M1 polarization and the effect of PGE2 on TNF-α production by lymph node cells from dogs with visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is caused by the intracellular parasite Leishmania infantum. Increased levels of arginase, nitric oxide (NO2 ) and prostaglandin E2 (PGE2 ) can play a regulatory role regarding the immune response in CVL cases. This study aimed to evaluate the arginase activity in adherent macrophages cultured from the lymph nodes of healthy and naturally infected dogs and to examine the NO2 and PGE2 levels in the supernatant of these cultures. In addition, the regulatory effect of PGE2 on the production of tumour necrosis factor (TNF-α) and interleukin-10 (IL-10) in supernatants from the total lymph node was observed in leucocyte cultures. The arginase activity was lower in the adherent macrophages cultured from the lymph nodes of naturally infected dogs and there were higher concentrations of NO2 and PGE2 in the supernatants of these cultures. Higher TNF-α and IL-10 concentrations were observed in supernatants from total lymph node leucocytes cultures, from infected dogs, and the presence of indomethacin only decreased TNF-α in the supernatant of these cultures. We conclude that the low arginase activity in macrophages suggested that M1 polarization and PGE2 were participating in the immune response and were increasing TNF-α in CVL.

Recombinant K28 antigen in ELISA in the diagnosis of canine visceral leishmaniosis.

Crude total antigen (CTA) from Leishmania infantum and recombinant antigen K39 (rK39) and recombinant antigen K28 (rK28) were compared using an ELISA for the diagnosis of canine visceral leishmaniosis (CVL). Forty-two blood samples from healthy dogs from a nonendemic area and 80 blood samples from an endemic area for dogs with visceral leishmaniosis (VL), confirmed with positive parasitological tests for Leishmania spp., were used in an ELISA. The parasitological diagnosis was chosen as a gold standard. The ELISA with rK28 antigen showed sensitivity of 100% and specificity of 100%, high agreement with CTA and rK39, indicating that the rK28 antigen is useful for ELISA serological diagnosis of CVL.

Interaction of small cationic peptides with intact basement membranes. A study using intrinsic optical signals of chick retinas.

UNLABELLED: Cationic peptides (polylysines and polyarginines) are being developed as drug delivery systems to nuclei. Therefore, a detailed description of tissue response changes upon the application of cationic peptides over intact basement membranes of excitable tissue is of interest in pharmacology. In this paper we examine the effects of two naturally occurring cationic peptides protamine (polyarginine) and crotamine (polylysine) on the optical profiles of retinal spreading depression waves (RSDs). This intrinsic optical signal (IOS), recorded non-invasively, provides information about dissipation of electrochemical gradients within the tissue and its metabolic consequences. Protamine at nanomolar range brought the tissue excitability to collapse without any signs of acute toxicity whereas crotamine, a known myotoxin from rattlesnake, decreased the tissue transparency and changed markedly the optical profiles of RSDs. Also, fluorescent crotamine was incorporated to Müller cells in a few minutes, suggesting a close membrane interaction. The optical changes brought about by crotamine were easily washed off. By contrast, the excitability collapse in presence of protamine lasted for at least two hours. CONCLUSIONS: we concluded that crotamine has fusogenic properties that alters ion transport in excitable tissue. Protamine effect seems to be similar to its effect on basement membrane of epithelium due to its property of making heteropolymers with heparan sulfate. The clinical syndrome expressed in mice after crotamine injection suggested excitotoxic CNS effects confirmed by the isolated retina experiments.

Effects of P-MAPA immunomodulator on Toll-like receptor 2, ROS, nitric oxide, MAPKp38 and IKK in PBMC and macrophages from dogs with visceral leishmaniasis.

