Evaluation of encapsulated anethole and carvone in lambs artificially- and naturally-infected with Haemonchus contortus.

Molecules from natural sources, such as essential oils, have shown activity against parasites in vitro, but have not yet been explored extensively in vivo. Anethole and carvone (10% each), encapsulated with 80% of a solid matrix, referred to as EO (encapsulated oils), were tested in vivo in 2 experiments. In Experiment 1: Lambs were artificially infected with multidrug resistant Haemonchus contortus, or left uninfected, and treated (or not) with 50 mg/kg bw (body weight) of EO in a controlled environment. Thirty-two male lambs were kept in individual cages for a period of 45 days, after which animals were evaluated for parasitological, hematological, toxicological, and nutritional parameters. After 45 days of treatment, EO at 50 mg/kg bw provided a significant (P ≤ 0.05) reduction in fecal egg count (FEC). Although FEC was reduced, animals from both treatments had similar counts of total adult worms. The low FEC was caused probably by a significant reduction (P ≤ 0.05) in both male worm size and female fecundity. Dry matter intake of uninfected controls was significantly (P ≤ 0.05) reduced, although no toxicity was observed in treated animals. Thus, in Experiment 2, conducted for five months we used an EO dose of 20 mg/kg bw. Thirty-four weaned lambs, free of parasites, were divided in two groups and kept in collective pens. One group received EO at 20 mg/kg bw mixed with concentrate for 5 months and the other was kept as a control group (CTL). Parasitological and hematological parameters as well as body weight were evaluated. In the first 2.5 months, CTL and EO groups were confined, and both presented similar clinical parameters. Then, animals were allotted to graze on contaminated pastures to acquire natural infection for the next 2.5 months. The infection was patent after 25 days and both groups had similar decreases in weight gain, increases in FEC, and decreases in blood parameters. Coprocultures from CTL and EO groups established that parasite population was 90% Haemonchus sp. We concluded that the technology of encapsulation is safe and practical to deliver to lambs at the farm level and anethole and carvone at 50 mg/kg bw caused a significant decrease in FEC and, consequently, in pasture contamination by free living stages of H. contortus. However, EO at 20 mg/kg bw was not effective to prevent or treat sheep naturally-infected with gastrointestinal nematodes.

Inclusion complex and nanoclusters of cyclodextrin to increase the solubility and efficacy of albendazole.

Albendazole (ABZ), a benzimidazole widely used to control gastrointestinal parasites, is poorly soluble in water, resulting in variable and incomplete bioavailability. This has favored the appearance ABZ-resistant nematodes and, consequently, an increase in its clinical ineffectiveness. Among the pharmaceutical techniques developed to increase drug efficacy, cyclodextrins (CDs) and other polymers have been extensively used with water-insoluble pharmaceutical drugs to increase their solubility and availability. Our objective was to prepare ABZ formulations, including ß-cyclodextrin (ßCD) or hydroxypropyl-ß-cyclodextrin (HPßCD), associated or not to the water-soluble polymer polyvinylpyrrolidone (PVP). These formulations had their solubility and anthelmintic effect both evaluated in vitro. Also, their anthelmintic efficacy was evaluated in lambs naturally infected with gastrointestinal nematodes (GIN) through the fecal egg count (FEC) reduction test. In vitro, the complex ABZ/HPßCD had higher solubility than ABZ/ßCD. The addition of PVP to the complexes increased solubility and dissolution rates more effectively for ABZ/HPßCD than for ABZ/ßCD. In vivo, 48 lambs naturally infected with GIN were divided into six experimental groups: control, ABZ, ABZ/ßCD, ABZ/ßCD-PVP, ABZ/HPßCD, and ABZ/HPßCD-PVP. Each treated animal received 10 mg/kg of body weight (based on the ABZ dose) for three consecutive days. After 10 days of the last administered dose, treatment efficacy was calculated. The efficacy values were as follows: ABZ (70.33%), ABZ/ßCD (85.33%), ABZ/ßCD-PVP (82.86%), ABZ/HPßCD (78.37%), and ABZ/HPßCD-PVP (43.79%). In vitro, ABZ/HPßCD and ABZ/HPßCD-PVP had high solubility and dissolution rates. In vivo, although the efficacies of ABZ/ßCD, ABZ/ßCD-PVP, and ABZ/HPßCD increased slightly when compared to pure ABZ, this increase was not significant (P > 0.05).

