Edgar Badell

Disruption of the langerin/CD207 Gene Abolishes Birbeck Granules without a Marked Loss of Langerhans Cell Function

ABSTRACT Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin − / − mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin − / − mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin − / − LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin − / − mice were not impaired in their capacity to process native OVA protein for I-A b -restricted presentation to CD4+ T lymphocytes or for H-2K b -restricted cross-presentation to CD8+ T lymphocytes. langerin − / − mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin − / − and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin − / − C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.

Evaluation of the Andromas Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Aerobically Growing Gram-Positive Bacilli

ABSTRACT Matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

A murine DC-SIGN homologue contributes to early host defense against Mycobacterium tuberculosis.

The C-type lectin dendritic cell−specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) mediates the innate immune recognition of microbial carbohydrates. We investigated the function of this molecule in the host response to pathogens in vivo, by generating mouse lines lacking the DC-SIGN homologues SIGNR1, SIGNR3, and SIGNR5. Resistance to Mycobacterium tuberculosis was impaired only in SIGNR3-deficient animals. SIGNR3 was expressed in lung phagocytes during infection, and interacted with M. tuberculosis bacilli and mycobacterial surface glycoconjugates to induce secretion of critical host defense inflammatory cytokines, including tumor necrosis factor (TNF). SIGNR3 signaling was dependent on an intracellular tyrosine-based motif and the tyrosine kinase Syk. Thus, the mouse DC-SIGN homologue SIGNR3 makes a unique contribution to protection of the host against a pulmonary bacterial pathogen.

Plasmidic versus Insertional Cloning of Heterologous Genes in Mycobacterium bovis BCG: Impact on In Vivo Antigen Persistence and Immune Responses

ABSTRACT Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag(p26) genes from the simian immunodeficiency virus (SIV) SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopy-replicating vector, and the expression levels of both nef and gag in the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of the nef-gag operon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, the nef-gag operon was inserted site specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes even though a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude as that of the replicative rBCG strain containing several copies of the genes.

TLR4- and TLR2-Mediated B Cell Responses Control the Clearance of the Bacterial Pathogen, Leptospira interrogans

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira interrogans that are transmitted by asymptomatic infected rodents. Leptospiral lipoproteins and LPS have been shown to stimulate murine cells via TLRs 2 and 4. Host defense mechanisms remain obscure, although TLR4 has been shown to be involved in clearing Leptospira. In this study, we show that double (TLR2 and TLR4) knockout (DKO) mice rapidly died from severe hepatic and renal failure following Leptospira inoculation. Strikingly, the severe proinflammatory response detected in the liver and kidney from Leptospira-infected DKO mice appears to be independent of MyD88, the main adaptor of TLRs. Infection of chimeric mice constructed with wild-type and DKO mice, and infection of several lines of transgenic mice devoid of T and/or B lymphocytes, identified B cells as the crucial lymphocyte subset responsible for the clearance of Leptospira, through the early production of specific TLR4-dependent anti-Leptospira IgMs elicited against the leptospiral LPS. We also found a protective tissue compartmentalized TLR2/TLR4-mediated production of IFN-γ by B and T lymphocytes, in the liver and kidney, respectively. In contrast, the tissue inflammation observed in Leptospira-infected DKO mice was further characterized to be mostly due to B lymphocytes in the liver and T cells in the kidney. Altogether these findings demonstrate that TLR2 and TLR4 play a key role in the early control of leptospirosis, but do not directly trigger the inflammation induced by pathogenic Leptospira.

Multilocus Sequence Typing Identifies Evidence for Recombination and Two Distinct Lineages of Corynebacterium diphtheriae

ABSTRACT We describe the development of a multilocus sequence typing (MLST) scheme for Corynebacterium diphtheriae, the causative agent of the potentially fatal upper respiratory disease diphtheria. Global changes in diphtheria epidemiology are highlighted by the recent epidemic in the former Soviet Union (FSU) and also by the emergence of nontoxigenic strains causing atypical disease. Although numerous techniques have been developed to characterize C. diphtheriae, their use is hindered by limited portability and, in some instances, poor reproducibility. One hundred fifty isolates from 18 countries and encompassing a period of 50 years were analyzed by multilocus sequence typing (MLST). Strain discrimination was in accordance with previous ribotyping data, and clonal complexes associated with disease outbreaks were clearly identified by MLST. The data produced are portable, reproducible, and unambiguous. The MLST scheme described provides a valuable tool for monitoring and characterizing endemic and epidemic C. diphtheriae strains. Furthermore, multilocus sequence analysis of the nucleotide data reveals two distinct lineages within the population of C. diphtheriae examined, one of which is composed exclusively of biotype belfanti isolates and the other of multiple biotypes.

