Description of high rates of unapparent and simultaneous multiple dengue virus infection in a Colombian jungle settlement.

Dengue disease statistics is mainly based on consulting patients with febrile illness, but misdiagnosed and asymptomatic cases are important to measure dengue epidemiology in endemic areas. The main objective of this work was to determine the prevalence of IgM and IgG antibodies or NS1 antigen and viral RNA in a group of healthy volunteers from an isolated village in Colombian Chocó rain forest. It found 51.7% of virologically PCR confirmed asymptomatic cases, despite low IgM seroprevalence. It was confirmed that all four serotypes are in the circulation and in 17.2% of individuals it detected natural coinfections of two or three different serotypes simultaneously. This is the first report in Colombia evaluating viremia in asymptomatic volunteers. These findings pose a big concern about the transmission of dengue virus by asymptomatic individuals because they can spread the virus without take appropriate control measures.

Biochemical comparison of venoms from young Colombian Crotalus durissus cumanensis and their parents

Crotalus durissus cumanensis, a rattlesnake endemic to Colombia and Venezuela, is considered one of the most lethal snake species in Latin America. The aim of the present study was to compare the protein content and biological activity of the venom obtained from eight specimens of C. durissus cumanensis, namely two adults from different localities of Colombia and six offspring born in captivity. Protein profiles of crude venoms were analyzed by SDS-PAGE and RP-HPLC, and biological activities were evaluated for lethality, edema, defibrination, hemolytic and coagulant activities to assess individual venoms of adults and a pool of young snake venoms. Transient edema appeared rapidly after venom inoculation, whereas hemorrhagic effect was not observed. Differences in protein profiles, lethality, hemolytic, coagulant and defibrinating activities between both adult snake venoms were observed; those from the mother snake exhibited higher activities. Venoms from young snakes were similar to the one obtained from the mother, but the coagulant effect was stronger in offspring venoms. Notably, biological effects of the father snake venom were not comparable to those previously described for C. durissus cumanensis from Venezuela and C. durissus terrificus from Brazil, confirming the high variability of the venom from Crotalus species.

Human cytokine responses to meso-endemic malaria on the Pacific Coast of Colombia.

Studies of naturally-acquired immunity to malaria in endemic regions provide the potential for a greater understanding of the regulation of human immune responses to the malarial parasite. However, little is known about the acquisition of malaria-specific immunity in regions of unstable, meso-endemic or hypo-endemic transmission. Cytokine profiles - patterns in the expression of interleukin-4 (IL-4), interleukin-10, interleukin-12, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) - were therefore studied during the natural acquisition of immunity to Plasmodium falciparum and P. vivax among individuals from Buenaventura, a meso-endemic region on the Pacific Coast of Colombia. In general, specific type-1 immune responses, characterized by IFN-gamma expression, were more likely to develop during P. falciparum infection, whereas pro-inflammatory cytokine profiles (with TNF-alpha expression) were observed more frequently among the P. vivax infections. Type-2 cytokine profiles, characterized by dominant IL-4 expression, were infrequent. Expression of IL-4 probably occurs primarily after prolonged exposure to parasites (which would, by definition, not be common in a meso-endemic region).

Immunity and physio-pathogenesis of human malaria / Inmunidad y fisiopatogénesis de la malaria humana

Malaria is currently considered one of the most health-threatening diseases of humans producing hundreds of million clinical cases every year in the tropical and subtropical regions of the world. Together Plasmodium falciparum and P. vivax a re responsible for most of the 500 millions cases produced yearly. Significant efforts are being made to identify the immune mechanisms involved both in protection and physio-pathogenesis of the disease as well as to develop protective vaccines. Both innate and acquired immune mechanisms participate in malaria protection and an important degree of immunity is developed in individuals permanently exposed to malaria in endemic areas, however it is not sufficient to induce sterile protection in the host. Several of the most important clinical complications such as cerebral malaria, are associated with the failure of host defences to control parasite replication, sequestration of parasitised red blood cells in several organs and the excessive secretion of pro -inflammatory cytokines. Although vaccination of animals and human volunteers has demonstrated the feasibility of the induction of sterile immunity, several vaccine candidates are intended to diminish the disease. We review here the current concepts about the immune re s p o n s e s induced by malaria, their potential in disease induction and some of the most relevant vaccine candidates (AU)