Doctors' awareness concerning primary immunodeficiencies in Brazil.

BACKGROUND: PIDs are a heterogeneous group of genetic illnesses, and delay in their diagnosis is thought to be caused by a lack of awareness among physicians concerning PIDs. The latter is what we aimed to evaluate in Brazil. METHODS: Physicians working at general hospitals all over the country were asked to complete a 14-item questionnaire. One of the questions described 25 clinical situations that could be associated with PIDs and a score was created based on percentages of appropriate answers. RESULTS: A total of 4026 physicians participated in the study: 1628 paediatricians (40.4%), 1436 clinicians (35.7%), and 962 surgeons (23.9%). About 67% of the physicians had learned about PIDs in medical school or residency training, 84.6% evaluated patients who frequently took antibiotics, but only 40.3% of them participated in the immunological evaluation of these patients. Seventy-seven percent of the participating physicians were not familiar with the warning signs for PIDs. The mean score of correct answers for the 25 clinical situations was 48.08% (±16.06). Only 18.3% of the paediatricians, 7.4% of the clinicians, and 5.8% of the surgeons answered at least 2/3 of these situations appropriately. CONCLUSIONS: There is a lack of medical awareness concerning PIDs, even among paediatricians, who have been targeted with PID educational programmes in recent years in Brazil. An increase in awareness with regard to these disorders within the medical community is an important step towards improving recognition and treatment of PIDs.

Combined effects of Limonene and Ivermectin on P-glycoprotein-9 gene expression of lambs Infected with Haemonchus contortus.

Although ivermectin (IVM) has a wide spectrum and long half-life, its frequent use as an anthelmintic for the last 42 years led to its worldwide tolerance by Haemonchus contortus. We evaluated the combination of limonene (LIM), a P-glycoprotein (Pgp) modulator, with IVM in lambs infected with a multidrug-resistant H. contortus. Twenty-four male Dorper lambs were artificially infected with two doses (seven days apart) of 8000 infective larvae of a multidrug-resistant isolate of H. contortus. The infection was patent 25 days later. Fifteen days before treatment with IVM (DAY -15), animals were divided into 4 groups: Infected-untreated control (CTL), IVM, LIM, and LIM+IVM. From DAY -15 to DAY + 14, groups LIM and LIM+IVM received 200 mg/kg of body weight/day of LIM via oral. On DAY 0, a single dose of IVM at 200 µg/kg of body weight was administered orally to groups IVM and LIM+IVM. On DAY + 7 and DAY + 14, fecal egg counts (FEC) were performed and on DAY + 14 animals were euthanized for total worm count (TWC), worm length, fecundity of females, and Pgp-9 gene expression. On DAY + 7, group LIM+IVM had 96.29% efficacy based on Fecal Egg Count Reduction TEST (FECRT) and a highly significant reduction in FEC (P = 0.0005) when compared to CTL. On DAY + 14, the efficacy of LIM+IVM was 82.87% on FECRT, although no differences were found among groups for FEC, TWC, worm length, or Pgp-9 gene expression. Female worms from the CTL group had higher egg counts in their uterus when compared to LIM. No differences were found for hematological or biochemical parameters, body weight, or weight gain among groups. Thus, LIM given daily at 200 mg/kg was safe for animals and, when combined with IVM, decreased egg shedding and could reduce pasture contamination, although it was unable to kill multidrug-resistant H. contortus.

Rumen fermentation and production effects of Origanum vulgare L. leaves in lactating dairy cows.

