The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.

Prevalence and genotyping of hepatitis C virus in blood donors in the state of Pará, Northern Brazil

Given the scarcity of epidemiological information on hepatitis C virus (HCV) infection in Northern Brazil, we determined the prevalence and genotypic frequency in blood donors in the state of Pará (PA). Blood samples from all of the blood donors at the Fundação HEMOPA (blood bank of PA) from 2004-2006 were screened for the presence of antibodies to anti-HCV and samples seroreactive to anti-HCV were further tested for HCV RNA using real-time PCR. In total, 116 HCV-RNA samples were genotyped, based on maximum likelihood phylogenetic analyses, using BioEdit, Modelgenerator, PHYML and FigTree software. The population consisted of 242,726 volunteers who donated blood from 2004-2006; the most common subgroup was males between the ages of 18-29 years old (37.30%). Within the whole group, 1,112 blood donors (0.46%) had indeterminate or positive serology; among these, 28.78% were males whose ages ranged from 18-29 years. A diagnosis of chronic HCV infection was confirmed for 304 donors (60.20% males; 66.45% were 30-49 years old), resulting in a prevalence of HCV RNA in 0.13% of the samples (304 of 242,726). HCV genotyping revealed a high frequency of genotype 1 (108/116) followed by genotype 3 (8/116). This study found HCV infection to be relatively infrequent in PA; genotype 1 was most commonly isolated. This information can help guide prevention and control policies aimed at efficient diagnosis and control measures.

Rural tourism: a risk factor for schistosomiasis transmission in Brazil

This paper reports an outbreak of acute schistosomiasis among 38 tourists who rented a country house in the district of Igarapé, the metropolitan region of Belo Horizonte, Brazil, during a holiday period in 2006. A total number of 32 individuals were positive for Schistosoma mansoni. Results of stool examinations revealed individual S. mansoni egg counts per gram of faeces (epg) ranging from 4-768 epg with a geometric mean egg count of 45. The most frequent clinical symptoms were abdominal pain (78.1%), headache (75%), fever (65.6%), dry cough (65.2%) and both diarrhoea and asthenia (59.4%). A malacological survey of the area, where 22 specimens of Biomphalaria glabrata were collected, revealed three (13.6%) specimens eliminating Schistosoma cercariae. This investigation re-confirms a recently described pattern of schistosomiasis infection, resulting in the acute form of the disease and connected to rural tourism, which contributes to the spread of the disease among the middle-class and into non-endemic areas. The lack of specific knowledge about acute schistosomiasis among health services causes an increased number of unnecessary diagnostic procedures and delays in accurate diagnosis and treatment, resulting in considerable discomfort for the patients.

Ecological patterns of blood-feeding by kissing-bugs (Hemiptera: Reduviidae: Triatominae)

Host use by vectors is important in understanding the transmission of zoonotic diseases, which can affect humans, wildlife and domestic animals. Here, a synthesis of host exploitation patterns by kissing-bugs, vectors of Chagas disease, is presented. For this synthesis, an extensive literature review restricted to feeding sources analysed by precipitin tests was conducted. Modern tools from community ecology and multivariate statistics were used to determine patterns of segregation in host use. Rather than innate preferences for host species, host use by kissing-bugs is influenced by the habitats they colonise. One of the major limitations of studies on kissing-bug foraging has been the exclusive focus on the dominant vector species. We propose that expanding foraging studies to consider the community of vectors will substantially increase the understanding of Chagas disease transmission ecology. Our results indicate that host accessibility is a major factor that shapes the blood-foraging patterns of kissing-bugs. Therefore, from an applied perspective, measures that are directed at disrupting the contact between humans and kissing-bugs, such as housing improvement, are among the most desirable strategies for Chagas disease control.

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-β1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.

Immunodetection of Helicobacter sp. and the associated expression of ABO blood group antigens in the gastric mucosa of captive and free-living New World primates in the Amazon region

The histo-blood group ABH antigens were first described in humans. These antigens are only present on erythrocytes from great apes and humans, while in more primitive animals they are found in tissues and body fluids. The ABH antigens are mainly distributed in tissues exposed to the external environment and potentially serve as ligands for pathogens or inhibitors of tissue connections. The objective of this paper was two-fold: (i) to determine the presence of Helicobacter sp. in the gastric mucosa of 16 captive and 24 free-living New World monkeys and (ii) to evaluate the presence of histopathological alterations related to bacterial infection and the associated expression of ABH antigens in the tissue. Stomach tissues from 13 species of monkey were assessed using haematoxylin-eosin and modified Gram staining (Hucker) methods. An immunohistochemical analysis of the tissue revealed the presence of infectious bacteria that were characteristic of the genus Helicobacter sp. The results demonstrate that various species of monkey might be naturally infected with the Helicobacter sp. and that there is an increased susceptibility to infection. This study serves as a comparative analysis of infection between human and non-human primates and indicates the presence of a new species of Helicobacter.

