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.

Studies of the small bowel surface by scanning electron microscopy in infants with persistent diarrhea

We describe the ultrastructural abnormalities of the small bowel surface in 16 infants with persistent diarrhea. The age range of the patients was 2 to 10 months, mean 4.8 months. All patients had diarrhea lasting 14 or more days. Bacterial overgrowth of the colonic microflora in the jejunal secretion, at concentrations above 10(4) colonies/ml, was present in 11 (68.7%) patients. The stool culture was positive for an enteropathogenic agent in 8 (50.0%) patients: for EPEC O111 in 2, EPEC O119 in 1, EAEC in 1, and Shigella flexneri in 1; mixed infections due to EPEC O111 and EAEC in 1 patient, EPEC O119 and EAEC in 1 and EPEC O55, EPEC O111, EAEC and Shigella sonnei in 1. Morphological abnormalities in the small bowel mucosa were observed in all 16 patients, varying in intensity from moderate 9 (56.3%) to severe 7 (43.7%). The scanning electron microscopic study of small bowel biopsies from these subjects showed several surface abnormalities. At low magnification (100X) most of the villi showed mild to moderate stunting, but on several occasions there was subtotal villus atrophy. At higher magnification (7,500X) photomicrographs showed derangement of the enterocytes; on several occasions the cell borders were not clearly defined and very often microvilli were decreased in number and height; in some areas there was a total disappearance of the microvilli. In half of the patients a mucus-fibrinoid pseudomembrane was seen partially coating the enterocytes, a finding that provides additional information on the pathophysiology of persistent diarrhea.

Behavioral and electroencephalographic analysis of seizures induced by intrahippocampal injection of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

In this study, the behavioral and electroencephalographic (EEG) analysis of seizures induced by the intrahippocampal injection in rats of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera, was determined. The first alterations occurred during microinjection of granulitoxin (8 µg) into the dorsal hippocampus and consisted of seizure activity that began in the hippocampus and spread rapidly to the occipital cortex. This activity lasted 20-30 s, and during this period the rats presented immobility. During the first 40-50 min after its administration, three to four other similar short EEG seizure periods occurred and the rats presented the following behavioral alterations: akinesia, facial automatisms, head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements. Within 40-50 min, the status epilepticus was established and lasted 8-12 h. These results are similar to those observed in the acute phase of the pilocarpine model of temporal lobe epilepsy and suggest that granulitoxin may be a useful tool not only to study the sodium channels, but also to develop a new experimental model of status epilepticus.

Central injection of captopril inhibits the blood pressure response to intracerebroventricular choline

In the present study, we investigated the involvement of the brain renin-angiotensin system in the effects of central cholinergic stimulation on blood pressure in conscious, freely moving normotensive rats. In the first step, we determined the effects of intracerebroventricular (icv) choline (50, 100 and 150 µg) on blood pressure. Choline increased blood pressure in a dose-dependent manner. In order to investigate the effects of brain renin-angiotensin system blockade on blood pressure increase induced by choline (150 µg, icv), an angiotensin-converting enzyme inhibitor, captopril (25 and 50 µg, icv), was administered 3 min before choline. Twenty-five µg captopril did not block the pressor effect of choline, while 50 µg captopril blocked it significantly. Our results suggest that the central renin-angiotensin system may participate in the increase in blood pressure induced by icv choline in normotensive rats.

Ethidium bromide-induced demyelination of the sciatic nerve of adult Wistar rats

Peripheral nerve ultrastructure was assessed after single or multiple local injections of the intercalating dye ethidium bromide. Thirty-four adult Wistar rats of both sexes were divided into five groups and maintained in a controlled environment with rat chow and water ad libitum throughout the experiment. The experimental animals were injected with 1 µl of 0.1% ethidium bromide in 0.9% saline into the central third of the left sciatic nerve 1 (group 1), 2 (group 2), 4 (group 3), 6 (group 4) or 8 (group 5) times. In groups 2 to 5 the injections were made at 28-day intervals. Control animals received the same amount of 0.9% saline. The animals were killed at different times after injection: group 1 at 7 days (2 rats) and 15 days (2 rats); for groups 2, 3, 4 and 5, all rats were killed 10 days after the last injection and the lesions were investigated by light and transmission electron microscopy. In the acute lesions, intoxicated Schwann cells showed a vacuolated cytoplasm and separation of the sheaths from the axon. Myelin sheaths underwent progressive vesiculation and subsequent segmental demyelination. Myelin debris were withdrawn by macrophages and remyelination by Schwann cells was prominent. With the increase in the number of injections collagen fibers also increased in number and progressively enveloped smaller numbers of remyelinated axons composing new fascicles. Wallerian degeneration of fibers apparently not affected by ethidium bromide was more intense in the nerves from groups 4 and 5. The peripheral nerve repairs itself after demyelinating challenges with a profusion of collagen fibers and new fasciculations. This experimental model is valid to mimic recurrent demyelinating neuropathies.

