Minimal doses of hydroxyurea for sickle cell disease

The use of hydroxyurea (HU) can improve the clinical course of sickle cell disease. However, several features of HU treatment remain unclear, including the predictability of drug response and determination of adequate doses, considering positive responses and minimal side effects. In order to identify adequate doses of HU for treatment of sickle cell disease, 10 patients, 8 with sickle cell anemia and 2 with Sß thalassemia (8SS, 2Sß), were studied for a period of 6 to 19 months in an open label dose escalation trial (10 to 20 mg kg-1 day-1). Hemoglobin (Hb), fetal hemoglobin (Hb F) and mean corpuscular volume (MCV) values and reticulocyte, neutrophil and platelet counts were performed every two weeks during the increase of the HU dose and every 4 weeks when the maximum HU dose was established. Reduction in the number of vasoocclusive episodes was also considered in order to evaluate the efficiency of the treatment. The final Hb and Hb F concentrations, and MCV values were significantly higher than the initial values, while the final reticulocyte and neutrophil counts were significantly lower. There was an improvement in the concentration of Hb (range: 0.7-2.0 g/dl) at 15 mg HU kg-1 day-1, but this concentration did not increase significantly when the HU dose was raised to 20 mg kg-1 day-1. The concentration of Hb F increased significantly (range: 1.0-18.1%) when 15 mg HU was used, and continued to increase when the dose was raised to 20 mg kg-1 day-1. The final MCV values increased 11-28 fl (femtoliters). However, reticulocyte (range: 51-205 x 109/l) and neutrophil counts (range: 9.5-1.3 x 109/l) obtained at this dose were significantly lower than those obtained with 15 mg kg-1 day-1. All patients reported a decrease in frequency or severity of vasoocclusive episodes. These results suggest that a hydroxyurea dose of 15 mg kg-1 day-1 seems to be adequate for treatment of sickle cell disease in view of the minimal side effects observed and the improvement in laboratory and clinical parameters.

CALIBRATION AND USE OF AN IN-HOUSE ANTI-MEASLES IgG STANDARD SERUM

For the purpose of research a large quantity of anti-measles IgG working reference serum was needed. A pool of sera from five teenagers was prepared and named Alexandre Herculano (AH). In order to calibrate the AH serum, 18 EIA assays were performed testing in parallel AH and the 2nd International Standard 1990, Anti-Measles Antibody, 66/202 (IS) in a range of dilutions (from 1/50 to 1/25600). A method which compared parallel lines resulting from the graphic representation of the results of laboratory tests was used to estimate the power of AH relative to IS. A computer programme written by one of the authors was used to analyze the data and make potency estimates. Another method of analysis was used, comparing logistic curves relating serum concentrations with optical density by EIA. For that purpose an existing computer programme (WRANL) was used. The potency of AH relative to IS, by either method, was estimated to be 2.4. As IS has 5000 milli international units (mIU) of anti-measles IgG per millilitre (ml), we concluded that AH has 12000 mIU/ml.

Trypanosoma cruzi parasitemia observed in immunocompromised patients: the importance of the artificial xenodiagnosis

Trypanosoma cruzi parasitemia observed in immunocompromised patients (transplant or positive HIV) occurred more frequently by the artificial xenodiagnosis method (10/38) compared with hemoculture (2/38), given the same quantity of blood. Other ways of diagnosis, like mice inoculation (5/38), QBC and buffy coat (2/38), were evaluated also. This result showed the importance of the artificial xenodiagnosis. The other techniques increased only one more patient positive.