A lactating cow trial was conducted to study the effects of dietary addition of oregano leaf material (Origanum vulgare L.; OV; 0, control vs. 500 g/d) on ruminal fermentation, methane production, total tract digestibility, manure gas emissions, N metabolism, organoleptic characteristics of milk, and dairy cow performance. Eight primiparous and multiparous Holstein cows (6 of which were ruminally cannulated) were used in a crossover design trial with two 21-d periods. Cows were fed once daily. The OV material was top-dressed and mixed with a portion of the total mixed ration. Cows averaged 80 ± 12.5 d in milk at the beginning of the trial. Rumen pH, concentration of total and individual volatile fatty acids, microbial protein outflow, and microbial profiles were not affected by treatment. Ruminal ammonia-N concentration was increased by OV compared with the control (5.3 vs. 4.3mM). Rumen methane production, which was measured only within 8h after feeding, was decreased by OV. Intake of dry matter (average of 26.6 ± 0.83 kg/d) and apparent total tract digestibly of nutrients did not differ between treatments. Average milk yield, milk protein, lactose, and milk urea nitrogen concentrations were unaffected by treatment. Milk fat content was increased and 3.5% fat-corrected milk yield tended to be increased by OV, compared with the control (3.29 vs. 3.12% and 42.4 vs. 41.0 kg/d, respectively). Fat-corrected (3.5%) milk feed efficiency and milk net energy for lactation (NE(L)) efficiency (milk NE(L) ÷ NE(L) intake) were increased by OV compared with the control (1.64 vs. 1.54 kg/kg and 68.0 vs. 64.4%, respectively). Milk sensory parameters were not affected by treatment. Urinary and fecal N losses, and manure ammonia and methane emissions were unaffected by treatment. Under the current experimental conditions, supplementation of dairy cow diets with 500 g/d of OV increased milk fat concentration, feed and milk NE(L) efficiencies, and tended to increase 3.5% fat-corrected milk yield. The sizable decrease in rumen methane production with the OV supplementation occurred within 8h after feeding and has to be interpreted with caution due to the large within- and between-animal variability in methane emission estimates. The OV was introduced into the rumen as a pulse dose at the time of feeding, thus most likely having larger effect on methane production during the period when methane data were collected. It is unlikely that methane production will be affected to the same extent throughout the entire feeding cycle.

Synergistic interaction of ten essential oils against Haemonchus contortus in vitro.

Anthelmintic resistance in sheep gastrointestinal nematodes is a worldwide problem. Multi-drug resistant haemonchosis is the most serious impediment for small ruminant systems, and there are no new drug candidates currently under development. Molecules from natural sources have demonstrated anthelmintic activity against parasites. In this work, the monoterpenoids carvacrol, carvone, cineole, linalool, limonene, and thymol and the phenylpropanoids cinnamaldehyde, anethole, vanillin, and eugenol were assessed individually or in mixtures of ten binary, three ternary, and three quaternary combinations using the in vitro egg hatch assay with eggs of a multi-drug resistant strain of Haemonchus contortus. The main objective of this study was to identify the most effective interaction among essential oils with the greatest individual anthelmintic efficacy and to determine the most powerful combinations. The essential oils were ranked by their 50% lethal concentration (LC50) as follows (mg/mL): cinamaldehyde (0.018), anethole (0.070), carvone (0.085), carvacrol (0.11), thymol (0.13), linalool (0.29), vanillin (0.57), eugenol (0.57), cineole (4.74), and limonene (207.5). Quantification of synergism, additive effect, and antagonism were calculated for binary, ternary, and quaternary combinations. The best anthelmintic effect resulting from synergistic activity among 16 different combinations was for cinnamaldehyde:carvacrol (CL50 0.012mg/mL) and anethole:carvone (CL50 0.013mg/mL). These results indicate that these binary combinations would be promising to be tested in sheep infected with H. contortus.

Terminalia catappa: Chemical composition, in vitro and in vivo effects on Haemonchus contortus.

Haemonchus contortus is the most important nematode in small ruminant systems, and has developed tolerance to all commercial anthelmintics in several countries. In vitro (egg hatch assay) and in vivo tests were performed with a multidrug strain of Haemonchus contortus using Terminalia catappa leaf, fruit pulp, and seed extracts (in vitro), or pulp and seed powder in lambs experimentally infected with H. contortus. Crude extracts from leaves, fruit pulp and seeds obtained with 70% acetone were lyophilized until used. In vitro, the extracts had LC50=2.48µg/mL (seeds), LC50=4.62µg/mL (pulp), and LC50=20µg/mL (leaves). In vitro, seed and pulp extracts had LC50 similar to Thiabendazole (LC50=1.31µg/mL). Condensed tannins were more concentrated in pulp extract (183.92g of leucocyanidin/kg dry matter) than in either leaf (4.6g) or seed (35.13g) extracts. Phytochemical tests established that all extracts contained alkaloids, flavonoids, saponins, phenols, and terpenoids. Based on these results, in vivo tests were performed to evaluate the anthelmintic activity of T. catappa whole fruit (pulp+seed) powder. Male Santa Ines lambs were artificially infected with multidrug-resistant H. contortus and divided, according to similar fecal egg count (FEC) and weight, into two groups: Control (infected/untreated) and treated (infected/treated with whole fruit powder). Whole fruit powder was mixed with concentrate and provided at 2g/kg of body weight (BW) for five days. After treatment, parasitological analysis (FEC and egg hatch assay), renal profile (urea and creatinine), liver profile (aspartate aminotransferase) and BW were determined. In vitro (based on LC50), seed/pulp extracts had ovicidal effect similar to Thiabendazole but whole fruit powder had no anthelmintic effect on adult nematodes in the abomasum. We discuss the plausible causes of the lack of in vivo activity.

