Maria Cristina Dos-Santos

Local inflammatory events induced by Bothrops atrox snake venom and the release of distinct classes of inflammatory mediators.

Bothrops atrox is responsible for most accidents involving snakes in the Brazilian Amazon and its venom induces serious systemic and local effects. The local effects are not neutralized effectively by commercial antivenoms, resulting in serious sequelae in individuals bitten by this species. This study investigates the local inflammatory events induced in mice by B. atrox venom (BaV), such as vascular permeability, leukocyte influx and the release of important inflammatory mediators such as cytokines, eicosanoids and the chemokine CCL-2, at the injection site. The effect of BaV on cyclooxygenase (COX-1 and COX-2) expression was also investigated. The results showed that intraperitoneal (i.p.) injection of BaV promoted a rapid and significant increase in vascular permeability, which reached a peak 1 h after venom administration. Furthermore, BaV caused leukocyte infiltration into the peritoneal cavity between 1 and 8 h after i.p. injection, with mononuclear leukocytes (MNs) predominating in the first 4 h, and polymorphonuclear leukocytes (PMNs) in the last 4 h. Increased protein expression of COX-2, but not of COX-1, was detected in leukocytes recruited in the first and fourth hours after injection of BaV. The venom caused the release of eicosanoids PGD₂, PGE₂, TXA₂ and LTB₄, cytokines TNF-α, IL-6, IL-10 and IL-12p70, but not IFN-γ, and chemokine CCL-2 at different times. The results show that BaV is able to induce an early increase in vascular permeability and a leukocyte influx to the injection site consisting mainly of MNs initially and PMNs during the later stages. These phenomena are associated with the production of cytokines, the chemokine CCL-2 and eicosanoids derived from COX-1 and COX-2.

A eficácia do antiveneno botrópico-crotálico na neutralização das principais atividades do veneno de Bothrops jararacussu

A mionecrose e um dos efeitos causados pelo veneno de Bothrops jararacussu. Uma miotoxina com homologia estrutural a fosfolipase A2 (PLA2), mas sem atividade enzimatica, foi isolada desse veneno. O veneno de Crotalus durissus terrificus apresenta tambem atividade miotoxica, que vem sendo atribuida a crotoxina e a PLA2 (crotoxina B), o componente basico do complexo crotoxina. O veneno de Bothrops jararacussu apresenta tres proteinas, que tem identidade imunologica com a PLA2 da crotoxina. O presente trabalho comparou a eficiencia dos antivenenos polivalentes comerciais produzidos pelo Instituto Butantan - o antiveneno botropico (AB) e o antiveneno botropico-crotalico (AB/C) - na neutralizacao das atividades letal, hemorragica, coagulante e miotoxica do veneno de B. jararacussu. Os dois antivenenos neutralizaram de maneira semelhante a atividade hemorragica, mas o AB/C foi tres vezes mais potente que o AB em neutralizar a acao miotoxica e duas vezes mais potente na neutralizacao da letalidade e na acao coagulante do veneno de B. jararacussu. Os dados sugerem que a utilizacao do AB/C pode ser vantajosa no tratamento de pacientes picados por serpentes dessa especie

Psychological stress and its influence on salivary flow rate, total protein concentration and IgA, IgG and IgM titers.

The hypothalamic-pituitary-adrenal and sympathetic-adrenomedullary axes are the main systems activated in response to stress. Alterations in salivary components and flow rate have been associated with oral health problems and psychological stress. Objectives: The aim of the present study was to investigate the influence of psychological stress on salivary flow, total protein concentration and IgG, IgM and IgA concentrations. Methods: Thirty-eight medical students, average age of 21.4 ± 2.1 years and enrolled in the 2nd to 5th years of their course, took part voluntarily in the study which involved two different periods: the first after vacations and the second during the final exams (a gap of 4 months). An Oral Health Questionnaire and the Lipp Inventory of Stress Symptoms for Adults (ISSL) were applied during both these periods. The flow rate, total protein concentration and immunoglobulin titers of saliva samples, collected after stimulation and stored in a container with protease inhibitor, were measured. Results: Analysis of the ISSL showed that 42.1% (n = 16) of the students had stress during the post-vacation period, and 44.7% (n = 17) during the final exams. The students’ salivary flow rate was significantly lower during the latter period than during the post-vacation period (p < 0.0001), regardless of the presence or absence of psychological stress as measured by the ISSL. There was a reduction in salivary flow rate and a consequent reduction in total protein concentration during the exam period (p = 0.0058). However, during both periods of the study there was no significant difference in total salivary protein concentration between the groups of students with or without psychological stress according to the ISSL (p > 0.05). IgG predominated over IgA and IgM (p < 0.001) during both study periods, regardless of the presence or absence of psychological stress. The study period and the presence of stress influenced the secretion of salivary immunoglobulins. IgM titers during the post-vacation period (p = 0.0044), and IgA (p = 0.028), IgG (p = 0.022) and IgM (p = 0.0075) titers during the final exams were higher in students with symptoms of psychological stress. Conclusions: Although the immunoglobulin titers were high, there was a reduction in the students’ salivary flow rates and a consequent reduction in total protein concentrations.

