A short history of innate immunity

Innate immunity refers to the mechanisms responsible for the first line of defense against pathogens, cancer cells and toxins. The innate immune system is also responsible for the initial activation of the body's specific immune response (adaptive immunity). Innate immunity was studied and further developed in parallel with adaptive immunity beginning in the first half of the 19th century and has been gaining increasing importance to our understanding of health and disease. In the present overview, we describe the main findings and ideas that contributed to the development of innate immunity as a continually expanding branch of modern immunology. We start with the toxicological studies by Von Haller and Magendie, in the late 18th and early 19th centuries, and continue with the discoveries in invertebrate immunity that supported the discovery and characterization of lipopolysaccharide (LPS) and pattern recognition receptors that led to the development of the pattern recognition and danger theory.

Intravenous whole blood transfusion results in faster recovery of vascular integrity and increased survival in experimental cerebral malaria

BACKGROUND Cerebral malaria is a lethal complication of Plasmodium falciparum infections in need of better therapies. Previous work in murine experimental cerebral malaria (ECM) indicated that the combination of artemether plus intraperitoneal whole blood improved vascular integrity and increased survival compared to artemether alone. However, the effects of blood or plasma transfusion administered via the intravenous route have not previously been evaluated in ECM. OBJECTIVES To evaluate the effects of intravenous whole blood compared to intravenous plasma on hematological parameters, vascular integrity, and survival in artemether-treated ECM. METHODS Mice with late-stage ECM received artemether alone or in combination with whole blood or plasma administered via the jugular vein. The outcome measures were hematocrit and platelets; plasma angiopoietin 1, angiopoietin 2, and haptoglobin; blood-brain barrier permeability; and survival. FINDINGS Survival increased from 54% with artemether alone to 90% with the combination of artemether and intravenous whole blood. Intravenous plasma lowered survival to 18%. Intravenous transfusion provided fast and pronounced recoveries of hematocrit, platelets, angiopoietins levels and blood brain barrier integrity. MAIN CONCLUSIONS The outcome of artemether-treated ECM was improved by intravenous whole blood but worsened by intravenous plasma. Compared to prior studies of transfusion via the intraperitoneal route, intravenous administration was more efficacious.

Oswaldo Gonçalves Cruz: the character, the scientist, the academician

Abstract The present work analyses some particular aspects of Oswaldo Cruz's unique biography, valuing his work, which was built along a successful physician and scientist professional trajectory and also as a courageous and fortunate formulator of public health policies and of fight strategies against the epidemics that seasonally affected the city of Rio de Janeiro at the beginning of the 20th century. The authors also dwell on his legacy as Head scientist and manager of the Institute that bears his name and became the template for experimental research and medicine in Brazil and the bedrock of the Fundação Oswaldo Cruz, one of the most important Brazilian Institutions devoted to teaching, research, development and production in health. This heritage made possible to overcome the existing dissensions between doctors and scientists to build a sanitary movement committed to the major health problems in Brazil. Finally, the paper explores some features of the character and reports some of his moments during his passage, as a Full Academician, at the Brazilian Academia Nacional de Medicina.

A morning with Louis Pasteur: a short history of the "clean hands" / Uma manhã com Louis Pasteur: uma breve história das "mãos limpas" / Una mañana con Louis Pasteur: una pequeña historia sobre las "manos limpias"

Abstract: This article examines the story of Louis Pasteur from the point of view of a classic movie presented at the Weekly Seminars of the "Oswaldo Cruz Institute", at the end of the 2017 activities. Although very old, the movie The Story of Louis Pasteur (Warner Bros., 1936) inspired spectators and gave rise to an energetic debate that led the authors to decide for publishing the comments of the Seminar Coordinator, the guest commentator and the audience. The movie communicates to the public the legacy of one of the greatest precursors of the public health history using also fictional characters. The article presents the reliable passages in Pasteur's biography and the fictional ones, without disrespecting the production of the creators of cinematographic work. The major merit of the movie, one of the first steps towards the policy of scientific diffusion, is to disclose the importance of vaccines and hand hygiene to prevent infectious diseases. The authors argue that the film-maker impeccably captured the scientist's tenacity in the relentless search for discoveries and Pasteur's idea that only persistent work can lead to rewarding results, remembering that the context created by previous researchers enabled Pasteur to establish new paradigms. Finally, the authors cite movie passages illustrating realities that are still in force: (i) the inertial resistance of science to new paradigms, illustrated by the medical-scientific community opposing to simple practices proposition, such as washing hands and boiling instruments, and (ii) the excessive confidence, and even arrogance, of some specialists, instead of serenity and humility that arise from committed study and accumulated knowledge.

