Carlos A. Sorgi

Circulating Interleukin-6 and High-Sensitivity C-Reactive Protein Decrease After Periodontal Therapy in Otherwise Healthy Subjects

BACKGROUND Periodontal disease has been associated with many chronic inflammatory systemic diseases, and a common chronic inflammation pathway has been suggested for these conditions. However, few studies have evaluated whether periodontal disease, in the absence of other known inflammatory conditions and smoking, affects circulating markers of chronic inflammation. This study compared chronic inflammation markers in control individuals and patients with periodontal disease and observed whether non-surgical periodontal therapy affected inflammatory disease markers after 3 months. METHODS Plasma and serum of 20 controls and 25 patients with periodontal disease were obtained prior to and 3 months after non-surgical periodontal therapy. All patients were non-smokers, they did not use any medication, and they had no history or detectable signs and symptoms of systemic diseases. Periodontal and systemic parameters included probing depth, bleeding on probing, clinical attachment level, hematologic parameters, as well as the following inflammatory markers: interleukin (IL)-6, high-sensitivity C-reactive protein (hs-CRP), CD40 ligand, monocyte chemoattractant protein (MCP)-1, soluble P-selectin (sP-selectin), soluble vascular adhesion molecule (sVCAM)-1, and soluble intercellular adhesion molecule (sICAM)-1. RESULTS There were no differences in the hematologic parameters of the patients in the control and periodontal disease groups. Among the tested inflammatory markers, IL-6 concentrations were higher in the periodontal disease group at baseline compared to the controls (P = 0.006). Therapy was highly effective (P <0.001 for all the analyzed clinical parameters), and a decrease in circulating IL-6 and hs-CRP concentrations was observed 3 months after therapy (P = 0.001 and P = 0.006, respectively). Our results also suggest that the CD40 ligand marker may have been different in the control and periodontal disease groups prior to the therapy (P = 0.009). CONCLUSIONS In apparently otherwise healthy patients, periodontal disease is associated with increased circulating concentrations of IL-6 and hs-CRP, which decreased 3 months after non-surgical periodontal therapy. With regard to the CD40 ligand, MCP-1, sP-selectin, sVCAM-1, and sICAM-1, no changes were seen in the periodontal disease group between baseline and 3 months after therapy.

Leukotrienes play a role in the control of parasite burden in murine strongyloidiasis

It is clear that leukotrienes mediate inflammatory response; new aspects of leukotriene function have recently been described. In this study, we demonstrate that leukotrienes are key chemical mediators in the control of parasite burdens in mice infected with Strongyloides venezuelensis. High leukotriene levels were detected in the lungs and small intestines of Swiss mice. In infected Swiss mice treated with MK886, a leukotriene synthesis inhibitor, numbers of adult worms, and eggs/g/feces were greater than in infected-only animals. The MK886 treatment inhibited leukotriene B4 production in the lungs and small intestines, albeit on different postinfection days. Similarly, parasite burdens and eggs/g/feces were greater in 5-lipoxygenase−/− mice than in wild-type animals. These observation were confirmed by histopathological study of the duodena. We subsequently observed significant lower numbers of eosinophils and mononuclear cells in the blood, peritoneal cavity fluid, and bronchoalveolar lavage fluid of Swiss mice treated with MK886. In the lung parenchyma of infected animals, MK886 significantly inhibited synthesis of IL-5 at the beginning of infection, whereas levels of IL-12 increased progressively throughout the postinfection period. However, levels of leukotriene C4, PGE2, TNF-α, IL-3, IL-4, IFN-γ, and IL-10 were comparable between the treated and untreated groups. Nevertheless, IgE and IgG1 (but not IgG2a) synthesis was also significantly inhibited by MK886 administration. Therefore, in S. venezuelensis-infected mice, adult worm and egg burdens are leukotriene dependent. These findings indicate potential immunostimulatory strategies involving leukotriene administration, and may serve as an alert to physicians treating Strongyloides stercoralis-infected patients presenting asthma-like symptoms because use of 5-lipoxygenase inhibitors may worsen the infection.

Circulating matrix metalloproteinase-8 (MMP-8) and MMP-9 are increased in chronic periodontal disease and decrease after non-surgical periodontal therapy.

