Malaria transmission blocking immunity and sexual stage vaccines for interrupting malaria transmission in Latin America

Malaria is a vector-borne disease that is considered to be one of the most serious public health problems due to its high global mortality and morbidity rates. Although multiple strategies for controlling malaria have been used, many have had limited impact due to the appearance and rapid dissemination of mosquito resistance to insecticides, parasite resistance to multiple antimalarial drug, and the lack of sustainability. Individuals in endemic areas that have been permanently exposed to the parasite develop specific immune responses capable of diminishing parasite burden and the clinical manifestations of the disease, including blocking of parasite transmission to the mosquito vector. This is referred to as transmission blocking (TB) immunity (TBI) and is mediated by specific antibodies and other factors ingested during the blood meal that inhibit parasite development in the mosquito. These antibodies recognize proteins expressed on either gametocytes or parasite stages that develop in the mosquito midgut and are considered to be potential malaria vaccine candidates. Although these candidates, collectively called TB vaccines (TBV), would not directly stop malaria from infecting individuals, but would stop transmission from infected person to non-infected person. Here, we review the progress that has been achieved in TBI studies and the development of TBV and we highlight their potential usefulness in areas of low endemicity such as Latin America.

Population genetic analysis of large sequence polymorphisms in Plasmodium falciparum blood-stage antigens

Plasmodium falciparum, the causative agent of human malaria, invades host erythrocytes using several proteins on the surface of the invasive merozoite, which have been proposed as potential vaccine candidates. Members of the multi-gene PfRh family are surface antigens that have been shown to play a central role in directing merozoites to alternative erythrocyte receptors for invasion. Recently, we identified a large structural polymorphism, a 0.58 Kb deletion, in the C-terminal region of the PfRh2b gene, present at a high frequency in parasite populations from Senegal. We hypothesize that this region is a target of humoral immunity. Here, by analyzing 371 P. falciparum isolates we show that this major allele is present at varying frequencies in different populations within Senegal, Africa, and throughout the world. For allelic dimorphisms in the asexual stage antigens, Msp-2 and EBA-175, we find minimal geographic differentiation among parasite populations from Senegal and other African localities, suggesting extensive gene flow among these populations and/or immune-mediated frequency-dependent balancing selection. In contrast, we observe a higher level of inter-population divergence (as measured by F(st)) for the PfRh2b deletion, similar to that observed for SNPs from the sexual stage Pfs45/48 loci, which is postulated to be under directional selection. We confirm that the region containing the PfRh2b polymorphism is a target of humoral immune responses by demonstrating antibody reactivity of endemic sera. Our analysis of inter-population divergence suggests that in contrast to the large allelic dimorphisms in EBA-175 and Msp-2, the presence or absence of the large PfRh2b deletion may not elicit frequency-dependent immune selection, but may be under positive immune selection, having important implications for the development of these proteins as vaccine candidates. (C) 2009 Elsevier B.V. All rights reserved.

Molecular and morphological data support the existence of a sexual cycle in species of the genus Paracoccidioides

The genus Paracoccidioides includes the thermodimorphic species Paracoccidioides brasiliensis and P. lutzii, both of which are etiologic agents of paracoccidioidomycosis, a systemic mycosis that affects humans in Latin America. Despite the common occurrence of a sexual stage among closely related fungi, this has not been observed with Paracoccidioides species, which have thus been considered asexual. Molecular evolutionary studies revealed recombination events within isolated populations of the genus Paracoccidioides, suggesting the possible existence of a sexual cycle. Comparative genomic analysis of all dimorphic fungi and Saccharomyces cerevisiae demonstrated the presence of conserved genes involved in sexual reproduction, including those encoding mating regulators such as MAT, pheromone receptors, pheromone-processing enzymes, and mating signaling regulators. The expression of sex-related genes in the yeast and mycelial phases of both Paracoccidioides species was also detected by realtime PCR, with nearly all of these genes being expressed preferentially in the filamentous form of the pathogens. In addition, the expression of sex-related genes was responsive to the putative presence of pheromone in the supernatants obtained from previous cocultures of strains of two different mating types. In vitro crossing of isolates of different mating types, discriminated by phylogenetic analysis of the α-box (MAT1-1) and the high-mobility-group (HMG) domain (MAT1-2), led to the identification of the formation of young ascocarps with constricted coiled hyphae related to the initial stage of mating. These genomic and morphological analyses strongly support the existence of a sexual cycle in species of the genus Paracoccidioides. © 2013, American Society for Microbiology.

