Marcus V. Andrade

IL-33 induces a hyporesponsive phenotype in human and mouse mast cells.

IL-33 is elevated in afflicted tissues of patients with mast cell (MC)–dependent chronic allergic diseases. Based on its acute effects on mouse MCs, IL-33 is thought to play a role in the pathogenesis of allergic disease through MC activation. However, the manifestations of prolonged IL-33 exposure on human MC function, which best reflect the conditions associated with chronic allergic disease, are unknown. In this study, we found that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to Ag. This reduction required >72 h exposure to IL-33 for onset and 1–2 wk for reversion following IL-33 removal. This hyporesponsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation, including Ag-mediated calcium mobilization and cytoskeletal reorganization, potentially as a consequence of downregulation of the expression of phospholipase Cγ1 and Hck. These findings suggest that IL-33 may play a protective, rather than a causative, role in MC activation under chronic conditions and, furthermore, reveal regulated plasticity in the MC activation phenotype. The ability to downregulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease.

Permissive Hypotension and Desmopressin Enhance Clot Formation

BACKGROUND Experimental studies of uncontrolled hemorrhage demonstrated that permissive hypotension (PH) reduces blood loss, but its effect on clot formation remains unexplored. Desmopressin (DDAVP) enhances platelet adhesion promoting stronger clots. We hypothesized PH and DDAVP have additive effects and reduce bleeding in uncontrolled hemorrhage. METHODS Rabbits (n = 42) randomized as follows: sham; normal blood pressure (NBP) resuscitation; PH resuscitation-60% baseline mean arterial pressure; NBP plus DDAVP 1 hour before (DDAVP NBP) or 15 minutes after beginning of shock (DDAVP T1 NBP); and PH plus DDAVP 1 hour before (DDAVP PH) or 15 minutes after beginning of shock (DDAVP T1 PH). Fluid resuscitation started 15 minutes after aortic injury and ended at 85 minutes. Intraabdominal blood loss was calculated, aortic clot sent for electron microscopy. Activated partial thromboplastin time, platelet count, thromboelastometry, arterial blood gases, and complete blood count were performed at baseline and 85 minutes. Analysis of variance was used for comparison. RESULTS NBP received more fluid volume and had greater intraabdominal blood loss. DDAVP, when administered preshock, significantly reduced blood loss in NBP and fluid requirement when given postshock. Platelets, arterial blood gas, complete blood count, and activated partial thromboplastin time were similar at 85 minutes. NBP delayed clot formation and worsened thrombodynamic potential on thromboelastometry, whereas PH and DDAVP improved. Electron microscopy showed lack of fibrin on NBP clots, whereas DDAVP and PH clots displayed exuberant fibrin/platelet aggregates. DDAVP NBP presented intermediate clots. CONCLUSION PH reduced bleeding and improved hemostasis compared with normotensive resuscitation. DDAVP given preshock exerted similar effects with normotensive resuscitation.

Permissive hypotension does not reduce regional organ perfusion compared to normotensive resuscitation: animal study with fluorescent microspheres

IntroductionThe objective of this study was to investigate regional organ perfusion acutely following uncontrolled hemorrhage in an animal model that simulates a penetrating vascular injury and accounts for prehospital times in urban trauma. We set forth to determine if hypotensive resuscitation (permissive hypotension) would result in equivalent organ perfusion compared to normotensive resuscitation.MethodsTwenty four (n=24) male rats randomized to 4 groups: Sham, No Fluid (NF), Permissive Hypotension (PH) (60% of baseline mean arterial pressure - MAP), Normotensive Resuscitation (NBP). Uncontrolled hemorrhage caused by a standardised injury to the abdominal aorta; MAP was monitored continuously and lactated Ringer’s was infused. Fluorimeter readings of regional blood flow of the brain, heart, lung, kidney, liver, and bowel were obtained at baseline and 85 minutes after hemorrhage, as well as, cardiac output, lactic acid, and laboratory tests; intra-abdominal blood loss was assessed. Analysis of variance was used for comparison.ResultsIntra-abdominal blood loss was higher in NBP group, as well as, lower hematocrit and hemoglobin levels. No statistical differences in perfusion of any organ between PH and NBP groups. No statistical difference in cardiac output between PH and NBP groups, as well as, in lactic acid levels between PH and NBP. NF group had significantly higher lactic acidosis and had significantly lower organ perfusion.ConclusionsHypotensive resuscitation causes less intra-abdominal bleeding than normotensive resuscitation and concurrently maintains equivalent organ perfusion. No fluid resuscitation reduces intra-abdominal bleeding but also significantly reduces organ perfusion.