Recommendations for Vaccination in Adult Patients with Systemic Inflammatory Rheumatic Diseases from the Portuguese Society of Rheumatology.

BACKGROUND: Serious infections are a major cause of morbidity and mortality in systemic inflammatory rheumatic disease (SIRD) patients. Although vaccination may prevent numerous infections, vaccination uptake rates are low in this group of patients. OBJECTIVES: To develop evidence-based recommendations for vaccination in SIRD patients. METHODS: We searched MEDLINE (until 31 October 2014) and EMBASE (until 14 December 2014) databases, as well as the ACR and EULAR congress abstracts (2011-2014). Patients with any systemic inflammatory rheumatic disease were included and all vaccines were considered. Any safety and efficacy outcomes were admitted. Search results were submitted to title and abstract selection, followed by detailed review of suitable studies. Data were subsequently pooled according to the type of vaccine and the SIRD considered. Results were presented and discussed by a multidisciplinary panel and systematic literature review (SLR)-derived recommendations were voted according to the Delphi method. The level of agreement among rheumatologists was assessed using an online survey. RESULTS: Eight general and seven vaccine-specific recommendations were formulated. Briefly, immunization status should routinely be assessed in all SIRD patients. The National Vaccination Program should be followed and some additional vaccines are recommended. To maximize the efficacy of vaccination, vaccines should preferably be administered 4 weeks before starting immunosuppression or, if possible when disease activity is controlled. Non-live vaccines are safe in SIRD, including immunosuppressed patients. The safety of live attenuated vaccines in immunosuppressed patients deserves further ascertainment, but might be considered in particular situations. DISCUSSION: The present recommendations combine scientific evidence with the multidisciplinary expertise of our taskforce panel and attained desirable agreement among Portuguese rheumatologists. Vaccination recommendations need to be updated on a regular basis, as more scientific data regarding vaccination efficacy and safety, emergent infectious threats, new vaccines as well as new immunomodulatory therapies become available.

Filamentous cross-bridges link intermediate filaments to the nuclear pore complexes.

Intermediate filament-nuclear matrix interactions were studied in cultured rat ventral prostate cells and isolated rat uterine epithelial cells. Cytokeratin filaments were identified by immunoelectron microscopy. In addition to conventional thin section of Triton X-100 treated cells, subcellular residues composed of intermediate filaments and nuclear matrix were critical-point dried and platinum-carbon replicated. The results demonstrate the presence of a previously unrecognized type of filamentous cross-bridges that link intermediate filaments to the nuclear pore complexes.

Immunocytochemical study of tissue distribution and hormonal control of chondroitin-, dermatan- and keratan sulfates from rodent uterus.

The distribution patterns of rat and mouse uterine glycosaminoglycans (GAGs), as well as their modulation by estradiol (E2) and/or progesterone (P), were investigated using monoclonal antibodies (MABs) directed against chondroitin- (CS)/dermatan sulfates (DS), keratan sulfate (KS) and a trophoblast GAG. The localization of GAGs in relation to collagens (I, IV and VI) and fibronectin was also analyzed. We found that uterine GAGs are differentially distributed in the endometrium and myometrium, in a pattern that is species-related. CS-containing proteoglycans (PGs) occur between collagen bundles and fibroblasts, at the periphery of the latter, and in basement membrane zones (BMZs), in a pattern resembling that of collagen VI. BMZs contain preferentially CS-PGs bearing 4-sulfated disaccharides adjacent to the core protein. DS-PGs are mostly associated with collagen bundles. E2 and/or P elicit distinct modifications on the above described pattern, which are also species-related. The simultaneous administration of E2 and P changes the prevalent sulfation of the disaccharides adjacent to the core protein of stromal CS-PGs. In the mouse, an unsulfated intracellular epitope appears following E2 (or E2P) administration, mostly in epithelial cells. In the rat, KS and the trophoblast GAG are E2-dependent and down-regulated by P. The functional significance of the hormone-induced GAG changes, namely the possible role of the E2-dependent KS in implantation, are discussed.

