The recurrence of leprosy reactional episodes could be associated with oral chronic infections and expression of serum IL-1, TNF-α, IL-6, IFN-γ and IL-10

The aim of this study was to determine whether the presence of leprosy reactional episodes could be associated with chronic oral infection. Thirty-eight leprosy patients were selected and divided into 2 groups: group I - 19 leprosy patients with oral infections, and group II - 19 leprosy patients without oral infections. Ten patients without leprosy, but presenting oral infections, were assigned to the control group. Leprosy patients were classified according to Ridley and Jopling classification and reactional episodes of the erythema nodosum type or reversal reaction were identified by clinical and histopathological features associated with serum IL-1, TNF-α, IL-6, IFN-γ and IL-10 levels. These analyses were performed immediately before and 7 days after the oral infection elimination. Patients from group I presenting oral infections reported clinical improvement of the symptoms of reactional episodes after dental treatment. Serum IL-1, TNF-α, IL-6, IFN-γ and IL-10 levels did not differ significantly before and after dental treatment as determined by the Wilcoxon test (p>0.05). Comparison of the 2 groups showed statistically significant differences in IL-1 and IL-6 at baseline and in IL-1, IL-6 and IL-10 on the occasion of both collections 7 days after therapy. Serum IL-6 and IL-10 levels in group I differed significantly at baseline compared to control (Mann-Whitney test; p<0.05). These results suggest that oral infection could be involved as a maintenance factor in the pathogenesis of leprosy reactional episodes.

Pulp capping materials exert an effect on the secretion of IL-1β and IL-8 by migrating human neutrophils

Pulp repair is a complex process whose mechanisms are not yet fully understood. The first immune cells to reach the damaged pulp are neutrophils that play an important role in releasing cytokines and in phagocytosis. The objective of this study was to analyze the effect of different pulp-capping materials on the secretion of interleukin-1 beta (IL-1β) and interleukin-8 (IL-8) by migrating human neutrophils. Neutrophils were obtained from the blood of three healthy donors. The experimental groups were calcium hydroxide [Ca(OH)2], an adhesive system (Single Bond), and mineral trioxide aggregate (MTA). Untreated cells were used as control. Transwell chambers were used in performing the assays to mimic an in vivo situation of neutrophil chemotaxis. The pulp-capping materials were placed in the lower chamber and the human neutrophils, in the upper chamber. The cells were counted and the culture medium was assayed using ELISA kits for detecting and quantifying IL-1β and IL8. The data were compared by ANOVA followed by Tukey's test (p < 0.05). The secretion of IL-8 was significantly higher in all groups in comparison to the control group (p < 0.05). The adhesive system group showed higher IL-8 than the MTA group (p < 0.05). The secretion of IL-1β was significantly greater only in the MTA group (p < 0.001). It was concluded that only MTA is able to improve the secretion of IL-1β, and all materials tested increased IL-8 secretion. These results combined with all the other biological advantages of MTA indicate that it could be considered the material of choice for dental pulp capping.

Comparison of direct immunofluorescence, conventional cell culture and polymerase chain reaction techniques for detecting respiratory syncytial virus in nasopharyngeal aspirates from infants

A total of 316 samples of nasopharyngeal aspirate from infants up to two years of age with acute respiratory-tract illnesses were processed for detection of respiratory syncytial virus (RSV) using three different techniques: viral isolation, direct immunofluorescence, and PCR. Of the samples, 36 (11.4%) were positive for RSV, considering the three techniques. PCR was the most sensitive technique, providing positive findings in 35/316 (11.1%) of the samples, followed by direct immunofluorescence (25/316, 7.9%) and viral isolation (20/315, 6.3%) (p < 0.001). A sample was positive by immunofluorescence and negative by PCR, and 11 (31.4%) were positive only by RT-PCR. We conclude that RT-PCR is more sensitive than IF and viral isolation to detect RSV in nasopharyngeal aspirate specimens in newborn and infants.

Survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction

This work reports a survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction (PCR). Seventy pampas deer were captured in the dry season and surveyed using PCR, microscopic agglutination test (MAT) (n = 51) and by both techniques (n = 47). PCR detected infections in two pampas deer and MAT detected infections in three. Through sequencing and phylogenetic analyses, the PCR-amplified fragment detected in deer was identified as Leptospira interrogans. Serovars Pomona and Butembo were detected using MAT and the highest titre was 200 for serovar Pomona. Epidemiological aspects of the findings are discussed.

Performance of transport and selective media for swine Bordetella bronchiseptica recovery and it comparison to polymerase chain reaction detection

Three comparative assays were performed seeking to improve the sensitivity of the diagnosis of Bordetella bronchiseptica infection analyzing swine nasal swabs. An initial assay compared the recovery of B. bronchiseptica from swabs simultaneously inoculated with B. bronchiseptica and some interfering bacteria, immersed into three transport formulations (Amies with charcoal, trypticase soy broth and phosphate buffer according to Soerensen supplemented with 5% of bovine fetal serum) and submitted to different temperatures (10ºC and 27ºC) and periods of incubation (24, 72 and 120 hours). A subsequent assay compared three selective media (MacConkey agar, modified selective medium G20G and a ceftiofur medium) for their recovery capabilities from clinical specimens. One last assay compared the polymerase chain reaction to the three selective media. In the first assay, the recovery of B. bronchiseptica from transport systems was better at 27ºC and the three formulations had good performances at this temperature, but the collection of qualitative and quantitative analysis indicated the advantage of Amies medium for nasal swabs transportation. The second assay indicated that MacConkey agar and modified G20G had similar results and were superior to the ceftiofur medium. In the final assay, polymerase chain reaction presented superior capability of B. bronchiseptica detection to culture procedures.

Interleukin-22 forms dimers that are recognized by two interleukin-22R1 receptor chains

Interleukin-22 (IL-22) is a class 2 cytokine whose primary structure is similar to that of interleukin 10 (IL-10) and interferon-gamma (IFN-gamma). IL-22 induction during acute phase immune response indicates its involvement in mechanisms of inflammation. Structurally different from IL-10 and a number of other members of IL-10 family, which form intertwined inseparable V-shaped dimers of two identical polypeptide chains, a single polypeptide chain of IL-22 folds on itself in a relatively globular structure. Here we present evidence, based on native gel electrophoresis, glutaraldehyde cross-linking, dynamic light scattering, and small angle x-ray scattering experiments, that human IL-22 forms dimers and tetramers in solution under protein concentrations assessable by these experiments. Unexpectedly, low-resolution molecular shape of IL-22 dimers is strikingly similar to that of IL-10 and other intertwined cytokine dimeric forms. Furthermore, we determine an ab initio molecular shape of the IL-22/IL-22R1 complex which reveals the V-shaped IL-22 dimer interacting with two cognate IL-22R1 molecules. Based on this collective evidence, we argue that dimerization might be a common mechanism of all class 2 cytokines for the molecular recognition with their respective membrane receptor. We also speculate that the IL-22 tetramer formation could represent a way to store the cytokine in nonactive form at high concentrations that could be readily converted into functionally active monomers and dimers upon interaction with the cognate cellular receptors.

Cell-Free Antigens from Paracoccidioides brasiliensis Drive IL-4 Production and Increase the Severity of Paracoccidioidomycosis

The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.

Therapeutic Efficacy of Cintredekin Besudotox (IL13-PE38QQR) in Murine Lung Fibrosis Is Unaffected by Immunity to Pseudomonas aeruginosa Exotoxin A

Background: We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE. Methodology/Principal Findings: Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice. Conclusions: Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.

Immunomodulatory effects of recombinant BCG expressing pertussis toxin on TNF-alpha and IL-10 in a bladder cancer model

Background: Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a rBCG-S1PT strain that induced a stronger cellular immune response than BCG. This preclinical study was designed to compare the modulatory effects of BCG and rBCG-S1PT on bladder TNF-alpha and IL-10 expression and to evaluate antitumour activity. Methods: For Experiment I, the MB49 bladder cancer cell line was used in C57BL/6 mice. Chemical cauterization of the bladder was performed to promote intravesical tumor implantation. Mice were treated by intravesical instillation with BCG, rBCG-S1PT or PBS once a week for four weeks. After 35 days the bladders were removed and weighed. TNF-

