Human_Leishmaniasis@cytokines.bahia.br

The cell-mediated immune response is critical in the resistance to and recovery from leishmaniasis. Cytokines are central elements in mounting an immune response and have received a great deal of attention in both human and experimental leishmaniasis. IFN-g is responsible for macrophage activation leading to leishmanicidal mechanisms. Understanding the balance of cytokines that lead to enhanced production of or synergize with IFN-g, and those cytokines that counterbalance its effects is fundamental for developing rational immunotherapeutic or immunoprophylactic approaches to leishmaniasis. Here we focus on the cytokine balance in human leishmaniasis, particularly IL-10 as an IFN-g opposing cytokine, and IL-12 as an IFN-g inducer. The effects of these cytokines were evaluated in terms of several parameters of the human immune response. IL-10 reduced lymphocyte proliferation, IFN-g production and cytotoxic activity of responsive human peripheral blood mononuclear cells. Neutralization of IL-10 led to partial restoration of lymphoproliferation, IFN-g production and cytotoxic activity in unresponsive visceral leishmaniasis patients. IL-12 also restored the responses of peripheral blood mononuclear cells from visceral leishmaniasis patients. The responses obtained with IL-12 are higher than those obtained with anti-IL-10, even when anti-IL-10 is combined with anti-IL-4

Interferon gamma is a key cytokine in lung phase immunity to schistosomes but what is its precise role?

Vaccination of mice with radiation-attenuated cercariae of Schistosoma mansoni induces a high level of protection against challenge with normal larvae. The immune effector mechanism, which operates in the lungs, is a cell-mediated delayed-type hypersensitivity response and involves the formation of a tight focus of mononuclear cells around embolised larvae. CD4+ T cells with Th1 characteristics are a major component of the infiltrate. They secrete abundant interferon gamma (IFNg) upon antigen stimulation in vitro, whilst in vivo neutralisation of the cytokine results in 90% abrogation of immunity. IFNg can induce a large number of genes and an attempt has been made to identify the ones which are essential components of the effector mechanism. Inducible nitric oxide synthase (iNOS) is such a candidate and nitric oxide (NO) is produced by cultures of airway leucocytes from the lungs of vaccinated mice post-challenge. However, the continued resistance of mice with a disrupted iNOS gene indicates that NO has only a minor role in the protective response. Mice with a disrupted IFNg receptor gene have been used to dissect the role of the cytokine. After vaccination and challenge, CD4+ T cells from the pulmonary interstitium have reduced levels of ICAM-1 and LFA-1 expression, compared to wild-type animals, which coincides with a reduced cohesiveness of foci. However, immunity is not significantly impaired in mice with a disrupted ICAM-1 gene, and focus formation is normal. Similarly, a role has not been found for CD2/CD48 interactions in cell aggregation. Possible IFNg-inducible molecules yet to be fully investigated include other ligand-receptor pairs, chemokines, and tumour necrosis factor a.

Interleukin-12 and protective immunity to schistosomes

The attenuated vaccine against Schistosoma mansoni induces Th1-mediated protective immunity and we have sought to identify a role for IL-12 in this model. Elevated levels of IL-12 (p40 mRNA) were detected in the lymph nodes (LN) and the lungs of vaccinated mice, whilst treatment of vaccinated mice with anti-IL-12 antibodies decreased the ratio of IFNg:IL-4 secreted by in vitro-cultured LN cells. However, there was only marginal abrogation of the level of resistance in these mice. Soluble antigens from the lung-stage of the parasite (SLAP) appeared to be efficient stimulators of IFNg and IL-12 secretion. These antigens when used to immunise mice in conjunction with IL-12 as an adjuvant, elicited a polarised Th1 response with abundant IFNg secretion but no IL-4. This immunisation regime also induced significant protection against reinfection, whereas inoculation of mice with SLAP alone did not. The induction of a dominant Th1 response using SLAP + IL-12 probably operates via IFNg production by natural killer (NK) cells stimulated by IL-12, since in vivo ablation of NK cells using anti-NK1.1 antibody reduced CD4+-dependent IFNg production from cultured LN cells by over 97%. Nevertheless, in mice with a genetic disruption of the IFNg receptor, administration of SLAP + IL-12 induced levels of IFNg equal to those in wild-type mice, thus showing that in this model IL-12 can directly prime T cells independent of IFNg. Clearly, IL-12 has a critical role in protective immunity to schistosomes and it may aid the development of an effective vaccine against this disease

Cytokines as determinants of resistance and pathology in human Schistosoma mansoni infection