Immunocytochemical localization of corticosteroid-binding globulin (CBG) in guinea pig hepatocytes.

The nature of corticosteroid-binding globulin (CBG)-producing cells was studied in guinea pig liver by immunoperoxidase cytochemistry in light and electron microscopies. In light microscopy, CBG was detected in the cytoplasm of hepatocytes from pregnant and nonpregnant guinea pigs. The CBG-stained hepatocytes were more numerous in the peripheral regions of the lobules and around the portal space. In electron microscopy, CBG was associated with the hepatocyte rough endoplasmic reticulum cisternae. Patches of dense deposits were occasionally seen in the perinuclear cisternae and in cisternae identified as part of the Golgi apparatus. No deposits were seen in the smooth endoplasmic reticulum or any other cell organelles. Kupffer and bile duct cells proved to be negative to CBG. These findings demonstrate that the rough endoplasmic reticulum is the site of CBG synthesis in the guinea pig hepatocyte and confirm the hepatic origin of CBG, previously shown by biochemical methods. The distribution of CBG was also studied by light microscopy in other tissues from pregnant guinea pigs. No CBG was detected inside cells from muscle, heart, lung, kidney, ovary, uterus, or placenta. CBG was only detected in vascularized zones (glomeruli in the kidney, perifollicular capillary network and corpora lutea of the ovary, and connective tissue separating the myometrium layers of the uterus).

Reevaluation of fibronectin-collagen interactions in tissues: an immunocytochemical and immunochemical study.

We studied the distribution of fibronectin (FN) in rat uterus, rat tail tendon, and rooster comb, using LM and EM immunocytochemistry. Special attention was paid to the interaction of FN with collagen (COL). Various labeling protocols and dot-blot experiments were performed to confirm the results. Under conditions of labeling specificity, FN distribution over native COL fibrils was usually sparse, especially when these were organized into thick bundles. No labeling was observed over section surfaces of COL fibrils when postembedding methods were used, which indicates that no FN is present within these fibrils. Under conditions in which exogenous FN could react with tissues, e.g., when preincubation with normal serum for background blocking was performed, artifactual staining appeared over COL. Such a reaction also occurred when anti-FN antiserum completely blocked by liquid-phase adsorption was used. Therefore, the FN present in soluble FN-anti-FN immune complexes must have still been able to react with COL. The artifactual labeling was, in all cases, almost exclusively localized on the section surfaces of COL fibrils. These results suggest that FN has a very low affinity for the surface of native COL fibrils.

Immunoquantitative analysis of artemisinin from Artemisia annua using polyclonal antibodies.

Artemisinin was derivatized to dihydroartemisinin carboxymethylether in three steps, without disturbing the peroxide bridge, and then linked to either thyroglobulin (TGB) or bovine serum albumin (BSA). The artemisinin-TGB and -BSA conjugates were injected in female New Zealand rabbits but only the artemisinin-TGB conjugate generated polyclonal antibodies. An enzyme-linked immunosorbent assay (ELISA) was developed and the specificity of the antibodies was confirmed by comparison with pre-immune serum and by competitive assays using different dilutions of artemisinin standards. Although anti-artemisinin antibodies cross-reacted with artemisitene and dihydroartemisinin at all dilutions used, cross-reaction with deoxyartemisinin, artemisinic acid, and arteannuin B occurred only at high concentrations. ELISA successfully detected artemisinin from crude extracts in concentrations as low as 1.5 ng ml-1; and was epsilon 400-fold more sensitive than the HPLC-EC. The ELISA successfully detected and quantified artemisinin in different organs of greenhouse-grown plants and in eight clones of Artemisia annua grown in tissue culture but artemisinin was overestimated owing to cross-reactivity of the antibodies with artemisinin-related compounds present in the samples. Despite overestimation of artemisinin content, the correlations between ELISA and HPLC-EC were r = 0.92 when samples were diluted 100 times, and r = 0.90 when samples were diluted 500 times, indicating that ELISA is a potential tool for screening large A. annua populations.

Micronucleus induction in mussels exposed to okadaic acid.