Resumo: O artigo examina a história de Louis Pasteur do ponto de vista de um filme clássico que foi projetado durante os Seminários Semanais do Instituto Oswaldo Cruz no final das atividades de 2017. Embora bastante antigo, o filme The Story of Louis Pasteur (Warner Bros., 1936) inspirou os espectadores e deu lugar a um debate animado que levou os autores a decidir publicar os comentários da Coordenadora dos Seminários, do debatedor convidado e do público. O filme incorpora personagens reais e fictícios para comunicar ao público o legado de um dos maiores precursores da história da saúde pública. O artigo destaca os episódios reais da biografia de Pasteur e também os ficcionais, sem desmerecer o trabalho dos criadores cinematográficos. O principal mérito do filme, um dos primeiros passos de uma política de divulgação científica, é de revelar a importância das vacinas e da higiene das mãos na prevenção das doenças infecciosas. Os autores argumentam que o cineasta retratou primorosamente a tenacidade do grande cientista na busca incansável por descobertas, além de sua ideia de que somente o trabalho persistente pode levar a resultados recompensadores, lembrando que o contexto criado por pesquisadores anteriores permitiu que Pasteur estabelecesse paradigmas novos. Finalmente, os autores citam trechos do filme que ilustram realidades que persistem até os dias de hoje: (i) a tendência da ciência de resistir, por inércia, aos paradigmas novos, exemplificada pela oposição da comunidade de ciência médica à proposição de práticas simples como a lavagem de mãos e a fervura de instrumentos e (ii) a confiança excessiva, e até arrogância, de alguns especialistas, em vez da serenidade e da humildade que nascem da pesquisa dedicada e do conhecimento acumulado.

Resumen: Este artículo examina la historia de Louis Pasteur desde el punto de vista de una película clásica, presentada en los Seminarios Semanales del Instituto Oswaldo Cruz, al final de las actividades de 2017. A pesar de ser muy antigua, la película The Story of Louis Pasteur (Warner Bros., 1936) inspiró a los espectadores y provocó un animado debate que condujo a los autores a la decisión de publicar los comentarios de la Coordinadora de los Seminarios, así como los del comentarista invitado y de la audiencia. Utilizando también personajes de ficción, la película transmitía al público el legado de uno de los más grandes precursores de la historia de la salud pública. El artículo señala pasajes fidedignos en la biografía de Pasteur y ficticios, sin menoscabar el trabajo de los creadores cinematográficos. El mayor mérito de la película, uno de los primeros pasos hacia la política de divulgación científica, es revelar la importancia de las vacunas y de la higiene de las manos para prevenir enfermedades infecciosas. Los autores enfatizan que el director de la película capturó impecablemente la tenacidad del científico en su búsqueda sin descanso de descubrimientos, así como su idea de que solo un trabajo persistente podía conducir a resultados gratificantes, recordando que el contexto creado por investigadores previos permitieron a Pasteur establecer nuevos paradigmas. Finalmente, los autores citan pasajes de la película que ilustran realidades todavía muy vigentes: (i) la resistencia por inercia de la ciencia a nuevos paradigmas, ilustrados por la oposición de la comunidad médico-científica hacia la propuesta de prácticas simples, tales como lavarse las manos y hervir los instrumentos, así como (ii) la excesiva confianza, e incluso arrogancia, de algunos especialistas, en lugar de la serenidad y humildad que surgen del estudio realizado y el conocimiento acumulado.

Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria

BACKGROUND The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination. OBJECTIVE Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy. METHODS The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision. FINDINGS The linearity in SYBR™ Green and TaqMan™ systems was 106 and 102 copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR. CONCLUSION Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors.

Lack of quadruple and quintuple mutant alleles associated with sulfadoxine-pyrimethamine resistance in Plasmodium vivax isolates from Brazilian endemic areas