Transmission immunity in malaria: reflections on the underlying immune mechanisms during natural infections and following artificial immunization

Malaria transmission-blocking immunity has been studied in natural malaria infections in man, during infections in animals and following artificial immunization of animals with sexual stage malaria parasites. Effective immunity, which prevents infectivity of a malarial infection to mosquitoes, has been observed under all of these circumstances. Two general types of effector mechanism have been identified. One is an antibody mediated mechanism which acts against the extracellular sexual stages of the parasite within the midgut of a blood feeding mosquito. The other is a cytokine mediated mechanism which inactivates the gametocytes of the parasites while in the circulation of the vertebrate host. Both effects have been observed during natural infections and following artificial immunization. The basis of induction of transmission-blocking immunity, including the nature of the memory for such immunity, however, may be very different in different host/parasite systems and during natural infection of following artificial immunization. Following artificial immunization a strong immune memory for transmission blocking immunity has been observed in animal systems. By contrast, following natural infections in man immune memory for transmission blocking immunity has been found to be weak and short lived if it occurs at all. It is suggested that the immunogens which induce natural transmission blocking immunity may be CD4+ independent.

Effects of genetic alterations in arbuscular mycorrhizal fungi on plant growth and on their response to a change of host

Abstract :The majority of land plants form the symbiosis with arbuscular mycorrhizal fungi (AMF). The AM symbiosis has existed for hundreds of millions of years but little or no specificity seems to have co- evolved between the partners and only about 200 morphospecies of AMF are known. The fungi supply the plants most notably with phosphate in exchange for carbohydrates. The fungi improve plant growth, protect them against pathogens and herbivores and the symbiosis plays a key role in ecosystem productivity and plant diversity. The fungi are coenocytic, grow clonally and no sexual stage in their life cycle is known. For these reasons, they are presumed ancient asexuals. Evidence suggests that AMF contain populations of genetically different nucleotypes coexisting in a common cytoplasm. Consequently, the nucleotype content of new clonal offspring could potentially be altered by segregation of nuclei at spore formation and by genetic exchange between different AMF. Given the importance of AMF, it is surprising that remarkably little is known about the genetics and genomics of the fungi.The main goal of this thesis was to investigate the combined effects of plant species differences and of genetic exchange and segregation in AMF on the symbiosis. This work showed that single spore progeny can receive a different assortment of nucleotypes compared to their parent and compared to other single spore progeny. This is the first direct evidence that segregation occurs in AMF. We then showed that both genetic exchange and segregation can lead to new progeny that differentially alter plant growth compared to their parents. We also found that genetic exchange and segregation can lead to different development of the fungus during the establishment of the symbiosis. Finally, we found that a shift of host species can differentially alter the phenotypes and genotypes of AMF progeny obtained by genetic exchange and segregation compared to their parents.Overall, this study confirms the multigenomic state of the AMF Glomus intraradices because our findings are possible only if the fungus contains genetically different nuclei. We demonstrated the importance of the processes of genetic exchange and segregation to produce, in a very short time span, new progeny with novel symbiotic effects. Moreover, our results suggest that different host species could affect the fate of different nucleotypes following genetic exchange and segregation in AMF, and can potentially contribute to the maintenance of genetic diversity within AMF individuals. This work brings new insights into understanding how plants and fungi have coevolved and how the genetic diversity in AMF can be maintained. We recommend that the intra-ir1dividual AMF diversity and these processes should be considered in future research on this symbiosis.Résumé :La majorité des plantes terrestres forment des symbioses avec les champignons endomycorhiziens arbusculaires (CEA). Cette symbiose existe depuis plusieurs centaines de millions d'années mais peu ou pas de spécificité semble avoir co-évoluée entre les partenaires et seulement 200 morpho-espèces de CEA sont connues. Le champignon fournit surtout aux plantes du phosphate en échange de carbohydrates. Le champignon augmente la croissance des plantes, les protège contre des pathogènes et herbivores et la symbiose joue un rôle clé dans la productivité des écosystèmes et de la diversité des plantes. Les CEA sont coenocytiques, se reproduisent clonalement et aucune étape sexuée n'est connue dans leur cycle de vie. Pour ces raisons, ils sont présumés comme anciens asexués. Des preuves suggèrent que les CEA ont des populations de nucleotypes différents coexistant dans un cytoplasme commun. Par conséquent, le contenu en nucleotype des nouveaux descendants clonaux pourrait être altéré par la ségrégation des noyaux lors de la fonnation des spores et par l'échange génétique entre différents CEA. Etant donné l'importance des CEA, il est surprenant que si peu soit connu sur la génétique et la génomique du champignon.Le principal but de cette thèse a été d'étudier les effets combinés de différentes espèces de plantes et des mécanismes d'échange génétique et de ségrégation chez les CEA sur la symbiose. Ce travail a montré que chaque nouvelle spore produite pouvait recevoir un assortiment différent de noyaux comparé au parent ou comparé à d'autres nouvelles spores. Ceci est la première preuve directe que la ségrégation peut se produire chez les CEA. Nous avons ensuite montré qu'à la fois l'échange génétique et la ségrégation pouvaient mener à de nouveaux descendants qui altèrent différemment la croissance des plantes, comparé à leurs parents. Nous avons également trouvé que l'échange génétique et la ségrégation pouvaient entraîner des développements différents du champignon pendant l'établissement de la symbiose. Pour finir, nous avons trouvé qu'un changement d'espèce de l'hôte pouvait altérer différemment les phénotypes et génotypes des descendants issus d'échange génétique et de ségrégation, comparé à leurs parents.Globalement, cette étude confirme l'état multigénomique du CEA Glumus intraradices car nous résultats sont possibles seulement si le champignon possède des noyaux génétiquement différents. Nous avons démontrés l'importance des mécanismes d'échange génétique et de ségrégation pour produire en très peu de temps de nouveaux descendants ayant des effets symbiotiques nouveaux. De plus, nos résultats suggèrent que différentes espèces de plantes peuvent agir sur le devenir des nucleotypes après l'échange génétique et la ségrégation chez les CEA, et pourraient contribuer à la maintenance de la diversité génétique au sein d'un même CEA. Ce travail apporte des éléments nouveaux pour comprendre comment les plantes et les champignons ont coévolué et comment la diversité génétique chez les CEA peut être maintenue. Nous recommandons de considérer la diversité génétique intra-individuelle des CEA et ces mécanismes lors de futures recherches sur cette symbiose.