Rat hepatocyte invasion by Listeria monocytogenes and analysis of TNF-alpha role in apoptosis

Listeria monocytogenes, etiological agent of severe human foodborne infection, uses sophisticated mechanisms of entry into host cytoplasm and manipulation of the cellular cytoskeleton, resulting in cell death. The host cells and bacteria interaction may result in cytokine production as Tumor Necrosis Factor (TNF) alpha. Hepatocytes have potential to produce pro-inflammatory cytokines as TNF-alpha when invaded by bacteria. In the present work we showed the behavior of hepatocytes invaded by L. monocytogenes by microscopic analysis, determination of TNF-alpha production by bioassay and analysis of the apoptosis through TUNEL technique. The presence of bacterium, in ratios that ranged from 5 to 50,000 bacteria per cell, induced the rupture of cellular monolayers. We observed the presence of internalized bacteria in the first hour of incubation by electronic microscopy. The levels of TNF-alpha increased from first hour of incubation to sixth hour, ranging from 0 to 3749 pg/mL. After seven and eight hours of incubation non-significant TNF-alpha levels decrease occurred, indicating possible saturation of cellular receptors. Thus, the quantity of TNF-alpha produced by hepatocytes was dependent of the incubation time, as well as of the proportion between bacteria and cells. The apoptosis rate increased in direct form with the incubation time (1 h to 8 + 24 h), ranging from 0 to 43%, as well as with the bacteria : cells ratio. These results show the ability of hepatocyte invasion by non-hemolytic L. monocytogenes, and the main consequences of this phenomenon were the release of TNF-alpha by hepatocytes and the induction of apoptosis. We speculate that hepatocytes use apoptosis induced by TNF-alpha for release bacteria to extracellular medium. This phenomenon may facilitate the bacteria destruction by the immune system.

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 µ L and photomicrographied. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10² promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.

The liver in secondary syphilis clinical, laboratory and histopathological investigation of 22 patients without jaundice

22 patients with a history of syphilitic contact, skin lesions and positive serology were evaluated by physical examination, tiver function tests and líver biopsy for evidence of hepatic lesions secondary to treponema infection. Only minimal evidences of hepatic damage were revealed by clinical examination and liver function tests. On biopsy 21 cases of NSRH were noted with one case of gramioma formation. No spirochaetes were found, so these findings could not be attributed to a direct action of the treponema.

Reflux esophagitis and gastroesophageal reflux disease: a cross-sectional study of gastroesophageal reflux disease patients by age group

The purpose of this study was to explore the relationship between the intensity of acid reflux and severity of esophageal tissue damage in a cross-sectional study of patients with gastroesophageal reflux disease (GERD). Seventy-eight patients with were selected in accordance with the strict 24-hour ambulatory esophageal pHmetry (24h-pHM) criteria and distributed into three age groups: Group A: 14 - 24 years of age. Group B: 25 - 54; and Group C: 55 - 64. The 24h-pHM was carried out in accordance with DeMeester standardization, and the Savary-Miller classification for the diagnosis of reflux esophagitis was used. The groups were similar in 24h-pHM parameters (p > 0.05), having above normal values. For the study group as a whole, there was no correlation between age group and intensity of acid reflux, and there was no correlation between intensity of acid reflux and severity of esophageal tissue damage. However, when the same patients were sub-grouped in accordance with the depth of their epithelial injury and then distributed into age groups, there was a significant difference in esophagitis without epithelial discontinuity. Younger patients had less epithelial damage than older patients. Additionally, although there was a significant progression from the least severe to the moderate stages of epithelial damage among the age groups, there was no apparent difference among the age groups in the distribution between the moderate stages and most severe stages. The findings support the conclusion that the protective response of individuals to acid reflux varies widely. Continued aggression by acid reflux appears to lead to the exhaustion of individual mechanisms of epithelial protection in some patients, but not others, regardless of age or duration of the disease. Therefore, the diagnosis and follow-up of GERD should include both measurements of the quantity of refluxed acid and an assessment of the damage to the esophageal epithelium.