BACKGROUND AND OBJECTIVE Brazil is responsible for a large number of Plasmodium vivax cases in America. Given the emergence of P. vivax parasites resistant to chloroquine and the effectiveness of antifolates in vivax malaria treatment together with a correlation between mutations in P. vivax dhfr and dhps genes and SP treatment failure, the point mutations in these genes were investigated. METHODS Blood samples from 54 patients experiencing vivax malaria symptomatic episodes in the Amazonian Region were investigated. Genomic DNA was extracted using a DNA extraction kit (QIAGENTM). Nested polymerase chain reaction (PCR) amplification was carried out followed by Sanger sequencing to detect single nucleotide polymorphisms (SNPs). FINDINGS All tested isolates showed non-synonymous mutations in pvdhfr gene: 117N (54/54, 100%) and 58R (25/54, 46%). Double mutant allele 58R/117N (FRTNI, 28%) was the most frequent followed by triple mutant alleles (58R/117N/173L, FRTNL, 11%; 58R/61M/117N, FRMNI, 5% 117N/173L, FSTNL, 4%) and quadruple mutant allele (58R/61M/117N/173L, FRMNL, 2%). A single mutation was observed at codon C383G in pvdhps gene (SGKAV, 48%). CONCLUSION No evidence of molecular signatures associated with P. vivax resistance to SP was observed in the Brazilian samples.

Chloroquine and mefloquine resistance profiles are not related to the circumsporozoite protein (CSP) VK210 subtypes in field isolates of Plasmodium vivax from Manaus, Brazilian Amazon

BACKGROUND The central repetitive region (CRR) of the Plasmodium vivax circumsporozoite surface protein (CSP) is composed of a repetitive sequence that is characterised by three variants: VK210, VK247 and P. vivax-like. The most important challenge in the treatment of P. vivax infection is the possibility of differential response based on the parasite genotype. OBJECTIVES To characterise the CSP variants in P. vivax isolates from individuals residing in a malaria-endemic region in Brazil and to profile these variants based on sensitivity to chloroquine and mefloquine. METHODS The CSP variants were determined by sequencing and the sensitivity of the P. vivax isolates to chloroquine and mefloquine was determined by Deli-test. FINDINGS Although five different allele sizes were amplified, the sequencing results showed that all of the isolates belonged to the VK210 variant. However, we observed substantial genetic diversity in the CRR, resulting in the identification of 10 different VK210 subtypes. The frequency of isolates that were resistant to chloroquine and mefloquine was 11.8 and 23.8%, respectively. However, we did not observe any difference in the frequency of the resistant isolates belonging to the VK210 subtypes. MAIN CONCLUSION The VK210 variant is the most frequently observed in the studied region and there is significant genetic variability in the CRR of the P. vivax CSP. Moreover, the antimalarial drug sensitivity profiles of the isolates does not seem to be related to the VK210 subtypes.

Synthesis and molecular modelling studies of pyrimidinones and pyrrolo[34-d]-pyrimidinodiones as new antiplasmodial compounds

BACKGROUND Malaria is responsible for 429,000 deaths per year worldwide, and more than 200 million cases were reported in 2015. Increasing parasite resistance has imposed restrictions to the currently available antimalarial drugs. Thus, the search for new, effective and safe antimalarial drugs is crucial. Heterocyclic compounds, such as dihydropyrimidinones (DHPM), synthesised via the Biginelli multicomponent reaction, as well as bicyclic compounds synthesised from DHPMs, have emerged as potential antimalarial candidates in the last few years. METHODS Thirty compounds were synthesised employing the Biginelli multicomponent reaction and subsequent one-pot substitution/cyclisation protocol; the compounds were then evaluated in vitro against chloroquine-resistant Plasmodium falciparum parasites (W2 strain). Drug cytotoxicity in baseline kidney African Green Monkey cells (BGM) was also evaluated. The most active in vitro compounds were evaluated against P. berghei parasites in mice. Additionally, we performed an in silico target fishing approach with the most active compounds, aiming to shed some light into the mechanism at a molecular level. RESULTS The synthetic route chosen was effective, leading to products with high purity and yields ranging from 10-84%. Three out of the 30 compounds tested were identified as active against the parasite and presented low toxicity. The in silico study suggested that among all the molecular targets identified by our target fishing approach, Protein Kinase 3 (PK5) and Glycogen Synthase Kinase 3β (GSK-3β) are the most likely molecular targets for the synthesised compounds. CONCLUSIONS We were able to easily obtain a collection of heterocyclic compounds with in vitro anti-P. falciparum activity that can be used as scaffolds for the design and development of new antiplasmodial drugs.

On frogs, toxins and true friendship: an atypical case report

The authors report a series of events including the scientific interest for poisonous dendrobates of French Guiana, the human confrontation with the immensity of the evergreen rainforest, the fragility of the best-prepared individuals to a rough life, and the unique and very special manifestation of a solid friendship between two experts and enthusiasts of outdoor life. In the evergreen forest of South America, as in many other scientific field expeditions, everything may suddenly go wrong, and nothing can prepare researchers to accidents that may occur in a succession of uncontrollable errors once the first mistake is done. This is what happened during an expedition in search for dendrobates by an experienced forest guide and naturalist. The authors decided to report the story, considering that it deserved to be brought to the attention of those interested in venomous animals and toxins, in order to illustrate the potential danger of dealing with these organisms.