Recombination in Glomus intraradices, a supposed ancient asexual arbuscular mycorrhizal fungus

Background: Arbuscular mycorrhizal fungi (AMF) are important symbionts of most plant species, promoting plant diversity and productivity. This symbiosis is thought to have contributed to the early colonisation of land by plants. Morphological stasis over 400 million years and the lack of an observed sexual stage in any member of the phylum Glomeromycota led to the controversial suggestion of AMF being ancients asexuals. Evidence for recombination in AMF is contradictory. Results: We addressed the question of recombination in the AMF Glomus intraradices by sequencing 11 polymorphic nuclear loci in 40 morphologically identical isolates from one field. Phylogenetic relationships among genotypes showed a reticulate network pattern providing a rationale to test for recombination. Five statistical tests predicted multiple recombinant regions in the genome of a core set of isolates. In contrast, five clonal lineages had fixed a large number of differences. Conclusion: Our data show that AMF from one field have undergone recombination but that clonal lineages coexist. This finding has important consequences for understanding AMF evolution, co-evolution of AMF and plants and highlights the potential for commercially introduced AMF inoculum recombining with existing local populations. Finally, our results reconcile seemingly contradictory studies on whether AMF are clonal or form recombining populations.

Genetic divergence among and within Colletotrichum lindemuthianum races assessed by RAPD

Genetic divergence within and among races of Colletotrichum lindemuthianum was determined using RAPD markers. In addition to the different races of the fungus three isolates of the sexual stage of Colletotrichum lindemuthianum (Glomerella cingulata f.sp. phaseoli) were included in this study. The band patterns generated using 11 primers produced 133 polymorphic bands. The polymorphic bands were used to determine genetic divergence among and within the pathogen races. The isolates analyzed were divided into six groups with 0.75 relative similarity. Group VI, formed by three isolates of the sexual phase of Colletotrichum lindemuthianum, was the most divergent. Races previously determined using differential cultivars did not correlate with the results obtained using RAPD markers.

A microestrutura e o desenvolvimento dos órgãos genitais masculinos de preás (Galea spixii) criados em cativeiro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Variação periódica da triiodotironina (T3) plasmática e sua ação na reprodução induzida do matrinxã, Brycon cephalus (Günther, 1869) em cativeiro