Effect of perimuscular injection of Bothrops jararacussu venom on plasma creatine kinase levels in mice: influence of dose and volume

The effect of dose and volume of a perimuscular injection of Bothrops jararacussu venom on myonecrosis of skeletal muscle was studied in mice. An increase of the venom dose (0.25 to 2.0 µg/g) at a given volume (50 µl) resulted in an increase in plasma creatine kinase (CK) levels 2 h after injection. Plasma CK activity increased from the basal level of 129.27 ± 11.83 (N = 20) to 2392.80 ± 709.43 IU/l (N = 4) for the 1.0 µg/g dose. Histological analysis of extensor digitorum longus muscle 4 h after injection showed lesion of peripheral muscle fibers, disorganization of the bundles or the complete degeneration of muscle fibers. These lesions were more extensive when higher doses were injected. Furthermore, an increase in volume (12.5 to 100 µl) by dilution of a given dose (0.5 µg/g) also increased plasma CK levels from 482.31 ± 122.79 to 919.07 ± 133.33 IU/l (N = 4), respectively. These results indicate that care should be taken to standardize volumes and sites of venom injections.

Role of nitric oxide of the median preoptic nucleus (MnPO) in the alterations of salivary flow, arterial pressure and heart rate induced by injection of pilocarpine into the MnPO and intraperitoneally

We investigated the effect of L-NAME, a nitric oxide (NO) inhibitor and sodium nitroprusside (SNP), an NO-donating agent, on pilocarpine-induced alterations in salivary flow, mean arterial blood pressure (MAP) and heart rate (HR) in rats. Male Holtzman rats (250-300 g) were implanted with a stainless steel cannula directly into the median preoptic nucleus (MnPO). Pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO induced an increase in salivary secretion (P<0.01). Pilocarpine (1, 2, 4, 8, 16 mg/kg) ip also increased salivary secretion (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO or ip (1, 2, 4, 8, 16 mg/kg) increased salivary secretion (P<0.01). SNP (30 µg) injected into the MnPO or ip prior to pilocarpine attenuated salivary secretion (P<0.01). Pilocarpine (40 µg) injection into the MnPO increased MAP and decreased HR (P<0.01). Pilocarpine (4 mg/kg body weight) ip produced a decrease in MAP and an increase in HR (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine potentiated the increase in MAP and reduced HR (P<0.01). SNP (30 µg) injected into the MnPO prior to pilocarpine attenuated (100%) the effect of pilocarpine on MAP, with no effect on HR. Administration of L-NAME (40 µg) into the MnPO potentiated the effect of pilocarpine injected ip. SNP (30 µg) injected into the MnPO attenuated the effect of ip pilocarpine on MAP and HR. The present study suggests that in the rat MnPO 1) NO is important for the effects of pilocarpine on salivary flow, and 2) pilocarpine interferes with blood pressure and HR (side effects of pilocarpine), that is attenuated by NO.

Modeling Front End Electronics for a Gas Electron Multiplier Detector

The Large Hadron Collider (LHC) in The European Organization for Nuclear Research (CERN) will have a Long Shutdown sometime during 2017 or 2018. During this time there will be maintenance and a possibility to install new detectors. After the shutdown the LHC will have a higher luminosity. A promising new type of detector for this high luminosity phase is a Triple-GEM detector. During the shutdown these detectors will be installed at the Compact Muon Solenoid (CMS) experiment. The Triple-GEM detectors are now being developed at CERN and alongside also a readout ASIC chip for the detector. In this thesis a simulation model was developed for the ASICs analog front end. The model will help to carry out more extensive simulations and also simulate the whole chip before the whole design is finished. The proper functioning of the model was tested with simulations, which are also presented in the thesis.