La malaria es considerada en la actualidad como una de las enfermedades más amenazantes para la salud del hombre, por cuanto produce cientos de millones de casos clínicos cada año en regiones tropicales y subtropicales del mundo. El Plasmodium falciparum y P. vivax son responsables de más de 500 millones de casos clínicos al año. Por todo ello, se están realizando esfuerzos muy grandes para identificar los mecanismos inmunes involucrados tanto en la protección como en la fisio-patología de la enfermedad, así como para el desarrollo de vacunas protectoras. Se ha demostrado que tanto los mecanismos de inmunidad innata como adquirida, participan en la protección contra la malaria y que los individuos permanentemente expuestos a malaria en las zonas endémicas, desarrollan un grado significativo de inmunidad. Sin embargo, la inmunidad nunca es suficiente para inducir la protección estéril del huésped. Muchas de las complicaciones clínicas como la malaria cerebral, están asociadas con una fallas para controlar la multiplicación del Plasmodium, el secuestro de glóbulos rojos parasitados en varios órganos y una excesiva secreción de citocinas pro-inflamatorias. La vacunación de animales y de voluntarios humanos ha demostrado la factibilidad de inducir una inmunidad estéril, sin embargo, actualmente se están tratando de encontrar vacunas para prevenir las manifestaciones clínicas. Aquí revisamos el estado actual del conocimiento sobre la respuesta inmune inducida por la infección, su papel en la producción de la enfermedad y algunos de los candidatos a vacunas más relevantes (AU)

Plasmodium vivax malaria vaccine development.

Plasmodium vivax represents the most widespread malaria parasite worldwide. Although it does not result in as high a mortality rate as P. falciparum, it inflicts debilitating morbidity and consequent economic impact in endemic communities. In addition, the relapsing behavior of this malaria parasite and the recent resistance to anti-malarials contribute to making its control more difficult. Although the biology of P. vivax is different from that of P. falciparum and the human immune response to this parasite species has been rather poorly studied, significant progress is being made to develop a P. vivax-specific vaccine based on the information and experience gained in the search for a P. falciparum vaccine. We have devoted great effort to antigenically characterize the P. vivax CS protein and to test its immunogenicity using the Aotus monkey model. Together with other groups we are also assessing the immunogenicity and protective efficacy of the asexual blood stage vaccine candidates MSP-1 and DBP in the monkey model, as well as the immunogenicity of Pvs25 and Pvs28 ookinete surface proteins. The transmission-blocking efficacy of the responses induced by these latter antigens is being assessed using Anopheles albimanus mosquitoes. The current status of these vaccine candidates and other antigens currently being studied is described.

Variants of the Plasmodium vivax circumsporozoite protein (VK210 and VK247) in Colombian isolates.

Phenotypic diversity has been described in the central repeated region of the circumsporozoite protein (CSP) from Plasmodium vivax. Two sequences VK210 (common) and VK247 (variant) have been found widely distributed in P. vivax isolates from several malaria endemic areas around the world. A third protein variant called P. vivax-like showing a sequence similar to the simian parasite P. simioovale has also been described. Here, using an immunofluorescent test and specific monoclonal antibodies, we assessed the presence of two of these protein variants (VK210 and VK247) in laboratory produced sporozoite. Both sequences were found in parasite isolates coming from different geographic regions of Colombia. Interestingly, sporozoites carrying the VK247 sequence were more frequently produced in Anopheles albimanus than sporozoites with the VK210 sequence. This difference in sporozoites production was statistically significant (p <0.05, Kruskal-Wallis); not correlation was found with parameters as the total number of parasites or gametocytes in blood from human donors used to feed mosquitoes. Previous studies in the same region have shown a higher prevalence of anti-VK210 antibodies which in theory may suggest their role in blocking the development of sporozoites carrying the CSP VK210 sequence.

