Purification and characterisation of a haemorrhagic fraction from the venom of the Uracoan rattlesnake Crotalus vegrandis.

Uracoan rattlesnake (Crotalus vegrandis) venom was subjected to chromatographic, electrophoretic, biochemical and in vivo haemorrhagic analysis. A haemorrhagic toxin (Uracoina-1) active on skin at the site of inoculation in mice was purified by Mono Q2 anion-exchange chromatography and size exclusion (SE) high-performance liquid chromatography. The purified preparation was a protein of M(r) 58,000 as revealed by sodium dodecyl sulphate--polyacrylamide gel electrophoresis under denatured conditions and with silver staining. The use of EDTA, EGTA and 1,10-phenanthroline inhibited haemorrhagic and proteolytic activities. Inhibitors of serine proteinases such as PMSF and TCLK had no effect on the haemorrhagic fraction. Uracoina-1 hydrolyses casein, hide powder azure and fibrinogen have an optimal pH of 8.2. It rapidly digests the A alpha-chain of fibrinogen. Thermal denaturation of Uracoina-1 after exposure at 60 degrees C for 15 min led to inactivation of the haemorrhagic activity. In addition, Uracoina-1 is myotoxic, lacking haemolytic, defibrinating and lethal effects. The N-terminal amino acid sequence (20 residues) was determined.

Ergosterone-coupled Triazol molecules trigger mitochondrial dysfunction, oxidative stress, and acidocalcisomal Ca2+ release in Leishmania mexicana promastigotes.

The protozoan parasite Leishmania causes a variety of sicknesses with different clinical manifestations known as leishmaniasis. The chemotherapy currently in use is not adequate because of their side effects, resistance occurrence, and recurrences. Investigations looking for new targets or new active molecules focus mainly on the disruption of parasite specific pathways. In this sense, ergosterol biosynthesis is one of the most attractive because it does not occur in mammals. Here, we report the synthesis of ergosterone coupled molecules and the characterization of their biological activity on Leishmania mexicana promastigotes. Molecule synthesis involved three steps: ergosterone formation using Jones oxidation, synthesis of Girard reagents, and coupling reaction. All compounds were obtained in good yield and high purity. Results show that ergosterone-triazol molecules (Erg-GTr and Erg-GTr2) exhibit an antiproliferative effect in low micromolar range with a selectivity index ~10 when compared to human dermic fibroblasts. Addition of Erg-GTr or Erg-GTr2 to parasites led to a rapid [Ca2+]cyt increase and acidocalcisomes alkalinization, indicating that Ca2+ was released from this organelle. Evaluation of cell death markers revealed some apoptosis-like indicators, as phosphatidylserine exposure, DNA damage, and cytosolic vacuolization and autophagy exacerbation. Furthermore, mitochondrion hyperpolarization and superoxide production increase were detected already 6 hours after drug addition, denoting that oxidative stress is implicated in triggering the observed phenotype. Taken together our results indicate that ergosterone-triazol coupled molecules induce a regulated cell death process in the parasite and may represent starting point molecules in the search of new chemotherapeutic agents to combat leishmaniasis.

Chemotherapy of malaria and resistance to antimalarial drugs in Guayana area, Venezuela.

Resistance to antimalarial chemotherapy is one of the greatest difficulties for the control of malaria transmission. Seventy patients with Plasmodium falciparum malaria were included in a study of resistance to chloroquine and sulfadoxine-pyrimethamine therapy. Resistance levels RI, RII, and RIII were established. Eighteen infections (51%) cleared after chloroquine treatment and did not recur within 28 days of follow-up; these were classified as sensitive. Ten infections (29%) were resistant at the RI level. Resistance at level RII was observed in 5 (14%) cases, and RIII resistance was demonstrated in 2 infections (6%). With sulfadoxine-pyrimethamine, 28 (80%) infections were classified as sensitive. Six infections (17%) showed resistance at level RII, and 1 (3%) infection was resistant at the RI level. Resistance at level RIII was not observed. In a microtest for chloroquine and sulfadoxine-pyrimethamine sensitivity in vitro, schizont development was accomplished successfully in 70 blood samples. In vitro resistance to chloroquine was demonstrated in 15 of 70 (21%) of all isolates. Eight of 70 (11%) of all isolates showed resistance to sulfadoxine-pyrimethamine. Diversity of response of P. falciparum to the studied antimalarial drugs in the Guayana area of Venezuela is considered a problem restricting the control of malaria in this geographical area. A constant evaluation program monitoring P. falciparum drug sensitivity is necessary for preserving the efficacy of the established treatment.