Some toxins present in the marine environment are capable of inducing mutagenicity and/or carcinogenicity. Among these toxins, okadaic acid (OA) is gaining considerable interest since it induces DNA based modifications at low concentrations and accumulates in filter-feeding marine animals, including those used for human consumption. This study aims to evaluate the genotoxicity of OA in the haemocytes of the mussel Perna perna, using the micronucleus assay. Fifty-four mussels were separated into three groups of 18 animals. One group received 0.3 microg of OA diluted in 10 microl of ethanol and ultrapure water while the other groups were considered as controls and were exposed to a solvent plus seawater mixture. A significantly higher frequency of micronuclei was observed in haemocytes from the OA-exposed group. There were no statistical differences between the two control groups.

Expression of neural cell adhesion molecule immunoreactivity in hypertrophic myocardium.

Myocardial neural cell adhesion molecule (N-CAM) is temporally regulated, being expressed during cardiac morphogenesis and innervation and suppressed in the adult heart. We have investigated the plasticity of N-CAM expression in hypertrophic muscle using the rat model of chronic hypoxia to selectively induce right ventricular hypertrophy over a 14 day time course. Sarcolemmal and intercalated disc N-CAM immunostaining was more extensive in the ventricular myocardium of hypoxic rats compared to normoxic controls. Quantitative assessment of the immunoreactivity in tissue extracts demonstrated a selective increase in the amount of N-CAM immunoreactivity in the hypertrophic myocardium of the right ventricle of rats exposed to hypoxia and this was associated with an increase of the 125 kDa isoform. We conclude that myocardial hypertrophy may be a factor influencing N-CAM expression in the heart and adhesion molecules may have a role in cardiac remodelling.

The elastic system fibers.

Using the tannic acid-glutaraldehyde fixation it was possible to clearly visualize, with reproducible results, the elastic fiber amorphous material because of its density. The microfibrillar component was also evidenced with a good performance. With this technique the ultrastructural patterns of the oxytalan and elaunin fibers were also demonstrated. The oxytalan fibers appeared as a bundle of microfibrils similar to elastic microfibrils without amorphous material. The elaunin fibers presented a dispersed amorphous material intermingled among the microfibrils. It was suggested by ultrastructural evidences that oxytalan and elaunin fibers may represent interruption in successive phases in the development of the elastic fibers.

Iron from complex salts and its bioavailability to rats.

Complex iron compounds are being used as iron suppliers. Bioavailability of their iron was tested through several parameters. A biological iron prophylactic study in rats was simultaneously carried out with three of these iron complexes products: ferric orthophosphate, iron sodium EDTA and iron glycine chelate. Their iron biodisponibility was compared to that of ferrous sulfate. Five low iron diets were prepared according to AOAC method. Four of them were enriched to a level of 20 mg Fe/kg, with ferrous sulfate and the 3 iron complexes salts under study. These diets were offered to 5 groups of 6 weanling rats each in an iron prophylactic test. Food intake was measured during 5 weeks, weight checked weekly, blood and liver collected for analyses. Weight gain, hemoglobin, hematocrit, transferrin saturation, iron hemoglobin, biodisponibility and relative iron biological values were calculated. The prophylactic iron rat assay proved to be a feasible and practical model to test and compare iron salts biodisponibility. NaFeEDTA and iron aminochelate produced similar results as ferrous sulfate and their iron has a high biological value for the rats. Orthophosphate iron had lower biological value when compared to the reference standard ferrous sulfate and the other complex products studied.

Does a circadian variation occur in myocardial ischemia over 48 hours in patients with unstable angina?

OBJECTIVE: To study the incidence of and variation in myocardial ischemia over 48 hours in patients with unstable angina. METHODS: Thirty-nine patients with unstable angina underwent long-term electrocardiography for 48 hours. The number of events and the period of time of ischemia (in minutes) were analyzed for the 48 hours, in two periods of 24 hours, and in periods of 4 hours. RESULTS: We analyzed 1755.8 hours of monitoring tapes, and ischemic episodes were detected in 18 (46.2%) patients, corresponding to 173 ischemic episodes, allowing the evaluation of 1304 minutes of ischemia.only 4 of which were (2.2%) symptomatic, Considering the entire period of time of recording and the predetermined time intervals, we observed a higher number of ischemic episodes (38) and a longer duration of ischemia (315.4 minutes) between 11:00 am and 3:00 pm. However, no significant differences occurred among the values in the different intervals. CONCLUSION: Long-term electrocardiography over 48 hours showed a high incidence (97.8%) of silent ischemic episodes in patients with unstable angina. No evidence of a circadian variation of myocardial ischemia in unstable angina was observed.