Behavioural responses to human skin extracts and antennal phenotypes of sylvatic first filial generation and long rearing laboratory colony Rhodnius prolixus

Chagas disease is a major public health issue and is mainly spread by Triatominae insects (Hemiptera: Reduviidae). Rhodnius prolixus is the main vector species in Northern South America. Host-seeking behaviour in R. prolixus is mediated by different compounds that are produced by and emanate from the host or microbiota on the host's skin. We tested the behavioural responses of sylvatic first filial generation (F1) and colony insects to extracts of human skin with a dual choice olfactometer. In addition, we compared the antennal phenotypes in both populations. No statistical differences were found between the two populations at the behavioural level. Both showed a preference for face and feet extracts and this effect was abolished for face extracts after treatment with an antibacterial gel. The observation of the antennal phenotype showed that there were differences between both groups in the total length, total surface area and number and density of bristles. However, the number and density of chemoreceptive sensilla (basiconic and thin and thick-walled trichoids) and the total density of sensilla did not show statistically significant differences. These results demonstrate that colony insects, which have only been fed with living hens for the last 30 years, are attracted by human skin extracts in a similar way as F1 sylvatic insects.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

Neutralization of bitis parviocula (Ethiopian mountain adder) venom by the south african institute of medical research (SAIMR) antivenom

BACKGROUND: The Ethiopian mountain adder (Bitis parviocula) is a viperid known only from a few locations in southwestern Ethiopia. METHODS: a total of 30 µg of B. arietans and B. parviocula venoms were run on a 10-20% Tricine gel. To assay lethality dose fifty (LD50), five groups of eight mice for each venom were used. Hemorrhagic activity for crude venom was tested. Fibrinogenolytic activity of crude venom was measured using (2.5 mg/mL) of fibrinogen solution and (0.03 mg/mL) of crude venom. Gelatinase activity of the venom was tested on a Kodak X-OMAT TM film. Crude venoms of B. parviocula and B. arietans were tested for their abilities to affect clotting time, clotting rate and platelet function on whole human blood. RESULTS: The (SAIMR) antivenom was confirmed in this study to neutralize the lethal activity of venom from Bitis parviocula. The ED50s of SAIMR antivenom on B. parviocula and B. arietans neutralized half of 18.2 and 66.7 mg of venom, respectively. The hemorrhagic activities (MHDs) of B. parviocula and B. arietans were 0.88 and 1.7 µg, respectively. Bitis arietans and B. parviocula venoms degradated α and β chains at different times. The γ chains remained unaffected. Bitis parviocula venom did not exhibit gelatinase activity, while B. arietans had a MGD of 6.9 µg. At 3 mg/mL, the crude venoms of B. parviocula and B. arietans did not significantly affect clotting time or clotting rate. CONCLUSIONS: The SAIMR antivenom is very effective in neutralizing the venom of B. parviocula and should be considered in treating envenomations by these snakes.

Evaluation of pathogenic fungi occurrence in traumatogenic structures of freshwater fish

INTRODUCTION: Fungal infections in human skin, such as sporotrichosis, can occur after fish induced trauma. This work aimed to identify fungi in freshwater fish that are pathogenic to humans. METHODS: Extraction of dental arches from Serrassalmus maculatus (piranha) and Hoplias malabaricus (wolf fish), stings from Pimelodus maculatus (mandis catfish), dorsal fin rays from Plagioscion spp. (corvina) and Tilapia spp., for culture in Mycosel agar. Some cultures were submitted to DNA extraction for molecular identification by sequencing ITS-5.8S rDNA. RESULTS: Cultures identified most yeast as Candida spp., while sequencing also permitted the identification of Phoma spp. and Yarrowia lipolytica. CONCLUSIONS: While the search for S. schenckii was negative, the presence of fungus of the genera Phoma and Candida revealed the pathogenic potential of this infection route. The genus Phoma is involved in certain forms of phaeohyphomycosis, a subcutaneous mycosis caused by dematiaceous fungi, with reports of infections in human organs and systems. Traumatizing structures of some freshwater fish present pathogenic fungi and this may be an important infection route that must be considered in some regions of Brazil, since there are a large number of a fisherman in constant contact with traumatogenic fish.

Uma estação biológica para o estudo dos mosquitos e dos outros animaes silvestres relacionados com a febre amarela