High-density lipoprotein prevents SAA-induced production of TNF-α in THP-1 monocytic cells and peripheral blood mononuclear cells

In this study, we evaluated whether human serum and lipoproteins, especially high-density lipoprotein (HDL), affected serum amyloid A (SAA)-induced cytokine release. We verified the effects of SAA on THP-1 cells in serum-free medium compared to medium containing human serum or lipoprotein-deficient serum. SAA-induced tumour necrosis factor-alpha (TNF-α) production was higher in the medium containing lipoprotein-deficient serum than in the medium containing normal human serum. The addition of HDL inhibited the SAA-induced TNF-α release in a dose-dependent manner. This inhibitory effect was specific for HDL and was not affected by low-density lipoprotein or very low-density lipoprotein. In human peripheral blood mononuclear cells, the inhibitory effect of HDL on TNF-α production induced by SAA was less pronounced. However, this effect was significant when HDL was added to lipoprotein-deficient medium. In addition, a similar inhibitory effect was observed for interleukin-1 beta release. These findings confirm the important role of HDL and support our previous hypothesis that HDL inhibits the effects of SAA during SAA transport in the bloodstream. Moreover, the HDL-induced reduction in the proinflammatory activity of SAA emphasizes the involvement of SAA in diseases, such as atherosclerosis, that are characterized by low levels of HDL.

The residual efficacy of a cypermethrin pour-on formulation applied on goats on the mortality and blood intake of Triatoma infestans

Triatoma infestans is the main vector of Trypanosoma cruzi, the aetiological agent of Chagas disease in the Gran Chaco region of South America. As a frequent blood meal source for triatomine bugs, domestic goats play a key role in the eco-epidemiology of Chagas disease. The aim of this study was to evaluate the mortality and blood intake of T. infestans fed on goats that had been treated with different doses of pour-on insecticide. Third-instar nymphs were fed on goats that had been treated with 0 cc, 5 cc, 10 cc or 15 cc of a pour-on formulation of cypermethrin. The exposure of T. infestans to animals treated at different post-application intervals revealed a residual activity of the insecticide. The mortality rate in the treated groups was higher than in the control groups until 30 days post-insecticide application (p = 0.03), except in the group treated with 5 cc, in which no mortality was detected after seven days of insecticide application. Rainfall affected the triatomicide effect, reducing the time of residual activity. The cypermethrin pour-on treatment decreased the blood intake of T. infestans. Thirty days after the cypermethrin application, nymph mortality was 16% (± 13) with both doses (10 cc and 15 cc). The 15 cc dose did not result in higher insect mortality or increased persistence compared to the 10 cc dose.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

Blood shizonticidal activities of phenazines and naphthoquinoidal compounds against Plasmodium falciparum in vitro and in mice malaria studies

Due to the recent advances of atovaquone, a naphthoquinone, through clinical trials as treatment for malarial infection, 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite Plasmodium falciparum. These compounds include 2-hydroxy-3-methylamino naphthoquinones (2-9), lapachol (10), nor-lapachol (11), iso-lapachol (12), phthiocol (13) and phenazines (12-20). Their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines. Compounds 2 and 5 showed the highest activity against P. falciparum chloroquine-resistant blood-stage parasites (clone W2), indicated by their low inhibitory concentration for 50% (IC50) of parasite growth. The therapeutic potential of the active compounds was evaluated according to the selectivity index, which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro IC50. Naphthoquinones 2 and 5, with activities similar to the reference antimalarial chloroquine, were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive. Based on their in vitro and in vivo activities, compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study.

