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Abstract Background Thalidomide is an anti-inflammatory and anti-angiogenic drug currently used for the treatment of several diseases, including erythema nodosum leprosum, which occurs in patients with lepromatous leprosy. In this research, we use DNA microarray analysis to identify the impact of thalidomide on gene expression responses in human cells after lipopolysaccharide (LPS) stimulation. We employed a two-stage framework. Initially, we identified 1584 altered genes in response to LPS. Modulation of this set of genes was then analyzed in the LPS stimulated cells treated with thalidomide. Results We identified 64 genes with altered expression induced by thalidomide using the rank product method. In addition, the lists of up-regulated and down-regulated genes were investigated by means of bioinformatics functional analysis, which allowed for the identification of biological processes affected by thalidomide. Confirmatory analysis was done in five of the identified genes using real time PCR. Conclusions The results showed some genes that can further our understanding of the biological mechanisms in the action of thalidomide. Of the five genes evaluated with real time PCR, three were down regulated and two were up regulated confirming the initial results of the microarray analysis.

Endoplasmic reticulum (ER) stress triggers the integrated ER-stress response (IERSR) that ensures cellular survival of ER stress and represents a primordial form of innate immunity. We investigated the role of IERSR during Leishmania amazonensis infection. Treatment of RAW 264.7 infected macrophages with the ER stress-inducing agent thapsigargin (TG; 1 µM) increased L. amazonensis infectivity in an IFN1-a receptor (IFNAR)-dependent manner. In Western blot assays, we showed that L. amazonensis activates the inositol-requiring enzyme (IRE1)/ X-box binding protein (XBP)-1-splicing arms of the IERSR in host cells. In chromatin immunoprecipitation (ChIP) assays, we showed an increased occupancy of enhancer and promoter sequences for the Ifnb gene by XBP1 in infected RAW 264.7 cells. Knocking down XBP1 expression by transducing RAW 264.7 cells with the short hairpin XBP1 lentiviral vector significantly reduced the parasite proliferation associated with impaired translocation of phosphorylated IFN regulatory transcription factor (IRF)-3 to the nucleus and a decrease in IFN1-β expression. Knocking down XBP1 expression also increased NO concentration, as determined by Griess reaction and reduced the expression of antioxidant genes, such as heme oxygenase (HO)-1, that protect parasites from oxidative stress. We conclude that L. amazonensis activation of XBP1 plays a critical role in infection by protecting the parasites from oxidative stress and increasing IFN1-β expression. [ABSTRACT FROM AUTHOR]

Leishmania parasites utilize adaptive evasion mechanisms in infected macrophages to overcome host defenses and proliferate. We report here that the PERK/eIF2α/ATF4 signaling branch of the integrated endoplasmic reticulum stress response (IERSR) is activated by Leishmania and this pathway is important for Leishmania amazonensis infection. Knocking down PERK or ATF4 expression or inhibiting PERK kinase activity diminished L. amazonensis infection. Knocking down ATF4 decreased NRF2 expression and its nuclear translocation, reduced HO-1 expression and increased nitric oxide production. Meanwhile, the increased expression of ATF4 and HO-1 mRNAs were observed in lesions derived from patients infected with the prevalent related species L.(V.) braziliensis. Our data demonstrates that Leishmania parasites activate the PERK/eIF2α/ATF-4 pathway in cultured macrophages and infected human tissue and that this pathway is important for parasite survival and progression of the infection.

The evolution of Leishmania infection depends on the balance between microbicidal and suppressor macrophage functions. Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a classic antiviral protein, is able to regulate a number of signaling pathways and macrophage functions. We investigated the possible role of PKR in the modulation of Leishmania infection. Our data demonstrated that Leishmania amazonensis infection led to PKR activation and increased PKR levels. Consistently, in macrophages from PKR knockout 129Sv/Ev mice and RAW-264.7 cells stably expressing a dominant-negative (DN) construct of PKR (DN-PKR), L. amazonensis infection was strongly reduced. The treatment of infected macrophages with the synthetic double-stranded RNA poly(LC), a potent PKR inductor, increased L. amazonensis intracellular proliferation. This effect was reversed by 2-aminopurine (2-AP), a pharmacological inhibitor of PKR, as well as by the expression of DN-PKR. NO release induced by dsRNA treatment was inhibited by L. amazonensis through NF-κB modulation. PKR activation induced by dsRNA also resulted in IL-10 production, whose neutralization with specific antibody completely abrogated L. amazonensis proliferation. Our data demonstrated a new role of PKR in protozoan parasitic infection through IL-10 modulation. [ABSTRACT FROM AUTHOR]