Coactivation of the shoulder and arm muscles during closed kinetic chain exercises on an unstable surface

Introduction: The purpose of this study was to compare the electromyography index of muscle coactivation of the following muscle pairs: posterior deltoid and pectoralis major (PD/PM); triceps brachii and biceps brachii (TB/BB); and serratus anterior and upper trapezius (SA/UT) during three different closed kinetic chain exercises (wall-press, bench-press and push-up) on an unstable surface at the maximal load. Methods: A total of 20 healthy sedentary men participated in the study. Integral linear values were obtained from three sustained contractions of six seconds each for the three proposed exercises. Mean coactivation index values were compared using the mixed-effects linear model, with a five percent significance level. Results: Electromyography indexes of muscle coactivation showed significant differences for the PD/PM and TB/BB muscle pairs. No differences were found between exercises for the SA/UT muscle pair. Conclusion: Our results seem to differ from those of previous studies, which reported that the similarity in exercises performed is responsible for the comparable muscle activation levels.

IL-17 Produced during Trypanosoma cruzi Infection Plays a Central Role in Regulating Parasite-Induced Myocarditis

Background: Chagas disease is a neglected disease caused by the intracellular parasite Trypanosoma cruzi. Around 30% of the infected patients develop chronic cardiomyopathy or megasyndromes, which are high-cost morbid conditions. Immune response against myocardial self-antigens and exacerbated Th1 cytokine production has been associated with the pathogenesis of the disease. As IL-17 is involved in the pathogenesis of several autoimmune, inflammatory and infectious diseases, we investigated its role during the infection with T. cruzi. Methodology/Principal Findings: First, we detected significant amounts of CD4, CD8 and NK cells producing IL-17 after incubating live parasites with spleen cells from normal BALB/c mice. IL-17 is also produced in vivo by CD4(+), CD8(+) and NK cells from BALB/c mice on the early acute phase of infection. Treatment of infected mice with anti-mouse IL-17 mAb resulted in increased myocarditis, premature mortality, and decreased parasite load in the heart. IL-17 neutralization resulted in increased production of IL-12, IFN-gamma and TNF-alpha and enhanced specific type 1 chemokine and chemokine receptors expression. Moreover, the results showed that IL-17 regulates T-bet, ROR gamma t and STAT-3 expression in the heart, showing that IL-17 controls the differentiation of Th1 cells in infected mice. Conclusion/Significance: These results show that IL-17 controls the resistance to T. cruzi infection in mice regulating the Th1 cells differentiation, cytokine and chemokine production and control parasite-induced myocarditis, regulating the influx of inflammatory cells to the heart tissue. Correlations between the levels of IL-17, the extent of myocardial destruction, and the evolution of cardiac disease could identify a clinical marker of disease progression and may help in the design of alternative therapies for the control of chronic morbidity of chagasic patients.

BALB/c Mice Infected with Antimony Treatment Refractory Isolate of Leishmania braziliensis Present Severe Lesions due to IL-4 Production

Background: Leishmania braziliensis is the main causative agent of cutaneous leishmaniasis in Brazil. Protection against infection is related to development of Th1 responses, but the mechanisms that mediate susceptibility are still poorly understood. Murine models have been the most important tools in understanding the immunopathogenesis of L. major infection and have shown that Th2 responses favor parasite survival. In contrast, L. braziliensis-infected mice develop strong Th1 responses and easily resolve the infection, thus making the study of factors affecting susceptibility to this parasite difficult. Methodology/Principal Findings: Here, we describe an experimental model for the evaluation of the mechanisms mediating susceptibility to L. braziliensis infection. BALB/c mice were inoculated with stationary phase promastigotes of L. braziliensis, isolates LTCP393(R) and LTCP15171(S), which are resistant and susceptible to antimony and nitric oxide (NO), respectively. Mice inoculated with LTCP393(R) presented larger lesions that healed more slowly and contained higher parasite loads than lesions caused by LTCP15171(S). Inflammatory infiltrates in the lesions and production of IFN-gamma, TNF-alpha, IL-10 and TGF-beta were similar in mice inoculated with either isolate, indicating that these factors did not contribute to the different disease manifestations observed. In contrast, IL-4 production was strongly increased in LTCP393(R)-inoculated animals and also arginase I (Arg I) expression. Moreover, anti-IL-4 monoclonal antibody (mAb) treatment resulted in decreased lesion thickness and parasite burden in animals inoculated with LTCP393(R), but not in those inoculated with LTCP15171(S). Conclusion/Significance: We conclude that the ability of L. braziliensis isolates to induce Th2 responses affects the susceptibility to infection with these isolates and contributes to the increased virulence and severity of disease associated with them. Since these data reflect what happens in human infection, this model could be useful to study the pathogenesis of the L. braziliensis infection, as well as to design new strategies of therapeutic intervention.