BACKGROUND Periodontal disease shares risk factors with cardiovascular diseases and other systemic inflammatory diseases. The present study was designed to assess the circulating matrix metalloproteinases (MMPs) from chronic periodontal disease patients and, subsequently, after periodontal therapy. METHODS We compared the plasma concentrations of MMP-2, MMP-3, MMP-8, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2, and total gelatinolytic activity in patients with periodontal disease (n=28) with those of control subjects (n=22) before and 3 months after non-surgical periodontal therapy. RESULTS Higher plasma MMP-3, MMP-8, and MMP-9 concentrations were found in periodontal disease patients compared with healthy controls (all P<0.05), whereas MMP-2, TIMP-1, and TIMP-2 levels were not different. Treatment decreased plasma MMP-8 and MMP-9 concentrations by 35% and 39%, respectively (both P<0.02), while no changes were found in controls. MMP-2, MMP-3, TIMP-1, and TIMP-2 remained unaltered in both groups. Plasma gelatinolytic activity was higher in periodontal disease patients compared with controls (P<0.001) and decreased after periodontal therapy (P<0.05). CONCLUSIONS This study showed increased circulating MMP-8 and MMP-9 levels and proteolytic activity in periodontal disease patients that decrease after periodontal therapy. The effects of periodontal therapy suggest that it may attenuate inflammatory chronic diseases.

Histoplasma capsulatum Cell Wall β-Glucan Induces Lipid Body Formation through CD18, TLR2, and Dectin-1 Receptors: Correlation with Leukotriene B4 Generation and Role in HIV-1 Infection

Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of worldwide significance. Infection with Hc produces a broad spectrum of diseases and may progress to a life-threatening systemic disease, particularly in individuals with HIV infection. Resolution of histoplasmosis is associated with the activation of cell-mediated immunity, and leukotriene B4 plays an important role in this event. Lipid bodies (LBs) are increasingly being recognized as multifunctional organelles with roles in inflammation and infection. In this study, we investigated LB formation in histoplasmosis and its putative function in innate immunity. LB formation in leukocytes harvested from Hc-infected C57BL/6 mice peaks on day 2 postinfection and correlates with enhanced generation of lipid mediators, including leukotriene B4 and PGE2. Pretreatment of leukocytes with platelet-activating factor and BLT1 receptor antagonists showed that both lipid mediators are involved in cell signaling for LB formation. Alveolar leukocytes cultured with live or dead Hc also presented an increase in LB numbers. The yeast alkali-insoluble fraction 1, which contains mainly β-glucan isolated from the Hc cell wall, induced a dose- and time-dependent increase in LB numbers, indicating that β-glucan plays a signaling role in LB formation. In agreement with this hypothesis, β-glucan-elicited LB formation was inhibited in leukocytes from 5-LO−/−, CD18low and TLR2−/− mice, as well as in leukocytes pretreated with anti-Dectin-1 Ab. Interestingly, human monocytes from HIV-1-infected patients failed to produce LBs after β-glucan stimulation. These results demonstrate that Hc induces LB formation, an event correlated with eicosanoid production, and suggest a role for these lipid-enriched organelles in host defense during fungal infection.

Tityus serrulatus venom and toxins Ts1, Ts2 and Ts6 induce macrophage activation and production of immune mediators

Scorpion envenomation induces a systemic immune response, and neurotoxins of venom act on specific ion channels, modulating neurotransmitter release or activity. However, little is known about the immunomodulatory effects of crude venom from scorpion Tityus serrulatus (TsV) or its toxins (Ts1, Ts2 and Ts6) in combination with lipopolysaccharide (LPS). To investigate the immunomodulatory effects of TsV and its toxins (Ts1, Ts2 and Ts6), J774.1 cells were stimulated with different concentrations (25, 50 and 100 μg/mL) of venom or toxins pre-stimulated or not with LPS (0.5 μg/mL). Macrophage cytotoxicity was assessed, and nitric oxide (NO) and cytokine production were analyzed utilizing the culture supernatants. TsV and its toxins did not produce cytotoxic effects. Depending on the concentrations used, TsV, Ts1 and Ts6 stimulated the production of NO, interleukin (IL)-6 and tumor necrosis factor (TNF)-α in J774.1 cells, which were enhanced under LPS co-stimulation. However, LPS + Ts2 inhibited NO, IL-6 and TNF-α production, and Ts2 alone stimulated the production of IL-10, suggesting an anti-inflammatory activity for this toxin. Our findings are important for the basic understanding of the mechanisms involved in macrophage activation following envenomation; additionally, these findings may contribute to the discovery of new therapeutic compounds to treat immune-mediated diseases.

