Flying under the radar - impact and factors influencing asymptomatic DENV infections.

The clinical outcome of DENV and other Flaviviruses infections represents a spectrum of severity that ranges from mild manifestations to severe disease, which can ultimately lead to death. Nonetheless, most of these infections result in an asymptomatic outcome that may play an important role in the persistent circulation of these viruses. Also, although little is known about the mechanisms that lead to these asymptomatic infections, they are likely the result of a complex interplay between viral and host factors. Specific characteristics of the infecting viral strain, such as its replicating efficiency, coupled with host factors, like gene expression of key molecules involved in the immune response or in the protection against disease, are among crucial factors to study. This review revisits recent data on factors that may contribute to the asymptomatic outcome of the world's widespread DENV, highlighting the importance of silent infections in the transmission of this pathogen and the immune status of the host.

Implementation of an infection control program with emphasis on cohorting to patients with carbapenemase-producing Enterobacteriaceae. The experience of 2 years in a tertiary teaching hospital in northern Portugal.

BACKGROUND: The emergence of carbapenemase-producing Enterobacterales (CPE) represents a major public health threat. Our purpose was to evaluate a surveillance and cohorting program implemented in patients infected or carriers of CPE. METHODS: A prospective registry of CPE carriers or infected patients was analyzed from October 2015 until December 2017. All inpatients presenting with CPE were included in a hospital cohort with dedicated healthcare staff and contact precaution measures. RESULTS: A total of 480 patients were identified, of which 15.8% (n = 76) were infected. Men comprised 56.7% of the cohort (n = 272) and 69.2% (n = 332) were elderly. About 46.3% (n = 222) had a previous hospital admission and 81.7% (n = 392) had at least 1 antibiotic course in the previous 90 days. There was a decline in infected patients in 2017. Periodic and admission screenings accounted for 63% and 74% of cases in 2016 and 2017, with increased detection rate comparing with contact/investigation screenings. In 2017, significantly fewer patients were identified outside the admission/point of prevalence screening (P = .009). In 2017 the proportion of invasive carbapenem-resistant Klebsiella pneumoniae amongst CPE in our center was below the national average (2016: 13.3% vs 5.2%; 2017: 6.6% vs 8.6%). A reduction of the consumption of carbapenems was also observed in 2017. CONCLUSION: The implementation of the program has increased the number of patients identified by the preventive method and stabilized the emergence of new CPE cases. Furthermore, the program cohort compared well with the national picture, with a lower number of infected patients and a lower proportion of carbapenem-resistant K pneumoniae in invasive specimens. These indicators reflect the added value of the CPE surveillance and cohorting program.

Transcranial direct current stimulation based metaplasticity protocols in working memory.

BACKGROUND: It has been already shown that delivering tDCS that are spaced by an interval alters its impact on motor plasticity. These effects can be explained, based on metaplasticity in which a previous modification of activity in a neuronal network can change the effects of subsequent interventions in the same network. But to date there is limited data assessing metaplasticity effects in cognitive functioning. OBJECTIVES: The aim of this study was to test several tDCS-based metaplasticity protocols in working memory (WM), by studying the impact of various interstimulation intervals in the performance of a 3-back task. METHODS: Fifteen healthy volunteers per experiment participated in this study. Experiments 1 and 2 tested an anodal tDCS-induced metaplasticity protocol (1 mA, 10 + 10') with 3 interstimulation intervals (10, 30, and 60 min). Experiment 3 determined the effects of a similar protocol-with a 10-min interval between two sessions of cathodal tDCS or anodal plus cathodal tDCS (1 mA, 10 + 10'). RESULTS: Two consecutive sessions of anodal tDCS delivered with a 10 min interval between them did not improve WM performance (P = .095). This effect remained the same if the interval was increased to 30 or 60 min. In contrast, when a 10 min interval was given between two consecutive cathodal tDCS sessions, performance in the 3 back task increased (P = .042). CONCLUSIONS: These results suggest that the polarity effects of tDCS on working memory are dependent on the previous level of activity of the recruited neural population.