Endotoxin tolerance and cross-tolerance in mast cells involves TLR4, TLR2 and FcεR1 interactions and SOCS expression: perspectives on immunomodulation in infectious and allergic diseases

BackgroundThe study of the endotoxin tolerance phenomenon in light of the recently defined roles of mast cells and toll-like receptors as essential components of the innate immune response and as orchestrators of acquired immunity may reveal potentially useful mechanisms of immunomodulation of infectious and allergic inflammatory responses, such as sepsis or asthma. Here we evaluated the phenomenon of direct tolerance of endotoxins, as well as the induction of cross-tolerance and synergism by stimulation with toll-like receptor-2 (TLR2) and FcεR1 agonists, in murine mast cells prestimulated with lipopolysaccharide (LPS). Additionally, we evaluated some stimulatory and inhibitory signaling molecules potentially involved in these phenomena.MethodsMC/9 cells and primary bone marrow-derived mast cells obtained from C57BL/6 and TLR4-/- knock-out mice were sensitized to DNP-HSA (antigen) by incubation with DNP-IgE and were prestimulated with LPS for 18 hr prior to stimulation. Cultures were stimulated with LPS or Pam3Cys-Ser-(Lys)4 3HCl (P3C), a TLR2 agonist, individually or in combination with antigen. The production of IL-6 and TNFα, the phosphorylation of NFκB and p38 MAPK, and the expression of TLR4 and SOCS-1 and -3 were analyzed.ResultsWe found that production of TNFα and IL-6 in murine mast cells that have been pretreated with LPS and challenged with TLR4 (LPS) or -2 (P3C) agonists was reduced, phenomena described as endotoxin tolerance (LPS) and cross-tolerance (P3C), respectively. The expression of TLR4 was not affected by LPS pretreatment. Our results show that the FcεR1 agonist DNP-HSA (antigen) interacts synergistically with LPS or P3C to markedly enhance production of cytokines (TNFα and IL-6). This synergistic effect with LPS and P3C was also attenuated by LPS pretreatment and was mediated by TLR4. These results may be attributed to the reduction in phosphorylation of the mitogen-activated protein kinase (MAPK), p38, and the transcription factor NFκB, as well as to an increase in the expression of the suppressors of cytokine signaling (SOCS)-1 and -3 proteins in LPS-pretreated mast cells.ConclusionsThese findings can be explored with respect to the modulation of inflammatory responses associated with infectious and allergic processes in future studies.

Toll-like receptor activation and mechanical force stimulation promote the secretion of matrix metalloproteinases 1, 3 and 10 of human periodontal fibroblasts via p38, JNK and NF-kB

Matrix metalloproteinases (MMPs) are known to play a key role during orthodontic treatment leading to periodontal remodelling and tooth movement. MMPs may be induced by mechanical forces. However, the role played by toll-like receptors (TLRs) in modulating the effects of the mechanical force on periodontal fibroblasts is not known. To investigate the interaction between mechanical force and TLR stimulation, primary cultures of human periodontal fibroblasts were submitted to centrifugation in the presence of LPS and Pam3Cys, which are known TLR-4 and TLR-2 ligands, respectively. The expression of MMP-1, -2, -3, -8, -9, -10 and -13; TIMP (Tissue Inhibitor of Metalloproteinases) -1, -2 and -4; TNF-α (Tumour Necrosis Factor alpha); IL-1β (Interleukin 1 beta); ERK 1/2 (Extracellular Signal-Regulated Kinase 1/2); p38; JNK (c-jun N-terminal Kinase); IRAK1 (Interleukin-1 Receptor-Associated Kinase); and NF-κB (Nuclear Factor kappa B) were measured by antibody array, ELISA and immunoblotting methods. The activation of TLRs associated with centrifugation induced an increase in the secretion of MMPs 1, 3 and 10, with no increase in TNF-α or IL-1β. An increase in the phosphorylation of the MAP kinases p38 and JNK and the transcription factor NF-κB, without an increase in TIMPs was also observed. These findings suggest that the secretion of MMPs by cultured periodontal fibroblasts that is induced by combined TLR activation and mechanical force stimulation is regulated via the p38, JNK and NF-κB pathways. The increased secretion of MMPs by TLR activation may be an important factor that should be considered during orthodontic treatment.