Antivenom activity of opossum (Didelphis marsupialis) serum fraction.

We have found an opossum serum fraction of approximately 97,000 mol. wt to be highly proficient in inactivating the haemorrhagic and proteolytic fractions of Bothrops lanceolatus venom. This antivenom substance, isolated from opossum serum or a synthetic peptide based on the aforementioned protein, would probably be useful in the medical management of Bothrops accidents.

Detection of glutamate dehydrogenase enzyme activity in Plasmodium falciparum infection.

We describe the separation of an active glutamate dehydrogenase [GDH (NADP+)] enzyme from the plasma of patients with P. falciparum infection using columns of sepharose anti-GDH (NADP+) of Proteus spp. The activity of this enzyme was also detected in P. falciparum culture supernatant. The parasitic origin of this enzyme was suggested by western blot analysis using anti-P. falciparum culture supernatant and anti-whole parasite antibodies. The differential inhibition of the P. falciparum GDH (NADP+) indicates that some epitopes recognised by the antibodies in both preparations may be different. The determination of P. falciparum GDH (NADP+) activity could be developed into a specific technique for the diagnosis of falciparum malaria.

Characterization of Plasmodium falciparum glutamate dehydrogenase-soluble antigen.

The major aim of this study was to characterize a soluble Plasmodium falciparum antigen from the plasma of malaria-infected humans and Plasmodium falciparum culture supernatants, using immunoabsorbent techniques and Western blotting. An Mr 60-kDa protein was isolated from the plasma of patients with Plasmodium falciparum malaria by affinity chromatography using rabbit anti-Proteus spp GDH(NADP+) serum as ligand. This protein, present in plasma of patients with acute Plasmodium falciparum infection, in Plasmodium falciparum culture supernatants, and in immune complexes, was tested with Plasmodium falciparum malaria hyperimmune serum from patients living in hyperendemic areas and rabbit anti-Proteus spp GDH(NADP+) serum prepared in the laboratory. In this report, we describe the results of a study showing that parasite GDH(NADP+) can be used to detect the presence of Plasmodium falciparum. It appears that this technique permits the chromatographic detection of a Plasmodium falciparum excretion antigen that may be used in the production of monoclonal antibodies to improve immunodiagnostic assays for the detection of antigenemia, and opens the possibility of its use as a non-microscopic screening method.

A 33-35 kDa circulating antigen from Plasmodium falciparum.

Isolation and characterisation of Plasmodium falciparum (Welch, 1897) soluble antigens from infected patient plasma, Western blotting, thermal stability and ELISA assays using hyperimmune IgG-antimalaria antibodies was the main objective of this work. A circulating antigen of approximately Mr 33-35 kDa with good specificity and antigenicity, in the plasma of malarial patients was shown. Heating at 100 degrees C did not destroy its antigenicity. When fractions highly enriched in the 33-35 kDa proteins were used in ELISAs, a seroreactivity in plasma obtained from primary-infected individuals was found. Controls from normal patients were always negative. The antigenic characteristics suggest that it may be included within the group of new described Plasmodium soluble antigens.

Cardiac ultrastructural alterations in mice inoculated with Tityus discrepans (Buthidae) venom.

In this work we have studied the cardiologic clinical manifestations appearing in response to toxic aggression by Tityus discrepans venom, such as hypertension, hypotension, tachycardia and pulmonary acute oedema. These depend on changes in the organisation of cellular and subcellular components of cardiac tissues and probably correspond with the damage found in envenomed humans. To evaluate cardiac tissue subcellular response to Tityus discrepans venom, male C57/B1 adult mice were randomised into two groups: envenomed mice were intraperitoneally injected at a dose of 5 mg/Kg of body weight and controls received saline solution. Samples from cardiac tissue were prepared for electron microscopy study and observed in a Hitachi-300. The most relevant cardiac ultrastructural findings in this model showed diffuse disarray of the myofibrils and abnormal pattern of the bands in the sarcomera, contractile element disorganisation, degeneration of fibres and loss of the characteristic sarcomeric structure given the appearance of a lax tissue. One of the most prominent features was the presence of a remarkable perinuclear oedema and the perinuclear cistern exhibited indentations over its whole arrangement. The vascular endothelium of the microvessels exhibited alterations with evident cytoplasmic projections toward the lumen of the vessel. Mitochondria presented a condensed conformation. All findings were degenerative signs of the contractile apparatus. We suggest that any cardiac tissue damage produced by toxins present in this venom are responsible for some of the clinical manifestations in envenomed animals and patients.

Ultrastructural alterations in cortex of adrenal gland caused by the toxic effect of bee (Apis mellifera) venom.