The outbreak of the jungle or forest yellow fever, through the adapta¬tion, quite recently of the yellow fever virus o the forest mosquitoes, brou¬ght the necessity of ecological researches on hese mosquitoes, as well as on the wild animals they bite, some of them being susceptible to the desease. This has been done by the special yellow fever Service of the State of Sao Paulo, in a special Biological Station in Perús, São Paulo, which has been built in the midst of the jungle. This station was made with plain materials, and covered with straw, but was confortable enough for the technical work, i nthe early months of 1938. During the months in which the investigations were being carried on, the following interesting results were obtained: 1. As we have already pointed out in other places, the forest mosquitoes biting us during daytime, are always new born insects, having not yet sucked blood, as it is the general rule with all mosquitoes, and therefore also, with the anopheles and stegomyia, and this explains why nobody gets malaria or yellow fever, transmitted by anofeles or by aedes aegypti during the day. We think therefore, the jungle yellow fever, got during daytime is not due to the infected jungle or forest mosquito biting, but to infection through the human skin coming into close contact with tre virus, which the forest mosquitoes lay with their dejections, on the leaves of the trees where they remain sitting du¬ring the day. 2. As it is the rule with anopheles, stegomyia and other mosquitoes, the insects once having sucked blood, take nocturnal habits and, therefore, bite us, only during the night, so it happens with the forest mosquito, and insects with developped eggs and blood in stomach have been caught within the sta¬tion house, during the night. During the day, these mosquitoes do not bite, but remain quite still on the leaves of the trees, in the damp parts of the woods. 3. Jungle or forest mosquitoes can easely bite wild animals, some with more avidity then ethers, as it has bee npointed out to the opossum (didei-phis) and other animals. They also bite birds having very thin skin and only exceptionally, cold bloods animals. 5. Is has hot been possible to ascertain how forest mosquitoes are able to live, from onde season to another, through winter, when temperature drops near and even below zero. They have not been found in holes of the terrain, of trees and of animals, as it is the rule in cold countries. During winter, in the forest, it is possible to find larvs in the holes of bambus and trees full of water. As wild animals do not harbour the yellow fever virus for a long time in their body, it is diffcult to explain how the desease lasts from one season to another. Many ecological features on the mosquito, remains yet to be explained and therefore it in necessary to go on with the investigations, in bio¬logical stations, such as that one built up in Perús, São Paulo.

All-around care for patients with Chagas disease: a challenge for the XXI century

In 1987, the University of Pernambuco's Oswaldo Cruz Hospital in Recife, Brazil opened its Chagas Disease and Heart Failure Outpatient Clinic with the aim of providing its patients all-around care through adoption of a biopsychosocial model of care. All-around care involves caring for the patient as a whole human being in the context of the biological, psychological and social factors present, which are an inherent part of the human condition. One prerequisite for the proposed model of care is the participation of a multidisciplinary team of trained technical staff committed to this framework. Although the main focus of the service is on care, teaching and research are also an important part of its work. The Pernambuco Association of Chagas Disease Patients is guided by the same model of care and has been carrying out educational activities relating to the disease, its treatment and support for patients and family members for several years. This Association plays an important role in advocating to public authorities on behalf of patients. The accumulated experience of the past 22 years has shown us that a broad vision of health care can help clinicians and policy makers to make decisions that are more in tune with the everyday reality of the patient, which in turn has a positive impact on adherence to treatment and quality of life.

Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal, cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme

Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.

Rove beetles of medical importance in Brazil (Coleoptera, Staphylinidae, Paederinae)

Rove beetles of medical importance in Brazil (Coleoptera, Staphylinidae, Paederinae). The rove beetles of the genus Paederus Fabricius, 1775 are the most important group within Coleoptera causing dermatitis around the world. The medical importance of Paederus depends on its toxic hemolymph released when these beetles are crushed on human skin. The effects are mainly dermatitis linearis and some sporadic cases of conjunctivitis. In Brazil seven species of Paederus are known to cause dermatitis: P. amazonicus Sharp, 1876, P. brasiliensis Erichson, 1840, P. columbinus Laporte, 1835, P. ferus Erichson, 1840, P. mutans Sharp, 1876, P. protensus Sharp, 1876 stat. rev., and Paederus rutilicornis Erichson, 1840. Paederus mutans and P. protensus are for the first time recorded as of medical importance, whereas the record of P. rutilicornis in Brazil is doubtful. All seven species are redescribed and a dichotomous key is provided. The geographic distributions of all species are documented. The results provided here include the most recent and relevant taxonomic revision of Paederus of the Neotropical region, the first identification key for Brazilian species and the increase of recorded species of medical importance in the world.

Fibrinolytic action on fresh human clots of whole body extracts and two semipurified fractions from Lonomia achelous caterpillar

The severe bleeding diathesis produced by intoxication with the venom of Lonomia achelous caterpillars is characterized by prolonged bleeding from superficial skin wounds as well as massive hemorrhage into body cavities. The aim of the present study was to evaluate the effect of the crude venom and its fibrinolytic fractions on in vitro lysis of whole blood clots. Venom fractions with fibrinolytic activity were obtained by gel filtration chromatography on Sephadex G75 using imidazole buffer, pH 7.4, at a flow rate of 24 ml/h. Four peaks with fibrinolytic activity were obtained by this method. The highest activity was found in the first two peaks (both peaks were used for the experiments). The results show that the caterpillar venom degraded the preformed clots at a slower rate than plasmin. In addition, plasma protease inhibitors of the fibrinolytic system (a2-antiplasmin, a2-macroglobulin, PAI, etc.) only weakly inhibited the lytic effect of the caterpillar venom. These characteristics, as well as the pattern of fibrinogen degradation products, the delay period on fibrin plate lysis and amidolytic activity on chromogenic substrate, reported previously, indicate that the caterpillar enzymes are different from plasmin and trypsin.