Variants of the Plasmodium vivax circumsporozoite protein (VK210 and VK247) in Colombian isolates

Phenotypic diversity has been described in the central repeated region of the circumsporozoite protein (CSP) from Plasmodium vivax. Two sequences VK210 (common) and VK247 (variant) have been found widely distributed in P. vivax isolates from several malaria endemic areas around the world. A third protein variant called P. vivax-like showing a sequence similar to the simian parasite P. simio-ovale has also been described. Here, using an immunofluorescent test and specific monoclonal antibodies, we assessed the presence of two of these protein variants (VK210 and VK247) in laboratory produced sporozoite. Both sequences were found in parasite isolates coming from different geographic regions of Colombia. Interestingly, sporozoites carrying the VK247 sequence were more frequently produced in Anopheles albimanus than sporozoites with the VK210 sequence. This difference in sporozoites production was statistically significant (p <0.05, Kruskal-Wallis); not correlation was found with parameters as the total number of parasites or gametocytes in blood from human donors used to feed mosquitoes. Previous studies in the same region have shown a higher prevalence of anti-VK210 antibodies which in theory may suggest their role in blocking the development of sporozoites carrying the CSP VK210 sequence

HLA-A*0201 restricted CD8+ T-lymphocyte responses to malaria: identification of new Plasmodium falciparum epitopes by IFN-gamma ELISPOT.

The role of antigen specific CD8+ T-lymphocytes in mediating protection against sporozoite-induced malaria has been well established in murine models. In humans, indirect evidence has accumulated suggesting a similar protective role for antigen-specific CD8+ T-lymphocytes. Nevertheless, the low frequency of circulating specific cells together with the lack of sensitive methods to quantify them has hampered the direct assessment of their function. Using a combination of short-term cell culture and IFN-gamma ELISPOT, we studied CD8+ T-lymphocyte responses to a panel of HLA-A*0201 binding peptides. In addition to confirming the response to already described epitopes, we also identified five new CD8+ T-lymphocyte epitopes. These epitopes are presented in pre-erythrocytic stages gene products of Plasmodium falciparum 7G8 strain and correspond to the following protein segments: circumsporozoite (CS) 64-72, 104-113, 299-308 and 403-411; liver stage antigen (LSA-1) repeat region; sporozoite surface protein 2 or thrombospondin related anonymous protein (SSP2/TRAP) 78-88 and 504-513. Four of these peptides are conserved amongst all published sequences of P. falciparum strains. We conclude that the modified IFN-gamma ELISPOT assay is a sensitive technique to monitor antigen-specific CD8+ T-lymphocyte responses in human malaria which may help in the improvement and assessment of the efficacy of malaria subunit vaccines.

Molecular characterization of HLA class I in Colombians carrying HLA-A2: high allelic diversity and frequency of heterozygotes at the HLA-B locus.

Polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) typing was used to analyze HLA class I A, B and C alleles in three different Colombian populations. Fifty-nine samples were from Hispano-American Mestizos living in the urban areas of Cali (referred to here as Aso population). Forty-four and thirty samples were from the African Black populations of Zacarias (Zac) and Punta Soldado (PS), respectively. Samples were selected for expression of HLA-A2 by monoclonal antibody staining and allele-specific hybridization, and their HLA-A2 subtype distribution has been reported previously. Although only a limited number of samples was analyzed, the data suggest the existence of a remarkable degree of HLA class I polymorphism in the populations studied, with representatives of most serological classes. Despite their common African origin, the populations Zac and PS, both resident in malaria endemic regions, showed some striking differences in allelic distribution for all three class I loci. Furthermore, the samples from Aso and PS, but not Zac, showed a low percentage of blank alleles at the HLA-B locus (0 and 0.4%, respectively), suggesting the possibility of a heterozygote advantage for HLA-B alleles in Colombian populations.

Mapping and comparison of the B-cell epitopes recognized on the Plasmodium vivax circumsporozoite protein by immune Colombians and immunized Aotus monkeys.

Plasma samples of individuals from two malaria-endemic villages on the Colombian Pacific coast and synthetic peptides representing different fragments of the central and flanking regions of the Plasmodium vivax circumsporozoite protein (CSP) were used to perform a fine mapping of the B-cell epitopes on the whole CSP. In addition, the immunogenicity of long polypeptides corresponding to the amino (N) and carboxyl (C) regions was evaluated in Aotus monkeys. The epitopes recognized after natural infection of humans and after immunization of Aotus with these synthetic peptides were compared. Human samples more frequently contained specific antibodies to the central region. The type-I repeat region of the CSP was predominantly recognized by the human sera (by 68% of those from the village of Zacarías and 75% of those from Bajo Calima), a significantly smaller population reacting with the type-II repeat (20% and 11%, respectively). Most of the sera reacting with the type-I repeat recognized the minimal epitope AGDR. Although the N- and C-terminal polypeptides were both highly immunogenic in Aotus and induced long-lasting antibodies, titres of antibodies to the C-terminal polypeptide were higher than those of antibodies to the N-terminal. Competitive inhibition assays performed using human and monkey plasma allowed the identification of dominant B-cell epitopes on sequence 71-90 (p8) from the amino region and sequence 332-361 (p24/p25) from the carboxyl region. The high prevalence of naturally induced antibodies to the three epitopes, the possible functional role of the corresponding sequences, and the high immunogenicity of these epitopes in Aotus could be of great importance in the development of a malaria vaccine based on P. vivax CSP.

