Preliminary results in the immunodiagnosis of tuberculosis in children based on T cell responses to ESAT-6 and PPD antigens

The aim of this work was to study the difference in interferon gamma (IFN-gamma) production by T lymphocytes after early secretory antigen target 6 (ESAT-6) or purified protein derivate (PPD) stimulation in whole blood culture supernatants from children with suspected tuberculosis (TB) disease (n = 21), latent TB infection (n = 16) and negative controls (NC) (n = 22) from an endemic area in Brazil. The concentration of IFN-gamma (pg/ml) was measured by enzyme linked immunosorbent assay and the differences in the IFN-gamma levels for each group were compared and evaluated using an unpaired Student's t-test; p values < 0.05 were considered significant. Measurement of IFN-gamma levels after ESAT-6 stimulation raised the possibility of early diagnosis in the latent TB group (p = 0.0030). Nevertheless, the same group showed similar responses to the NC group (p > 0.05) after PPD stimulation. The IFN-gamma assay using ESAT-6 as an antigenic stimulus has the potential to be used as a tool for the immunodiagnosis of early TB in children.

Immune and inflammatory responses to Leishmania amazonensis isolated from different clinical forms of human leishmaniasis in CBA mice

Leishmania amazonensis causes different diseases depending on the host and parasitic virulence factors. In this study, CBA mice were infected with L. amazonensis isolates from patients with localized (Ba125), diffuse cutaneous (Ba276) or visceral leishmaniasis (Ba109). Mice infected with Ba125 and Ba276 progressed rapidly and lesions displayed an infiltrate rich in parasitized macrophages and were necrotic and ulcerated. Ba109 induced smaller lesions and a mixed inflammatory infiltrate without necrosis or ulceration. Ba109 induced an insidious disease with lower parasite load in CBA mice, similar to human disease. Levels of IFN-γ, IL-4 and IL-10 did not differ among the groups. Because all groups were unable to control the infection, expression of IL-4 associated with low production of IFN-γ in the early phase of infection may account for susceptibility, but others factors may contribute to the differences observed in inflammatory responses and infection progression. Evaluation of some parasitic virulence factors revealed that Ba276 exhibits higher ecto-ADPase and 5'-nucleotidase activities compared to the Ba109 and Ba125 strains. Both Ba276 and Ba125 had higher arginase activity in comparison to Ba109. Finally, these data suggest that the differences in enzyme activities among parasites can account for differences in host inflammatory responses and infection progression.

Modulation of Trypanosoma cruzi-specific T-cell responses after chemotherapy for chronic Chagas disease

The aim of this review is to describe the contributions of the knowledge of T-cell responses to the understanding of the physiopathology and the responsiveness to etiological treatment during the chronic phase of Chagas disease.T-helper (Th)1 and interleukin (IL)-10Trypanosoma cruzi-specific T-cells have been linked to the asymptomatic phase or to severe clinical forms of the disease, respectively orvice versa, depending on the T. cruziantigen source, the patient’s location and the performed immunological assays. Parasite-specific T-cell responses are modulated after benznidazole (BZ) treatment in chronically T. cruzi-infected subjects in association with a significant decrease in T. cruzi-specific antibodies. Accumulating evidence has indicated that treatment efficacy during experimental infection with T. cruziresults from the combined action of BZ and the activation of appropriate immune responses in the host. However, strong support of this interaction in T. cruzi-infected humans remains lacking. Overall, the quality of T-cell responses might be a key factor in not only disease evolution, but also chemotherapy responsiveness. Immunological parameters are potential indicators of treatment response regardless of achievement of cure. Providing tools to monitor and provide early predictions of treatment success will allow the development of new therapeutic options.

Biomarkers of therapeutic responses in chronic Chagas disease: state of the art and future perspectives

The definition of a biomarker provided by the World Health Organization is any substance, structure, or process that can be measured in the body, or its products and influence, or predict the incidence or outcome of disease. Currently, the lack of prognosis and progression markers for chronic Chagas disease has posed limitations for testing new drugs to treat this neglected disease. Several molecules and techniques to detect biomarkers inTrypanosoma cruzi-infected patients have been proposed to assess whether specific treatment with benznidazole or nifurtimox is effective. Isolated proteins or protein groups from different T. cruzistages and parasite-derived glycoproteins and synthetic neoglycoconjugates have been demonstrated to be useful for this purpose, as have nucleic acid amplification techniques. The amplification of T. cruziDNA using the real-time polymerase chain reaction method is the leading test for assessing responses to treatment in a short period of time. Biochemical biomarkers have been tested early after specific treatment. Cytokines and surface markers represent promising molecules for the characterisation of host cellular responses, but need to be further assessed.