The role of different cytokines in the peripheral blood mononuclear cell (PBMC) proliferative response and in in vitro granuloma formation was evaluated in a cross-sectional study with patients with the different clinical forms and phases of Schistosoma mansoni infection, as well as a group of individuals "naturally" resistant to infection named normal endemic (NE). The blockage of IL-4 and IL-5 using anti-IL-4 and anti-IL-5 antibodies significantly reduced the PBMC proliferative response to soluble egg (SEA) and adult worm (SWAP) antigens in acute (ACT), chronic intestinal (INT) and hepatosplenic (HS) patients. Similar results were obtained in the in vitro granuloma formation. Blockage of IL-10 had no significant effect on either assay using PBMC from ACT or HS. In contrast, the addition of anti-IL-10 antibodies to PBMC cultures from INT patients significantly increased the proliferative response to SEA and SWAP as well as the in vitro granuloma formation. Interestingly, association of anti-IL-4 and anti-IL-10 antibodies did not increase the PBMC proliferative response of these patients, suggesting that IL-10 may act by modulating IL-4 and IL-5 secretion. Addition of recombinant IL-10 decreased the proliferative response to undetectable levels when PBMC from patients with the different clinical forms were used. Analysis of IFN-g in the supernatants showed that PBMC from INT patients secreted low levels of IFN-g upon antigenic stimulation. In contrast, PBMC from NE secreted high levels of IFN-g. These data suggest that IL-10 is an important cytokine in regulating the immune response and possibly controlling morbidity in human schistosomiasis mansoni, and that the production of IFN-g may be associated with resistance to infection.

Quantitative receptor radioautography in the study of receptor-receptor interactions in the nucleus tractus solitarii

The nucleus tractus solitarii (NTS) in the dorsomedial medulla comprises a wide range of neuropeptides and biogenic amines. Several of them are related to mechanisms of central blood pressure control. Angiotensin II (Ang II), neuropeptide Y (NPY) and noradrenaline (NA) are found in the NTS cells, as well as their receptors. Based on this observation we have evaluated the modulatory effect of these peptide receptors on a2-adrenoceptors in the NTS. Using quantitative receptor radioautography, we observed that NPY and Ang II receptors decreased the affinity of a2-adrenoceptors for their agonists in the NTS of the rat. Cardiovascular experiments agreed with the in vitro data. Coinjection of a threshold dose of Ang II or of the NPY agonists together with an ED50 dose of adrenergic agonists such as NA, adrenaline and clonidine counteracted the depressor effect produced by the a2-agonist in the NTS. The results provide evidence for the existence of an antagonistic interaction between Ang II at1 receptors and NPY receptor subtypes with the a2-adrenoceptors in the NTS. This receptor interaction may reduce the transduction over the a2-adrenoceptors which can be important in central cardiovascular regulation and in the development of hypertension

Immunoregulatory and anti-inflammatory effects of n-3 polyunsaturated fatty acids

1. Fish oils are rich in the long-chain n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids. Linseed oil and green plant tissues are rich in the precursor fatty acid, a-linolenic acid (18:3n-3). Most vegetable oils are rich in the n-6 PUFA linoleic acid (18:2n-6), the precursor of arachidonic acid (20:4n-6). 2. Arachidonic acid-derived eicosanoids such as prostaglandin E2 are pro-inflammatory and regulate the functions of cells of the immune system. Consumption of fish oils leads to replacement of arachidonic acid in cell membranes by eicosapentaenoic acid. This changes the amount and alters the balance of eicosanoids produced. 3. Consumption of fish oils diminishes lymphocyte proliferation, T-cell-mediated cytotoxicity, natural killer cell activity, macrophage-mediated cytotoxicity, monocyte and neutrophil chemotaxis, major histocompatibility class II expression and antigen presentation, production of pro-inflammatory cytokines (interleukins 1 and 6, tumour necrosis factor) and adhesion molecule expression. 4. Feeding laboratory animals fish oil reduces acute and chronic inflammatory responses, improves survival to endotoxin and in models of autoimmunity and prolongs the survival of grafted organs. 5. Feeding fish oil reduces cell-mediated immune responses. 6. Fish oil supplementation may be clinically useful in acute and chronic inflammatory conditions and following transplantation. 7. n-3 PUFAs may exert their effects by modulating signal transduction and/or gene expression within inflammatory and immune cells.