Inhibition of the inflammatory and coagulant action of Bothrops atrox venom by the plant species Marsypianthes chamaedrys.

AIM OF THE STUDY This study investigated the efficacy of Marsypianthes chamaedrys Vahl (Lamiaceae) inflorescence and leaf extracts in inhibiting the inflammatory and coagulant actions of Bothrops atrox venom. MATERIALS AND METHODS Marsypianthes chamaedrys, which is used in Brazil as a folk medicine to treat snakebites and local inflammatory reactions, was tested in vitro to determine its ability to block indirect phospholipase A(2) and direct coagulant activities and in vivo to determine its ability to inhibit leukocyte migration and cytokine release. RESULTS In vitro, Marsypianthes chamaedrys showed antiphospholipase A(2) and anticoagulant activities; the latter activity was also confirmed by prothrombin time (PT) and activated partial thromboplastin time (aPTT) in the absence of venom. Of the extracts used, those obtained from the crushed plant had the greater inhibitory activity in in vitro tests, showing that biological activity is affected by the way extracts are obtained. In vivo, Marsypianthes chamaedrys inhibited leukocyte migration and the release of the proinflammatory cytokines IL-6 and TNF-α without altering the concentration of the anti-inflammatory cytokine IL-10. CONCLUSIONS As specific antivenoms are not effective in neutralizing the local action of Bothrops venoms, characterization of the anti-inflammatory mechanisms induced by Marsypianthes chamaedrys is of vital importance if the extracts of this plant species are to be used in future as adjuvants in the treatment of snakebites.

Plants used to treat snakebites in Santarém, western Pará, Brazil: An assessment of their effectiveness in inhibiting hemorrhagic activity induced by Bothrops jararaca venom

ETHNOPHARMACOLOGICAL RELEVANCE The poor distribution and limited availability of antivenoms in Brazil have led to greater use of plants to treat snakebites. Very often such plants are the only alternative available to riverside communities. MATERIALS AND METHODS Direct questionnaire-based interviews were conducted with members of the Cucurunã, São Pedro and Alter do Chão communities in Santarém, Pará, Brazil. For each of the 12 most frequently mentioned species aqueous extracts were prepared and the phytochemical profiles determined by thin layer chromatography. The concentrations of phenolic compounds (tannins and flavonoids) in the aqueous extracts were determined by colorimetric assays. To assess inhibition of the hemorrhagic activity of Bothrops jararaca venom, solutions containing the venom mixed with aqueous extracts in the ratios 1:12 and 1:48 were tested (w/w). SDS-PAGE and Western blot were used to assess the action of the extracts on Bothrops jararaca venom. RESULTS In all, 24 plants belonging to 19 families were mentioned in the survey as being used to treat snakebites. Leaves (84%), seeds (60.9%) and inner bark (53%) were cited as the most frequently used parts in folk remedies, which were usually prepared in the form of a decoction (62.5%), tincture (45%) or maceration (22.5%). Hemorrhage induced by Bothrops jararaca venom was completely inhibited by aqueous extracts of Bellucia dichotoma, Connarus favosus, Plathymenia reticulata and Philodendron megalophyllum, which had a high phenolic content and contained condensed and hydrolyzable tannins. The results of SDS-PAGE showed that some venom protein bands were not visible when the venom was preincubated with the extracts that had completely inhibited hemorrhagic activity of the venom. Western blot showed that the extracts did not have any enzymatic action on the proteins in the venom as it failed to detect low-molecular-weight bands, which are indicative of possible enzymatic cleavage. CONCLUSIONS Traditional use of plants to treat snakebites is a common practice in the western region of Pará, Brazil. Our findings show that some plant extracts were able to inhibit snake venom-induced hemorrhage in vitro. In vivo studies are being carried out to validate the traditional use of these species to treat snakebites.

Cell phenotyping in saliva of individuals under psychological stress

Total leukocytes, NK cells, B and T lymphocytes present in the saliva of medical students with or without stress were quantified by flow cytometry in 10,000 events. The symptoms of psychological stress were monitored with Lipps Inventory of Stress Symptoms for Adults (ISSL). No significant differences were observed in the number of cells phenotyped in students with and those without psychological stress. However, a negative correlation was observed between the number of NK cells and T lymphocytes in students with stress (r=-0.8173; p=0.0058), suggesting that innate immunity is predominant in the adaptation phase.

The influence of a TNF gene polymorphism on the severity of rheumatoid arthritis in the Brazilian Amazon.