Effects of the 2, 4-D herbicide on gills epithelia and liver of the fish Poecilia vivipara / Efeitos do herbicida 2, 4-D no epitélio das brânquias e no fígado do peixe Poecilia vivípara

The 2,4-dichlorophenoxyacetic acid, usually named 2,4-D is one of the most widely used herbicides in the world. Acute toxicity of 2,4-D herbicide was investigated through its effects on guppies (Poecilia vivipara Bloch et Schneider 1801). Fish were exposed to the herbicide at concentrations of 10, 20 and 40µl per liter of water for 24 hours to determine its effects on gills and liver epithelia. The estimated LC50 was 34.64µl of 2,4-D per liter of water. Histochemical analyses and Feulgen's reaction were conducted to detect glycoconjugates and DNA, respectively, in gills and liver epithelia. Histochemistry revealed qualitative variations of glycoconjugates present on mucous cells and granules. The four types of mucous cells contained neutral granules, acids, or both. Increasing amounts of syalomucins were observed from the control group to the group exposed to the highest concentration of 2,4-D, suggesting increased mucous viscosity and the formation of plaques that could inhibit gas exchange and osmoregulation. Lamellar fusion observed in the group exposed to 40µl of 2,4-D suggests a defense mechanism. Hepatocytes showed vacuolization in the 10 and 20µl/L groups. The 40 µl/L group showed normal hepatocytes as well as changed ones, many Ito cells, micronuclei, and nuclear swelling. These effects may be associated with toxicity or adaptative processes to cellular stress. The data from this study indicates the importance of assessing similar risks to aquatic species and suggests that Poecilia vivipara is an adequate biological model for analysis of environmental contamination.

A toxicidade aguda do herbicida 2,4-D foi investigada através dos efeitos no peixe Poecilia vivípara (Bloch et Schneider, 1801). Grupos de peixes foram expostos ao herbicida nas concentrações de 10, 20 e 40µl por litro de água, durante 24 horas. As brânquias e o fígado foram estudados. A concentração letal média (CL50) do herbicida para a espécie em questão foi de 34,64µl/l. Foram realizadas colorações histoquímicas e coloração de Feulgen para identificar glicoconjugados e DNA, respectivamente, nos tecidos acima citados. Os métodos histoquímicos revelaram os tipos de glicoconjugados presentes nas células mucosas e nos grânulos. Os quatro tipos de células mucosas apresentaram glicoconjugados neutros, ácidos, ou ambos em um mesmo tipo celular. Observou-se a presença crescente de sialomucinas do grupo controle até o grupo exposto a maior concentração de 2,4-D, sugerindo aumento da viscosidade do muco e, consequentemente, formação de placas que impedem as trocas gasosas e a osmorregulação. A fusão lamelar observada no grupo exposto a 40µl de 2,4-D sugere ser um mecanismo de defesa. Os hepatócitos apresentaram processo de vacuolização nos grupos 10 e 20µl/l. No grupo de 40µl/l, observou-se a presença de células de Ito, micronúcleos e hepatócitos normais e outros com edema nuclear. Este estudo indica a importância da avaliação de riscos semelhantes a espécies aquáticas e sugere a espécie Poecilia vivipara como modelo biológico adequado para análises de contaminação ambiental.

Malaria infections: what and how can mice teach us.

Malaria imposes a horrific public health burden - hundreds of millions of infections and millions of deaths - on large parts of the world. While this unacceptable health burden and its economic and social impact have made it a focal point of the international development agenda, it became consensual that malaria control or elimination will be difficult to attain prior to gain a better understanding of the complex interactions occurring between its main players: Plasmodium, the causative agent of disease, and its hosts. Practical and ethical limitations exist regarding the ability to carry out research with human subjects or with human samples. In this review, we highlight how rodent models of infection have contributed significantly during the past decades to a better understanding of the basic biology of the parasite, host response and pathogenesis.

Host-cell sensors for Plasmodium activate innate immunity against liver-stage infection.

Before they infect red blood cells and cause malaria, Plasmodium parasites undergo an obligate and clinically silent expansion phase in the liver that is supposedly undetected by the host. Here, we demonstrate the engagement of a type I interferon (IFN) response during Plasmodium replication in the liver. We identified Plasmodium RNA as a previously unrecognized pathogen-associated molecular pattern (PAMP) capable of activating a type I IFN response via the cytosolic pattern recognition receptor Mda5. This response, initiated by liver-resident cells through the adaptor molecule for cytosolic RNA sensors, Mavs, and the transcription factors Irf3 and Irf7, is propagated by hepatocytes in an interferon-α/ß receptor-dependent manner. This signaling pathway is critical for immune cell-mediated host resistance to liver-stage Plasmodium infection, which we find can be primed with other PAMPs, including hepatitis C virus RNA. Together, our results show that the liver has sensor mechanisms for Plasmodium that mediate a functional antiparasite response driven by type I IFN.