Leishmania (L.) chagasi is the etiologic agent of visceral leishmaniasis (VL) that can be transmitted to humans and dogs. VL in Brazil represents a serious public health problem; therefore, it is important to study new alternatives to treat infected dogs. In dogs, the therapeutic arsenal against canine VL is limited. The immunomodulator protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) improves immunocompetence when the immune system is impaired, but its dependence on Toll-like receptors (TLRs) and the mechanisms involved in immune response remain unclear. The in vitro action of P-MAPA on the expression of TLR2 and TLR4, reactive oxygen species (ROS), nitric oxide (NO) and p38 mitogen-activated protein kinase (p38 MAPK) and IKK phosphorylation was studied in mononuclear cells from peripheral blood and macrophages from healthy and Leishmania-infected dogs. The PBMC or macrophages were isolated and cultured with different concentrations of P-MAPA (20,100 and 200 µg/ml) in a humid environment at 37°C with 5% CO(2). Observation revealed that Leishmania-infected dogs showed a decrease in TLR2 in macrophages compared with healthy dogs and in induction with P-MAPA. ROS were increased in PBMCs from Leishmania spp.-infected dogs compared with healthy dogs and P-MAPA improved ROS production. NO production was increased in culture supernatant from macrophages stimulated by P-MAPA in both healthy and Leishmania spp. infected dogs. Treatment of macrophages from healthy dogs with immunomodulatory P-MAPA induced p38 MAPK and IKK phosphorylation, suggesting signal transduction by this pathway. These findings suggest that P-MAPA has potential as a therapeutic drug in the treatment of canine visceral leishmaniasis.

Improvement in clinical signs and cellular immunity of dogs with visceral leishmaniasis using the immunomodulator P-MAPA.

This study investigated the immunotherapeutic potential of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride immuno-modulator (P-MAPA) on canine visceral leishmaniasis. Twenty mongrel dogs presenting clinical symptoms compatible with leishmaniasis and diagnosis confirmed by the detection of anti-leishmania antibodies were studied. Ten dogs received 15 doses of the immunomodulator (2.0 mg/kg) intramuscularly, and 10 received saline as a placebo. Skin and peripheral blood samples were collected following administration of the immunomodulator. The groups were followed to observe for clinical signals of remission; parasite load in the skin biopsies using real-time PCR, the cytokines IL-2, IL-10 and IFN-γ in the supernatant of peripheral blood mononuclear cells stimulated in vitro with either total promastigote antigen or phytohemagglutinin measured by capture ELISA, and changes in CD4⁺ and CD8⁺ T cell subpopulations evaluated by flow cytometry. Comparison between the groups showed that treatment with the immunomodulator promoted improvement in clinical signs and a significant reduction in parasite load in the skin. In peripheral blood mononuclear cell cultures, supernatants showed a decrease in IL-10 levels and an increase in IL-2 and IFN-γ. An increase in CD8⁺ T cells was observed in peripheral blood. In addition, the in vitro leishmanicidal action of P-MAPA was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and no leishmanicidal activity was detected. These findings suggest that P-MAPA has potential as an immunotherapeutic drug in canine visceral leishmaniasis, since it assists in reestablishing partial immunocompetence of infected dogs.

Gyroxin and its biological activity: effects on CNS basement membranes and endothelium and protease-activated receptors.

Gyroxin is a glycoprotein isolated from rattlesnake venom, with known thrombin-like serine protease properties and behavioral action in the CNS. The mechanism of the latter has eluded experimenters for three decades. In this paper about the in vitro chick retina we demonstrate an excitotoxic CNS action of Gyroxin by observing retinal Intrinsic Optical Signals (IOS). These show sudden dynamic changes in the intact tissue due to gyroxin action. The very fast kinetics of this response precludes deep tissue penetration by the protein, a mechanism of tissue response described here for the first time. At nanomolar concentrations, Gyroxin alters profoundly the optical profiles of retinal spreading depression waves (RSDs), suggesting modulation of ionic transport and metabolism. This effect is reversible in contrast with the acute cell lysis induced with gyroxin pulses at higher concentration. Because there may be more than one target of Gyroxine at the retinal inner limiting membrane, additional biochemical assays were performed to study a possible Na/K-ATPase blockade and PAR receptor activation. We conclude that the Gyroxin interaction with basement membranes of CNS and endothelium triggers conformational phase transitions at basement membranes, with multiple functional consequences.

Evaluation of TNF-α, IL-4, and IL-10 and parasite density in spleen and liver of L. (L.) chagasi naturally infected dogs.

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite can cause visceral leishmaniasis, which can also be transmitted to humans. Cytokines are key elements of the host immune response against Leishmania spp. To investigate whether tumor necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 are associated with pattern infection in dogs, these cytokines were quantified in the spleen and liver of dogs naturally infected with L. (L.) chagasi, with or without clinical manifestations, and their levels were correlated with the parasite load verified in these organs. A total of 40 adult dogs naturally infected with L. (L.) chagasi were assessed, together with 12 uninfected control dogs. Samples from spleen and liver were used to determine the cytokine levels by capture ELISA and for quantifying parasite load by real-time PCR. Statistical analysis was performed using the minimum Chi square method and group means were compared using the Tukey test. TNF-α, IL-4 and IL-10 levels in infected dogs were higher than in control groups; the liver was the main cytokine-producing organ during infection. The level of splenic TNF-α showed correlation with parasite load and may represent an important marker for infection process evolution, with the participation of IL-10. These results may contribute to a clearer understanding of the immune response in dogs infected with L. (L.) chagasi, which may lead to the development of prophylactic or preventive measures for these animals.

