Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Background: Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods: Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results: Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion: 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs. © 2009 Ishikawa et al; licensee BioMed Central Ltd.

Role of periodontal pathogenic bacteria in RANKL-mediated bone destruction in periodontal disease

Accumulated lines of evidence suggest that hyperimmune responses to periodontal bacteria result in the destruction of periodontal connective tissue and alveolar bone. The etiological roles of periodontal bacteria in the onset and progression of periodontal disease (PD) are well documented. However, the mechanism underlying the engagement of periodontal bacteria in RANKL-mediated alveolar bone resorption remains unclear. Therefore, this review article addresses three critical subjects. First, we discuss earlier studies of immune intervention, ultimately leading to the identification of bacteria-reactive lymphocytes as the cellular source of osteoclast-induction factor lymphokine (now called RANKL) in the context of periodontal bone resorption. Next, we consider (1) the effects of periodontal bacteria on RANKL production from a variety of adaptive immune effector cells, as well as fibroblasts, in inflamed periodontal tissue and (2) the bifunctional roles (upregulation vs. downregulation) of LPS produced from periodontal bacteria in a RANKL-induced osteoclast-signal pathway. Future studies in these two areas could lead to new therapeutic approaches for the management of PD by down-modulating RANKL production and/or RANKL-mediated osteoclastogenesis in the context of host immune responses against periodontal pathogenic bacteria. © 2010 Mikihito Kajiya et al.

Anti-inflammatory activity of Lychnophora passerina, Asteraceae (Brazilian arnica)

Ethnopharmacological relevance: Lychnophora passerina (Asteraceae), popularly known as arnica, is used to treat inflammation, pain, rheumatism, contusions, bruises and insect bites in Brazilian traditional medicine. Materials and methods: The anti-inflammatory activity of crude ethanolic extract of aerial parts of L. passerina and its ethyl acetate and methanolic fractions had their abilities to modulate the production of NO, TNF-α and IL-10 inflammatory mediators in LPS/IFN-γ-stimulated J774.A1 macrophages evaluated. Moreover, the crude ethanolic extract and derived fractions were also in vivo assayed by carrageenan-induced paw oedema in mice. Results: In vitro assays showed remarkable anti-inflammatory activity of L. passerina crude ethanolic extract (EE) and its ethyl acetate (A) and methanolic (M) fractions, through the inhibition of production of NO and TNF-α inflammatory mediators and induction of production of IL-10 anti-inflammatory cytokine. In vivo assays showed anti-inflammatory activity for EE 10% ointment, similar to the standard drug diclofenac gel. The A and M fraction ointments 20% presented anti-inflammatory activity. Conclusion: The results obtained showed that possible anti-inflammatory effects of EE and its A and M fractions may be attributed to inhibition pro-inflammatory cytokines production, TNF-α and NO and to increased IL-10 production. EE, A and M ointments showed topical in vivo anti-inflammatory activity. The in vivo anti-inflammatory activity of EE of L. passerina may be related to synergistic effects of different substances in the crude extract. Therefore, traditional use of aerial parts of L. passerina in the inflammatory conditions could be beneficial to treat topical inflammatory conditions, as evidenced by the present study. © 2011 Elsevier B.V. All rights reserved.

Mast cells modulate the inflammatory process in endotoxin-induced uveitis

Purpose: To investigate the role of mast cells and annexin-A1 (Anxa1) in endotoxin-induced uveitis (EIU). Methods: EIU was induced by injection of lipopolysaccharide (LPS) into the paws of rats, which were then sacrificed after 24 and 48 h. To assess EIU in the absence of mast cells, groups of animals were pretreated with compound 48/80 (c48/80) and sacrificed after 24 h after no treatment or EIU induction. The eyes were used for histological studies and the aqueous humor (AqH) pool was used for the analysis of transmigrated cells and Anxa1 levels. In inflammatory cells, Anxa1 expression was monitored by immunohistochemistry. Results: After 24 h, rats with EIU exhibited degranulated mast cells, associated with elevated numbers of infiltrating leukocytes and the high expression of Anxa1 in the AqH and the neutrophils. After 48 h of EIU, the mast cells were intact, indicating granule re-synthesis, and there was a reduction of neutrophil transmigration and an increase in the number of mononuclear phagocytic cells in ocular tissues. Anxa1 expression was decreased in neutrophils but increased in mononuclear phagocytic cells. In the animals pretreated with c48/80 and subjected to EIU, mast cells responded to this secretagogue by degranulating and few transmigrated neutrophils were observed. Conclustions: We report that mast cells are a potential source of pharmacological mediators that are strongly linked to the pathophysiology of EIU, and the endogenous protein Anxa1 is a mediator in the homeostasis of the inflammatory process with anti-migratory effects on leukocytes, which supports further studies of this protein as an innovative therapy for uveitis. © 2011 Molecular Vision.