Brazilian medical publications: citation patterns for Brazilian-edited and non-Brazilian literature

Today, the quality of a scientific article depends on the periodical in which it is published and on the number of times the article is cited in the literature. In Brazil, the criteria for the evaluation of this scientific production are improving. However, there is still some resistance, with authors arguing that Brazilian publications must be preferentially addressed to the national readers and, therefore, they should ideally be written in Portuguese. In order to determine the kind of scientific journals cited in the reference lists of articles published in medical periodicals edited in Brazil, in the present study we determine the rate of Portuguese/English citations. Three issues of 43 periodicals (19 indexed in SciELO, 10 in PubMed, 10 in LILACS, and 4 in the ISI-Thompson base) of different medical specialties were analyzed, and the number of both Portuguese and English citations in the reference list of each article was recorded. The results showed that in Brazilian-edited journals the mean number of citations/article was 20.9 +/- 6.9 and the percentage of citations of international non-Brazilian periodicals was 86.0 +/- 11.2%. Of the latter, 94.4 +/- 7.0 are indexed by ISI-Thompson. Therefore, we conclude that Brazilian medical scientists cite the international non-Brazilian periodicals more than the national journals, and most of the cited papers are indexed by ISI-Thompson.

Evaluation of Serum Levels of Chemokines during Interferon-β Treatment in Multiple Sclerosis Patients

Background: The molecules that provide access to activated T cells in the CNS, including chemokines, have been considered to be a crucial step in the pathogenesis of multiple sclerosis (MS).Aims: In this study, we investigated serial serum chemokine levels in patients with relapsing-remitting MS over 1 year and the association of these chemokine levels with treatment regimens, lesions on MRI and patients’ characteristics.Methods: Serum CXCL9, CXCL10, CCL2, CCL4 and CCL5 levels were evaluated using ELISA every 2 months for a year in 28 healthy controls and 28 MS patients during their treatment with interferon (IFN)-β. Patients under-went MRI and were evaluated using the Expanded Disability Status Scale (EDSS) at the first and final evaluations.Results: CXCL10 serum levels were higher in MS patients compared with controls, were positively correlated with T2 lesions on MRI and were slightly increased during relapses. Treatment with IFNβ-1a or IFNβ-1b was associated with increased CXCL10 levels when evaluated more than 36 hours after subcutaneous injection. The CXCL9 levels were higher after MS relapse. There was significant variability in CCL4 and CCL5 levels in the serial evaluations, associated with gender and treatment. CCL2 levels were higher in treated MS patients than healthy controls, particularly among those patients with a stable form of the disease.Conclusion: Serum is a feasible resource for searching for an immunological marker in MS. Peripheral chemokine levels correlated in different ways with IFNβ therapy and with disease and patient characteristics.Clinical trial registration number: ISRCTN45526724.

Mast cells control insulitis and increase Treg cells to confer protection against STZ-induced type 1 diabetes in mice.