Expression of cytokine genes in Aotus monkeys immunized with synthetic and recombinant Plasmodium vivax and P. falciparum antigens.

Cytokine responses in human host-protective immunity to malaria have yet to be completely elucidated. No data appear to exist on the cytokine patterns in non-human primate models immunized with malarial antigens. Expression of mRNA transcripts of 10 cytokines, the adhesion molecule ICAM-1 and inducible nitric oxide synthase (iNOS) in peripheral-blood mononuclear cells (PBMC) from nine Aotus monkeys was analysed by reverse-transcriptase PCR. Five of the monkeys had been immunized with multiple-antigen peptides (MAP) of the Plasmodium vivax circumsporozoite protein and two with constructs of the P. falciparum merozoite surface protein-1 (MSP-1). The other two monkeys served as non-immunized controls. PBMC were cultured for 24 h after stimulation with phytohaemagglutinin mitogen, MAP and MSP-1 antigens. Elevated expression of interleukin-6 (IL-6), IL-10, IL-12, tumour necrosis factor-alpha (TNF-alpha), TNF-beta and iNOS was seen in response to the MAP. Monkeys immunized with either P. falciparum MSP r190L or synthetic 190L peptides expressed predominantly the type-1 cytokines (IL-1 beta, IL-12, interferon-gamma, TNF-alpha, TNF-beta) characteristic of splenic, cell-mediated activity with macrophage activation and nitric oxide production.

Serologic responses to recombinant Plasmodium vivax Duffy binding protein in a Colombian village.

The Plasmodium vivax Duffy binding protein (DBP) is essential during merozoite invasion into human erythrocytes. Because of its biological importance, the DBP is also seen to have potential use as a malaria blood-stage vaccine. We have used a soluble recombinant DBP (rDBP) containing the functional ligand domain to assess the natural immunogenicity of DBP in a low-endemic vivax malaria region. Human sera from adult residents from a Colombian village with unstable vivax malaria transmission reacted specifically with the rDBP as determined by ELISA. There was a significant positive correlation between increased antibody response (average, median, and percent positives) and age of patients, although the level of responses did vary considerably in their reactivity to the rDBP from negative to very high level within each age group. These data confirm previous findings on the serologic reactivity of the DBP in exposed populations and that immunologic boosting to the DBP occurs in malaria-endemic regions even with low-level transmission.

Immunogenicity of four Plasmodium falciparum preerythrocytic antigens in Aotus lemurinus monkeys.

Aotus lemurinus monkeys were immunized with pools of either lipid-tailed peptides injected in PBS or peptides in Montanide ISA-51, all derived from four Plasmodium falciparum pre-erythrocytic antigens, namely, LSA1, LSA3, SALSA, and STARP. These formulations were well tolerated. Their immunogenicity was demonstrated by the induction of both B- and T-cell responses to most of the peptides studied (of the 12, 10 induced antibody production, 9 induced T-cell proliferative responses, and all 12 induced gamma interferon secretion). Immune responses proved to be long lasting, since some were still detectable 210 days after immunization. Of particular importance is the fact that B- and T-cell responses elicited in this way by synthetic peptides were specific for native parasite proteins on P. falciparum sporozoites and liver stage parasites.

Prevention of preeclampsia by linoleic acid and calcium supplementation: a randomized controlled trial.