Leukotrienes Are Potent Adjuvant during Fungal Infection: Effects on Memory T Cells

Leukotrienes (LTs) are potent lipid mediators involved in the control of host defense. LTB4 induces leukocyte accumulation, enhances phagocytosis and bacterial clearance, and increases NO synthesis. LTB4 is also important in early effector T cell recruitment that is mediated by LTB4 receptor 1, the high-affinity receptor for LTB4. The aims of this study were to evaluate whether LTs are involved in the secondary immune response to vaccination in a murine model of Histoplasma capsulatum infection. Our results demonstrate that protection of wild-type mice immunized with cell-free Ags from H. capsulatum against histoplasmosis was associated with increased LTB4 and IFN-γ production as well as recruitment of memory T cells into the lungs. In contrast, cell-free Ag-immunized mice lacking 5-lipoxygenase−/−, a critical enzyme involved in LT synthesis, displayed a marked decrease on recruitment of memory T cells to the lungs associated with increased synthesis of TGF-β as well as IL-10. Strikingly, these effects were associated with increased mortality to 5-lipoxygenase−/−-infected mice. These data establish an important immunomodulatory role of LTs, in both the primary and secondary immune responses to histoplasmosis.

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.

Anti-Inflammatory Effects of Ellagic Acid on Acute Lung Injury Induced by Acid in Mice

Acute lung injury (ALI) is characterized by alveolar edema and uncontrolled neutrophil migration to the lung, and no specific therapy is still available. Ellagic acid, a compound present in several fruits and medicinal plants, has shown anti-inflammatory activity in several experimental disease models. We used the nonlethal acid aspiration model of ALI in mice to determine whether preventive or therapeutic administration of ellagic acid (10 mg/kg; oral route) could interfere with the development or establishment of ALI inflammation. Dexamethasone (1 mg/kg; subcutaneous route) was used as a positive control. In both preventive and therapeutic treatments, ellagic acid reduced the vascular permeability changes and neutrophil recruitment to the bronchoalveolar lavage fluid (BALF) and to lung compared to the vehicle. In addition, the ellagic acid accelerated the resolution for lung neutrophilia. Moreover, ellagic acid reduced the COX-2-induced exacerbation of inflammation. These results were similar to the dexamethasone. However, while the anti-inflammatory effects of dexamethasone treatment were due to the reduced activation of NF-κB and AP-1, the ellagic acid treatment led to reduced BALF levels of IL-6 and increased levels of IL-10. In addition, dexamethasone treatment reduced IL-1β. Together, these findings identify ellagic acid as a potential therapeutic agent for ALI-associated inflammation.

Silencing of mitochondrial alternative oxidase gene of Aspergillus fumigatus enhances reactive oxygen species production and killing of the fungus by macrophages

We previously demonstrated that conidia from Aspergillus fumigatus incubated with menadione and paraquat increases activity and expression of cyanide-insensitive alternative oxidase (AOX). Here, we employed the RNA silencing technique in A. fumigatus using the vector pALB1/aoxAf in order to down-regulate the aox gene. Positive transformants for aox gene silencing of A. fumigatus were more susceptible both to an imposed in vitro oxidative stress condition and to macrophages killing, suggesting that AOX is required for the A. fumigatus pathogenicity, mainly for the survival of the fungus conidia during host infection and resistance to reactive oxygen species generated by macrophages.

TLR2, TLR4 and CD14 recognize venom-associated molecular patterns from Tityus serrulatus to induce macrophage-derived inflammatory mediators.

Scorpion sting-induced human envenomation provokes an intense inflammatory reaction. However, the mechanisms behind the recognition of scorpion venom and the induction of mediator release in mammalian cells are unknown. We demonstrated that TLR2, TLR4 and CD14 receptors sense Tityus serrulatus venom (TsV) and its major component, toxin 1 (Ts1), to mediate cytokine and lipid mediator production. Additionally, we demonstrated that TsV induces TLR2- and TLR4/MyD88-dependent NF-κB activation and TLR4-dependent and TLR2/MyD88-independent c-Jun activation. Similar to TsV, Ts1 induces MyD88-dependent NF-κB phosphorylation via TLR2 and TLR4 receptors, while c-Jun activation is dependent on neither TLR2 nor TLR4/MyD88. Therefore, we propose the term venom-associated molecular pattern (VAMP) to refer to molecules that are introduced into the host by stings and are recognized by PRRs, resulting in inflammation.