Mycobacterium bovis BCG-infected neutrophils and dendritic cells cooperate to induce specific T cell responses in humans and mice

Neutrophils are increasingly thought to modulate dendritic cell (DC) functions. We investigated the role of the neutrophil‐DC partnership in the response to Mycobacterium bovis BCG–the vaccine used against tuberculosis. We compared neutrophil‐DC crosstalk in humans and mice, searching for functional differences. In both species, neutrophils captured fluorescent BCG‐enhanced green fluorescent protein (EGFP) and were more phagocytic than DC. Non‐apoptotic BCG‐infected neutrophils clustered with immature DC, establishing intimate contacts with dendrites, at which fluorescent intact bacilli were observed. Physical interactions between neutrophils and DC were required for DC activation. Human BCG‐infected DC produced interleukin (IL)‐10, an inhibitory cytokine, whereas DC exposed to BCG‐infected neutrophils produced low to undetectable amounts of the cytokine. Mouse BCG‐infected neutrophils induced sustained IL‐2 production by DC. Human DC exposed to BCG‐infected neutrophils stimulated recall T cell reactivity from vaccinated donors. Mouse DC infected with recombinant ovalbumin (OVA)‐producing BCG (rBCGova) elicited proliferation of TCR‐OVA‐transgenic CD4 and CD8 T cells. Moreover, exposing DC to rBCGova‐infected neutrophils enhanced OVA presentation. Thus, in mice and humans, neutrophils help DC to cross‐present BCG antigens to T cells. Our results suggest that this “ménage à trois” involving neutrophils, DC and T cells plays a major role in the immune response to BCG.

Mycobacterium bovis Bacillus Calmette-Guérin Vaccination Mobilizes Innate Myeloid-Derived Suppressor Cells Restraining In Vivo T Cell Priming Via IL-1R–Dependent Nitric Oxide Production

Early immune response to the largely used Mycobacterium bovis bacillus Calmette-Guérin (BCG) intradermal vaccine remains ill defined. Three days after BCG inoculation into the mouse ear, in addition to neutrophils infiltrating skin, we observed CD11b+Ly-6CintLy-6G− myeloid cells. Neutrophil depletion markedly enhanced their recruitment. These cells differed from inflammatory monocytes and required MyD88-dependent BCG-specific signals to invade skin, whereas neutrophil influx was MyD88 independent. Upon BCG phagocytosis, CD11b+Ly-6CintLy-6G− cells produced NO, which required the IL-1 receptor. Despite NO production, they were unable to kill BCG or the nonpathogenic Mycobacterium smegmatis. However, they markedly impaired T cell priming in the draining lymph node. Their elimination by all-trans retinoid acid treatment increased the number of IFN-γ–producing CD4 T cells. Thus, BCG vaccination recruits innate myeloid-derived suppressor cells, akin to mouse tumor-infiltrating cells. These propathogenic cells dampen the early T cell response and might facilitate BCG persistence.

Human malaria in immunocompromised mice: new in vivo model for chemotherapy studies.

ABSTRACT We have recently designed a new Plasmodium falciparummouse model and documented its potential for the study of immune effector mechanisms. In order to determine its value for drug studies, we evaluated its response to existing antimalarial drugs compared to that observed in humans. Immunocompromised BXN (bg/bg xid/xid nu/nu) mice were infected with either the sensitive NF54 strain or the multiresistant T24 strain and then treated with chloroquine, quinine, mefloquine, or dihydroartemisinin. A parallelism was observed between previously reported human responses and P. falciparum-parasitized human red blood cell (huRBC)–BXN mouse responses to classical antimalarial drugs, measured in terms of speed of decrease in parasitemia and of morphological alterations of the parasites. Mice infected with the sensitive strain were successfully cured after treatment with either chloroquine or mefloquine. In contrast, mice infected with the multiresistant strain failed to be cured by chloroquine or quinine but thereafter responded to dihydroartemisinin treatment. The speed of parasite clearance and the morphological alterations induced differed for each drug and matched previously reported observations, hence stressing the relevance of the model. These data thus suggest that P. falciparum-huRBC–BXN mice can provide a valuable in vivo system and should be included in the short list of animals that can be used for the evaluation of P. falciparum responses to drugs.

Protection against tuberculosis induced by oral prime with Mycobacterium bovis BCG and intranasal subunit boost based on the vaccine candidate Ag85B-ESAT-6 does not correlate with circulating IFN-γ producing T-cells

The potent IFN-gamma inducing fusion antigen Ag85B-ESAT-6 (85B6) is a lead subunit candidate to improve current vaccination against Mycobacterium tuberculosis (Mtb). The recombinant M. bovis BCG strain Myc3504 was constructed to secrete 85B6. It was based on commercial BCG strain Moreau Rio de Janeiro (BCG(MoWT)) which remains available for human oral administration. Myc 3504 induced higher levels of 85B6-specific IFN-gamma circulating T-cells as compared to BCG(MoWT). A novel needle-free mucosal immunization regimen combining oral prime with Myc3504 or BCG(MoWT) with intranasal boost with LTK-63-adjuvanted 85B6 was compared to subcutaneous prime-boost immunization. Strikingly whereas parenteral immunization induced sustained levels of 85B6-specific IFN-gamma secretion by circulating T-cells, mucosal regimens induced barely detectable IFN-gamma. Despite this, mice and guinea pigs immunized with the mucosal regimens were as efficiently protected against aerosol Mtb challenge as parenterally immunized animals. After Mtb challenge, anti-ESAT-6 IFN-gamma responses sharply increased in non-vaccinated mice as a hallmark of infection. Parenterally immunized mice that controlled Mtb infection, displayed anti-ESAT-6 IFN-gamma responses as high as non-immunized infected mice, compromising the possible use of ESAT-6 as a diagnostic tool. Interestingly, in mucosally immunized mice that were equally protected, post-challenge ESAT-6-specific IFN-gamma T-cell response remained low.