O matrinxã, Brycon cephalus, espécie nativa oriunda da Bacia Amazônica, apresenta características adequadas para a piscicultura. Entretanto, trata-se de peixe reofílico, sendo necessário manejo adequado para induzir à reprodução. O objetivo deste trabalho foi caracterizar o perfil da triiodotironina (T3) plasmática no matrinxã, durante 16 meses (outubro/97 a janeiro/99), relacionando-o com a maturação sexual, além de testar a ação do T3 associado ao extrato pituitário de carpa na reprodução induzida da espécie. O experimento foi conduzido no Centro Nacional de Pesquisa de Peixes Tropicais - CEPTA, Pirassununga, SP, e no Departamento de Morfologia e Fisiologia Animal, da Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal - UNESP. Foram amostrados, mensalmente, 8 a 12 peixes, de ambos os sexos, totalizando 173 animais, de onde retirou-se sangue para dosagem do T3 plasmático. As gônadas foram removidas para análise histológica com identificação do sexo e determinação do estádio de maturação. Analisaram-se testículos e ovários de 161 peixes em corte transversal, tendo predominado machos (63,35%) e o estádio sexual imaturo entre ambos. A concentração plasmática de T3 foi maior de dezembro a janeiro, para machos e fêmeas, coincidindo com o período de maior atividade reprodutiva da espécie e maiores temperaturas da água. em janeiro/99, reprodutores de matrinxã foram induzidos com extrato de pituitária de carpa (EPC) associado à administração de T3 (20 mg/kg em 0,1 mL de suspensão oleosa). Os resultados sugeriram que o T3 atuou sinergicamente à gonadotropina do extrato hipofisário e que o tratamento agudo de triiodotironina com o EPC pode estimular o eixo hipotálamo-hipófise-ovário. O tempo de eclosão das larvas provenientes das fêmeas tratadas com o T3 foi menor e o crescimento inicial e a sobrevivência dessas larvas, maiores.

Caracterização anatômica foliar de clones de seringueira (Hevea spp.) visando resistência ao Microcyclus ulei

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Malva sylvestris, novo hospedeiro de Puccinia heterospora no Brasil

Observou-se a ocorrência de ferrugem em Malva sylvestris, causada por Puccinia heterospora, no Município de Garanhuns (PE). Esta espécie fúngica é microcíclica e apresenta apenas o estádio sexual, com a produção de teliósporos, com morfologia variada, podendo produzir, em menor número, teliósporos bicelulares do tipo Puccinia e, predominantemente teliósporos unicelulares do tipo Uromyces (mesosporos). A produção abundante de mesosporos é uma das características morfológicas mais marcantes da espécie. Essa ferrugem possui ampla distribuição geográfica, infectando plantas pertencentes à família Malvaceae. Este representa o primeiro registro dessa ferrugem sobre o gênero Malva no Brasil. Uma amostra de referência foi depositada no Herbário Dimitri Sucre Benjamin do Instituto de Pesquisas Jardim Botânico do Rio de Janeiro (JBRJ-RB). São apresentadas a descrição da morfologia do fungo com base na amostra coletada, ilustrações e comentários sobre a distinção entre essa espécie e outras morfologicamente semelhantes. Palavras-chave: ferrugem, Malvaceae, Pucciniales, teliósporos.

Malaria’s Eve: Evidence of a recent population bottleneck throughout the world populations of Plasmodium falciparum

We have analyzed DNA sequences from world-wide geographic strains of Plasmodium falciparum and found a complete absence of synonymous DNA polymorphism at 10 gene loci. We hypothesize that all extant world populations of the parasite have recently derived (within several thousand years) from a single ancestral strain. The upper limit of the 95% confidence interval for the time when this most recent common ancestor lived is between 24,500 and 57,500 years ago (depending on different estimates of the nucleotide substitution rate); the actual time is likely to be much more recent. The recent origin of the P. falciparum populations could have resulted from either a demographic sweep (P. falciparum has only recently spread throughout the world from a small geographically confined population) or a selective sweep (one strain favored by natural selection has recently replaced all others). The selective sweep hypothesis requires that populations of P. falciparum be effectively clonal, despite the obligate sexual stage of the parasite life cycle. A demographic sweep that started several thousand years ago is consistent with worldwide climatic changes ensuing the last glaciation, increased anthropophilia of the mosquito vectors, and the spread of agriculture. P. falciparum may have rapidly spread from its African tropical origins to the tropical and subtropical regions of the world only within the last 6,000 years. The recent origin of the world-wide P. falciparum populations may account for its virulence, as the most malignant of human malarial parasites.

Molecular markers reveal cryptic sex in the human pathogen Coccidioides immitis.