Bee accidents incidence is underestimated because many people do not consult to the physicians. Here it is described for the first time the severe mice adrenal gland damage induced by Apis mellifera venom. Biopsy specimens were obtained from mice adrenal gland and after sample preparation observed in Hitachi H-7100 electron microscope. In this work the ultrastructural analysis showed, 6 h after injection, a non homogeneous smooth endothelial reticulum, and in some places loss of plasma membrane. The fenestrae spaces were bigger and detritus in the capillary lumen were observed. Erythrocytes were seen in a cortical cell. After 48 h of venom injection, expanded fenestrae were observed. Capillary basal membrane was interrupted. Myelin-like figures and autophagic vacuoles were noticed. Swollen smooth endoplasmic reticulum elements and endothelial unfolding to the light were seen. Moreover, swollen Golgi and mitochondria were observed, in some places forming myelinic-like figures. At 144 h after venom injection, widened spaces were noticed in capillary fenestrae. Cellular section showed swollen and lost smooth endoplasmic reticulum elements. Smooth endoplasmic reticulum tubules disappearance suggested non steroidogenesis. In conclusion, we suggest that some of the bee envenoming human clinical manifestations, as is observed in mice, are determined by suprarenal gland damage produced by toxins present in this venom.

Cellular and subcellular changes in muscle, neuromuscular junctions and nerves caused by bee (Apis mellifera) venom.

Biopsy specimens of cervico-scutular muscles obtained from animals injected with bee crude venom were prepared for electron microscopy studies. At 6 h from Apis mellifera venom injection, in mice under transmission electron microscopy, the muscular fibres presented different atrophy levels with increment of the intermyofibrillar spaces. Tubules and sarcoplasmic reticulum elements were altered, in some places only tubular fragments and an increment of the intermyofibrillar spaces were noticed as well as loss of fibre regularity and prominent triads. In subsarcolemma region, areas lacking myofibrils and mitochondria damages were observed. Muscular segmental necrosis and atrophy areas were observed. Neuromuscular junctions were altered. The number of synaptic vesicles was very variable and synaptic clefts showed irregularities. A decrease in the number and arrangement of the synaptic clefts, as well as free polysomes, suggesting regeneration processes, were also observed. The myelinic nerves exhibited in the axon or in the wall vacuolisation areas. The presence of severe muscular lesions, the finding of venom activities in the presynaptic region and the detection of damages in the neuromuscular junctions at different chronological stages of our experiments indicate that the bee venom is highly toxic for neuromuscular structures.

An intent of correlating clinical manifestations and ultrastructural lung changes in mice inoculated with Tityus discrepans (Buthidae) venom.

Investigation of the pathogenesis of human pulmonary damage caused by the toxic aggression by Tityus discrepans venom relies to a considerable extent upon the use of animal models. In this work, authors examine the correlation of clinical manifestations and changes in the organisation of cellular and subcellular pulmonary components in the murine model that probably resembles the damage found in envenomed humans. To evaluate lung subcellular response to Tityus discrepans venom, male C57/Bl adult mice were randomised into two groups: envenomed mice were intraperitoneally injected at a dose of 5 mg/Kg of weight and controls received saline solution. Lung samples were processed by electronic microscopy techniques and observed in a Hitachi-300. Ultrastructural findings in pulmonary tissue showed a partial denudation of its epithelial cells; the basal membrane was irregular and swollen. In the interstice there were fibroblasts with multiple cytoplasmatic projections and abundant extracellular material. Adjacent to the interstice bronchiolar areas with cells showing disperse nuclear chromatin were appreciated. There were losses of the intercellular unions and a clear separation among the cellular plasmatic membranes was observed. In conclusion, we suggest that some of the clinical manifestations of scorpion envenoming may be determined by ultrastructural lung damage produced by toxins present in this venom.

Kidney structural and ultrastructural pathological changes induced by uracoan rattlesnake (Crotalus vegrandis Klauber 1941) venom.