An enhanced genetic algorithm to solve the static and multistage transmission network expansion planning

An enhanced genetic algorithm (EGA) is applied to solve the long-term transmission expansion planning (LTTEP) problem. The following characteristics of the proposed EGA to solve the static and multistage LTTEP problem are presented, (1) generation of an initial population using fast, efficient heuristic algorithms, (2) better implementation of the local improvement phase and (3) efficient solution of linear programming problems (LPs). Critical comparative analysis is made between the proposed genetic algorithm and traditional genetic algorithms. Results using some known systems show that the proposed EGA presented higher efficiency in solving the static and multistage LTTEP problem, solving a smaller number of linear programming problems to find the optimal solutions and thus finding a better solution to the multistage LTTEP problem. Copyright © 2012 Luis A. Gallego et al.

Aerocistite aguda induzida por tioglicolato, lipolisacarídeo e Aeromona hydrophila inativada em Piaractus mesopotamicus: Efeitos hematológicos

The effects of swim bladder injection with thioglycolate, Escherichia coli lipopolysaccharide (LPS) and heat-inactivated Aeromonas hydrophila were assessed on hematological responses in pacu, Piaractus mesopotamicus (Characidae). A quantitative assessment was done on erythrocytes, thrombocytes e leucocytes at 6, 24, and 48 h pos-injection of the inflammatory agents and compared with fish injected with saline solution (control). Fish injected with inactivated A. hydrophila showed a reduction of erythrocytes and hemoglobin, whereas the hematocrit increased 6 h pos-injection. The results show that thioglycolate and LPS also induced a reduction on hemoglobin and an increase on the hematocrit. The thrombocytes count decreased 6 h post A. hydrophila injection, whereas increased 48 hours post LPS injection. The leukocytes count increased after 6 h post A. hydrophila injection, while the lymphocytes and PAS-positive granular leukocytes (PAS-LG) count decreased after 24 h post injection. In fish injected with thioglycolate or with LPS showed an increase in the LG-PAS counts when compared to A. hydrophila or control groups. The monocytes count was not affected by the different inflammatory agents.

Reduced in vitro immune response on titania nanotube arrays compared to titanium surface

Material surfaces that provide biomimetic cues, such as nanoscale architectures, have been shown to alter cell/biomaterial interactions. Recent studies have identified titania nanotube arrays as strong candidates for use in interfaces on implantable devices due to their ability to elicit improved cellular functionality. However, limited information exists regarding the immune response of nanotube arrays. Thus, in this study, we have investigated the short- and long-term immune cell reaction of titania nanotube arrays. Whole blood lysate (containing leukocytes, thrombocytes and trace amounts of erythrocytes), isolated from human blood, were cultured on titania nanotube arrays and biomedical grade titanium (as a control) for 2 hours and 2 and 7 days. In order to determine the in vitro immune response on titania nanotube arrays, immune cell functionality was evaluated by cellular viability, adhesion, proliferation, morphology, cytokine/chemokine expression, with and without lipopolysaccharide (LPS), and nitric oxide release. The results presented in this study indicate a decrease in short- and long-term monocyte, macrophage and neutrophil functionality on titania nanotube arrays as compared to the control substrate. This work shows a reduced stimulation of the immune response on titania nanotube arrays, identifying this specific nanoarchitecture as a potentially optimal interface for implantable biomedical devices. © 2013 The Royal Society of Chemistry.