Simple flow cytometric detection of haemozoin containing leukocytes and erythrocytes for research on diagnosis, immunology and drug sensitivity testing.

BACKGROUND: Malaria pigment (haemozoin, Hz) has been the focus of diverse research efforts. However, identification of Hz-containing leukocytes or parasitized erythrocytes is usually based on microscopy, with inherent limitations. Flow cytometric detection of depolarized Side-Scatter is more accurate and its adaptation to common bench top flow cytometers might allow several applications. These can range from the ex-vivo and in-vitro detection and functional analysis of Hz-containing leukocytes to the detection of parasitized Red-Blood-Cells (pRBCs) to assess antimalarial activity. METHODS: A standard benchtop flow cytometer was adapted to detect depolarized Side-Scatter. Synthetic and Plasmodium falciparum Hz were incubated with whole blood and PBMCs to detect Hz-containing leukocytes and CD16 expression on monocytes. C5BL/6 mice were infected with Plasmodium berghei ANKA or P. berghei NK65 and Hz-containing leukocytes were analysed using CD11b and Gr1 expression. Parasitized RBC from infected mice were identified using anti-Ter119 and SYBR green I and were analysed for depolarized Side Scatter. A highly depolarizing RBC population was monitored in an in-vitro culture incubated with chloroquine or quinine. RESULTS: A flow cytometer can be easily adapted to detect depolarized Side-Scatter and thus, intracellular Hz. The detection and counting of Hz containing leukocytes in fresh human or mouse blood, as well as in leukocytes from in-vitro experiments was rapid and easy. Analysis of CD14/CD16 and CD11b/Gr1 monocyte expression in human or mouse blood, in a mixed populations of Hz-containing and non-containing monocytes, appears to show distinct patterns in both types of cells. Hz-containing pRBC and different maturation stages could be detected in blood from infected mice. The analysis of a highly depolarizing population that contained mature pRBC allowed to assess the effect of chloroquine and quinine after only 2 and 4 hours, respectively. CONCLUSIONS: A simple modification of a flow cytometer allows for rapid and reliable detection and quantification of Hz-containing leukocytes and the analysis of differential surface marker expression in the same sample of Hz-containing versus non-Hz-containing leukocytes. Importantly, it distinguishes different maturation stages of parasitized RBC and may be the basis of a rapid no-added-reagent drug sensitivity assay.

Accumulation of Plasmodium berghei-infected red blood cells in the brain is crucial for the development of cerebral malaria in mice.

Cerebral malaria is the most severe complication of human infection with Plasmodium falciparum. It was shown that Plasmodium berghei ANKA-induced cerebral malaria was prevented in 100% of mice depleted of CD8+ T cells 1 day prior to the development of neurological signs. However, the importance of parasites in the brains of these mice was never clearly investigated. Moreover, the relevance of this model to human cerebral malaria has been questioned many times, especially concerning the relative importance of leukocytes versus parasitized erythrocytes sequestered in the brain. Here, we show that mice protected from cerebral malaria by CD8+ T-cell depletion have significantly fewer parasites in the brain. Treatment of infected mice with an antimalarial drug 15 to 20 h prior to the estimated time of death also protected mice from cerebral malaria without altering the number of CD8+ T cells in the brain. These mice subsequently developed cerebral malaria with parasitized red blood cells in the brain. Our results clearly demonstrated that sequestration of CD8+ T cells in the brain is not sufficient for the development of cerebral malaria in C57BL/6 mice but that the concomitant presence of parasitized red blood cells is crucial for the onset of pathology. Importantly, these results also demonstrated that the experimental cerebral malaria model shares many features with human pathology and might be a relevant model to study its pathogenesis.

Cerebral malaria and the hemolysis/methemoglobin/heme hypothesis: shedding new light on an old disease.

Malaria causes more than 1 million deaths every year with cerebral malaria (CM) being a major cause of death in Sub-Saharan African children. The nature of the malaria-associated pathogenesis is complex and multi-factorial. A unified hypothesis involving sequestration of infected red blood cells, systemic host inflammatory response and hemostasis dysfunction has been proposed to explain the genesis of CM. In this review, we discuss the role of hemolysis, methemoglobin and free heme in CM, brought to light by our recent studies in mice as well as by other studies in humans.