Reduced schedule of human anti-rabies immunization with Fuenzalida & Palacios vaccine: additional data

It was reevaluated a reduced schedule for anti-rabies post-exposure immunization with newborn mice nervous tissue vaccine (Fuenzalida 8c Palacios) in a group of 30 non exposed volunteers. The vaccine was administered by intramuscular injections on days zero, 2, 4, 16 and 27, in the deltoid area. Antibody levels were determinated by a simplified serum neutralization microtest on days zero, 16 and 37. On days 16 and 37 the antibody levels of the whole group was >0.5 IU/ml and >1.0 IU/ml, respectively. The cell mediated immunity was precociously detected (on day 4) by the delayed type hipersensitivity skin test. Our results show that this reduced schedule elicited an early and effective humoral and cellular immune response. However it is necessary other studies with larger groups of vaccinees in order to obtain definitive conclusion.

EPIDEMIOLOGICAL SURVEY ON CANINE POPULATION WITH THE USE OF IMMUNOLEISH SKIN TEST IN ENDEMIC AREAS OF HUMAN AMERICAN CUTANEOUS LEISHMANIASIS IN THE STATE OF RIO DE JANEIRO, BRAZIL

A survey for canine tegumentary leishmaniasis (CTL) has been carried out between 1986 and 1993 in seven endemic localities for American cutaneous leishmaniasis in the State of Rio de Janeiro. 270 dogs have been examined for their clinical aspects, the development of delayed hypersensitivity (DHS) with Immunoleish antigen and with immunofluorescent antibody research of IgG (IF). 28.2% of them had ulcer lesions and 3.3% had scars. The lesions consisted of single (39.5%) and mucocutaneous lesions (31.6%), multiple cutaneous (25.0%) and mucocutaneous lesions associated with cutaneous ulcers (4.0%). Twelve (15.8%) isolates from biopsies were analyzed by zimodeme and schizodeme and identified as L. (V.) braziliensis. The overall prevalence of canine infection that was evaluated with the skin test was of 40.5% and with IF it was of 25.5%. Both tests showed a high positive rate with relation to the animals with mucosal lesions, as in the case of human mucocutaneous leishmaniasis. The comparison of the two tests showed the skin test to have a better performance although there was no statistical difference (p>0.05) between them. The proportional sensitivity and specificity was of 84.0% and 74.0%, respectively. The Immunoleish skin test and IF are useful tools to be employed in CTL field epidemiological surveys.

Evaluation of the human immunodeficiency virus type 1 and 2 antibodies detection in dried whole blood spots (dbs) samples

Human Immunodeficiency Vírus Type 1 and 2 antibodies detection was performed in 457 dried whole blood spots samples (S&S 903). Q-Preven HIV 1+2 was the screening test used. The results were compared with the gold standard serum tests by ELISA (Cobas Core e Axsym HIV1/2 gO) and imunofluorescence was the definitive confirmatory test. The samples were obtained from the Hospital Nossa Senhora da Conceição in Porto Alegre, RS - Brazil, through whole blood transfer to filter paper card and sent to Caxias do Sul, RS - Brazil where the tests were performed. The dried whole blood spot stability was evaluated with two different panels. The first one was composed of five negative and five positive samples stored at room temperature, 4 ºC, -20 ºC and -70 ºC, while the second was composed of two negative and three positive samples stored at 37 ºC (humidity <50%). Each sample was screened every week for six weeks. These measurement results didn't show variation during the study period. The detected sensibility was 100%, specificity was 99.6%, the positive predictive value was 99.5% and negative predictive values were 100%. The results demonstrated high performance characteristics, opening a new perspective of dried whole blood spot utilization in HIV screening diagnosis.

Spleen cell proliferation during and after skin myiasis by human bot fly Dermatobia hominis

Spleen cells from mice were examined at 5, 10, 15, 20 and 25 days post-infection (dpi) with Dermatobia hominis larva and at 5, 10, 15, 30 and 60 days post-larval emergence (dple). Cell proliferation in vitro assays were carried out with RPMI-1640 medium and larval secretory product (LSP) of D. hominis at 5, 10, 15, 20 and 25 days. When each group of mice was tested against each medium, significance was only seen for 25 dpi, with increasing order: LSP-10 d, -25 d, -5 d, -20 d, -15 d and RPMI. Significant results were also observed when each medium was tested against mice at each dpi or dple. Each dple group vs. each medium produced significant results only for 10 dple, with increasing order: LSP-5 d, -20 d, -25 d, -10 d, -15 d and RPMI. Comparative tests were also carried out between groups to refine certain observations. The LSPs were also analyzed using SDS-PAGE. The results prove that myiasis caused depletion of spleen cells, particularly under the effect of the LSP-10 and -15, but the cells tended to increase up to 60 dple. This in vitro assay may represent the real systemic immune response in the relationship LSP-D. hominis-host.