Inverting ionospheric D-region electron density pro-file from riometer measurement

The aim of this work is to invert the ionospheric electron density profile from Riometer (Relative Ionospheric opacity meter) measurement. The newly Riometer instrument KAIRA (Kilpisjärvi Atmospheric Imaging Receiver Array) is used to measure the cosmic HF radio noise absorption that taking place in the D-region ionosphere between 50 to 90 km. In order to invert the electron density profile synthetic data is used to feed the unknown parameter Neq using spline height method, which works by taking electron density profile at different altitude. Moreover, smoothing prior method also used to sample from the posterior distribution by truncating the prior covariance matrix. The smoothing profile approach makes the problem easier to find the posterior using MCMC (Markov Chain Monte Carlo) method.

Consequences of cerebroventricular insulin injection on renal sodium handling in rats: effect of inhibition of central nitric oxide synthase

In the present study, we investigated the effects of acute intracerebroventricular (icv) insulin administration on central mechanisms regulating urinary sodium excretion in simultaneously centrally NG-nitro-L-arginine methylester (L-NAME)-injected unanesthetized rats. Male Wistar-Hannover rats were randomly assigned to one of five groups: a) icv 0.15 M NaCl-injected rats (control, N = 10), b) icv dose-response (1.26, 12.6 and 126 ng/3 µL) insulin-injected rats (N = 10), c) rats icv injected with 60 µg L-NAME in combination with NaCl (N = 10) or d) with insulin (N = 10), and e) subcutaneously insulin-injected rats (N = 5). Centrally administered insulin produced an increase in urinary output of sodium (NaCl: 855.6 ± 85.1 Δ%/min; 126 ng insulin: 2055 ± 310.6 Δ%/min; P = 0.005) and potassium (NaCl: 460.4 ± 100 Δ%/min; 126 ng insulin: 669.2 ± 60.8 Δ%/min; P = 0.025). The urinary sodium excretion response to icv 126 ng insulin microinjection was significantly attenuated by combined administration of L-NAME (126 ng insulin: 1935 ± 258.3 Δ%/min; L-NAME + 126 ng insulin: 582.3 ± 69.6 Δ%/min; P = 0.01). Insulin-induced natriuresis occurred by increasing post-proximal sodium excretion, despite an unchanged glomerular filtration rate. Although the rationale for decreased urinary sodium excretion induced by combined icv L-NAME and insulin administration is unknown, it is tempting to suggest that perhaps one of the efferent signals triggered by insulin in the CNS may be nitrergic in nature.

GM1 improves neurofascin155 association with lipid rafts and prevents rat brain myelin injury after hypoxia-ischemia

White matter injury characterized by damage to myelin is an important process in hypoxic-ischemic brain damage (HIBD). Because the oligodendrocyte-specific isoform of neurofascin, neurofascin 155 (NF155), and its association with lipid rafts are essential for the establishment and stabilization of the paranodal junction, which is required for tight interaction between myelin and axons, we analyzed the effect of monosialotetrahexosyl ganglioside (GM1) on NF155 expression and its association with lipid rafts after HIBD in Sprague-Dawley rats, weighing 12-15 g, on day 7 post-partum (P7; N = 20 per group). HIBD was induced on P7 and the rats were divided into two groups: one group received an intraperitoneal injection of 50 mg/kg GM1 three times and the other group an injection of saline. There was also a group of 20 sham-operated rats. After sacrifice, the brains of the rats were removed on P30 and studied by immunochemistry, SDS-PAGE, Western blot analysis, and electron microscopy. Staining showed that the saline group had definite rarefaction and fragmentation of brain myelin sheaths, whereas the GM1 group had no obvious structural changes. The GM1 group had 1.9-2.9-fold more GM1 in lipid rafts than the saline group (fraction 3-6; all P < 0.05) and 0.5-2.4-fold higher expression of NF155 in lipid rafts (fraction 3-5; all P < 0.05). Injection of GM1 increased the content of GM1 in lipid rafts as well as NF155 expression and its lipid raft association in HIBD rat brains. GM1 may repair the structure of lipid rafts, promote the association of NF155 (or other important proteins) with lipid rafts, stabilize the structure of paranodes, and eventually prevent myelin sheath damage, suggesting a novel mechanism for its neuroprotective properties.