Vaccination with live Plasmodium yoelii blood stage parasites under chloroquine cover induces cross-stage immunity against malaria liver stage.

Immunity to malaria has long been thought to be stage-specific. In this study we show that immunization of BALB/c mice with live erythrocytes infected with nonlethal strains of Plasmodium yoelii under curative chloroquine cover conferred protection not only against challenge by blood stage parasites but also against sporozoite challenge. This cross-stage protection was dose-dependent and long lasting. CD4(+) and CD8(+) T cells inhibited malaria liver but not blood stage. Their effect was mediated partially by IFN-gamma, and was completely dependent of NO. Abs against both pre-erythrocytic and blood parasites were elicited and were essential for protection against blood stage and liver stage parasites. Our results suggest that Ags shared by liver and blood stage parasites can be the foundation for a malaria vaccine that would provide effective protection against both pre-erythrocytic and erythrocytic asexual parasites found in the mammalian host.

Improved isolation of murine hepatocytes for in vitro malaria liver stage studies.

BACKGROUND: Primary hepatocyte cultures are a valuable tool for the understanding of cellular and molecular phenomena occurring during malaria liver stage. This paper describes an improved perfusion/dissociation procedure to isolate hepatocytes from mouse liver that is suitable for malaria studies and allows reproducible preparation of primary hepatocytes with consistent cell yields and controlled purity. RESULTS: This protocol is a detailed description of a technique to isolate and culture mouse hepatocytes and represents an improvement over previous descriptions of hepatocyte isolation for malaria studies, regarding three technical aspects: (1) dissociation reagents choice; (2) cell separation gradient and (3) cell purity control. Cell dissociation was optimized for a specific collagenase digestion media. The cell dissociation step was improved by using a three-layer discontinuous gradient. A cell purity check was introduced to monitor the expression of CD95 on hepatocytes using flow cytometry methods. CONCLUSION: The procedure described allows reproducible recovery of one to three million hepatocytes per preparation with cell purity of about 90% as determined by FACS analysis. Completion of the protocol is usually achieved in about four hours per preparation and pooling is suggested for multiple preparations of larger number of cells.

Recombinant human IFN-alpha inhibits cerebral malaria and reduces parasite burden in mice.

Most C57BL/6 mice infected i.p. with Plasmodium berghei ANKA (PbA) die between 7 and 14 days with neurologic signs, and the remainder die later (>15 days) with severe anemia. Daily i.p. injections of a recombinant human IFN-alpha (active on mouse cells) prevented death by cerebral malaria (87% deaths in the control mice vs 6% in IFN-alpha-treated mice). The mechanisms of this IFN-alpha protective effect were multiple. IFN-alpha-treated, PbA-infected mice showed 1) a marked decrease in the number of PbA parasites in the blood mediated by IFN-gamma, 2) less sequestered parasites in cerebral vessels, 3) reduced up-regulation of ICAM-1 expression in brain endothelial cells, 4) milder rise of blood levels of TNF, 5) increased levels of IFN-gamma in the blood resulting from an increased production by splenic CD8+ T cells, and 6) fewer leukocytes (especially CD8+ T cells) sequestered in cerebral vessels. On the other hand, IFN-alpha treatment did not affect the marked anemia observed in PbA-infected mice. Survival time in IFN-alpha-treated mice was further increased by performing three blood transfusions over consecutive days.

Regulatory CD4+ CD25+ Foxp3+ T cells expand during experimental Plasmodium infection but do not prevent cerebral malaria.

Pathogenic CD8+ T cells are implicated in the physiopathological mechanisms leading to experimental cerebral malaria (CM) in Plasmodium berghei ANKA (PbA) infected mice. Therefore, we hypothesised that in CM susceptible mice the neuropathology could be, at least in part, the result of an inefficient control of pathogenic effector T cells by CD4+ CD25+ Treg cells. Remarkably, the number of CD4+ CD25high T cells expressing Foxp3 increased in the spleen during the course of infection. These cells displayed an activated phenotype and consistent with that, CD4+ CD25high Treg cells isolated from PbA-infected mice showed an enhanced regulatory activity in vitro. Surprisingly, these cells do not migrate to the brain at the time of neurological symptoms as the conventional CD4+ T cells do. CM was not exacerbated in anti-CD25 treated mice when infected with PbA one month after treatment, even if splenic CD8+ T cells expressing CD69 increased in these mice. Taken together, these results show that P. berghei infection leads to an increase of the number of splenic CD4+ CD25high Treg cells exhibiting in vitro suppressive function, but they do not seem to be involved in vivo in the protection against CM.