Abstract: We have showed that a phospholipase A2 isolated from Lachesis muta snake venom, denoted LM-PLA2-I, had some biological effects. Here, we examined its effects on lymphocytes. Pre-incubation of human peripheral blood lymphocytes with LM-PLA2-I plus phosphatidylcholine (PC) stimulated the natural killer (NK) activity. This was accompanied by DNA binding of nuclear transcription factor κB and the increase in PKC activity with translocation of the enzyme from the cytoplasma into the plasma membrane. These effects were reproduced when lymphocytes were pre-incubated with commercial lysophosphatidylcholine (LPC) and abolished by stausrosporin or p-bromophenacyl bromide. Evaluation of phosphorylated PKC isoforms showed that pre-incubation with LPC activated the autophosphorylation of the PKCζ isoform. Taken together, these results confirm that the enzymatic activity of the phospholipase A2 present in L. muta venom is for the biological activity of the snake venom, and strongly suggest that the LPC produced may be acting as a modulator of PKC isoforms. [Copyright &y& Elsevier]

Host invasion by pathogens is frequently associated with the activation of nuclear factor kappaB (NF-kappaB), which modulates the expression of genes involved in the immunological response of the host. However, pathogens may also subvert these mechanisms to secure their survival. We describe the effect of Leishmania amazonensis infection on NF-kappaB transcriptional factor activation in macrophages and the subsequent reduction in inducible nitric oxide synthase (iNOS) expression. L. amazonensis promastigote infection activates the p50/p50 NF-kappaB complex, a classic transcriptional repressor. Interestingly, L. amazonensis promotes the change of the classical p65/p50 NF-kappaB dimer induced by LPS, leading to the p50/p50 NF-kappaB complex activation in macrophages stimulated with LPS. Moreover, this parasite promotes the reduction of p65 total levels in infected macrophages. All these effects contribute to the observation that this parasite is able to restrain the NF-kappaB-dependent transcriptional activity induced by LPS. Strikingly, L. amazonensis reduces the mRNA levels of the iNOS in addition to protein expression and the production of nitric oxide in LPS-stimulated macrophages. Accordingly, as revealed by reporter-gene assays, L. amazonensis-induced iNOS repression requires NF-kappaB sites in the iNOS promoter region. In summary, our results suggest that L. amazonensis has developed an adaptive strategy to escape from host defense by activating the NF-kappaB repressor complex p50/p50. The activation of this specific host transcriptional response negatively regulates the expression of iNOS, favoring the establishment and success of L. amazonensis infection.

Mycobacterium leprae, the causative agent of leprosy, invades peripheral nerve Schwann cells, resulting in deformities associated with this disease. NF-kappaB is an important transcription factor involved in the regulation of host immune antimicrobial responses. We aimed in this work to investigate NF-kappaB signaling pathways in the human ST88-14 Schwannoma cell line infected with M. leprae. Gel shift and supershift assays indicate that two NF-kappaB dimers, p65/p50 and p50/p50, translocate to the nucleus in Schwann cells treated with lethally irradiated M. leprae. Consistent with p65/p50 and p50/p50 activation, we observed IkappaB-alpha degradation and reduction of p105 levels. The nuclear translocation of p50/p50 complex due to M. leprae treatment correlated with repression of NF-kappaB-driven transcription induced by TNF-alpha. Moreover, thalidomide inhibited p50 homodimer nuclear translocation induced by M. leprae and consequently rescues Schwann cells from NF-kappaB-dependent transcriptional repression. Here, we report for the first time that M. leprae induces NF-kappaB activation in Schwann cells and thalidomide is able to modulate this activation.

In the adult central nervous system (CNS), prominent reactive astrocytosis is seen in acute traumatic brain injury, neurodegenerative diseases and a variety of viral infections. Reactive astrocytes synthesize a number of factors that could play different roles in neuronal regeneration. In this study, the effects of thermal stress were evaluated on nuclear factor-kappaB (NF-kappaB) activation and proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) secretion in primary astrocytic cultures. The ability of HSP70-positive astrocytes to support or inhibit neurite outgrowth was investigated in neuron-astrocyte cocultures. Cultured astrocytes from cerebral cortex of rats were exposed to transient hyperthermia (42 degrees C/30 min) and incubated at 37 degrees C for different periods of recovery. During HSP70 accumulation, astrocytes extended large and thick processes associated to rearrangement of glial fibrillary acidic protein (GFAP) filaments and an increase in protein synthesis and GFAP, suggesting an astrogliosis event. A delay of NF-kappaB activation appeared closely related to TNF-alpha secretion by HSP70-positive astrocytes. These cells demonstrated a functional shift from neurite growth-promoting to non-permissive substrate. We also found that gliotoxin, a specific NF-kappaB inhibitor, partially abrogated the inhibitory ability of reactive astrocytes. These findings may suggest a involvement of NF-kappaB and TNF-alpha in modulating the failure of HSP70-positive astrocytes to provide functional support to neuritic outgrowth., (Copyright 2002 Elsevier Science B.V.)