In Vivo Hemozoin Kinetics after Clearance of Plasmodium berghei Infection in Mice

Hemozoin (Hz) is released into the blood stream after rupture of infected red blood cells (iRBCs) at the end of each parasite replication cycle. This free Hz is ingested by circulating and resident phagocytes. The presence of Hz in tissues after clearance of infection has been previously reported. Still, little is known about the kinetics of Hz in vivo, during and after Plasmodium infection. It is particularly important to understand Hz kinetics after malaria infections as it has been reported that Hz is associated with impairment of immune functions, including possible consequences for coinfections. Indeed, if Hz remains biologically active for prolonged periods of time inside immunocompetent cells, the potential consequences of such accumulation and presence to the immune system should be clarified. Here, using several independent methods to assess the presence of Hz, we report the long-term in vivo kinetics of Hz in diverse organs in a murine model of malaria infection

Malaria and tuberculosis co-infection : role for hemozoin immunosuppression?

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2014Malaria and tuberculosis (TB) endemic regions overlap considerably, especially in sub-Saharan Africa. Although it is very likely that co-infections occur in these regions, not much is known about malaria-TB co-infections in humans, and how the interplay between these two infections might affect the prognosis of co-infected individuals. Furthermore, multiple Plasmodium infections will likely result in accumulation of malaria pigment (hemozoin) in host organs. Hemozoin was first thought of as an inert waste product resulting from hemoglobin digestion. However, several studies have associated the presence of hemozoin with host immunosuppression. Thus, the subject of this thesis was the study of Malaria-Tuberculosis co-infections with a particular focus on a possible role for hemozoin induced immunosuppression. For this, protocols for hemozoin production and characterization were first established. Then, in vitro studies to investigate how hemozoin ingestion affected cellular functions of human peripheral blood mononuclear cells (PBMC) were performed. For the first time hemozoin effects at the single cell level were investigated by flow-cytometry, using a novel method developed by us, which uses Side Scatter Depolarization for hemozoin detection. Following in vitro studies, we investigated hemozoin dynamics in host tissues of two murine models, post-malaria clearance. Hemozoin deposition in host organs was evaluated both by flow-cytometry, as well as by observation of histological preparations. Finally, the murine model was used to evaluate the susceptibility of Plasmodium infected mice to subsequent tuberculosis infection. Tuberculosis infection was evaluated during acute malaria, and during chronic Plasmodium infection. Results from in vitro studies suggested that hemozoin impaired cell functions and that these effects could be propagated to non-hemozoin containing cells. Hemozoin-induced impairment decreased microbicidal activity, as shown by higher mycobacterial load in these cells. Our investigation of hemozoin kinetics in host organs revealed that hemozoin is dynamic within and between host organs, with very little signs of it being eliminated over time. Malaria-tuberculosis co-infection in vivo demonstrated that mice presenting with acute malaria, have a poor prognosis when co-infected with tuberculosis. Mice infected with tuberculosis during chronic malaria did not become as sick as mice during acute infection however, they still exhibited symptoms of malaria-induced anaemia. Thus, in our model of co-infection hemozoin did not seem to contribute significantly to immunosuppression of the host nor to increased susceptibility to tuberculosis infection. Overall, we present a novel method for the detection of hemozoin that allowed functional investigation of hemozoin-containing and non-hemozoin containing leukocytes at the single cell level, in the same sample. This might be useful for further studies using leukocytes from malarious patients, who usually only have a few percent of these cells in circulation. We also provide evidence for poor prognosis of malaria-tuberculosis co-infection. Furthermore, we demonstrated that following a malaria episode, hemozoin deposition in host organs did not seem to contribute to immunosuppression of the host nor to increased susceptibility to tuberculosis infection by M. bovis BCG. However, we observed that hemozoin-containing macrophages participated in granuloma