Bromeliad-associated mosquitoes from Atlantic forest in Santa Catarina Island, southern Brazil (Diptera, Culicidae), with new records for the State of Santa Catarina

Bromeliad-associated mosquitoes (Diptera: Culicidae) in Atlantic Forest in Florianópolis, Santa Catarina, southern Brazil, were studied, examining plants of Vriesea philippocoburgi Wawra and Aechmea lindenii (E. Morren) Baker var. lindenii at secondary Atlantic rain forest, and A. lindenii and Vriesea friburgensis Mez var. paludosa (L. B. Smith) at "restinga" per month, during 12 months. No immature forms of mosquitoes were collected from A. lindenii in the secondary forest. Collections obtained 368 immature mosquitoes, none of them from A. lindenii from rain forest. Culex (Microculex) spp. constituted 79.8% of the total, Wyeomyia (Phoniomyia) spp. 17.93%, and Anopheles (Kerteszia) cruzii (Dyar & Knab, 1908) only 1.36%. The study shows the great predominance of species of medical importance not yet proved, and the small number of immature stages of anopheline mosquitoes. The rainfall, but not the mean temperatures, significantly influenced the quantity of mosquitoes from V. philippocoburgi. Significant differences between the quantities of immature forms of all the bromeliad species were found, and the shape of the plants could be important to the abundance of mosquitoes. All six species of Cx. (Microculex) found are recorded for the first time in the State of Santa Catarina, and all six species of Wyeomyia (Phoniomyia) are recorded for the first time in bromeliads in this state.

Diversity and structure of microcrustacean assemblages (Cladocera and Copepoda) and limnological variability in perennial and intermittent pools in a semi-arid region, Bahia, Brazil

Temporary wetlands undergo recurrent drought due to the scarcity of water, which disrupts the hydrological connectivity with adjacent aquatic systems. However, some environments retain water for longer periods, allowing greater persistence of the community. The current study evaluated differences in the microcrustacean assemblages and limnological variability between perennial and intermittent pools in a semi-arid region of Brazil. The abiotic features (water temperature, pH, total alkalinity, electrical conductivity and depth) of intermittent pools were affected more than perennial pools due to loss of water volume. This may have contributed to a higher average richness and diversity index in some intermittent pools and differences in the structure of the assemblages. The lowest species richness and diversity were recorded where physical factors, such as a large quantity of suspended solids and variability in the electrical conductivity of the water and pH, make the environment unsuitable for these organisms. These results suggest that community development in intermittent pools is interrupted by the dry season; when the water returns, due to rainfall or rising groundwater, each pond undergoes a different process of colonization. In these circumstances, the biological importance of temporary aquatic environments is clear, since such pools provide shelters and have an important role in the maintenance of the regional diversity of aquatic environments.

Myeloid Metaplasia of Spleen in Hookworm Disease

We investigated, in the liver and the spleen of ten pures cases of ankylostomiasis haemocytopoietic elements. We verified the weight of spleen in 23 cases of individuals from 3 to 60 years old. In no case did we meet with haemopoietic cells in liver. In seven cases we found in spleen elements of the red series at an advanced evolutional stage (orthochromatic erythroblasts with pyknotic nucleus). In some of these cases we observed megakaryocytes and numerous eosinophilous myelocytes.The three cases which did not show any myeloid metaplasia in spleen were from individuals of over 50 years. Nevertheless, in another case of an individual 59 years old this metaplasia was verified. In individuals of over 20 years, the average weight of spleen in nine cases appeared to be equal to the normal weight. In 14 other cases, between 3 and 14 years of age, the weight of this organ was always sensibly higher than in normal individuals of the corresponding age. These results suggest the possibility of the myeloid metaplasia being the fact responsible for the weight increase of spleen in young individuals victimatized by hookworm anaemia. The remarkable proliferation of orthochromatic erythroblasts shows that the degree and quickness of blood regeneration after iron administration are due, essentially, to the great quantity of haemoglobin previously formed in the spleen and bone marrow of ankylostomized organisms.