Acute renal insufficiency related to acute tubular necrosis is the most important complication caused by crotalid bite. For structural and ultrastructural studies of renal tissue, mice injected with crude venom or C. vegrandis haemorrhagic fraction, and controls were tested. Light microscopy analysis of kidneys at 24 h after injection of crude venom showed only moderate alterations such as tubular epithelia microvacuolisation. After 120 h marked glomerular and tubular capillaries congestion and interstitial oedema were observed. At 24 h after Uracoina-1 i.p. injection, intense glomerular and peritubular capillaries congestion was observed. Electron microscopic analysis of kidneys 24 h after i.p. injection of crude venom showed, capillary endothelial cell debris and pleomorphic mitochondria. Loss of interdigitations regularity, abundant dense bodies and light widening of the basal membrane were observed. Autophagic vacuoles were present as well as endothelia unfolding to the lumen and altered forms of podocytes. At 48 h, augmented endothelia without fenestrae formation with sequestration of low optical density debris inside the protrusions were noticed. At 120 h, capillary residues with loss of the endothelium were present and the basal membrane was widened. At 15 days, the number of vesicles and vacuoles in the tubules was increased and only few interdigitations were noticed. Autophagic vacuoles and mitochondrial matrix low electron density were observed. At 120 h after injection of crude venom, vascular damage with loss of capillary cell structures and collagen fibres were observed. At 24 h of haemorrhagic fraction injection, presence of autophagic vacuoles and myelinic figures were noticed.

Liver ultrastructural pathology in mice envenomed with Uracoan rattlesnake (Crotalus vegrandis) venom.

In South America rattlesnake venom activities have not been entirely characterised. Some studies have shown haemorrhagic, myotoxic, and neurotoxic effects as manifestations of envenoming in experimental animals and humans. Biopsy specimens were obtained from liver and immediately fixed in situ and observed in Hitachi H-500 and H-7100 electron microscopes. In this work the ultrastructural analysis of experimental mice liver showed hepatocytes with increased lipid droplets content and significant vacuolation in areas of their cytoplasm limiting with the Disse space. Lysosomes and altered peroxisomes exhibiting a very dense electron content were also evident. Mitochondrial pleomorphism including cup-shaped and ring-shaped mitochondria were frequently found. The cristae were scarce or absent in the majority of mitochondria observed. The rough endoplasmic reticulum showed a preferentially disposition lining the outer mitochondrial membranes. In some section glycogen particles were scarce and lipofuchsin granules could be observed. Red blood cells showed endothelial cell adherence and, in many instances, the liver sinusoids were observed plugged with aggregated red blood cells. In conclusion, using an animal model that probably correlates well with the pathological effects found in envenomed humans, we have shown the severe hepatocellular alterations caused by this venom.

Pancreas ultrastructural alterations in mice inoculated with Tityus discrepans (Buthidae) venom.

The symptoms of scorpionic envenomation in mice appear almost immediately after intraperitoneal injection and are manifested by great agitation, hair bristling, accelerated respiration, salivation and lacrimation, vomits and diarrhoea. In this work we intend to correlate those clinical manifestations appearing in response to the toxic aggression by Tityus discrepans venom, to the cellular or subcellular alterations produced in the mouse pancreas, probably similar to those damages found in envenomed humans. To evaluate pancreas subcellular response to Tityus discrepans venom, male C57/Bl adult mice were randomised into two groups: envenomed were intraperitoneally injected (hypochondrial left region) at a dose of 5 mg/Kg weight and controls received saline solution. Samples after preparation were studied in a Hitachi-300 transmission electron microscope. The most relevant ultrastructural changes in pancreatic tissues were an increase in the nuclear heterochromatin, with a corresponding decrease of euchromatin. In the cytoplasm, rough endoplasmic reticulum exhibited zones of oedema, losing its organised aspect. The secretion granules presented smaller electron density and variability in dimensions. At higher magnification a nucleus with picnotic appearance, with indentation of its perinuclear cistern was observed. There was a mitochondrial degeneration, with destruction of the mitochondrial matrix and autophagic vacuoles in its interior. At 48 h the lesions became intensified, with an evident increase in the intercellular spaces.

Ultrastructural pathology in skeletal muscle of mice envenomed with Crotalus vegrandis venom.

In this ultrastructural study we examined skeletal muscle fibres from mice intraperitoneally inoculated with a sublethal dose of Crotalus vegrandis (rattlesnake) venom. The group of mice inoculated presented neurological symptoms characterised by respiratory failure and hind limbs paralysis. Skeletal muscle fibres showed different degrees of alterations. Most of them presented the characteristic pattern of necrosis in progress. Atrophied and hypercontracted fibres were frequently seen. Some atrophied and necrotic fibres showed several nucleoli-like bodies in the nucleus. In the atrophic and hypercontracted fibres, sarcoplasmic vacuolation and abnormal mitochondria with stacked cristae were observed. Areas of segmental necrosis were also frequently found. In connection with these altered muscle fibres, capillary abnormalities were detected. This study suggests that in envenomed mice respiratory failure symptoms may be related with muscle damage caused by Crotalus vegrandis venom components.

Salivary gland ultrastructural alterations in mice inoculated with Tityus discrepans (Buthidae) venom.

