Background Leishmania (L.) are intracellular protozoan parasites able to survive and replicate in the hostile phagolysosomal environment of infected macrophages. They cause leishmaniasis, a heterogeneous group of worldwide-distributed affections, representing a paradigm of neglected diseases that are mainly embedded in impoverished populations. To establish successful infection and ensure their own survival, Leishmania have developed sophisticated strategies to subvert the host macrophage responses. Despite a wealth of gained crucial information, these strategies still remain poorly understood. MicroRNAs (miRNAs), an evolutionarily conserved class of endogenous 22-nucleotide non-coding RNAs, are described to participate in the regulation of almost every cellular process investigated so far. They regulate the expression of target genes both at the levels of mRNA stability and translation; changes in their expression have a profound effect on their target transcripts.Methodology/Principal FindingsWe report in this study a comprehensive analysis of miRNA expression profiles in L. major-infected human primary macrophages of three healthy donors assessed at different time-points post-infection (three to 24 h). We show that expression of 64 out of 365 analyzed miRNAs was consistently deregulated upon infection with the same trends in all donors. Among these, several are known to be induced by TLR-dependent responses. GO enrichment analysis of experimentally validated miRNA-targeted genes revealed that several pathways and molecular functions were disturbed upon parasite infection. Finally, following parasite infection, miR-210 abundance was enhanced in HIF-1α-dependent manner, though it did not contribute to inhibiting anti-apoptotic pathways through pro-apoptotic caspase-3 regulation.Conclusions/SignificanceOur data suggest that alteration in miRNA levels likely plays an important role in regulating macrophage functions following L. major infection. These results could contribute to better understanding of the dynamics of gene expression in host cells during leishmaniasis.

Successful establishment of infection by bacterial pathogens requires fine-tuning of virulence-related genes. Quorum sensing (QS) is a global regulation process based on the synthesis of, detection of, and response to small diffusible molecules, called N-acyl-homoserine lactones (AHL), in gram-negative bacteria. In numerous species, QS has been shown to regulate genes involved in the establishment of pathogenic interactions with the host. Brucella melitensis produces N-dodecanoyl homoserine lactones (C 12 -HSL), which down regulate the expression of flagellar genes and of the virB operon (encoding a type IV secretion system), both of which encode surface virulence factors. A QS-related regulator, called VjbR, was identified as a transcriptional activator of these genes. We hypothesized that VjbR mediates the C 12 -HSL effects described above. vjbR alleles mutated in the region coding for the AHL binding domain were constructed to test this hypothesis. These alleles expressed in trans in a ⌬vjbR background behave as constitutive regulators both in vitro and in a cellular model of infection. Interestingly, the resulting B. melitensis strains, unable to respond to AHLs, aggregate spontaneously in liquid culture. Preliminary characterization of these strains showed altered expression of some outer membrane proteins and overproduction of a matrix-forming exopolysaccharide, suggesting for the first time that B. melitensis could form biofilms. Together, these results indicate that QS through VjbR is a major regulatory system of important cell surface structures of Brucella and as such plays a key role in host-pathogen interactions.Brucella spp. are gram-negative intracellular pathogens belonging to the ␣-2 proteobacteria group, like Agrobacterium, Rhizobium, and Rickettsia, which also live in close association with a eukaryotic host (46). Bacteria of the genus Brucella are the etiologic agents of brucellosis, a worldwide zoonosis affecting a broad range of mammals and triggering important economic losses (63). Three Brucella species, B. melitensis, B. abortus, and B. suis, are able to infect humans, causing a chronic, debilitating disease with severe, sometimes fatal outcomes.Brucellae are remarkably well adapted to the intracellular lifestyle, being able to invade and to survive within macrophages and nonprofessional phagocytes (17,51). This is one of the bases for the still poorly understood chronicity of brucellosis (26). This aptitude relies on the perturbation of vesicular trafficking and the creation of a unique intracellular replication niche derived from the endoplasmic reticulum (7,8).Brucella is confronted with very diverse environments and host defenses both in the extracellular milieu and inside host cells. It is thus expected that this pathogen has to sense external and internal signals to achieve successful adaptation throughout its infectious cycle. Among such systems, quorum sensing (QS), stringent response, and signal transduction through two-component regulators have been particularly well studied (fo...

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Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen Brucella melitensis, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of B. melitensis 16 M. A ΔvjbR mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of Brucella. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the B. melitensis 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of Brucella to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in Brucella and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of Brucella strains to the host environment.

The aims of this work were both to validate a sensitive and specific method to quantify tacrolimus (TAC) in liver biopsies after hepatic transplantation and to evaluate the predictive value of either tissue or blood TAC concentrations for rejection in 146 adult patients under a TAC-based immunosuppression. Trough blood levels were monitored daily during the hospital stay by immunoassay. Liver biopsies were routinely performed at day 7 posttransplantation. The tissue assay was developed by liquid chromatography-mass spectrometry. The limit of quantification was 5 pg/mg, with intra- and interassay precision ranging from 3.9% to 14.3% and 4.7% to 15.9%, respectively. The extraction efficiency was approximately 80%. TAC found in liver biopsies ranged from less than 5 up to 387 pg/mg. Blood TAC levels ranged from 2.7 to 19.3 ng/mL. Tissue levels displayed excellent correlation with liver histopathologic BANFF rejection score, whereas blood levels did not. Clinically significant rejections (BANFF scores> or = 6) were characterized by mean TAC tissue and blood concentration of 13.1 pg/mg and 7.6 ng/mL, respectively, whereas these mean values became, respectively, 74.9 pg/mg (P < 0.05) and 7.1 ng/mL (not significant) for nonclinically significant rejection episodes (BANFF < 6). In this study, hepatic tissue TAC concentrations were distributed in a wider range and displayed a significantly better correlation with the severity of the organ rejection than predose blood levels. A tissue TAC concentration less than 30 pg/mg is 89% sensitive and 98% specific to discriminate clinically significant cellular rejection. Further studies are required to better understand the factors affecting TAC distribution within liver tissue (such as carrier proteins and cytochrome genetic polymorphism, liver function, age, hepatic blood flow, type of organ transplanted, time posttransplantation) and to define its value in the treatment of liver allograft rejection.