PURPOSE The aim of this study was to investigate the influence of the TNF -308 G/A polymorphism in the promoter region of the tumor necrosis factor-α gene on the susceptibility and severity of rheumatoid arthritis (RA) in individuals from the Brazilian Amazon. METHODS A total of 323 individuals-192 healthy controls without arthritis and 131 individuals suffering from arthritis-were genotyped for this polymorphism using a methodology based on PCR-RFLP. RESULTS The frequency of the A allele (TNF2) in rheumatoid arthritis sufferers was not significantly higher than in the controls (p=0.926; OR=0.97; confidence interval 0.54-1.76). However, using a logistic regression model, when the patients were stratified according to whether the manifestations were preponderantly articular or systemic, there was a strong association between the TNF2 allele and systemic arthritis (p=0.001; OR=5.89; confidence interval=1.98-17.5) as well as the use of anti-TNF immunotherapy (p=0.023; OR=1.10; confidence interval=1.00-1.14). The main factors that were found to influence the risk of extra-articular disease were age greater than or equal to 60 years (p=0.008; OR=4.06; confidence interval=1.45-11.38), disease duration greater than 10 years (p=0.031; OR=3.10; confidence interval=1.11-8.63) and positive rheumatoid factor (p=0.035; OR=2.07; confidence interval=1.05-4.09). CONCLUSIONS These results suggest that the TNF2 allele is associated with the more serious forms of the disease in individuals from the Brazilian Amazon but not with a risk for developing RA.

Heparin-antivenom association: differential neutralization effectiveness in Bothrops atrox and Bothrops erythromelas envenoming

Heparin, in some regions of Brazil has been used in the treatment of bothropic accidents, but the data found in the literature are inconclusive about its effectiveness. The venoms of Bothrops atrox and of B. erythromelas were characterized according to their biological activities. The capacity of heparin in neutralizing these activities was tested with doses of 3 and 6 IU in isolated form and associated to Antibothropic Serum (ABS). It was verified that heparin, in doses of 3 and 6 IU, was not effective in neutralizing the desfibrinating and edema-forming activities of B. atrox venom and the hemorrhagic and coagulant actions of both venoms. Heparin diminished the effectiveness of the ABS in the neutralization of the hemorrhagic and edema-forming activities of the B. atrox venom. However, heparin in the 6 IU dose was capable of neutralize the edema-forming of the B. erythromelas and increase the effectiveness of the ABS. Heparin also neutralized the phospholipasic A2 activity of B. atrox (14.3%) and B. erythromelas (28.0%) venoms. For B. erythromelas venom, the associated treatment, heparin and ABS, was more effective in the neutralization of its lethal activity.

Opioids and the immune system: clinical relevance

BACKGROUND AND OBJECTIVES The increasing use of opioids for pain treatment is a reality in several countries and, therefore, unusual questions arise, such as the influence of opioids on immune responses. The present study aims to detail the immune response by exploring the influences of opiate effects on inflammatory response in experimental and clinical situations, as well as its importance in daily practice. CONTENT After reviewing the articles published in journals indexed in Medline, we found that immune response has been generally described, especially regarding its cellular aspect. Following this approach, we identified the mechanisms of endogenous opioid release, modulation of immune response to exogenous opioids in acute and chronic pain, always ending with the clinical implications and applicability in routine care. CONCLUSIONS Although several studies point to an immunosuppressive effect of opioids, the clinical relevance of these observations remains uncertain and only serves as a prerequisite for further investigations in this area. Definitive recommendations for the use of opioids in various situations of clinical practice regarding the immunological consequences of these drugs still cannot be provided until the present moment.

Avaliação da reação em cadeia da polimerase no diagnóstico da tuberculose pulmonar em pacientes indígenas e não indígenas

OBJECTIVE To evaluate the accuracy of bacteriological methods and of polymerase chain reaction (with primers specific for IS6110 of the Mycobacterium tuberculosis complex) in testing sputum samples from indigenous (Amerindian) and non-indigenous patients. METHODS A total of 214 sputum samples (154 from indigenous patients and 60 from non-indigenous patients) were analyzed in order to determine the accuracy of smear microscopy (direct and concentrated versions) for acid-fast bacilli, culture, and polymerase chain reaction. RESULTS Both microscopy methods presented low sensitivity in comparison with culture and polymerase chain reaction. Specificity ranged from 91% to 100%, the concentrated acid-fast smear technique being the least specific. Nontuberculous mycobacteria were isolated three times more frequently in samples from indigenous patients than in those from non-indigenous patients. False-positive and false-negative polymerase chain reaction results were more common in the indigenous population. CONCLUSION Positivity and isolation of nontuberculous mycobacteria in the acid-fast smear in conjunction with polymerase chain reaction positivity raise the following hypotheses: nontuberculous mycobacteria species with DNA regions homologous to, or even still possessing, the M. tuberculosis IS6110 exist in the Amazon; colonization of the oropharynx or of a tuberculous lesion accelerates the growth of the nontuberculous mycobacteria present in the sputum samples, making it impossible to isolate M. tuberculosis; A history of tuberculosis results in positivity for M. tuberculosis DNA. The absence of bacteriological positivity in the presence of polymerase chain reaction positivity raises questions regarding the inherent technical characteristics of the bacteriological methods or regarding patient history of tuberculosis.