Species-specific inhibition of cerebral malaria in mice coinfected with Plasmodium spp.

Recent epidemiological observations suggest that clinical evolution of Plasmodium falciparum infections might be influenced by the concurrent presence of another Plasmodium species, and such mixed-species infections are now known to occur frequently in residents of most areas of endemicity. We used mice infected with P. berghei ANKA (PbA), a model for cerebral malaria (CM), to investigate the influence of experimental mixed-species infections on the expression of this pathology. Remarkably, the development of CM was completely inhibited by the simultaneous presence of P. yoelii yoelii but not that of P. vinckei or another line of P. berghei. In the protected coinfected mice, the accumulation of CD8(+) T cells in the brain vasculature, a pivotal step in CM pathogenesis, was found to be abolished. Protection from CM was further found to be associated with species-specific suppression of PbA multiplication. These observations establish the concept of mixed Plasmodium species infections as potential modulators of pathology and open novel avenues to investigate mechanisms implicated in the pathogenesis of malaria.

Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment.

In this study we present the first systematic analysis of the immunity induced by normal Plasmodium yoelii sporozoites in mice. Immunization with sporozoites, which was conducted under chloroquine treatment to minimize the influence of blood stage parasites, induced a strong protection against a subsequent sporozoite and, to a lesser extent, against infected RBC challenges. The protection induced by this immunization protocol proved to be very effective. Induction of this protective immunity depended on the presence of liver stage parasites, as primaquine treatment concurrent with sporozoite immunization abrogated protection. Protection was not found to be mediated by the Abs elicited against pre-erythrocytic and blood stage parasites, as demonstrated by inhibition assays of sporozoite penetration or development in vitro and in vivo assays of sporozoite infectivity or blood stage parasite development. CD4(+) and CD8(+) T cells were, however, responsible for the protection through the induction of IFN-gamma and NO.

Hepatocyte growth factor and its receptor are required for malaria infection.

Plasmodium, the causative agent of malaria, must first infect hepatocytes to initiate a mammalian infection. Sporozoites migrate through several hepatocytes, by breaching their plasma membranes, before infection is finally established in one of them. Here we show that wounding of hepatocytes by sporozoite migration induces the secretion of hepatocyte growth factor (HGF), which renders hepatocytes susceptible to infection. Infection depends on activation of the HGF receptor, MET, by secreted HGF. The malaria parasite exploits MET not as a primary binding site, but as a mediator of signals that make the host cell susceptible to infection. HGF/MET signaling induces rearrangements of the host-cell actin cytoskeleton that are required for the early development of the parasites within hepatocytes. Our findings identify HGF and MET as potential targets for new approaches to malaria prevention.

Chemokine receptor CCR2 is not essential for the development of experimental cerebral malaria.

Infection with Plasmodium berghei ANKA induces cerebral malaria in susceptible mice. Brain-sequestered CD8(+) T cells are responsible for this pathology. We have evaluated the role of CCR2, a chemokine receptor expressed on CD8(+) T cells. Infected CCR2-deficient mice were as susceptible to cerebral malaria as wild-type mice were, and CD8(+) T-cell migration to the brain was not abolished.

On the pathogenic role of brain-sequestered alphabeta CD8+ T cells in experimental cerebral malaria.

Cerebral malaria (CM) develops in a small proportion of persons infected with Plasmodium falciparum and accounts for a substantial proportion of the mortality due to this parasite. The actual pathogenic mechanisms are still poorly understood, and in humans investigations of experimental CM are unethical. Using an established Plasmodium berghei-mouse CM model, we have investigated the role of host immune cells at the pathological site, the brain. We report in this study the detailed quantification and characterization of cells, which migrated and sequestered to the brain of mice with CM. We demonstrated that CD8(+) alphabeta T cells, which sequester in the brain at the time when neurological symptoms appear, were responsible for CM mortality. These observations suggest a mechanism which unifies disparate observations in humans.