Electrical Properties of Isolated Cardiomyocytes in a Rat Model of Thiamine Deficiency

In modern society, thiamine deficiency (TD) remains an important medical condition linked to altered cardiac function. There have been contradictory reports about the impact of TD on heart physiology, especially in the context of cardiac excitability. In order to address this particular question, we used a TD rat model and patch-clamp technique to investigate the electrical properties of isolated cardiomyocytes from epicardium and endocardium. Neither cell type showed substantial differences on the action potential waveform and transient outward potassium current. Based on our results we can conclude that TD does not induce major electrical remodeling in isolated cardiac myocytes in either endocardium or epicardium cells.

Cardiomyocyte dysfunction during the chronic phase of Chagas disease

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure.

Calcium handling by vascular myocytes in hypertension

Calcium ions (Ca2+) trigger the contraction of vascular myocytes and the level of free intracellular Ca2+ within the myocyte is precisely regulated by sequestration and extrusion mechanisms. Extensive evidence indicates that a defect in the regulation of intracellular Ca2+ plays a role in the augmented vascular reactivity characteristic of clinical and experimental hypertension. For example, arteries from spontaneously hypertensive rats (SHR) have an increased contractile sensitivity to extracellular Ca2+ and intracellular Ca2+ levels are elevated in aortic smooth muscle cells of SHR. We hypothesize that these changes are due to an increase in membrane Ca2+ channel density and possibly function in vascular myocytes from hypertensive animals. Several observations using various experimental approaches support this hypothesis: 1) the contractile activity in response to depolarizing stimuli is increased in arteries from hypertensive animals demonstrating increased voltage-dependent Ca2+ channel activity in hypertension; 2) Ca2+ channel agonists such as Bay K 8644 produce contractions in isolated arterial segments from hypertensive rats and minimal contraction in those from normotensive rats; 3) intracellular Ca2+ concentration is abnormally increased in vascular myocytes from hypertensive animals following treatment with Ca2+ channel agonists and depolarizing interventions, and 4) using the voltage-clamp technique, the inward Ca2+ current in arterial myocytes from hypertensive rats is nearly twice as large as that from myocytes of normotensive rats. We suggest that an alteration in Ca2+ channel function and/or an increase in Ca2+ channel density, resulting from increased channel synthesis or reduced turnover, underlies the increased vascular reactivity characteristic of hypertension

Monosodium glutamate (MSG)-obese rats develop glucose intolerance and insulin resistance to peripheral glucose uptake

Different levels of insulin sensitivity have been described in several animal models of obesity as well as in humans. Monosodium glutamate (MSG)-obese mice were considered not to be insulin resistant from data obtained in oral glucose tolerance tests. To reevaluate insulin resistance by the intravenous glucose tolerance test (IVGTT) and by the clamp technique, newborn male Wistar rats (N = 20) were injected 5 times, every other day, with 4 g/kg MSG (N = 10) or saline (control; N = 10) during the first 10 days of age. At 3 months, the IVGTT was performed by injecting glucose (0.75 g/kg) through the jugular vein into freely moving rats. During euglycemic clamping plasma insulin levels were increased by infusing 3 mU . kg-1 . min-1 of regular insulin until a steady-state plateau was achieved. The basal blood glucose concentration did not differ between the two experimental groups. After the glucose load, increased values of glycemia (P<0.001) in MSG-obese rats occurred at minute 4 and from minute 16 to minute 32. These results indicate impaired glucose tolerance. Basal plasma insulin levels were 39.9 ± 4 µU/ml in control and 66.4 ± 5.3 µU/ml in MSG-obese rats. The mean post-glucose area increase of insulin was 111% higher in MSG-obese than in control rats. When insulinemia was clamped at 102 or 133 µU/ml in control and MSG rats, respectively, the corresponding glucose infusion rate necessary to maintain euglycemia was 17.3 ± 0.8 mg . kg-1 . min-1 for control rats while 2.1 ± 0.3 mg . kg-1 . min-1 was sufficient for MSG-obese rats. The 2-h integrated area for total glucose metabolized, in mg . min . dl-1, was 13.7 ± 2.3 vs 3.3 ± 0.5 for control and MSG rats, respectively. These data demonstrate that MSG-obese rats develop insulin resistance to peripheral glucose uptake

Impaired beta-adrenergic response and decreased L-type calcium current of hypertrophied left ventricular myocytes in postinfarction heart failure