formation in response to tuberculosis infection. This might prove relevant in the context of a tuberculosis infection by the virulent strain M. tuberculosis, in which tighter control by immune cells is necessary to prevent tuberculosis dissemination.As regiões endémicas da malária e tuberculose sobrepõem-se consideravelmente, especialmente na África sub-Saariana. Embora seja bastante provável que co-infecções entre malária e tuberculose ocorram nestas regiões, pouco se sabe sobre a sua incidência, ou como a sua interação poderá afectar o prognóstico de indivíduos co-infectados. Episódios consecutivos de malária poderão levar à acumulação do pigmento malárico (hemozoína) nos órgãos do hospedeiro. Inicialmente, a hemozoína era considerada um sub-produto inerte, resultante da digestão da hemoglobina pelo parasita. No entanto, vários estudos têm associado a presença da hemozoína com imunossupressão do hospedeiro. Assim, o tema desta tese foi o estudo da co-infecção malária-tuberculose, com especial foco no possível papel da hemozoína na indução de imunossupressão no hospedeiro. Neste projecto, começou por estabelecer-se protocolos para produção e caracterização de hemozoína. Consequentemente, foram realizados estudos in vitro para investigar o impacto da ingestão de hemozoína nas funções celulares de células mononucleadas do sangue periférico (PBMC) humanos. Pela primeira vez, os efeitos da hemozoína foram investigados por citometria de fluxo, utilizando um novo método desenvolvido por nós, que utiliza a detecção da despolarização da luz para a identificação de células que contêm hemozoína. Após esta caracterização in vitro, a dinâmica da hemozoína em tecidos do hospedeiro após a infecção por Plasmodium foi investigada, usando dois modelos de ratinho. A acumulação de hemozoína em órgãos do hospedeiro foi avaliada por citometria de fluxo, e pela observação de preparações histológicas, ao longo do tempo. Finalmente, o modelo de murino foi usado para avaliar a susceptibilidade de ratinhos com malária a uma subsequente infecção por tuberculose. A infecção por tuberculose foi avaliada durante as fases aguda e crónica de malária. Os resultados dos estudos in vitro sugerem que a hemozoína compromete funções celulares importantes e que estes efeitos podem ser propagados a células que não têm hemozoína. A inibição de funções celulares levou à redução da capacidade microbicida, como mostrado pelo aumento da carga bacteriana nestas células. Relativamente à avaliação da cinética da hemozoína nos tecidos do hospedeiro, este estudo revelou que a hemozoína tem uma distribuição dinâmica tanto dentro do mesmo órgão, como entre IV diferentes órgãos do hospedeiro. Também se observou que, ao longo do tempo, a hemozoína não parece ser eliminada dos órgãos do hospedeiro. Os ensaios in vivo de co-infecção malária-tuberculose demonstraram que ratinhos com malária desenvolvem uma doença fulminante, quando co-infectados com tuberculose. Este fenótipo agravado não foi observado em ratinhos infectados com tuberculose durante uma infecção crónica de malária, ainda que estes apresentassem sinais de anemia associada à malária. Tendo em conta os resultados obtidos com o nosso modelo, a hemozoína não parece contribuir de forma significativa para a imunossupressão do hospedeiro nem para um aumento de susceptibilidade à infecção por tuberculose. Em resumo, este trabalho apresenta um novo método para a detecção de hemozoína. Este método permitiu a investigação de funções celulares em leucócitos com e sem hemozoína na mesma amostra, ao nível celular. Esta metodologia poderá ser aplicada noutros estudos, nomeadamente na análise de leucócitos com hemozoína de doentes com malária, cujas percentagens são normalmente baixas. Os resultados apresentados nesta tese também sugerem que co-infecções de malária-tuberculose resultam numa deterioração rápida do hospedeiro. No entanto, como observado durante a infecção crónica de malária, a deposição de hemozoína nos órgãos do hospedeiro não parece contribuir para um aumento da imunossupressão do hospedeiro, não tendo sido observada uma maior susceptibilidade à infecção por tuberculose. Ainda assim, foi observado que macrófagos com elevado conteúdo em hemozoína contribuem significativamente para a formação de granulomas, em resposta à infecção por M. bovis BCG. Isto pode ser relevante no contexto de uma infecção de tuberculose pela estirpe virulenta M. tuberculosis, em que é necessário um controlo mais restrito por células do sistema imunitário para prevenir a disseminação da tuberculose.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/61174/2009, projecto PIC/IC/83214/2007