Quantitative alterations in mast cell numbers in pancreatic lymph nodes (PLNs) have been reported to be associated with type 1 diabetes (T1D) progression, but their potential role during T1D remains unclear. In this study, we evaluated the role of mast cells in T1D induced by multiple low‐dose streptozotocin (MLD‐STZ) treatments, using two strains of mast cell‐deficient mice (W/Wv or Wsh/Wsh) and the adoptive transfer of mast cells. Mast cell deficient mice developed severe insulitis and accelerated hyperglycemia, with 100% of mice becoming diabetic compared to their littermates. In parallel, these diabetic mice had decreased numbers of T regulatory (Treg) cells in the PLNs. Additionally, mast cell deficiency caused a significant reduction in IL‐10, TGF‐β, and IL‐6 expression in the pancreatic tissue. Interestingly, IL‐6‐deficient mice are more susceptible to T1D associated with reduced Treg‐cell numbers in the PLNs, but mast cell transfer from wild‐type mice induced protection to T1D in these mice. Finally, mast cell adoptive transfer prior to MLD‐STZ administration conferred resistance to T1D, promoted increased Treg cells, and decreased IL‐17‐producing T cells in the PLNs. Taken together, our results indicate that mast cells are implicated in resistance to STZ‐induced T1D via an immunological tolerance mechanism mediated by Treg cells.

CD4+ and CD8+ Distribution Profile in Individuals Infected by Schistosoma mansoni

Rationale: Patients with chronic Schistosoma mansoni infection show lower anti‐soluble egg antigen (SEA) proliferation responses and higher responses to soluble worm antigen preparation (SWAP). Objective: To compare the activation status and proliferation response of peripheral blood mononuclear cells (PBMC) of infected (XTO) and egg‐negative individuals (NI) living in the same endemic area. Methods: XTO (n = 51) and NI individuals from the same geographical area (n = 37) and healthy blood donors (n = 22) were evaluated before and after stimulation with SEA and SWAP. The expression of activation markers (CD4+ HLADR+, CD8high+HLA‐DR+ and CD8+ CD28+) and proliferation assay was assessed by flow cytometry. Findings: PBMC from infected patients showed lower frequency of CD4+ but no change in CD8+ T cells when compared with the healthy donor group. The ratio CD4+/CD8+ was 1.3, 0.6 and 0.5 in healthy donors, infected and non‐infected individuals, respectively. The HLA‐DR+ expression on CD8+ was higher in PBMC from infected and non‐infected individuals than from healthy donors, but similar in both total lymphocytes and CD4+ populations. No intergroup proliferation response differences were observed in CD4+ and CD8+ PBMC unstimulated and stimulated with SEA and SWAP. The SEA but not SWAP‐stimulated cells showed a decrease in the expression of phosphorylated extracellular signal‐regulated kinase (ERK1/2). Conclusions: XTO and NI individuals living in the same area presented a smaller per cent of CD4+ and a higher per cent of CD8+ cells. The activation by either CD8high+HLA‐DR+ or CD8high+HLA‐DR+/CD8+ was enhanced and decreased in XTO and NI by CD8+ CD28+ and CD8+ CD28+/CD8+ when compared with healthy donor. ERK phosphorylation was attenuated in XTO and NI individuals when stimulated with SEA but not SWAP.

Mast cells are crucial in the resistance against Toxoplasma gondii oral infection

During oral infection, mucosal immunity assumes a predominant role. Here, we addressed the role of mast cells (MCs), which are mainly located in mucosa during oral infection with Toxoplasma gondii, using MC‐deficient (W/Wv) mice. We show that in the absence of MCs the resistance of W/Wv mice to oral infection was considerably reduced. W/Wv mice uniformly succumbed within 15 days of infection after administration of cysts of the ME49 strain of T. gondii. The rapid lethality of T. gondii in W/Wv mice correlated with a delayed Th1‐cell response, since IFN‐γ and IL‐12 levels peaked in the later phase of the infection. In vitro, BM‐derived MCs were able to recognize parasite lysate in a MyD88‐dependent way, reaffirming the role of this TLR adapter in immune responses to T. gondii. The importance of MCs in vivo was confirmed when W/Wv mice reconstituted with BM‐derived MCs from control mice retrieved an early strong Th1‐cell response and specially a significant IL‐12 production. In conclusion, MCs play an important role for the development of a protective immune response during oral infection with T. gondii.