Insulin-like growth factor and growth hormone receptor in postpartum lactating beef cows

The objective of this study was to evaluate the plasma concentrations of insulin-like growth factor-I (IGF-I), and the mRNA hepatic expression of IGF-I and of the growth hormone receptors GHR and GHR 1A, in postpartum beef cows. Four Angus and four crossbred (Angus x Nelore) postpartum suckled beef cows were used. Liver and blood samples were collected every 10 days, from calving to 40 days postpartum, for gene expression and for β-hydroxybutyrate and IGF-I assays, respectively. Samples for progesterone assay were collected every other day, from day 10 to 40 postpartum. Three cows ovulated before 40 days postpartum. IGF-I concentration was higher in Angus x Nelore than in Angus cows. There was no difference in the expression of GHR, GHR 1A and IGF-I according to breed or ovulatory status. IGF-I concentrations were higher in crossbred cows, but have not changed according to postpartum ovulatory status. Moreover, changes in postpartum IGF-I concentrations are not associated with changes in liver GHR, GHR 1A and IGF-I mRNA expression in either breed.

Transoperative refusion: a simple and safe method in emergency surgery

The objective is to reinforce the importance of blood reinfusion as a cheap, safe and simple method, which can be used in small hospitals, especially those in which there is no blood bank. Moreover, even with the use of devices that perform the collection and filtration of blood, more recent studies show that the cost-benefit ratio is much better when autologous transfusion is compared with blood transfusions, even when there is injury to hollow viscera and blood contamination. It is known that the allogeneic blood transfusion carries a number of risks to patients, among them are the coagulation disorders mediated by excess enzymes in the conserved blood, and deficiency in clotting factors, mainly the Factor V, the proacelerin. Another factor would be the risk of contamination with still unknown pathogens or that are not investigated during screening for selection of donors, such as the West Nile Fever and Creutzfeldt-Jacob, better known as "Mad Cow" disease. Comparing both methods, we conclude that blood autotransfusion has numerous advantages over heterologous transfusion, even in large hospitals. We are not against blood transfusions, just do not agree that the patient's own blood is discarded without making sure there will be enough blood in stock to get him out of the hemorrhagic shock.

Blood-gas and electrolyte values for Amazon parrots (Amazona aestiva)

The aim was to provide reference data for blood gas/acid-base status and electrolytes for non-anesthetized Amazon parrots (Amazona aestiva). Thirty-five adult parrots from Tietê ecologic park were utilized. Arterial blood (0.3ml) samples were anaerobically collected from the superficial ulnar artery in heparinized (sodium heparin) 1-ml plastic syringes. The samples were immediately analyzed through a portable analyzer (i-STAT*, Abbot, Illinois, USA) with cartridges (EG7+). These data were grouped in such a way as to present both mean and standard deviation: body weight (360±37g), respiratory rate (82±33 b/m), temperature (41.8±0.6°C), hydrogen potential (7.452±0.048), carbon dioxide partial pressure (22.1±4.0mmHg), oxygen partial pressure (98.1±7.6mmHg), base excess (-7.9±3.1), plasma concentration of bicarbonate ions (14.8±2.8mmol/L), oxygen saturation (96.2±1.1%), plasma concentration of sodium (147.4±2.2mmol/L), plasma concentration of potassium (3.5±0.53mmol/L), plasma concentration of calcium (0.8±0.28mmol/L), hematocrit (38.7±6.2%) and concentration of hemoglobin (13.2±2.1g/dl). This study led us to conclude that, although the results obtained showed hypocapnia and low values of bicarbonate and base excess, when compared to other avian species, these data are very similar. Besides, in spite of the equipment being approved only for human beings, it was considered simple and very useful in the analysis of avian blood samples. By using this equipment we were able to provide references data for non-anaesthetized Amazon parrots.

Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro?

Blood-derived products are commonly administered to horses and humans to treat many musculoskeletal diseases, due to their potential antioxidant and anti-inflammatory effects. Nevertheless, antioxidant effects have never been shown upon horse synovial fluid cells in vitro. If proved, this could give a new perspective to justify the clinical application of blood-derived products. The aim of the present study was to investigate the antioxidant effects of two blood-derived products - plasma (unconditioned blood product - UBP) and a commercial blood preparation (conditioned blood product - CBP)¹ - upon stimulated equine synovial fluid cells. Healthy tarsocrural joints (60) were tapped to obtain synovial fluid cells; these cells were pooled, processed, stimulated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and evaluated by flow cytometry for the production of reactive oxygen species (ROS). Upon addition of any blood-derived product here used - UBP and CBP - there was a significant decrease in the oxidative burst of synovial fluid cells (P<0.05). There was no difference between UBP and CBP effects. In conclusion, treatment of stimulated equine synovial cells with either UBP or CBP efficiently restored their redox equilibrium.