Toll-like receptor 2 knockdown modulates interleukin (IL)-6 and IL-8 but not stromal derived factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzymelinked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Anti-inflammatory mechanisms of the annexin A1 protein and its mimetic peptide Ac2-26 in models of ocular inflammation in vivo and in vitro

Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide's protective effects. Moreover, AnxA1-/- mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach. Copyright © 2013 by The American Association of Immunologists, Inc.

Proinflammatory profile of in vitro monocytes in the ageing is affected by lymphocytes presence

Background: Aging is associated with complex and constant remodeling of the immune function, resulting in an increasing susceptibility to infection and others diseases. The infections caused by Gram-negative microorganisms, present in nursing homes and hospitals, constitute one of the most common infections in the elderly, and are mainly combated by innate immune cells. Although the functions of innate immunity seem more preserved during aging than of adaptive immune mechanisms, two systems operate in an integrated way in the body, so that injury in one part of the immune system inevitably affects the other as they are part of a defensive network. The aim of this study was to investigate the in vitro production of proinflammatory (TNF-α, IL-6, IL-1β, CXCL-8 and MCP-1) and anti-inflammatory (TGF-β and IL-10) cytokines by monocytes, stimulated or not (basal) with lipopolysaccharide, from healthy young and elderly subjects. By means of PBMCs, we also studied if cytokine profile is altered in these different patient groups, in the presence of lymphocytes, under the same experimental conditions.Results: The monocytes from elderly presented higher basal production of TNF-α, MCP-1 and lower of TGF-β than young monocytes. PBMC showed similar cytokines production, irrespective age or stimulation presence. In the presence of lymphocytes, the spontaneous production of IL-10 was higher and of TGF-β was lower than monocytes, regardless of age. After LPS-stimulation, the presence of lymphocytes resulted in increased IL-6, IL-1β, MCP-1 and IL-10 and decreased CXCL-8 and TGF-β in comparison to pure culture of monocytes from young patients. With age, the same differences were observed, except for CXCL-8 and TGF-β which production was the same between monocytes and PBMC stimulated with LPS.Conclusion: These findings reinforce the systemic state of inflamm-aging frequently reported in elderly and considered a factor of susceptibility to numerous diseases. Still, the cytokine production from just monocytes of the elderly showed alterations, while in the lymphocyte presence not, suggesting an immunomodulator role of lymphocytes on monocytes. In addition, the differences between the production patterns by LPS-stimulated PBMC between young and elderly volunteers can be related with an imbalance in response against Gram-negative bacteria in throughout life. © 2013 Pinke et al.; licensee BioMed Central Ltd.

RBPI21 interacts with negative membranes endothermically promoting the formation of rigid multilamellar structures

rBPI21 belongs to the antimicrobial peptide and protein (AMP) family. It has high affinity for lipopolysaccharide (LPS), acting mainly against Gram-negative bacteria. This work intends to elucidate the mechanism of action of rBPI21 at the membrane level. Using isothermal titration calorimetry, we observed that rBPI21 interaction occurs only with negatively charged membranes (mimicking bacterial membranes) and is entropically driven. Differential scanning calorimetry shows that membrane interaction with rBPI21 is followed by an increase of rigidity on negatively charged membrane, which is corroborated by small angle X-ray scattering (SAXS). Additionally, SAXS data reveal that rBPI21 promotes the multilamellarization of negatively charged membranes. The results support the proposed model for rBPI21 action: first it may interact with LPS at the bacterial surface. This entropic interaction could cause the release of ions that maintain the packed structure of LPS, ensuring peptide penetration. Then, rBPI21 may interact with the negatively charged leaflets of the outer and inner membranes, promoting the interaction between the two bacterial membranes, ultimately leading to cell death. © 2013 Elsevier B.V.