Microgravity dependence of excitable biological and physicochemical media.

Neuronal tissue and especially the central nervous system (CNS) is an excitable medium. Self-organisation, pattern formation, and propagating excitation waves as typical characteristics in excitable media consequently have been found in neuronal tissue. The properties of such phenomena in excitable media do critically depend on the parameters (i.e., electromagnetic fields, temperature, chemical drugs) of the system and on small external forces to which gravity belongs. The spreading depression, a propagating excitation depression wave of neuronal activity, is one of the best described of the those wave phenomena in the CNS. Especially in the retina as a true part of the CNS it can be easily observed with optical techniques due to the high intrinsic optical signal of this tissue. Another of such waves in neuronal tissue is the propagating action potential in nerve fibres. In this paper, data from our laboratories concerning the influence of gravity on the velocity of propagating waves in excitable media are summarized mainly in terms of the retinal spreading depression and propagating action potentials. Additionally, we have used waves in gels of the Belousov-Zhabotinsky reaction as the physicochemical model system of biological activity as the properties of these waves follow the same theories as the spreading depression and action potentials and they have some striking similarities in wave behavior. Thus propagating Belousov-Zhabotinsky waves are described by their gravity dependence.

Wave onset in central gray matter - its intrinsic optical signal and phase transitions in extracellular polymers.

The brain is an excitable media in which excitation waves propagate at several scales of time and space. "One-dimensional" action potentials (millisecond scale) along the axon membrane, and spreading depression waves (seconds to minutes) at the three dimensions of the gray matter neuropil (complex of interacting membranes) are examples of excitation waves. In the retina, excitation waves have a prominent intrinsic optical signal (IOS). This optical signal is created by light scatter and has different components at the red and blue end of the spectrum. We could observe the wave onset in the retina, and measure the optical changes at the critical transition from quiescence to propagating wave. The results demonstrated the presence of fluctuations preceding propagation and suggested a phase transition. We have interpreted these results based on an extrapolation from Tasaki's experiments with action potentials and volume phase transitions of polymers. Thus, the scatter of red light appeared to be a volume phase transition in the extracellular matrix that was caused by the interactions between the cellular membrane cell coat and the extracellular sugar and protein complexes. If this hypothesis were correct, then forcing extracellular current flow should create a similar signal in another tissue, provided that this tissue was also transparent to light and with a similarly narrow extracellular space. This control tissue exists and it is the crystalline lens. We performed the experiments and confirmed the optical changes. Phase transitions in the extracellular polymers could be an important part of the long-range correlations found during wave propagation in central nervous tissue.

Correlation of the electrical and intrinsic optical signals in the chicken spreading depression phenomenon.

This paper presents some results on the correlation between the electrophysiological and intrinsic optical signals (IOS) of spreading depression waves in chicken retinae. We first show that the peak of the time derivative of the electrophysiological wave occurs precisely when the optical signal reaches the electrode tip. Second, by comparing bath applications of propranolol and glycerol it can be shown that the slow potential shift is not directly correlated to the intrinsic optical signal. Propranolol depresses the amplitude of the electrical wave, although the intrinsic optical signal continues being visible. On the other hand, we observe total absence of the IOS under glycerol, while the electrical wave is always present. Correlations of this kind are relevant for a deeper understanding of the underlying mechanisms of the spreading depression phenomenon.

Adult male involvement in reproductive health: an ethnographic study in a community of São Paulo City, Brazil.

The purpose of this research was to explore the involvement in reproductive health of adult men in a low-income community in São Paulo City, Brazil. The ethnonursing research method was used and data were collected through interviews with seven key and eight general informants. Lack of common sex education was found and informants' knowledge depended on their own families' involvement and individual life experience with machismo. Informants' behaviors and attitudes toward women were constructed by their own male-centered values. Leininger's culture care modes were used to explain culturally meaningful nursing care actions and decisions.

Control of the excitability of neuronal tissue by weak external forces.