Assessment of ventilatory neuromuscular drive in patients with obstructive sleep apnea

The presence of abnormalities of the respiratory center in obstructive sleep apnea (OSA) patients and their correlation with polysomnographic data are still a matter of controversy. Moderately obese, sleep-deprived OSA patients presenting daytime hypersomnolence, with normocapnia and no clinical or spirometric evidence of pulmonary disease, were selected. We assessed the ventilatory control and correlated it with polysomnographic data. Ventilatory neuromuscular drive was evaluated in these patients by measuring the ventilatory response (VE), the inspiratory occlusion pressure (P.1) and the ventilatory pattern (VT/TI, TI/TTOT) at rest and during submaximal exercise, breathing room air. These analyses were also performed after inhalation of a hypercapnic mixture of CO2 (DP.1/DPETCO2, DVE/DPETCO2). Average rest and exercise ventilatory response (VE: 12.2 and 32.6 l/min, respectively), inspiratory occlusion pressure (P.1: 1.5 and 4.7 cmH2O, respectively), and ventilatory pattern (VT/TI: 0.42 and 1.09 l/s; TI/TTOT: 0.47 and 0.46 l/s, respectively) were within the normal range. In response to hypercapnia, the values of ventilatory response (DVE/DPETCO2: 1.51 l min-1 mmHg-1) and inspiratory occlusion pressure (DP.1/DPETCO2: 0.22 cmH2O) were normal or slightly reduced in the normocapnic OSA patients. No association or correlation between ventilatory neuromuscular drive and ventilatory pattern, hypersomnolence score and polysomnographic data was found; however a significant positive correlation was observed between P.1 and weight. Our results indicate the existence of a group of normocapnic OSA patients who have a normal awake neuromuscular ventilatory drive at rest or during exercise that is partially influenced by obesity

Analysis of the DF508 mutation in a Brazilian cystic fibrosis population: comparison of pulmonary status of homozygotes with other patients

Sixty-one cystic fibrosis patients admitted for check-up or antibiotic treatment were enrolled for genetic and clinical evaluation. Genetic analysis was performed on blood samples stored on neonatal screening cards using PCR techniques to determine the presence of DF508 mutations. Clinical evaluation included Shwachman and Chrispin-Norman scores, age at onset of symptoms and diagnosis, spirometry, awake and sleep pulse oximetry, hyponychial angle measurement and presence of chronic Pseudomonas aeruginosa colonization. Eighteen patients (29.5%) were homozygous for the DF508 mutation, 26 (42.6%) had one DF508 mutation and 17 (27.9%) were noncarriers, corresponding to a 50.8% prevalence of the mutation in the whole population. Analysis by the Kruskal-Wallis test for comparison of genetic status with continuous variables or by the chi-square test and logistic regression for dichotomous variables showed no significant differences between any two groups for a = 0.05. We conclude that genetic status in relation to the DF508 mutation is not associated with pulmonary status as evaluated by the above variables

Cytokines produced by susceptible and resistant mice in the course of Paracoccidioides brasiliensis infection

Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and presents a wide spectrum of clinical manifestations. We established a genetically controlled murine model of PCM, where A/Sn mice develop an infection which mimics the benign disease (immune responses which favor cellular immunity) and B10.A animals present the progressive disseminated form of PCM (preferential activation of B cells and impairment of cellular immune responses). To understand the immunoregulatory phenomena associated with resistance and susceptibility in experimental PCM, A/Sn and B10.A mice were studied regarding antigen-elicited secretion of monokines (TNF-a and TGF-ß) and type-1 (IL-2 and IFN-g) and type-2 (IL-4,5,10) cytokines. Total lymph node cells from resistant mice infected ip with P. brasiliensis produced early and sustained levels of IFN-g and IL-2; type-2 cytokines (IL-4 and IL-5) started to appear 8 weeks after infection. In contrast, susceptible mice produced low levels of IFN-g concomitant with significant levels of IL-5 and IL-10 early in the infection. In the chronic phase of the disease, susceptible animals presented a transitory secretion of IL-2, and IL-4. In the pulmonary infection IL-4, IL-5 and IL-10 were preferentially detected in the lung cells washings of susceptible animals. After in vitro challenge with fungal antigens, normal peritoneal macrophages from B10.A mice secreted high levels of TGF-ß and low levels of TNF-a. In contrast, macrophages from A/Sn animals released high levels of TNF-a associated with a small production of TGF-ß. The in vivo depletion of IFN-g not only abrogated the resistance of A/Sn mice but also diminished the relative resistance of B10.A animals. The in vivo depletion of IL-4 did not alter the disease outcome, whereas administration of rIL-12 significantly enhanced resistance in susceptible animals. Taken together, these results suggest that an early secretion of high levels of TNF-a and IFN-g followed by a sustained secretion of IL-2 and IFN-g plays a dominant role in the resistance mechanisms to P. brasiliensis infection. In contrast, an early and ephemeral secretion of low levels of TNF-a and IFN-g associated with production of IL-5, IL-10 and TGF-ß characterizes the progressive disease of susceptible animals.