Evaluating Patellar Kinematics Through Magnetic Resonance Imaging During Open- and Closed-Kinetic-Chain Exercises

Purpose: To evaluate patellar kinematics of volunteers Without knee pain at rest and during isometric contraction in open- and closed-kinetic-chain exercises. Methods: Twenty individuals took part in this study. All were submitted to magnetic resonance imaging (MRI) during rest and voluntary isometric contraction (VIC) in the open anti closed kinetic chain at 15 degrees, 30 degrees, and 45 degrees of knee flexion. Through MRI and using medical e-film software, the following measurements were evaluated: sulcus angle, patellar-tilt angle, and bisect offset. The mixed-effects linear model was used for comparison between knee positions, between rest and isometric contractions, and between (he exercises. Results: Data analysis revealed that the sulcus angle decreased as knee flexion increased and revealed increases with isometric contractions in both the open and closed kinetic chain for all knee-flexion angles. The patellar-tilt angle decreased with isometric contractions in both the open and closed kinetic chain for every knee position. However, in the closed kinetic chain, patellar tilt increased significantly with the knee flexed at 15 degrees. The bisect offset increased with the knee flexed at 15 degrees during isometric contractions and decreased as knee flexion increased during both exercises. Conclusion: VIC in the last degrees of knee extension may compromise patellar dynamics. On the other hand, it is possible to favor patellar stability by performing muscle contractions with the knee flexed at 30 degrees and 45 degrees in either the open or closed kinetic chain.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1 beta maturation

Background: Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1 beta and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). Results: Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE(2) and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1 beta and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)alpha and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1 beta and PGE(2) did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-alpha and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1 beta was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE(2) production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion: These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1 beta maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE(2) production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

A simplified minimal residual disease polymerase chain reaction method at early treatment points can stratify children with acute lymphoblastic leukemia into good and poor outcome groups

Background Minimal residual disease is an important independent prognostic factor in childhood acute lymphoblastic leukemia. The classical detection methods such as multiparameter flow cytometry and real-time quantitative polymerase chain reaction analysis are expensive, time-consuming and complex, and require considerable technical expertise. Design and Methods We analyzed 229 consecutive children with acute lymphoblastic leukemia treated according to the GBTLI-99 protocol at three different Brazilian centers. Minimal residual disease was analyzed in bone marrow samples at diagnosis and on days 14 and 28 by conventional homo/heteroduplex polymerase chain reaction using a simplified approach with consensus primers for IG and TCR gene rearrangements. Results At least one marker was detected by polymerase chain reaction in 96.4%, of the patients. By combining the minimal residual disease results obtained on days 14 and 28, three different prognostic groups were identified: minimal residual disease negative on days 14 and 28, positive on day 14/negative on day 28, and positive on both. Five-year event-free survival rates were 85%, 75.6%,, and 27.8%, respectively (p<0.0001). The same pattern of stratification held true for the group of intensively treated children. When analyzed in other subgroups of patients such as those at standard and high risk at diagnosis, those with positive B-derived CD10, patients positive for the TEL/AML1 transcript, and patients in morphological remission on a day 28 marrow, the event-free survival rate was found to be significantly lower in patients with positive minimal residual disease on day 28. Multivariate analysis demonstrated that the detection of minimal residual disease on day 28 is the most significant prognostic factor. Conclusions This simplified strategy for detection of minimal residual disease was feasible, reproducible, cheaper and simpler when compared with other methods, and allowed powerful discrimination between children with acute lymphoblastic leukemia with a good and poor outcome.