In this work we have studied the possible relationships between clinical manifestations such as sialorrhea, appearing in response to the toxic aggression by Tityus discrepans venom, and the alterations or changes at cellular or subcellular levels in sub-maxillary salivary glands in the murine model. To evaluate salivary gland subcellular response to Tityus discrepans venom, male C57/Bl adult mice were randomised into two groups: a group of mice were intraperitoneally injected with Tityus discrepans venom at a dose of 5 mg/Kg of weight and controls received saline solution. In the salivary glands from the envenomed animals sub-cellular changes such as hyperchromatic nucleus, swollen rough endoplasmic reticulum, granules of different electron density, some of them practically transparent, nucleolus of low density and big size and electron-transparent cisterns were observed. Capillary wall was augmented in certain areas and thin in others. Endothelial cell infolding to the lumen was seen. The distribution of the vesicles and its density varied. Macrophages and plasmocytes were observed next to the damaged capillaries. Different electron density of cytoplasm was noticed. In conclusion, we suggest that sialorrhea is determined by salivary gland damage produced by toxins present in this venom.

Adrenal cortex alterations in mice infected with Plasmodium berghei.

The ultrastructural study of adrenal cortex from Plasmodium berghei infected mice showed different degrees of capillary wall alterations including disruption and widening of the fenestrae, capillaries packed with parasitized erythrocytes, necrosis of cortical cells, parasitized erythrocytes outside capillaries and in some instances inside cortical cell cytoplasm. Lymphocytes were also observed in degenerated cortical cells. Our results suggest that adrenal cortex lesions may be relevant in the etiopathogenesis of severe malaria.

Liver ultrastructural pathology in mice infected with Plasmodium berghei.

As liver can be an important target organ in malaria, we performed an ultrastructural study of hepatic alterations in the final stage of Plasmodium berghei infection in mice. Significant hepatocyte abnormalities were found. An elevated number of cells showed mitochondria with a high electron-dense matrix and multiple changes in shape and size, alterations in the structure of Golgi complex, swelling and disorganisation of both rough and smooth-surfaced endoplasmic reticulum, differently shaped peroxisome nucleoids, and disappearance of glycogen granules. In other areas the hepatocytes were significantly altered with diminished microvilli and exhibited myelin-like figures, autophagic vacuoles, abundant lipid droplets, and swollen mitochondria in their cytoplasm. Necrotic and atrophied hepatocytes with scarce microvilli in the Disse space and biliary canaliculi could be seen. Parasitised red blood cells and parasite debris were found inside degenerated hepatocytes. Alterations were also noticed in microvasculature, including thickened endothelial cells with swollen mitochondria, lysosomes and autophagic vacuoles in their cytoplasm. Our results demonstrate that hepatocyte damage is an important finding associated with the advanced stages of P. berghei malarial infection, which may lead to liver dysfunction in this disease.

Centipede (Scolopendra gigantea Linneaus 1758) envenomation in a newborn.

The first case of centipede (Scolopendra gigantea Linneaus 1758) envenomation in a newborn is reported. When first examined, approximately 6 hours after the bite, the 28-day-old girl was irritable, with uncontrollable cry and intense local pain, oedema, local hyperthermia, and blood clots at punctures. Uncontrollable crying in neonates should rise the possibility of an insect or arachnid sting.

[Epidemiology of sexually transmitted diseases in the mine area of Las Claritas, Bolivar State, Venezuela]. / Epidemiología de las enfermedades de transmisión sexual en la zona minera Las Claritas, Estado Bolívar, Venezuela.

The primordial objective of this study was to carry out an epidemiological analysis of sexually transmitted diseases (STD) in terms of sensed morbidity and real morbidity in the mining region of "Las Claritas", Bolivar State, Venezuela, during the third trimester of 1998. The epidemiological characteristics of time, people and place are interlaced, giving a relationship included within the Epidemiological Triad, that represents this work's conceptual framework; defining both, the determinant and the conditioning factors of the presence of STDs in the zone. The clinical interrogatory was partially steered towards the participant's sensed morbidity. A mostly feminine population is described (65%), with a low scholarly level in the general population and a high index of foreigners among the evaluated patients; with 48.72% of stable unions in the interviewees. From 166 evaluated patients, 27.70% referred to have had some STD episode, being syphilis and blenorrhagia the most frequently reported. In the survey, there was a general hush concerning the presence of STDs; but, key informants such as the medicine retailers, revealed a higher presence of these diseases, which are wrongly treated by automedication. These are important results, since they let opened a space for the epidemiological evaluation in the zone, in the various health, sanitary, environmental and social aspects and mainly, by the individual's quality of life expectations.