Effect of Nitrogen and Potassium Rates on Early Development of Macaw Palm

ABSTRACT The economic exploitation of macaw palm [Acrocomia aculeate(Jacq.) Lodd. ex Mart.] is currently in transition, from extractivism to agricultural cultivation, thus requiring studies on the fertilization of the crop. This study evaluated the response of three genotypes of macaw palm to increasing rates of nitrogen and potassium, grown in the field until the 2nd year and to establish reference contents of mineral nutrients in the leaf. The experiment was a split-plot randomized block design with five main treatments (N and K rates) and three secondary treatments (genotypes), with three replications, each plot containing three plants. Plant height, leaf number, vigor, and nutrient contents in leaf tissues were evaluated at the end of 2nd year of cultivation. Differential responses were observed among genotypes, indicating that some genotypes are more efficient in the use of mineral inputs. There was a differentiated and positive response to increasing side-dressed N and K rates in the vegetative development of macaw genotypes until the 2nd year of field cultivation, indicating variability in the species in terms of nutrient use efficiency. The N and K fertilization rate corresponding to 360 g N + 480 g K2O per plant, in four split applications over the two years of cultivation, was insufficient to induce maximum vegetative development in the three macaw genotypes. There was no variation in macro- and micronutrient contents in leaf dry matter of the three macaw genotypes.

Photosynthetic responses of young cashew plants to varying environmental conditions

The aim of this study was to characterize gas exchange responses of young cashew plants to varying photosynthetic photon flux density (PPFD), temperature, vapor-pressure deficit (VPD), and intercellular CO2 concentration (Ci), under controlled conditions. Daily courses of gas exchange and chlorophyll a fluorescence parameters were measured under natural conditions. Maximum CO2 assimilation rates, under optimal controlled conditions, were about 13 mmol m-2 s-1 , with light saturation around 1,000 mmol m-2 s-1. Leaf temperatures between 25ºC and 35ºC were optimal for photosynthesis. Stomata showed sensitivity to CO2, and a closing response with increasing Ci. Increasing VPD had a small effect on CO2 assimilation rates, with a small decrease above 2.5 kPa. Stomata, however, were strongly affected by VPD, exhibiting gradual closure above 1.5 kPa. The reduced stomatal conductances at high VPD were efficient in restricting water losses by transpiration, demonstrating the species adaptability to dry environments. Under natural irradiance, CO2 assimilation rates were saturated in early morning, following thereafter the PPFD changes. Transient Fv/Fm decreases were registered around 11h, indicating the occurrence of photoinhibition. Decreases of excitation capture efficiency, decreases of effective quantum yield of photosystem II, and increases in non-photochemical quenching were consistent with the occurrence of photoprotection under excessive irradiance levels.

Heliotropic responses of soybean cultivars at three phenological stages and under two water regimes

The objectives of this work were to determine the heliotropic movements of the upper trifoliates for two soybean cultivars, BR 16 and Embrapa 48, during a daily cycle, in three phenological stages and two water regimes, and to estimate the impact of irrigation and daily leaflet movements on agronomic characteristics and grain yield. Heliotropic movements were studied in three phenological stages: V4-V6, V7-V10, and R5 in irrigated and non-irrigated plots. For each stage, the leaflet elevation and azimuth were measured hourly. Under a low (V4-V6 stage) and mid (V7-V10 stage) leaf area index (LAI) the diaheliotropism was slightly more frequent and intensive in non-irrigated than in irrigated plants, only at early morning and late afternoon hours. At R5 stage (high LAI) the paraheliotropism of superior trifoliates was predominant and more intensive in non-irrigated plants. The heliotropic movements are correlated to carbon gain, but not to environment (light intensity or temperature), for measurements at 11h. 'Embrapa 48' expresses greater paraheliotropism than 'BR 16' at high LAI, while 'BR 16' displays lower heliotropic plasticity under irrigation. In spite of significant heliotropic differences, genotype and water availability treatments did not influence the final grain yield.

Flooding effects in seedlings of Cytharexyllum myrianthum Cham. and Genipa americana L.: responses of two neotropical lowland tree species

Six-month-old seedlings of Cytharexyllum myrianthum and Genipa americana, two common tree species in different flood-prone areas in Brazil, were flooded for up to 90 days to compare their survival and growth responses under these conditions. Seedlings of both species were found to be relatively tolerant to flooding but growth responses changed according to treatment and plant species. Growth of G. americana was reduced by flooding, showing a decrease in root and leaf dry mass, root/shoot ratio and height, without showing any adaptive morphological changes. On the other hand, growth of C. myrianthum seedlings was stimulated under flooding conditions, showing an increase in root dry mass, root/shoot ratio, height, stem diameter and some morphological changes in roots and stems, i.e., development of new roots and stem base hypertrophy. These results could be regarded as an experimental corroboration of the field observations, showing that these species could be indicated for restoration programs of some Neotropical wetlands.

