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.

Diversity and composition of dengue virus type 2 in Venezuela.

Dengue is a mosquito-borne disease caused by four closely related dengue virus (genus Flavivirus)serotypes (DENV-1­4). The clinical outcomes vary from mild febrile illness to life-threatening haemorrhagic manifestations. DENVs are endemic in the tropics and subtropics globally and currently no specific treatment or vaccines are available. In Venezuela, the American-Asian genotype of DENV-2 is the most prevalent and has been associated with severe disease outcomes.We aimed to follow-up the molecular epidemiology of DENV-2 in Venezuela to investigate if the evolution of the virus has remained the same throughout time or if the same dynamics documented in Brazil (hyperendemic co-circulation) also occurred. The results show that whereas the epidemiology of DENV in several endemic areas is characterized by serotype replacements through time, in Venezuela the American-Asian genotype DENV-2 has evolved into several genetic lineages and has remained in hyperendemic co-circulation with the other serotypes.

Aneurisma gigante de carótida derecha / Giant aneurysm of right carotid

Los aneurismas se definen como dilataciones localizadas y permanentes que suponen un incremento del calibre del vaso mayor del 50%. En la carótida interna son poco frecuentes, de etiología casi siempre aterosclerótica y aparecen con mayor frecuencia en mujeres mayores de 60 años. La porción cavernosa de la arteria carótida interna raramente sufre ruptura y tiene una clínica variada que incluye cefalea, diplopía, parálisis de los nervios oculomotores y dolor facial. Presentamos el caso clínico de un paciente del Hospital Vargas de Caracas con aneurisma gigante de arteria carótida derecha en seno cavernoso, quien inició de forma súbita cefalea holocraneana, acompañada de oftalmoplejía, midriasis, disminución de la agudeza visual y sin signos de irritación meníngea, que se manejó de forma médica conservadora con evolución satisfactoria

Aneurysms are defined as permanent expansions of the artery with an increase of arterial blood of 50 %. The ethiology is mostly atherosclerotic, and they appear more frequently in 60-year old women. The clinical presentation is more frequent with migraine, diplopya, paralysis of the oculomotor nerves and facial pain. We present a case of a patient with a gigantic aneurysm of the cavernous sinus of the carotid artery treated at the Hospital Vargas de Caracas, Venezuela. This patient had a sudden severe migraine, accompanied of ophthalmoplegia, mydriasis, impaired vision, without signs of meningeal irritation. The treatment was medical with satisfactory progress of the patient

Prostaglandin-induced programmed cell death in Trypanosoma brucei involves oxidative stress.

Recently, we reported the induction of a programmed cell death (PCD) in bloodstream forms of Trypanosoma brucei by prostaglandin D(2) (PGD(2)). As this prostanoid is readily metabolized in the presence of albumin, we were prompted to investigate if PGD(2) metabolites rather than PGD(2) itself are responsible for the observed PCD. In fact, J series metabolites, especially PGJ(2) and Delta(12)PGJ(2), were able to induce PCD more efficiently than PGD(2). However, the stable PGD(2) analog 17phenyl-trinor-PGD(2) led to the same phenotype as the natural PGD(2), indicating that the latter induces PCD as well. Interestingly, the intracellular reactive oxygen species (ROS) level increased significantly under J series metabolites treatment and, incubation with N-acetyl-L-cysteine or glutathione reduced ROS production and cell death significantly. We conclude that PGJ(2) and Delta(12)PGJ(2) formation within the serum represents a mechanism to amplify PGD(2)-induced PCD in trypanosomes via ROS production.

Prostaglandin D2 induces programmed cell death in Trypanosoma brucei bloodstream form.

African trypanosomes produce some prostanoids, especially PGD2, PGE2 and PGF2alpha (Kubata et al. 2000, J. Exp. Med. 192: 1327-1338), probably to interfere with the host's physiological response. However, addition of prostaglandin D2 (but not PGE2 or PGF2alpha) to cultured bloodstream form trypanosomes led also to a significant inhibition of cell growth. Based on morphological alterations and specific staining methods using vital dyes, necrosis and autophagy were excluded. Here, we report that in bloodstream form trypanosomes PGD2 induces an apoptosis-like programmed cell death, which includes maintenance of plasma membrane integrity, phosphatidylserine exposure, loss of mitochondrial membrane potential, nuclear chromatin condensation and DNA degradation. The use of caspase inhibitors cannot prevent the cell death, indicating that the process is caspase-independent. Based on these results, we suggest that PGD2-induced programmed cell death is part of the population density regulation as observed in infected animals.

