigatus. In neutropenic mice infected with wild-type A. fumigatus, increased pulmonary expression of E-selectin, cytokine-induced neutrophil chemoattractant (KC), and TNF-alpha occurred only when neutropenia had resolved. In nonneutropenic mice immunosuppressed with corticosteroids, A. fumigatus stimulated earlier pulmonary expression of E-selectin, VCAM-1, and KC, while expression of intercellular adhesion molecule 1 and TNF-alpha was suppressed. In both mouse models, expression of E-selectin and KC was associated with high pulmonary fungal burden, angioinvasion, and neutrophil adherence to endothelial cells. Therefore, the expression of leukocyte adhesion molecules and secretion of proinflammatory cytokines by endothelial cells in response to A. fumigatus could enhance the host defense against this organism by contributing to the recruitment of activated leukocytes to sites of angioinvasion.