The spreading depression (SD) is a pronounced example of excitation-depression waves in excitable media, to which neuronal tissue according to its structure and functions belongs. SD waves can especially easily be observed in the vertebrate retina which is neuronal tissue and a true part of the central nervous system (CNS). According to the high intrinsic optical signal (IOS) concomitant with the retinal spreading depression (rSD), it can be monitored with standard video imaging techniques, thus the retina has been used in our studies as a suitable model system for neuronal tissue in general. In particular, the control of wave set-up and propagation in excitable media by weak external forces is of high interest. Accordingly, the interaction of rSD waves with DC and AC electromagnetic fields of low amplitude and frequency and with gravity has been investigated in this study. The dependence of rSD-wave propagation velocity on the given parameters as one important indication of excitability control has been investigated in detail. Our results with rSD waves are partially compared to another well known excitable medium, the Belousov-Zhabotinsky reaction, where some data about the effects of electrical fields and gravity have already been published.

[Evaluation of rapid urease test stored in refrigerator]. / Avaliação do teste rápido da urease conservado em geladeira.

UNLABELLED: The rapid urease test is an accurate and cheap method, which results are readily available, and broadly used for routine Helicobacter pylori infection diagnosis. AIM: The evaluation of rapid urease test stored in refrigerator at 4 degrees C (SRUT) compared to regular rapid urease test. PATIENTS AND METHOD: Endoscopic biopsies were obtained from gastric antrum in 104 consecutive patients. Diagnosis of Helicobacter pylori infection was accomplished by rapid urease test, histology and rapid urease test stored (kept in refrigerator by a period ranging from 1 to 8 days). RESULTS: Infection was considered present if both rapid urease test and histology were positive. Helicobacter pylori was present in 45/104 patients (42%). Rapid urease test stored had specificity comparable to rapid urease test (93%), with sensitivity of 88%. CONCLUSION: We concluded that rapid urease test can be stored in refrigerator for up to 1 week, without loss of clinical applicability, and that can make the test even easier to use for routine Helicobacter pylori tests in a busy endoscopy unit.

Biphasic effects of melatonin on the propagation of excitation waves in the chicken retina.

The retinal spreading depression (SD) is a propagating wave in an excitable medium, the neuronal tissue of the retina. Its velocity is about 3 mm/min and it is accompanied by a variety of changes in the tissue, including electrical and optical events. The pronounced intrinsic optical signal (IOS) of the retinal SD makes it an extremely versatile tool for the investigation of the action of drugs on neuronal tissue and more specific on propagating excitation waves in neuronal tissue. Furthermore, in the last decade increasing evidence has been collected, which shows that SD waves are the basic mechanism of the aura in classical migraine. We have investigated the influence of melatonin on the propagation of retinal SD waves as it has been postulated to have protective effects on neuronal tissue. The results demonstrate that melatonin indeed slows down the retinal SD, however, only in a defined concentration range. Additionally, it changes the IOS of the wave.

Mathematical model of the CA1 region of the rat hippocampus.

A mathematical transcription of the intrinsic circuit of the CA1 region of the rat dorsal hippocampus was made and the model parameters adjusted according to experimental data from intracellular recordings and single channel kinetics. This model was able to simulate well the profile of the field potentials recorded extracellularly and the well known phenomenon of the paired-pulse depression. The results suggest that the depression of the second pulse, often interpreted in the literature as resulting from GABA(A) inhibition, can also be due to 'shunting' effects on the CA1 pyramids' membrane. The rhythmic oscillations of the field potential (EEG) was obtained as an emergent property of the network dynamics. The frequency of the field oscillation followed the main synaptic input in the region (Schaffer collaterals).

Distribution of gap junctions in the chicken retina.

Coupling between cells of neuronal tissue can be due to electrical or chemical synapses. The molecular basis of an electrical synapse is the gap junction channel. Gap junctions have been found between neurones and glial cells, however, in some tissue their presence in the membranes of different cell types is still under discussion. In the retina of vertebrates, which is a true part of the CNS, the presence of gap junctions in the specialised glial cells of the retina, the Müller cells is not clear for chicken. Since these cells span the whole retina vertically, for some tasks, like spatial buffering of potassium, such gap junctions would not be required, in contrast to other parts of the CNS. The spatial buffering of potassium among others plays an important role in the propagation of excitation-depression waves in neuronal tissue, especially in the chicken retina. However, gap junctions could be involved in creating an electrical syncitium of glial cells, which might also contribute to excitation-depression wave propagation. In this paper we present an about complete screening of the presence of gap junctions in the chicken retina, including the proof that the Müller cells of this retina do not have gap junctions. This finding is discussed considering the highly specialised morphological structure of the Müller cells of the chicken retina, which have an extremely extended endfeet tree.