Susceptibility of mosquito and tick cell lines to infection with various flaviviruses.

The genus Flavivirus consists of more than 70 virus species and subtypes, the majority of which are transmitted by mosquitoes or ticks, although some have no known vector (NKV). The ability of these viruses to infect cultured cells derived from mosquito or tick species offers a useful insight into the suitability of such vectors to harbour and replicate particular viruses. We undertook a comparative study of the susceptibility of mammalian Vero cells, a clonal mosquito cell line (C6/36) and recently developed cell lines derived from the ticks (Acari: Ixodidae) Ixodes ricinus (L.) (IRE/CTVM18), I. scapularis (Say) (ISE6), Rhipicephalus appendiculatus (Neumann) (RAE/CTVM1) and Amblyomma variegatum (Fabricius) (AVL/CTVM17) to infection with 13 flaviviruses (and one alphavirus) using immunofluorescence microscopy and plaque assay techniques. The C6/36 mosquito cell line was infected by all the mosquito-borne flaviviruses tested but not by NKV viruses or tick-borne viruses, with the exception of Langat virus (LGTV). The tick cell lines were susceptible to infection by all of the tick-borne viruses tested, as well as two mosquito-borne viruses, West Nile virus (WNV) and the alphavirus, Venezuelan equine encephalitis virus (VEEV), but not other mosquito-borne viruses or NKV viruses.

Molecular epidemiology of dengue virus type 3 in Venezuela.

During the past 40 years, dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS) have emerged in humans, with approximately 3 million cases reported and over 58 000 deaths. Dengue virus serotypes 1, 2 and 4 (DENV-1, -2 and -4) have been co-circulating in Venezuela for at least the past 10 years, causing minor or major outbreaks of dengue fever (DF) and DHF/DSS. The first recorded outbreak due to DENV-3 in Venezuela dates to 1964 and the virus then seems to have disappeared. However, DENV-3 re-appeared recently (in July, 2000) in Venezuela after 32 years of absence and produced a prolonged major outbreak, which, by the end of 2001, involved 83 180 cases of dengue, mostly DF (92 %). Previous phylogenetic studies revealed that the DENV-3 circulating during the 1960s Latin American outbreak was a genotype V virus. To gain a better understanding of the nature of the current epidemic, the complete sequence was determined of the envelope (E) gene of 15 Venezuelan DENV-3 viruses isolated during 2000 and 2001 from patients presenting with different disease severity. Sequence data were used in phylogenetic comparisons with global samples of DENV-3. Analysis revealed that the strain circulating in Venezuela is closely related to isolates that were previously present in Panama and Nicaragua in 1994 and since then have spread through Central American countries and Mexico. This study also confirms previous reports showing that the DENV-3 strain currently circulating in the Americas is related to the strain that caused DHF epidemics in Sri Lanka and India in 1989-1991 (genotype III). Finally, no evidence of the re-emergence of the strain that circulated in Venezuela in the late 1960s and 1970s (genotype V) was found.

Molecular pharmacology of chemo-resistant leishmania

Infectious diseases leishmaniosis among them, constitute a leading cause of death world wide, especially in the developing world, where they remain as an important cause of concern and has become a serious problem because of the everyday enhanced risk of co infection with HIV and the increasing frequency of resistance development of the parasites to the drug agents. Emergence of drug resistance is usually associated with changes in the expression of an specific membrane P-glycoprotein, but also includes physiological responses with high complexity. In the present review we summarize results which emphasize that the comprehesion of the molecular pharmacology of drug-resistant phenotype must include, as a way for identifying new strategies for the control of the disease, the understanding of the multiple biochemical and functional parasite mechanisms involved

Molecular epidemiology of dengue type 2 virus in Venezuela: evidence for in situ virus evolution and recombination.