Photosynthetic responses of tropical tree species from different successional groups under contrasting irradiance conditions

This study evaluated the photosynthetic responses of seven tropical trees of different successional groups under contrasting irradiance conditions, taking into account changes in gas exchange and chlorophyll a fluorescence. Although early successional species have shown higher values of CO2 assimilation (A) and transpiration (E), there was not a defined pattern of the daily gas exchange responses to high irradiance (FSL) among evaluated species. Cariniana legalis (Mart.) Kuntze (late secondary) and Astronium graveolens Jacq. (early secondary) exhibited larger reductions in daily-integrated CO2 assimilation (DIA) when transferred from medium light (ML) to FSL. On the other hand, the pioneer species Guazuma ulmifolia Lam. had significant DIA increase when exposed to FSL. The pioneers Croton spp. trended to show a DIA decrease around 19%, while Cytharexyllum myrianthum Cham. (pioneer) and Rhamnidium elaeocarpum Reiss. (early secondary) trended to increase DIA when transferred to FSL. Under this condition, all species showed dynamic photoinhibition, except for C. legalis that presented chronic photoinhibition of photosynthesis. Considering daily photosynthetic processes, our results supported the hypothesis of more flexible responses of early successional species (pioneer and early secondary species). The principal component analysis indicated that the photochemical parameters effective quantum efficiency of photosystem II and apparent electron transport rate were more suitable to separate the successional groups under ML condition, whereas A and E play a major role to this task under FSL condition.

Interruption of recently induced immune responses by oral administration of antigen

Interest in oral tolerance has been renewed in the last few years as a possibility of intervention in human autoimmune diseases. An obstacle in this direction is that, although easily induced in animals virgin of contact with the antigen, oral tolerance becomes hard to induce in previously immunized animals. The present results show that there is an early period after primary immunization in which prolonged oral exposure to the antigen may arrest ongoing immune responses. Beyond this period, oral exposures to the antigen become ineffective and may actually boost immune responses. The end of the susceptible period coincides with the emergence of free specific antibodies in serum. However, the previous administration of purified anti-ovalbumin antibodies (40 µg) was unable to block the induction of oral tolerance to ovalbumin in normal mice

Genetic responses against nitric oxide toxicity

The threat of free radical damage is opposed by coordinated responses that modulate expression of sets of gene products. In mammalian cells, 12 proteins are induced by exposure to nitric oxide (NO) levels that are sub-toxic but exceed the level needed to activate guanylate cyclase. Heme oxygenase 1 (HO-1) synthesis increases substantially, due to a 30- to 70-fold increase in the level of HO-1 mRNA. HO-1 induction is cGMP-independent and occurs mainly through increased mRNA stability, which therefore indicates a new NO-signaling pathway. HO-1 induction contributes to dramatically increased NO resistance and, together with the other inducible functions, constitutes an adaptive resistance pathway that also defends against oxidants such as H2O2. In E. coli, an oxidative stress response, the soxRS regulon, is activated by direct exposure of E. coli to NO, or by NO generated in murine macrophages after phagocytosis of the bacteria. This response is governed by the SoxR protein, a homodimeric transcription factor (17-kDa subunits) containing [2Fe-2S] clusters essential for its activity. SoxR responds to superoxide stress through one-electron oxidation of the iron-sulfur centers, but such oxidation is not observed in reactions of NO with SoxR. Instead, NO nitrosylates the iron-sulfur centers of SoxR both in vitro and in intact cells, which yields a form of the protein with maximal transcriptional activity. Although nitrosylated SoxR is very stable in purified form, the spectroscopic signals for the nitrosylated iron-sulfur centers disappear rapidly in vivo, indicating an active process to reverse or eliminate them.

Effect of the injection of an extract of Ascaris suum on macrophage activation during the early phase of Mycobacterium bovis BCG infection in C57Bl/6 mice