Coccidioides immitis, cause of a recent epidemic of "Valley fever" in California, is typical of many eukaryotic microbes in that mating and meiosis have yet to be reported, but it is not clear whether sex is truly absent or just cryptic. To find out, we have undertaken a population genetic study using PCR amplification, screening for single-strand conformation polymorphisms, and direct DNA sequencing to find molecular markers with nucleotide-level resolution. Both population genetic and phylogenetic analyses indicate that C. immitis is almost completely recombining. To our knowledge, this study is the first to find molecular evidence for recombination in a fungus for which no sexual stage has yet been described. These results motivate a directed search for mating and meiosis and illustrate the utility of single-strand conformation polymorphism and sequencing with arbitrary primer pairs in molecular population genetics.

The Perspectives of Gay, Bisexual, and Queer Adolescent Males with Parent-Child Sex Communication

Problem: Gay, bisexual, and queer (GBQ) adolescent males are disproportionately affected by negative sexual health outcomes compared to their heterosexual counterparts. Their sex education needs are not sufficiently addressed in the home and the larger ecological systems. The omission of their sex education needs at a time when they are forming a sexual identity during adolescence compels GBQ males to seek information in unsupervised settings. Evidence-based interventions aimed at ensuring positive sexual health outcomes through sex communication cannot be carried out with these youth as research on how parents and GBQ males discuss sex in the home has been largely uninvestigated.

Methods: This naturalistic qualitative study focused on the interpretive reports of 15- to 20-year-old GBQ males’ discussions about sex-related topics with their parents. From a purposive sample of 30 male adolescents who self-identified as GBQ, participants who could recall at least one conversation about sex with their parents were recruited for one-time interviews and card sorts. This strategy revealed, using Bronfenbrenners’ Bioecological Theory, their perceptions about sex communication in the context of their reciprocal relationship and the ecological systems that GBQ males and their parents navigate.

Results: Parents received poor ratings as sex educators, were generally viewed as not confident in their communication approach, and lacked knowledge about issues pertinent to GBQ sons. Nevertheless, participants viewed parents as their preferred source of sex information and recognized multiple functions of sex communication. The value placed by GBQ youth on sex communication underscores their desire to ensure an uninterrupted parent-child relationship in spite of their GBQ sexual orientation. For GBQ children, inclusive sex communication is a proxy for parental acceptance.

Results show that the timing, prompts, teaching aids, and setting of sex communication for this population are similar to what has been reported with heterosexual samples. However, most GBQ sons rarely had inclusive guidance about sex and sexuality that matched their attraction, behavior, and identities. Furthermore, the assumption of heterosexuality resulted in the early awareness of being different from their peers which led them to covertly search for sex information. The combination of assumed heterosexuality and their early reliance on themselves for applicable information is a missed parental opportunity to positively impact the health of GBQ sons. More importantly, due to the powerful reach of new media, there is a critical period of maximum receptiveness that has been identified which makes inclusive sex communication paramount in the pre-sexual stage for this population. Our findings also indicate that there are plenty of opportunities for systemic improvements to meet this population’s sexual education needs.

A microestrutura e o desenvolvimento dos órgãos genitais masculinos de preás (Galea spixii) criados em cativeiro

O preá do semiárido nordestino (Galea spixii) é um roedor pertencente à família Caviidae. São encontrados nas regiões da Caatinga e do Cerrado Brasileiro e se reproduz ao longo do ano, apresentando um período de gestação de 48 dias e uma ninhada de 2 a 4 crias. O objetivo deste estudo foi caracterizar histologicamente os componentes estruturais dos órgãos genitais de preás machos relacionando com a evolução cronológica destes órgãos na espécie. Foram utilizados para análise animais ao nascimento e aos 15, 30, 45, 60, 75, 90, 105, 120 e aos 150 dias de idade. Fragmentos do epidídimo, ducto deferente, pênis e glândulas sexuais acessórias foram coletados, fixados e processados para descrição em microscopia de luz. O epidídimo apresentou epitélio colunar simples e em cada fase sexual notou-se diferença quanto ao tamanho do lúmen tubular e à presença de espermatozóides no lúmen aos 45 dias de idade. O epitélio do ducto deferente no preá mostrou-se pseudo-estratificado colunar com crescente presença de estereocilios com o avanço da idade. A glândula vesicular no preá apresentou uma mucosa com pregueamento variado, de acordo com a fase do desenvolvimento sexual. A próstata mostrou-se pouco desenvolvida, com lúmen pequeno nos preás ao nascimento e aos 15 dias de idade; aos 45 dias mostrou-se com um pregueamento do epitélio variável. Os órgãos genitais masculinos do preá passaram por transformações morfológicas no decorrer da idade e com o desenvolvimento sexual, isso colaborou para a determinação do início da fase da puberdade, que na espécie em estudo foi aos 45 dias de idade.