Is flow cytometry better in counting malaria pigment-containing leukocytes compared to microscopy?

<p>Abstract</p> <p>Background</p> <p>Detection of malaria pigment (or haemozoin; Hz)-containing leukocytes may have prognostic relevance in malaria; however, studies reported conflicting results, with microscopic counts suggestive of being inaccurate and imprecise.</p> <p>Methods</p> <p>Numbers of Hz-containing leukocytes from a malaria patient obtained with a flow cytometer counting 50.000 gated events were compared with thin film microscopy as applied under field conditions.</p> <p>Results</p> <p>Flow cytometry identified 5.8% Hz-containing monocytes and 1.8% Hz-containing neutrophils. The microscopic examination yielded 10% and 13% of Hz-containing monocytes, as well as 0% and 0.5% of Hz-containing neutrophils for observers one and two, respectively.</p> <p>Conclusion</p> <p>Novel, robust and affordable cytometric methods should be evaluated in the field as they may assist in utilizing Hz-containing cells as clinically useful parameter.</p

The case for hypervirulence through gene deletion in Mycobacterium tuberculosis.

Deletion of genes in a pathogen is commonly associated with a reduction in its ability to cause disease. However, some rare cases have been described in the literature whereby deletion of a gene results in an increase in virulence. Recently, there have been several reports of hypervirulence resulting from gene deletion in Mycobacterium tuberculosis. Here, we explore this phenomenon in the context of the interaction between the pathogen and the host response

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

<p>Abstract</p> <p>Background</p> <p>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 <it>ex-vivo </it>and <it>in-vitro </it>detection and functional analysis of Hz-containing leukocytes to the detection of parasitized Red-Blood-Cells (pRBCs) to assess antimalarial activity.</p> <p>Methods</p> <p>A standard benchtop flow cytometer was adapted to detect depolarized Side-Scatter. Synthetic and <it>Plasmodium falciparum </it>Hz were incubated with whole blood and PBMCs to detect Hz-containing leukocytes and CD16 expression on monocytes. C5BL/6 mice were infected with <it>Plasmodium berghei </it>ANKA or <it>P. berghei </it>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 <it>in-vitro </it>culture incubated with chloroquine or quinine.</p> <p>Results</p> <p>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 <it>in-vitro </it>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.</p> <p>Conclusions</p> <p>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 <it>versus </it>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.</p

A two-step strategy for the complementation of M. tuberculosis mutants

The sequence of Mycobacterium tuberculosis, completed in 1998, facilitated both the development of genomic tools, and the creation of a number of mycobacterial mutants. These mutants have a wide range of phenotypes, from attenuated to hypervirulent strains. These phenotypes must be confirmed, to rule out possible secondary mutations that may arise during the generation of mutant strains. This may occur during the amplification of target genes or during the generation of the mutation, thus constructing a complementation strain, which expresses the wild-type copy of the gene in the mutant strain, becomes necessary. In this study we have introduced a two-step strategy to construct complementation strains using the Ag85 promoter. We have constitutively expressed dosR and have shown dosR expression is restored to wild-type level

A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis

The Mycobacterium tuberculosis TetR-type regulator Rv3574 has been implicated in pathogenesis as it is induced in vivo, and genome-wide essentiality studies show it is required for infection. As the gene is highly conserved in the mycobacteria, we deleted the Rv3574 orthologue in Mycobacterium smegmatis (MSMEG_6042) and used real-time quantitative polymerase chain reaction and microarray analyses to show that it represses the transcription both of itself and of a large number of genes involved in lipid metabolism. We identified a conserved motif within its own promoter (TnnAACnnGTTnnA) and showed that it binds as a dimer to 29 bp probes containing the motif. We found 16 and 31 other instances of the motif in intergenic regions of M. tuberculosis and M. smegmatis respectively. Combining the results of the microarray studies with the motif analyses, we predict that Rv3574 directly controls the expression of 83 genes in M. smegmatis, and 74 in M. tuberculosis. Many of these genes are known to be induced by growth on cholesterol in rhodococci, and palmitate in M. tuberculosis. We conclude that this regulator, designated elsewhere as kstR, controls the expression of genes used for utilizing diverse lipids as energy sources, possibly imported through the mce4 system

Effects of Lipid-Lowering Drugs on Vancomycin Susceptibility of Mycobacteria.

Tuberculosis is still a cause of major concern, partly due to the emergence of multidrug-resistant strains. New drugs are therefore needed. Vancomycin can target mycobacteria with cell envelope deficiency. In this study, we used a vancomycin susceptibility assay to detect drugs hampering lipid synthesis in Mycobacterium bovis BCG and in Mycobacterium tuberculosis We tested three drugs already used to treat human obesity: tetrahydrolipstatin (THL), simvastatin, and fenofibrate. Only vancomycin and THL were able to synergize on M. bovis BCG and on M. tuberculosis, although mycobacteria could also be inhibited by simvastatin alone. Lipid analysis allowed us to identify several lipid modifications in M. tuberculosis H37Rv treated with those drugs. THL treatment mainly reduced the phthiocerol dimycocerosate (PDIM) content in the mycobacterial cell wall, providing an explanation for the synergy, since PDIM deficiency has been related to vancomycin susceptibility. Proteomic analysis suggested that bacteria treated with THL, in contrast to bacteria treated with simvastatin, tried to recover, inducing, among other reactions, lipid synthesis. The combination of THL and vancomycin should be considered a promising solution in developing new strategies to treat multidrug-resistant tuberculosis.SCOPUS: ar.jinfo:eu-repo/semantics/publishe