Epidemic outbreaks of dengue fever (DF) were first recorded in Venezuela in 1978 and were followed by the emergence of dengue haemorrhagic fever (DHF) outbreaks in 1989. To gain a better understanding of the nature of these epidemics, the complete envelope (E) gene sequence of 34 Venezuelan dengue type 2 (DEN-2) viruses, isolated between 1997 and 2000 was determined. Of these isolates, 16 were from patients with DF and 17 were from patients diagnosed with DHF. There were no diagnostic sequence differences between them, suggesting that the E gene alone does not determine disease severity. These sequence data were also used in phylogenetic comparisons with a global sample of DEN-2 viruses, including strains collected previously from Venezuela. This analysis revealed that the ancestors of the Venezuelan viruses were Asian in origin, implying that a DEN-2 virus strain from this region was introduced into Venezuela and the wider Caribbean region during the late 1970s or the early 1980s. The phylogenetic trees further indicate that evolution of DEN-2 virus in Venezuela has occurred in situ, with differentiation into a number of distinct but co-circulating lineages, rather than the repeated introduction of new strains from other localities. By incorporating additional sequence data from the virus capsid, premembrane and membrane genes, evidence is provided that a single Venezuelan strain sequenced previously, designated Mara4, is a recombinant virus, incorporating genome sequence from Venezuelan and Asian parental viruses.

Hepatitis delta virus genotypes I and III circulate associated with hepatitis B virus genotype F In Venezuela.

The genotypes of hepatitis B (HBV) and delta (HDV) viruses circulating among Venezuelan Amerindian populations, where these viruses are endemic, were determined by sequencing of PCR amplified products from HBsAg positive sera. HDV genotype I (n = 7, 6 from West Amerindians), and III (n = 5, 4 from South Amerindians), were found. Only one HDV genotype I isolate was associated with HBV genotype D, 4 HDV genotype I and 2 HDV genotype III infected individuals were co-infected with HBV genotype F. The failure to detect the South American HDV genotype III in West Amerindians might be related to the outbreak of fulminant hepatitis with high mortality rate occurred between 1979 and 1982, probably affecting more the Amerindians infected with HDV genotype III. These results suggest the circulation of HDV genotype I among Amerindians, probably introduced through European immigrations, and that this HDV genotype is able to replicate in association with HBV genotype F.

Hepatitis B virus DNA in blood samples positive for antibodies to core antigen and negative for surface antigen.

Anti-hepatitis B core antigen (HBcAg)-positive hepatitis B surface antigen (HBsAg)-negative plasma samples from blood donors were tested by nested PCR. DNA positivity was more significantly associated with high levels of anti-HBcAg than with low levels of anti-HBsAg antibodies. Analysis of a dilution of anti-HBcAg antibodies might result in a more rational exclusion of anti-HBcAg-positive HBsAg-negative samples, reducing the number of donations discarded and enabling more countries to incorporate anti-HBcAg testing.

[Development of an enzyme assay for detection of hepatitis B virus core IGM antibodies]. / Desarrollo de un ensayo enzimático para la determinación de anticuerpos IgM anti-cápside del virus de la Hepatitis B.

Detection of IgM anti-core (anti-HBcAg) antibodies of Hepatitis B Virus (HBV) is an useful marker for hepatitis B virus (HBV) acute infection. The aim of this study was to perform an immunodiagnostic assay for the detection of IgM anti-HBcAg antibodies. Hepatitis B core antigen (HBcAg) was produced by a recombinant clone of Escherichia coli and used for the development of the immunoassay. An IgM capture enzyme immunoassay (EIA) was selected for the detection of IgM anti-HBcAg antibodies. A total of 110 human plasma or sera were tested by the capture EIA and a commercial assay. The capture EIA yielded 99% of sensitivity and 93% specificity, when compared with the commercial test. The capture EIA developed here is of interest for epidemiological studies, particularly for endemic regions in South America.

Clinical practice guidelines for the management of orbital cellulitis.

PURPOSE: A retrospective study was performed to review the management of periorbital and orbital cellulitis at Miami Children's Hospital, between January 1, 1993 and February 15, 1996. RESULTS: One hundred and one patients were included in this study. The variables analyzed in this study included age, sex, length of hospital stay, imaging studies, laboratory tests, and microbiology specimens collected. Patients were classified according to the modified Chandler classification. Average length of stay for our patient population was 4.5 days. Data regarding the radiologic studies, laboratory tests, and microbiology yield of specimens were analyzed. CONCLUSIONS: The incidence of orbital infection, manifested by lid swelling alone is much more common (stages I and II) than orbital infection involving postseptal findings (stages III, IV, and V); 84.16% compared with 15.84%, respectively. An updated approach and a general guideline for the management of periorbital and orbital cellulitis according to the clinical staging of the process is presented.