OBJECTIVE: To determine the effect of low doses of linoleic acid and calcium on prostaglandin (PG) levels and the efficacy of this treatment in the prevention of preeclampsia. METHODS: In a randomized, double-blind, placebo-controlled study we treated 86 primigravidas with risk factors for preeclampsia (high biopsychosocial risk [above 3 points], positive roll-over test, and high mean blood pressure [above 85 mmHg)] with daily doses of either 450 mg linoleic acid and 600 mg calcium (n=43) or 450 mg starch and 600 mg lactose placebo (n=43) during the third trimester of pregnancy. RESULTS: Four women in the experimental group (9.3%) developed preeclampsia compared with 16 (37.2%) controls (relative risk 0.25, 95% confidence interval 0.09, 0.69, P < .001). The median serum levels of PGE2 after 4 weeks of treatment increased by 106% in the experimental group (P=.03) and decreased by 33% in the control group (P=.02). The median ratio between thromboxane B2 and PGE2 decreased by 40% in the experimental group (P=.02) and increased by 18% in the control group (P=.14). No significant differences were observed in the median ratio between thromboxane B2 and 6-keto PGF1alpha in either group. No serious maternal or neonatal side effects of treatment occurred in either group. CONCLUSION: The administration of low daily doses of linoleic acid and calcium during the third trimester of pregnancy reduced the incidence of preeclampsia significantly in women at high risk, possibly by correcting the PGE2 levels.

Unstable, low-level transmission of malaria on the Colombian Pacific Coast.

The development of immune responses to malarial infection in inhabitants of endemic areas differs according to the level of exposure to the parasite. Adults living in a region where the level of malaria transmission is low (Colombia) have been shown to exhibit a similar response to each of the three regions of the circumsporozoite protein (the central repeated NANP region, and the flanking N- and C-termini). Conversely, donors exposed to a frequent sporozoite challenge in areas of high malaria transmission (Mali) exhibit antibodies predominantly to the NANP repeated domain. Malaria in the people of Zacarías, a community on the Pacific Coast of Colombia where malaria transmission is low and unstable, was the subject of the present study. Within a 9-year period, a negative correlation between rainfall and documented malaria cases was recorded for this area. Thick smears of blood samples of 319 individuals revealed that 8.5% had malarial infections. As most (67%) of the smear-positive cases were asymptomatic, it seems that, despite the low prevalence of malaria in this area, the establishment of clinical symptoms is attenuated, probably because of the acquisition of premunition. Within this region, the most commonly found Anopheles species (representing 61.1% of the mosquitoes caught) and that giving the highest monthly biting rate (4.0 bites/man) was An. neivai. Most (90%) of the human sera tested possessed antibodies to blood-stage forms of Plasmodium falciparum, and 18% had antibodies to sporozoites. More than half (58%) of the adults had been in contact with hepatitis B virus, 7.2% carried hepatitis B surface antigen, and syphilis was common but no subject was found to be seropositive for HIV. A better understanding of the dynamics of the different elements influencing malaria in areas of low, unstable transmission, such as the one described here, is essential for the design of new malaria-control strategies.

Synthetic polypeptides corresponding to the non-repeat regions from the circumsporozoite protein of Plasmodium falciparum: recognition by human T-cells and immunogenicity in owl monkeys.

Synthetic polypeptides encompassing the non-repeated regions of the circumsporozoite protein (CSP) of Plasmodium falciparum are very immunogenic in mice and are recognized by sera from donors living in regions where malaria is endemic, both in Africa and South America. Long polypeptides, encompassing the N- or C-terminal regions, have now been used to demonstrate peptide-specific T cells in donors living in an endemic area of Colombia. Although the N-terminal peptide (22-125) was recognized almost exclusively by donors from the endemic area, the patterns of recognition of the C-terminal peptide (289-390) in donors from endemic and non-endemic areas were similar and like the pattern with smaller peptides. The availability of the long polypeptides made it possible to compare T-cell responses to the non-repeated regions of the CSP with the presence of peptide-specific antibodies. No correlation was found and no antibodies were detected in donors from non-endemic regions. The long polypeptides also elicited strong antibody and T-cell responses in owl monkeys (Aotus lemurinus). The antibodies generated against the synthetic peptides in such monkeys also recognized sporozoites, the natural infective form of the parasite. The results emphasise the potential of the peptides tested as malaria-vaccine candidates. Not only are they recognized by humans at both the B- and T-cell level but they also elicit strong responses in monkeys and encompass several distinct T-cell epitopes, thus overcoming the limitations of specific, major-histocompatibility-complex restriction.