Infarct-induced heart failure is usually associated with cardiac hypertrophy and decreased ß-adrenergic responsiveness. However, conflicting results have been reported concerning the density of L-type calcium current (I Ca(L)), and the mechanisms underlying the decreased ß-adrenergic inotropic response. We determined I Ca(L) density, cytoplasmic calcium ([Ca2+]i) transients, and the effects of ß-adrenergic stimulation (isoproterenol) in a model of postinfarction heart failure in rats. Left ventricular myocytes were obtained by enzymatic digestion 8-10 weeks after infarction. Electrophysiological recordings were obtained using the patch-clamp technique. [Ca2+]i transients were investigated via fura-2 fluorescence. ß-Adrenergic receptor density was determined by [³H]-dihydroalprenolol binding to left ventricle homogenates. Postinfarction myocytes showed a significant 25% reduction in mean I Ca(L) density (5.7 ± 0.28 vs 7.6 ± 0.32 pA/pF) and a 19% reduction in mean peak [Ca2+]i transients (0.13 ± 0.007 vs 0.16 ± 0.009) compared to sham myocytes. The isoproterenol-stimulated increase in I Ca(L) was significantly smaller in postinfarction myocytes (Emax: 63.6 ± 4.3 vs 123.3 ± 0.9% in sham myocytes), but EC50 was not altered. The isoproterenol-stimulated peak amplitude of [Ca2+]i transients was also blunted in postinfarction myocytes. Adenylate cyclase activation through forskolin produced similar I Ca(L) increases in both groups. ß-Adrenergic receptor density was significantly reduced in homogenates from infarcted hearts (Bmax: 93.89 ± 20.22 vs 271.5 ± 31.43 fmol/mg protein in sham myocytes), while Kd values were similar. We conclude that postinfarction myocytes from large infarcts display reduced I Ca(L) density and peak [Ca2+]i transients. The response to ß-adrenergic stimulation was also reduced and was probably related to ß-adrenergic receptor down-regulation and not to changes in adenylate cyclase activity.

Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 ± 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 µM and a maximum increase of 51.2 ± 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.

Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

The nucleus tractus solitarius (NTS) plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs) in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 µM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM) amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV). The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration.

Immunoperoxidase technique in experimental chronic chagasic myocarditis

Chagas'disease has been described as the commonest form of chronic myocarditis. An immunologic pathogenesis has been discribed for this form of the disease. So far, no immunoperoxidase technique has been used for the detection of immunological deposits in chronic experimental Chagas'myocardiopathy. Forty-one Swiss mice, three months old were inoculated intraperitoneally with doses between 10 and 10(5) Tulahuen trypomastigotes. Mice were reinoculated one month after with doses between 10² and 10(5) and sacrificed at 6 (n=21) and 9 months (n=9) after the first inoculation. ECGs were recorded before sacrifice. Immunoperoxidase technique (peroxidase-antiperoxidase method), immunofluorescence (direct and indirect) as well as histological studies were performed in myocardiums and skeletal muscles of the surviving animals. The most sensitive methods for detecting chronic chagasic infection were the routine histologic studies (73%) and the ECGs 83% and 89% on 6 and 9 mo. post-infected mice, respectively. Myocardial involvement varied from interstitial mild focal lymphocyte infiltrates up to replacement of myocytes by loose connective tissue. Atrial myocardiums (21/23, 91%) were more affected than ventricles (9/23, 39%). Typical chagasic nests were rarely found. Skeletal muscle involvement (11/18 and 7/9) varied from mild to extensive lymphocyte and plasmacell infiltrates, and necrotic fibers. The involved antigen were shown in skeletal muscles by the immunoperoxidase technique as diffusely arranged granular intracytoplasmatic deposit for both IgC and total immunoglobulins. The coincidence between this technique and histologic muscle lesions was 11/18 (61(%) in 6 mo. and 6/8 (75%) at 9 mo. post-infection. In heart, delicate granular deposits of total immunoglobulins were seen diffusely arranged within the ventricular myocytes; coincidence between immunoperoxidase technique anl histologic involvement increased from 36 to 66% in animals sacrifeced 6 and 9 mo. post-infection. This strongly stressed the increase of immunologic phenomena with the chronification of infection. Concerning sensitivity, immunoperoxidase and direct immunofluorescence were highly sensitive in skeletal muscle (100%, p < 0.01). Conversely, direct immunofluorescence technique showed poor results in heart while immunoperoxidase increased its sensitivity from 21.4% (at 6 mo.) to 66.6% (at 9 mo.) post-infection (p < 0.001). Considering the necessity of obtaining an adequate vaccine in order to prevent this disease an experimental model like this, rendering immunological reactions as revealed by the immunoperoxidase technique, would be useful.