Injection of an Ascaris suum extract (Asc) affects both the humoral and cellular immune responses to unrelated antigens when it is co-administered with these antigens. In the present study we evaluated the effect of Asc on macrophage activation in the early phase of Mycobacterium bovis BCG (Pasteur strain TMCC 1173) infection in C57Bl/6 mice. C57Bl/6 mice were injected intraperitoneally (ip) with 0.1 mg BCG (BCG group) or BCG plus 1 mg Asc (BCG + Asc group). The peritoneal exudates were obtained at 2, 7 and 14 days after infection. The numbers of IFN-g-secreting cells were assessed by the ELISPOT assay. Nitric oxide (NO) production was measured by the Griess method and by the evaluation of NADPH diaphorase activity in the peritoneal exudates. The administration of Asc extract increased NADPH diaphorase activity (2 days: control = 0, BCG = 7%, BCG + Asc = 13%, and Asc = 4%; 7 days: control = 4, BCG = 13%, BCG + Asc = 21%, and Asc = 4.5%) and TNF-a levels (mean ± SD; 2 days: control = 0, BCG = 169 ± 13, BCG + Asc = 202 ± 37, and Asc = 0; 7 days: control = 0, BCG = 545 ± 15.5, BCG + Asc = 2206 ± 160.6, and Asc = 126 ± 26; 14 days: control = 10 ± 1.45, BCG = 9 ± 1.15, BCG + Asc = 126 ± 18, and Asc = 880 ± 47.67 pg/ml) in the early phase of BCG infection. Low levels of NO production were detected at 2 and 7 days after BCG infection, increasing at 14 days (mean ± SD; 2 days: control = 0, BCG = 3.7 ± 1.59, BCG + Asc = 0.82 ± 0.005, Asc = 0.48 ± 0.33; 7 days: control = 0, BCG = 2.78 ± 1.54, BCG + Asc = 3.07 ± 1.05, Asc = 0; 14 days: control = 0, BCG = 9.05 ± 0.53, BCG + Asc = 9.61 ± 0.81, Asc = 10.5 ± 0.2 (2 x 106) cells/ml). Furthermore, we also observed that Asc co-injection induced a decrease of BCG-colony-forming units (CFU) in the spleens of BCG-infected mice during the first week of infection (mean ± SD; 2 days: BCG = 1.13 ± 0.07 and BCG + Asc = 0.798 ± 0.305; 7 days: BCG = 1.375 ± 0.194 and BCG + Asc = 0.548 ± 0.0226; 14 days: BCG = 0.473 ± 0.184 and BCG + Asc = 0.675 ± 0.065 (x 102) CFU). The present data suggest that Asc induces the enhancement of the immune response in the early phase of BCG infection.

Fever induction pathways: evidence from responses to systemic or local cytokine formation

The immune and central nervous systems are functionally connected and interacting. The concept that the immune signaling to the brain which induces fever during infection and inflammation is mediated by circulating cytokines has been traditionally accepted. Administration of bacterial lipopolysaccharide (LPS) induces the appearance of a so-termed "cytokine cascade" in the circulation more or less concomitantly to the developing febrile response. Also, LPS-like fever can be induced by systemic administration of key cytokines (IL-1ß, TNF-alpha, and others). However, anti-cytokine strategies against IL-1ß or TNF-alpha along with systemic injections of LPS frequently lead to attenuation of the later stages of the febrile response but not of the initial phase of fever, indicating that cytokines are rather involved in the maintenance than in the early induction of fever. Within the last years experimental evidence has accumulated indicating the existence of neural transport pathways of immune signals to the brain. Because subdiaphragmatic vagotomy prevents or attenuates fever in response to intraperitoneal or intravenous injections of LPS, a role for vagal afferent nerve fibers in fever induction has been proposed. Also other sensory nerves may participate in the manifestation of febrile responses under certain experimental conditions. Thus, injection of a small dose of LPS into an artificial subcutaneous chamber results in fever and formation of cytokines within the inflamed tissue around the site of injection. This febrile response can be blocked in part by injection of a local anesthetic into the subcutaneous chamber, indicating a participation of cutaneous afferent nerve signals in the manifestation of fever in this model. In conclusion, humoral signals and an inflammatory stimulation of afferent sensory nerves can participate in the generation and maintenance of a febrile response.

Cardiovascular control in experimental diabetes

Several studies have reported impairment in cardiovascular function and control in diabetes. The studies cited in this review were carried out from a few days up to 3 months after streptozotocin administration and were concerned with the control of the circulation. We observed that early changes (5 days) in blood pressure control by different peripheral receptors were maintained for several months. Moreover, the impairment of reflex responses observed after baroreceptor and chemoreceptor stimulation was probably related to changes in the efferent limb of the reflex arc (sympathetic and parasympathetic), but changes also in the central nervous system could not be excluded. Changes in renal sympathetic nerve activity during volume expansion were blunted in streptozotocin-treated rats, indicating an adaptive natriuretic and diuretic response in the diabetic state. The improvement of diabetic cardiovascular dysfunction induced by exercise training seems to be related to changes in the autonomic nervous system. Complementary studies about the complex interaction between circulation control systems are clearly needed to adequately address the management of pathophysiological changes associated with diabetes.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.