Pharmacological modulation of the refractory period of retinal spreading depression.

Spreading depression (SD) is a propagating wave of neuronal activity in the central nervous system and may play a role in triggering classical migraine. The retina serves as a model system for examining the phenomenon of SD and the influence of various drugs on it. After a SD wave passes a new wave can not be elicited in the absolute refractory period of the tissue (about 2 min), this is followed by a relative refractory phase of about 20 min before complete recovery. The aim of the present study was to describe the effects of Ba2+, a blocker of glial cell K+ channels, octanol, a gap junction blocker and diethylbarbiturate, a GABA(A) chloride channel-activating drug on the modulation of the refractory period of the retinal SD and to examine the possible mechanisms underlying this modulation. Two properties of SD, which are highly sensitive to any changes in the experimental conditions, are the propagation velocity of the wave and the accompanying slow negative potential shift. We measured the propagation velocity and the field potential amplitude in the chicken retina as a function of the recovery state of the tissue under control conditions and compared them with measurements in the presence of Ba2+, octanol or diethylbarbiturate. Under these conditions the manner of the recovery of the tissue changed significantly. Although after blocking the glial (Müller) cell K+ channels with Ba2+ (200 microM), the curve of recovery of the propagation velocity to its maximum value has the same shape as under control conditions, the propagation velocity is reduced in the whole recovery period and in the recovered retina to 84% of the control velocity. The importance of electrical coupling in the refractory phase and in the recovered tissue was examined by adding octanol (1 mM) to the perfusion solution. In this case the relative recovery phase was shortened and the field potential amplitude (110% of control) and propagation velocity (112% of control) are increased in the completely recovered retina. With the GABA(A)-chloride channel-activating drug diethylbarbiturate (800 microM) the propagation velocity (112% of control) and the amplitude of the field potential (111% of control) in the complete recovered retina are increased, but this seems to have no influence on the refractory state.

Exogenous application of gangliosides changes the state of excitability of retinal tissue as demonstrated by retinal spreading depression experiments.

Gangliosides are amphiphilic, sialic acid-containing glycosphingolipids which are found preferentially in complex composition in the cellular membranes of the nervous system of vertebrates, including the vertebrate retina as well as in other membranes. They are always exposed to the extracellular side of the membranes. By virtue of the negative charges they carry at their headgroup, they contribute to the surface charge of the membrane and may affect ion distribution, mainly that of protons and calcium ions, at the outer side of the membranes. Using retinal spreading depression (RSD) as a tool, we show in this study that the addition of exogenous gangliosides to the extracellular space can change the state of excitability of the retinal tissue. In RSD experiments it reduces the propagation velocity as well as the intrinsic optical signal of RSD waves. These effects are concentration dependent (IC50 about 20 microM) and increase with the increasing negative charge of the ganglioside headgroup. As a possible mechanistic basis of the changes found, the change of the calcium homeostasis of the extracellular space by the exogenously added gangliosides is discussed. Gangliosides have been reported to be useful in the treatment of some neuropathological syndromes, including migraine, although experimental verification has not been possible up to now. Taking into account that the retina is a true part of the CNS, our data may be interpreted as the requested verification.

Arabidopsis thaliana class IV chitinase is early induced during the interaction with Xanthomonas campestris.

Endochitinases are widely distributed among higher plants, including a number of important crop species. They are generally considered to be involved in plant defence against potential pathogens. We have cloned a class IV chitinase gene (AtchitIV) from Arabidopsis thaliana. Southern blot analysis allowed the detection of two cross-hybridising genes in the A. thaliana genome. AtchitIV transcripts are detected in seedpods, but not in roots, inflorescence stems, leaves and flowers of healthy plants. The transcripts accumulated very rapidly in leaves after inoculation with Xanthomonas campestris. Maximum mRNA accumulation was reached one hour after infection and decreased to very low levels 72 hours after induction. This result suggests an involvement of AtchitIV in the initial events of the hypersensitive reaction. Nevertheless, A. thaliana plants transformed with the gus gene under the control of a class IV chitinase bean promoter, showed GUS activity in seed embryos. These data, together with the constitutive expression of the endogenous gene in the seedpods, points to additional physiological roles for this protein.