Therapeutical evaluation of different dose regimens of praziquantel in schistosomiasis mansoni, based on the quantitative oogram technique

A clinical trial involving 80 patients of both sexes, from ages 15 to 55, with chronic intestinal or hepatointestinal schistosomiasis mansoni, was carried out to evaluate the therapeutical efficacy of different dose regimens of praziquantel. The patients were randomly allocated into four groups with an equal number of cases and were then treated with one of the following dosages: 60 mg/kg for 1 day; 60 mg/kg daily for 2 days; 60 mg/kg daily for 3 days; and 30 mg/kg daily for 6 days. The assessment of parasitological cure was based on the quantitative oogram technique through rectal mucosa biopsies which were undertaken prior to, as well as, 1,2,4 and 6 months post-treatment. Concurrently, stool examinations according to the qualitative Hoffman, Pons & Janer (HPJ) and the quantitative Kato-Katz (K-K) methods were also performed. The best tolerability was observed with 30 mg/kg daily for 6 days whereas the highest incidence of side-effects (mainly dizziness and nausea) was found with 60 mg/kg daily for 3 days. No serious adverse drug reaction has occurred. The achieved cure rates were: 25% with 60 mg/kg for 1 day; 60% with 60 mg/kg daily for 2 days; 89.5% with 60 mg/kg daily for 3 days; and 90% with 30 mg/kg daily for 6 days. At the same time there has been a downfall of 64%, 73%, 87% and 84% respectively, in the median number of viable S. mansoni ova per gram of tissue. Thus, a very clear direct correlation between dose and effect could be seen. The corresponding cure rates according to stool examinations by HPJ were 39%, 80%, 100% and 95%; by K-K 89%, 100%, 100% and 100%. This discrepancy in results amongst the three parasitological methods is certainly due to their unequal accuracy. In fact, when the number of viable eggs per gram of tissue fell below 5,000 the difference in the percentage of false negative findings between HPJ (28%) and K-K (80%) became significative. When this number dropped to less than 2,000 the percentage of false negative results obtained with HPJ (49%) turned significant in relation to the oogram as well. In conclusion, it has been proven that praziquantel is a highly efficacious agent against S. mansoni infections. If administered at a total dose of 180 mg/kg divided into either 3 or 6 days, it yields a 90% cure rate. Possibly, one could reach 100% by increasing the total dose to 240 mg/kg. Furthermore, it was confirmed that the quantitative oogram technique is the most reliable parasitological method when evaluating the efficacy of new drugs in schistosomiasis mansoni.

Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA) using the larval excretory-secretory antigen of T. canis (TES), the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa). Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.

ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE

Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD) other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.

Evaluation of the formalin-Tween concentration technique for parasitic detection

The formalin-Tween sedimentation method was compared with the formalin-ether sedimentation for parasitic detection. Of a total 297 fecal specimens examined, 72.1% were positive. The formalin-tween technique was effective for ascertaining helminths, particularly Ascaris lumbricoides, Trichuris trichiura and hookworm eggs; however it has less capability for protozoa detection. This method is simple, inexpensive, less time consuming and highly sensitive when detecting the parasitic infection, particularly when focusing on helminth eggs.

Assessment of the rapid test based on an immunochromatography technique for detecting anti-Treponema pallidum antibodies

A rapid test based on an immunochromatography assay - Determine™ Syphilis TP (Abbott Lab.) for detecting specific antibodies to Treponema pallidum was evaluated against serum samples from patients with clinical, epidemiological and serological diagnosis of syphilis, patients with sexually transmitted disease other than syphilis, and individuals with negative serology for syphilis. The Determine™ test presented the sensitivity of 93.6%, specificity of 92.5%, and positive predictive value and negative predictive value of 95.2% and 93.7%, respectively. One serum sample from patient with recent latent syphilis showed a prozone reaction. Determine™ is a rapid assay, highly specific and easy to perform. This technique obviates the need of equipment and its diagnostic features demonstrate that it may be applicable as an alternative assay for syphilis screening under some emergency conditions or for patients living in remote localities.