On frogs, toxins and true friendship: an atypical case report

Abstract The authors report a series of events including the scientific interest for poisonous dendrobates of French Guiana, the human confrontation with the immensity of the evergreen rainforest, the fragility of the best-prepared individuals to a rough life, and the unique and very special manifestation of a solid friendship between two experts and enthusiasts of outdoor life. In the evergreen forest of South America, as in many other scientific field expeditions, everything may suddenly go wrong, and nothing can prepare researchers to accidents that may occur in a succession of uncontrollable errors once the first mistake is done. This is what happened during an expedition in search for dendrobates by an experienced forest guide and naturalist. The authors decided to report the story, considering that it deserved to be brought to the attention of those interested in venomous animals and toxins, in order to illustrate the potential danger of dealing with these organisms.

Malaria in Brazil: what happens outside the Amazonian endemic region

Brazil, a country of continental proportions, presents three profiles of malaria transmission. The first and most important numerically, occurs inside the Amazon. The Amazon accounts for approximately 60% of the nation’s territory and approximately 13% of the Brazilian population. This region hosts 99.5% of the nation’s malaria cases, which are predominantly caused by Plasmodium vivax (i.e., 82% of cases in 2013). The second involves imported malaria, which corresponds to malaria cases acquired outside the region where the individuals live or the diagnosis was made. These cases are imported from endemic regions of Brazil (i.e., the Amazon) or from other countries in South and Central America, Africa and Asia. Imported malaria comprised 89% of the cases found outside the area of active transmission in Brazil in 2013. These cases highlight an important question with respect to both therapeutic and epidemiological issues because patients, especially those with falciparum malaria, arriving in a region where the health professionals may not have experience with the clinical manifestations of malaria and its diagnosis could suffer dramatic consequences associated with a potential delay in treatment. Additionally, because the Anopheles vectors exist in most of the country, even a single case of malaria, if not diagnosed and treated immediately, may result in introduced cases, causing outbreaks and even introducing or reintroducing the disease to a non-endemic, receptive region. Cases introduced outside the Amazon usually occur in areas in which malaria was formerly endemic and are transmitted by competent vectors belonging to the subgenus Nyssorhynchus (i.e., Anopheles darlingi, Anopheles aquasalis and species of the Albitarsis complex). The third type of transmission accounts for only 0.05% of all cases and is caused by autochthonous malaria in the Atlantic Forest, located primarily along the southeastern Atlantic Coast. They are caused by parasites that seem to be (or to be very close to) P. vivax and, in a less extent, by Plasmodium malariae and it is transmitted by the bromeliad mosquito Anopheles (Kerteszia) cruzii. This paper deals mainly with the two profiles of malaria found outside the Amazon: the imported and ensuing introduced cases and the autochthonous cases. We also provide an update regarding the situation in Brazil and the Brazilian endemic Amazon.

Vascular dysfunction as a target for adjuvant therapy in cerebral malaria

Cerebral malaria (CM) is a life-threatening complication of Plasmodium falciparum malaria that continues to be a major global health problem. Brain vascular dysfunction is a main factor underlying the pathogenesis of CM and can be a target for the development of adjuvant therapies for the disease. Vascular occlusion by parasitised red blood cells and vasoconstriction/vascular dysfunction results in impaired cerebral blood flow, ischaemia, hypoxia, acidosis and death. In this review, we discuss the mechanisms of vascular dysfunction in CM and the roles of low nitric oxide bioavailability, high levels of endothelin-1 and dysfunction of the angiopoietin-Tie2 axis. We also discuss the usefulness and relevance of the murine experimental model of CM by Plasmodium berghei ANKA to identify mechanisms of disease and to screen potential therapeutic interventions.

Apoptosis of non-parasitised red blood cells in Plasmodium yoelii malaria

Recently, while studying erythrocytic apoptosis during Plasmodium yoelii infection, we observed an increase in the levels of non-parasitised red blood cell (nRBC) apoptosis, which could be related to malarial anaemia. Therefore, in the present study, we attempted to investigate whether nRBC apoptosis is associated with the peripheral RBC count, parasite load or immune response. To this end, BALB/c mice were infected with P. yoelii 17XL and nRBC apoptosis, number of peripheral RBCs, parasitaemia and plasmatic levels of cytokines, nitric oxide and anti-RBC antibodies were evaluated at the early and late stages of anaemia. The apoptosis of nRBCs increased at the late stage and was associated with parasitaemia, but not with the intensity of the immune response. The increased percentage of nRBC apoptosis that was observed when anaemia was accentuated was not related to a reduction in peripheral RBCs. We conclude that nRBC apoptosis in P. yoelii malaria appears to be induced in response to a high parasite load. Further studies on malaria models in which acute anaemia develops during low parasitaemia are needed to identify the potential pathogenic role of nRBC apoptosis.