Cardiovascular effects of the intracerebroventricular injection of adrenomedullin: roles of the peripheral vasopressin and central cholinergic systems

Our objective was to investigate in conscious Sprague-Dawley (6-8 weeks, 250-300 g) female rats (N = 7 in each group) the effects of intracerebroventricularly (icv) injected adrenomedullin (ADM) on blood pressure and heart rate (HR), and to determine if ADM and calcitonin gene-related peptide (CGRP) receptors, peripheral V1 receptors or the central cholinergic system play roles in these cardiovascular effects. Blood pressure and HR were observed before and for 30 min following drug injections. The following results were obtained: 1) icv ADM (750 ng/10 µL) caused an increase in both blood pressure and HR (DMAP = 11.8 ± 2.3 mmHg and ΔHR = 39.7 ± 4.8 bpm). 2) Pretreatment with a CGRP receptor antagonist (CGRP8-37) and ADM receptor antagonist (ADM22-52) blocked the effect of central ADM on blood pressure and HR. 3) The nicotinic receptor antagonist mecamylamine (25 µg/10 µL, icv) and the muscarinic receptor antagonist atropine (5 µg/10 µL, icv) prevented the stimulating effect of ADM on blood pressure. The effect of ADM on HR was blocked only by atropine (5 µg/10 µL, icv). 4) The V1 receptor antagonist [β-mercapto-β-β-cyclopentamethylenepropionyl¹, O-me-Tyr²,Arg8]-vasopressin (V2255; 10 µg/kg), that was applied intravenously, prevented the effect of ADM on blood pressure and HR. This is the first study reporting the role of specific ADM and CGRP receptors, especially the role of nicotinic and muscarinic central cholinergic receptors and the role of peripheral V1 receptors in the increasing effects of icv ADM on blood pressure and HR.

Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.

Accuracy of dose planning for prostate radiotherapy in the presence of metallic implants evaluated by electron spin resonance dosimetry

Radiotherapy is one of the main approaches to cure prostate cancer, and its success depends on the accuracy of dose planning. A complicating factor is the presence of a metallic prosthesis in the femur and pelvis, which is becoming more common in elderly populations. The goal of this work was to perform dose measurements to check the accuracy of radiotherapy treatment planning under these complicated conditions. To accomplish this, a scale phantom of an adult pelvic region was used with alanine dosimeters inserted in the prostate region. This phantom was irradiated according to the planned treatment under the following three conditions: with two metallic prostheses in the region of the femur head, with only one prosthesis, and without any prostheses. The combined relative standard uncertainty of dose measurement by electron spin resonance (ESR)/alanine was 5.05%, whereas the combined relative standard uncertainty of the applied dose was 3.35%, resulting in a combined relative standard uncertainty of the whole process of 6.06%. The ESR dosimetry indicated that there was no difference (P>0.05, ANOVA) in dosage between the planned dose and treatments. The results are in the range of the planned dose, within the combined relative uncertainty, demonstrating that the treatment-planning system compensates for the effects caused by the presence of femur and hip metal prostheses.

A dose-response curve for biodosimetry from a 6 MV electron linear accelerator

Biological dosimetry (biodosimetry) is based on the investigation of radiation-induced biological effects (biomarkers), mainly dicentric chromosomes, in order to correlate them with radiation dose. To interpret the dicentric score in terms of absorbed dose, a calibration curve is needed. Each curve should be constructed with respect to basic physical parameters, such as the type of ionizing radiation characterized by low or high linear energy transfer (LET) and dose rate. This study was designed to obtain dose calibration curves by scoring of dicentric chromosomes in peripheral blood lymphocytes irradiated in vitro with a 6 MV electron linear accelerator (Mevatron M, Siemens, USA). Two software programs, CABAS (Chromosomal Aberration Calculation Software) and Dose Estimate, were used to generate the curve. The two software programs are discussed; the results obtained were compared with each other and with other published low LET radiation curves. Both software programs resulted in identical linear and quadratic terms for the curve presented here, which was in good agreement with published curves for similar radiation quality and dose rates.