MyD88 or TRAM knockdown regulates interleukin (IL)-6, IL-8, and CXCL12 mRNA expression in human gingival and periodontal ligament fibroblasts

Background: In a previous report, it was shown that Toll-like receptor (TLR) 2 knockdown modulates interleukin (IL)-6 and IL-8 but not the chemokine CXCL12, an important mediator with inflammatory and proangiogenic effects, in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). This study investigates whether knocking down two important TLR adaptor molecules, such as myeloid differentiation protein 88 (MyD88) and TRIF-related adaptor molecule (TRAM), could affect mRNA expression of IL-6, IL-8, and CXCL12 in HGF and HPDLF. Methods: After small interfering (si) RNA-mediated silencing of MyD88 or TRAM, HGF and HPDLF were stimulated with Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) or two synthetic ligands of TLR2 (Pam2CSK4 and Pam3CSK4) for 6 hours. IL-6, IL-8, and CXCL12 mRNAs were evaluated by quantitative polymerase chain reaction. Results: Knockdown of MyD88 or TRAM partially impaired the IL-8 mRNA upregulation in both fibroblast subpopulations. Similarly, IL-6 upregulation was partially prevented by siMyD88 or siTRAM in HGF stimulated with Pg LPS, as well as in both fibroblast subtypes challenged with Pam2CSK4. Conversely, constitutive CXCL12 mRNA levels were upregulated by MyD88 or TRAM knockdown in non-stimulated cells. Conclusions: These results suggest that TLR adaptor molecules knockdown, such as MyD88 or TRAM, can decrease IL-6 and IL-8 mRNA and increase CXCL12 mRNA expression in HGF and HPDLF. This can be an important step for better understanding the mechanisms that control the inflammatory cytokine and chemokine expression, which in turn contributes to periodontal pathogenesis.

Immunomodulatory/anti-inflammatory effects of Baccharis dracunculifolia leaves

A possible immunomodulatory/anti-inflammatory effect of Baccharis dracunculifolia (Bd) and its major compound - caffeic acid (Ca) - on cytokines production (IL-1b, IL-6 and IL-10) by murine macrophages was investigated. Cells were incubated with Bd and Ca, and the inhibitory concentrations were tested before or after macrophages challenge with LPS. Bd and Ca stimulated IL-1b and inhibited IL-6 and IL-10 production. In LPS-challenge protocols, Bd prevented LPS action either before or after LPS challenge, whereas Ca prevented LPS effects only after LPS addition. Bd modulatory action on cytokines production may be at least in part mediated by Ca, since it has been shown to inhibit the transcription factor NF-kB. Further studies are still needed to evaluate Bd efficacy in inflammatory diseases, in order to explore its antiinflammatory activity in vivo. © 2013 Taylor & Francis.

Responses of melanocytes and melanomacrophages of Eupemphix nattereri (Anura: Leiuperidae) to Nle4, D-Phe7-α-melanocyte stimulating hormone and lipopolysaccharides

Melanocytes are found in various organs of ectothermic animals, playing a protective role against bacteria and free radicals. It is known that pigment cells from hematopoietic organs have immune functions. However, the role of visceral melanocytes is not well understood. Cutaneous melanocytes are responsive to α-melanocyte stimulating hormone (α-MSH), which is associated with the dispersion of melanin granules within melanocytes. α-MSH has also been reported to inhibit most forms of inflammatory responses by decreasing the pro-inflammatory cytokines and neutrophil migration. The present study evaluated the influence of an α-MSH analog (Nle4, D-Phe7-α-MSH) and lipopolysaccharides (LPS) from Escherichia coli on the liver and testicular tissues of the anuran Eupemphix nattereri. The tested hypotheses were: (i) the pigmented area will increase following hormone and LPS administration, (ii) pre-treatment with α-MSH will decrease the number of mast cells, and (iii) the hormone will have protective effects against LPS-induced responses. We found that hormone administration did not change hepatic pigmentation, but increased testicular pigmentation. Testicular pigmentation quickly increased after LPS administration, whereas there was a late response in the liver. The response of enhanced pigmentation was delayed and the number of mast cells decreased in animals previously treated with the α-MSH analog when compared to the LPS group. Hemosiderin and lipofuscin were found in melanomacrophages, but not in testicular melanocytes. Although both the liver and the testes of E. nattereri have pigmented cells, these are distinct in morphology, embryonic origin, and pigmentary substances. These differences may be responsible for the different responses of these cells to the α-MSH analog and LPS administration. © 2013 Elsevier GmbH.

Análise da reconstrução 3D a partir de um par estereoscópico HR-CCD/CBERS-2 usando dois modelos matemáticos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)