Absence of linkage between MHC and a gene involved in susceptibility to human schistosomiasis

Six hundred million people are at risk of infection by Schistosoma mansoni. MHC haplotypes have been reported to segregate with susceptibility to schistosomiasis in murine models. In humans, a major gene related to susceptibility/resistance to infection by S. mansoni (SM1) and displaying the mean fecal egg count as phenotype was detected by segregation analysis. This gene displayed a codominant mode of inheritance with an estimated frequency of 0.20-0.25 for the deleterious allele and accounted for more than 50% of the variance of infection levels. To determine if the SM1 gene segregates with the human MHC chromosomal region, we performed a linkage study by the lod score method. We typed for HLA-A, B, C, DR and DQ antigens in 11 informative families from an endemic area for schistosomiasis in Bahia, Brazil, by the microlymphocytotoxicity technique. HLA-DR typing by the polymerase chain reaction with sequence-specific primers (PCR-SSP) and HLA-DQ were confirmed by PCR-sequence-specific oligonucleotide probes (PCR-SSOP). The lod scores for the different q values obtained clearly indicate that there is no physical linkage between HLA and SM1 genes. Thus, susceptibility or resistance to schistosomiasis, as defined by mean fecal egg count, is not primarily dependent on the host's HLA profile. However, if the HLA molecule plays an important role in specific immune responses to S. mansoni, this may involve the development of the different clinical aspects of the disease such as granuloma formation and development of hepatosplenomegaly.

Pharmacokinetics and pharmacodynamics of propranolol in hypertensive patients after sublingual administration: systemic availability

The bioavailability of propranolol depends on the degree of liver metabolism. Orally but not intravenously administered propranolol is heavily metabolized. In the present study we assessed the pharmacokinetics and pharmacodynamics of sublingual propranolol. Fourteen severely hypertensive patients (diastolic blood pressure (DBP) ³115 mmHg), aged 40 to 66 years, were randomly chosen to receive a single dose of 40 mg propranolol hydrochloride by sublingual or peroral administration. Systolic (SBP) and diastolic (DBP) blood pressures, heart rate (HR) for pharmacodynamics and blood samples for noncompartmental pharmacokinetics were obtained at baseline and at 10, 20, 30, 60 and 120 min after the single dose. Significant reductions in BP and HR were obtained, but differences in these parameters were not observed when sublingual and peroral administrations were compared as follows: SBP (17 vs 18%, P = NS), DBP (14 vs 8%, P = NS) and HR (22 vs 28%, P = NS), respectively. The pharmacokinetic parameters obtained after sublingual or peroral drug administration were: peak plasma concentration (CMAX): 147 ± 72 vs 41 ± 12 ng/ml, P<0.05; time to reach CMAX (TMAX): 34 ± 18 vs 52 ± 11 min, P<0.05; biological half-life (t1/2b): 0.91 ± 0.54 vs 2.41 ± 1.16 h, P<0.05; area under the curve (AUCT): 245 ± 134 vs 79 ± 54 ng h-1 ml-1, P<0.05; total body clearance (CLT/F): 44 ± 23 vs 26 ± 12 ml min-1 kg-1, P = NS. Systemic availability measured by the AUCT ratio indicates that extension of bioavailability was increased 3 times by the sublingual route. Mouth paresthesia was the main adverse effect observed after sublingual administration. Sublingual propranolol administration showed a better pharmacokinetic profile and this route of administration may be an alternative for intravenous or oral administration.