Genetic polymorphisms in the glutamate-rich protein of Plasmodium falciparum field isolates from a malaria-endemic area of Brazil

The genetic diversity displayed by Plasmodium falciparum, the most deadly Plasmodium species, is a significant obstacle for effective malaria vaccine development. In this study, we identified genetic polymorphisms in P. falciparum glutamate-rich protein (GLURP), which is currently being tested in clinical trials as a malaria vaccine candidate, from isolates found circulating in the Brazilian Amazon at variable transmission levels. The study was performed using samples collected in 1993 and 2008 from rural villages situated near Porto Velho, in the state of Rondônia. DNA was extracted from 126 P. falciparum-positive thick blood smears using the phenol-chloroform method and subjected to a nested polymerase chain reaction protocol with specific primers against two immunodominant regions of GLURP, R0 and R2. Only one R0 fragment and four variants of the R2 fragment were detected. No differences were observed between the two time points with regard to the frequencies of the fragment variants. Mixed infections were uncommon. Our results demonstrate conservation of GLURP-R0 and limited polymorphic variation of GLURP-R2 in P. falciparum isolates from individuals living in Porto Velho. This is an important finding, as genetic polymorphisms in B and T-cell epitopes could have implications for the immunological properties of the antigen.

Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region has been found in the N-terminal R0 region of the protein. Herein, we describe the antiplasmodial activity of anti-GLURP antibodies present in the sera from individuals naturally exposed to malaria in a Brazilian malaria-endemic area. The anti-R0 antibodies showed a potent inhibitory effect on the growth of P. falciparum in vitro, both in the presence (ADCI) and absence (GI) of monocytes. The inhibitory effect on parasite growth was comparable to the effect of IgGs purified from pooled sera from hyperimmune African individuals. Interestingly, in the ADCI test, higher levels of tumour necrosis factor alpha (TNF-α) were observed in the supernatant from cultures with higher parasitemias. Our data suggest that the antibody response induced by GLURP-R0 in naturally exposed individuals may have an important role in controlling parasitemia because these antibodies are able to inhibit the in vitro growth of P. falciparum with or without the cooperation from monocytes. Our results also indicate that TNF-α may not be relevant for the inhibitory effect on P. falciparum in vitro growth.

Monoclonal auto-antibodies and sera of autoimmune patients react with Plasmodium falciparum and inhibit its in vitro growth

The relationship between autoimmunity and malaria is not well understood. To determine whether autoimmune responses have a protective role during malaria, we studied the pattern of reactivity to plasmodial antigens of sera from 93 patients with 14 different autoimmune diseases (AID) who were not previously exposed to malaria. Sera from patients with 13 different AID reacted against Plasmodium falciparum by indirect fluorescent antibody test with frequencies varying from 33-100 percent. In addition, sera from 37 AID patients were tested for reactivity against Plasmodium yoelii 17XNL and the asexual blood stage forms of three different P. falciparum strains. In general, the frequency of reactive sera was higher against young trophozoites than schizonts (p < 0.05 for 2 strains), indicating that the antigenic determinants targeted by the tested AID sera might be more highly expressed by the former stage. The ability of monoclonal auto-antibodies (auto-Ab) to inhibit P. falciparum growth in vitro was also tested. Thirteen of the 18 monoclonal auto-Ab tested (72 percent), but none of the control monoclonal antibodies, inhibited parasite growth, in some cases by greater than 40 percent. We conclude that autoimmune responses mediated by auto-Ab may present anti-plasmodial activity.

Chemoresistance of Plasmodium falciparum and Plasmodium vivax parasites in Brazil: consequences on disease morbidity and control

In Brazil, malaria still remains a clinically important febrile syndrome for local populations and travelers, occurring mostly in the Amazon Basin. This review aims to report the main efforts employed to control this disease since the 1940s and the emergence of Plasmodium falciparum and Plasmodium vivax chemoresistance to chloroquine and sulphadoxine-pyrimethamine among other drugs. Additionally, in vivo, in vitro and molecular studies as well as malaria chemoresistance consequences on disease morbidity and policy treatment guidelines were commented.