Competitive interactions between species of freshwater snails: I. Laboratory: Ia. General methodology

For the development of laboratory experiments on the competitive interacitons between freshwater snail populations, special snail rooms were set up in the main building of the Research Center "Aggeu Magalhães". In the current paper, the first of a series on this subject, the general methodology of the laboratory work is described in detail. Using indoor cement channels in which a uniform seminatural environment was created, interactions of freshwater snail populations can be studied with minimal interference of the usual variables. Controlled indoor environmental techniques, as described in the current paper, may also be utilized in different types of experiments in malacology, and represent a substantial technical advance in malacological work.

Infection of Oxydoras kneri Bleecker, 1862 (Pisces, Doradidae) by the acanthocephalan Paracavisoma impudica (Diesing, 1851) Kritcher, 1957

Infection of Oxydoras kneri by the acanthocephalan Paracavisoma impudica is described. The parasites do not penetrate deeply into the host gut wall and do not reach the muscularis layers. Host reaction is minimal, consisting of limited fibrosis around the proboscides. Haemorrhages and lymphocyte infiltration are not observed, and phagocytic cells are only present occasionally. Oxydoras kneri is a newly reported host for Paracavisoma impudica.

Experimental Infection of Aedes albopictus (Diptera: Culicidae) Larvae with the Xiphidiocercariae of a Hematolechid

Aedes albopictus larvae were exposed, either individually or in groups, to different concentrations of xiphidiocercariae of Haematoloechus sp. for parasitological studies. It was observed the acute lethal effect and some aspects of the host-parasite relationship, such as delay or progress in the host life cycle, the number and location of the metacercariae in the host, adult host malformations and the amount of metacercariae required to cause death. A delay in the cycle and a high mortality rate was in general observed. Inside the larvae, the metacercariae were found predominantly in the thorax, abdominal segments and in the head, along with a reduced number in the anal lobe and cervix. It was shown that in addition to the quantity of metacercariae present, their location in the larvae was also relevant in the determination of mortality and anomalies. Malformed adults developed from larvae containing from one to three metacercariae.

One-tube nested Polymerase Chain Reaction for detection of Chlamydia trachomatis

Here we present a one-tube nested PCR test, which allows the detection of minimal quantities of Chlamydia trachomatis in human fluids. This assay includes the use of an internal control to avoid false negative results due to the presence of inhibitors. The results obtained show that this assay is robust enough to be used for clinical diagnosis.

Effect of blood components, abdominal distension, and ecdysone therapy on the ultrastructural organization of posterior midgut epithelial cells and perimicrovillar membranes in Rhodnius prolixus

The effects of blood components, nerve-cord severance, and ecdysone therapy on the posterior midgut epithelial cells of 5th-instar Rhodnius prolixus nymphs 10 days after feeding were analyzed by transmission electron microscopy. Cutting the nerve-cord of the blood-fed insects partially reduced the development of microvilli and perimicrovillar membranes (PMM), and produced large vacuoles and small electrondense granules; insects fed on Ringer's saline diet exhibited well developed microvilli and low PMM production; swolled rough endoplasmatic reticulum and electrondense granules; Ringer's saline meal with ecdysone led to PMM development, glycogen particles, and several mitochondria in the cytoplasm; epithelial cells of the insects fed on Ringer's saline meal whose nerve-cord was severed showed heterogeneously distributed microvilli with reduced PMM production and a great quantity of mitochondria and glycogen in the cytoplasm; well developed microvilli and PMM were observed in nerve-cord severed insects fed on Ringer's saline meal with ecdysone; Ringer's saline diet containing hemoglobin recovered the release of PMM; and insects fed on human plasma showed slightly reduced PMM production, although the addition of ecdysone in the plasma led to a normal midgut ultrastructural organization. We suggest that the full development of microvilli and PMM in the epithelial cells depends on the abdominal distension in addition to ingestion of hemoglobin, and the release of ecdysone.