Cardiorespiratory adaptations induced by aerobic training in middle-aged men: the importance of a decrease in sympathetic stimulation for the contribution of dynamic exercise tachycardia

We investigated the effects of aerobic training on the efferent autonomic control of heart rate (HR) during dynamic exercise in middle-aged men, eight of whom underwent exercise training (T) while the other seven continued their sedentary (S) life style. The training was conducted over 10 months (three 1-h sessions/week on a field track at 70-85% of the peak HR). The contribution of sympathetic and parasympathetic exercise tachycardia was determined in terms of differences in the time constant effects on the HR response obtained using a discontinuous protocol (4-min tests at 25, 50, 100 and 125 watts on a cycle ergometer), and a continuous protocol (25 watts/min until exhaustion) allowed the quantification of the parameters (anaerobic threshold, VO2 AT; peak O2 uptake, VO2 peak; power peak) that reflect oxygen transport. The results obtained for the S and the T groups were: 1) a smaller resting HR in T (66 beats/min) when compared to S (84 beats/min); 2) during exercise, a small increase in the fast tachycardia (D0-10 s) related to vagal withdrawal (P<0.05, only at 25 watts) was observed in T at all powers; at middle and higher powers a significant decrease (P<0.05 at 50, 100 and 125 watts) in the slow tachycardia (D1-4 min) related to a sympathetic-dependent mechanism was observed in T; 3) the VO2 AT (S = 1.06 and T = 1.33 l/min) and VO2 peak (S = 1.97 and T = 2.47 l/min) were higher in T (P<0.05). These results demonstrate that aerobic training can induce significant physiological adaptations in middle-aged men, mainly expressed as a decrease in the sympathetic effects on heart rate associated with an increase in oxygen transport during dynamic exercise.

Nonspecific blockade of vascular free radical signals by methylated arginine analogues

Methylated arginine analogues are often used as probes of the effect of nitric oxide; however, their specificity is unclear and seems to be frequently overestimated. This study analyzed the effects of NG-methyl-L-arginine (L-NMMA) on the endothelium-dependent release of vascular superoxide radicals triggered by increased flow. Plasma ascorbyl radical signals measured by direct electron paramagnetic resonance spectroscopy in 25 rabbits increased by 3.8 ± 0.7 nmol/l vs baseline (28.7 ± 1.4 nmol/l, P<0.001) in response to papaverine-induced flow increases of 121 ± 12%. In contrast, after similar papaverine-induced flow increases simultaneously with L-NMMA infusions, ascorbyl levels were not significantly changed compared to baseline. Similar results were obtained in isolated rabbit aortas perfused ex vivo with the spin trap a-phenyl-N-tert-butylnitrone (N = 22). However, in both preparations, this complete blockade was not reversed by co-infusion of excess L-arginine and was also obtained by N-methyl-D-arginine, thus indicating that it is not related to nitric oxide synthase. L-arginine alone was ineffective, as previously demonstrated for NG-methyl-L-arginine ester (L-NAME). In vitro, neither L-arginine nor its analogues scavenged superoxide radicals. This nonspecific activity of methylated arginine analogues underscores the need for careful controls in order to assess nitric oxide effects, particularly those related to interactions with active oxygen species.

Mutations of androgen receptor gene in Brazilian patients with male pseudohermaphroditism

We describe the identification of point mutations in the androgen receptor gene in five Brazilian patients with female assignment and behavior. The eight exons of the gene were amplified by the polymerase chain reaction (PCR) and analyzed for single-strand conformation polymorphism (SSCP) to detect the mutations. Direct sequencing of the mutant PCR products demonstrated single transitions in three of these cases: G®A in case 1, within exon C, changing codon 615 from Arg to His; G®A in case 2, within exon E, changing codon 752 from Arg to Gln, and C®T in case 3, within exon B, but without amino acid change.

Fibrinolytic action on fresh human clots of whole body extracts and two semipurified fractions from Lonomia achelous caterpillar

The severe bleeding diathesis produced by intoxication with the venom of Lonomia achelous caterpillars is characterized by prolonged bleeding from superficial skin wounds as well as massive hemorrhage into body cavities. The aim of the present study was to evaluate the effect of the crude venom and its fibrinolytic fractions on in vitro lysis of whole blood clots. Venom fractions with fibrinolytic activity were obtained by gel filtration chromatography on Sephadex G75 using imidazole buffer, pH 7.4, at a flow rate of 24 ml/h. Four peaks with fibrinolytic activity were obtained by this method. The highest activity was found in the first two peaks (both peaks were used for the experiments). The results show that the caterpillar venom degraded the preformed clots at a slower rate than plasmin. In addition, plasma protease inhibitors of the fibrinolytic system (a2-antiplasmin, a2-macroglobulin, PAI, etc.) only weakly inhibited the lytic effect of the caterpillar venom. These characteristics, as well as the pattern of fibrinogen degradation products, the delay period on fibrin plate lysis and amidolytic activity on chromogenic substrate, reported previously, indicate that the caterpillar enzymes are different from plasmin and trypsin.