Prospecting potential links between PRRSV infection susceptibility of alveolar macrophages and other respiratory infectious agents present in conventionally reared pigs.

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is one of the main component of the porcine respiratory disease complex (PRDC), which strongly impact the pig production. Although PRRSV is often considered as a primary infection that eases subsequent respiratory coinfections, the possibility that other PRDC components may facilitate PRRSV infection has been largely overlooked. The main cellular targets of PRRSV are respiratory macrophages among them alveolar macrophages (AM) and pulmonary intravascular macrophages (PIM). AM, contrarily to PIM, are directly exposed to the external respiratory environment, among them co-infectious agents. In order to explore the possibility of a co-infections impact on the capacity of respiratory macrophages to replicate PRRSV, we proceed to in vitro infection of AM and PIM sampled from animals presenting different sanitary status, and tested the presence in the respiratory tract of these animals of the most common porcine respiratory pathogens (PCV2, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma floculare, Pasteurella multocida, Bordetella bronchiseptica, Streptoccocus suis). In this exploratory study with a limited number of animals, no statistic differences were observed between AM and PIM susceptibility to in vitro PRRSV infection, nor between AM coming from animals presenting very contrasting respiratory coinfection loads.

The respiratory DC/macrophage network at steady-state and upon influenza infection in the swine biomedical model.

Human and mouse respiratory tracts show anatomical and physiological differences, which will benefit from alternative experimental models for studying many respiratory diseases. Pig has been recognized as a valuable biomedical model, in particular for lung transplantation or pathologies such as cystic fibrosis and influenza infection. However, there is a lack of knowledge about the porcine respiratory immune system. Here we segregated and studied six populations of pig lung dendritic cells (DCs)/macrophages (Mθs) as follows: conventional DCs (cDC) 1 and cDC2, inflammatory monocyte-derived DCs (moDCs), monocyte-derived Mθs, and interstitial and alveolar Mθs. The three DC subsets present migratory and naive T-cell stimulation capacities. As observed in human and mice, porcine cDC1 and cDC2 were able to induce T-helper (Th)1 and Th2 responses, respectively. Interestingly, porcine moDCs increased in the lung upon influenza infection, as observed in the mouse model. Pig cDC2 shared some characteristics observed in human but not in mice, such as the expression of FCɛRIα and Langerin, and an intra-epithelial localization. This work, by unraveling the extended similarities of the porcine and human lung DC/Mθ networks, highlights the relevance of pig, both as an exploratory model of DC/Mθ functions and as a model for human inflammatory lung pathologies.

[Prognostic value of S-100B protein plasma measurement after cardiac arrest]. / Intérêt pronostique du dosage de la protéine S-100B sérique au décours d'un arrêt cardiaque en milieu extrahospitalier : données préliminaires françaises.

S-100B protein is selectively synthesized by glial cells, and is released in biological fluids after acute brain damage. We analyzed initial levels and evolution of plasma S-100B protein concentrations after resuscitated cardiopulmonary arrest (CPA). S-100B levels were determined in 27 subjects at the time of CPA (H0) then 12, 24 and 48 h after resuscitation. Initial levels of S-100B and kinetics revealed that: 1) 95% the of subjects with a concentration of protein S-100B greater than 0.80 microg/L at H0 did not survive; 2) 62% of subjects with a concentration of protein S-100B lower than 0.80 microg/L at H0 survived; 3) 100% of subjects with a protein S-100B level lower than 0.80 microg/L at H0 and whose evolution kinetics of S-100B levels showed a decrease survived; 4) 100% of the subjects whose S-100B levels increased from H12 died. In summary, this study suggests that the threshold of 0.80 microg/L for S-100B plasma levels at H0 could be predictive for the outcome of the CPA, when associated with the kinetic study of S-100B plasma concentration.

Differential expression of the efflux pumps P-glycoprotein and multidrug resistance-associated protein in human monocyte-derived dendritic cells.

P-glycoprotein (P-gp), an ATP-binding cassette (ABC) drug efflux pump, has been recently shown to play an important role in the physiology of Langherans cells, a subtype of dendritic cells (DC) found in the skin. The present study was designed to investigate expression and activity of P-gp and of multidrug resistance-associated protein (MRP), another ABC efflux pump sharing numerous substrates with P-gp, in human monocyte-derived DC. Immunolabeling experiments and dye efflux assays indicated that such cells displayed elevated levels of MRP activity and expression when compared to those present in parental monocytes. Generation of DC from monocytes in the presence of the MRP inhibitor indomethacin did not, however, alter the capacity of DC to stimulate allogeneic T cells proliferation in mixed lymphocyte reaction. In addition, indomethacin did not inhibit the up-regulation of the CD1a, a marker occurring during the differentiation of monocytes into DC. In contrast to that of MRP, functional expression of P-gp was not detected in monocyte-derived DC. Such antigen presenting cells that constitute a promising tool for antitumor vaccinal therapy therefore display differential expression of the efflux pumps P-gp and MRP.

HLA-DR mediated cell death is associated with, but not induced by TNF-alpha secretion in APC.

Tumor necrosis factor alpha (TNFalpha) is a pleiotropic cytokine involved in inflammatory responses which can trigger both cell apoptosis and cell activation. In antigen presenting cells (APC), TNFalpha increased antigen presentation, notably by up-regulation of HLA class II expression. In addition to their role in antigen presentation, HLA-DR molecules transduce intracellular signals which lead to cytokine up-regulation or cell death. We have previously observed that the susceptibility of APC to HLA-DR mediated apoptosis increase throughout their maturation. We therefore investigated the relationship between TNFalpha production and susceptibility to HLA-DR-mediated apoptosis of different APC. The hematopoietic progenitor cell line (KG1), monocytic cell line (THP-1), monocyte-derived dendritic cell (DC), and B-lymphoid cell line (Raji) have been studied. We report that apoptosis susceptibility and spontaneous TNFalpha release are correlated in these different cells. However, while autocrine TNFalpha production was critical for DC maturation, upregulation of TNFalpha release after HLA-DR crosslinking was not observed and neutralization of endogenous TNFalpha did not modify HLA-DR-mediated apoptosis. These data reveal that HLA-DR mediated apoptosis susceptibility and spontaneous TNFalpha release are regulated in a parallel manner and that while TNFalpha may induce maturation of APC to an "apoptosis sensitive" stage, there is no direct role for TNFalpha in HLA-DR-mediated apoptosis of APC.

HLA-DR-mediated apoptosis susceptibility discriminates differentiation stages of dendritic/monocytic APC.

Professional APC are characterized by their ability to present peptide via HLA class II in the presence of costimulatory molecules (CD40, CD80, and CD86). The efficiency of Ag presentation can be classed as follows: mature dendritic cells (DC) are most efficient, immature DC and macrophages are intermediate, and monocytes are considered poor APC. There is a large body of evidence demonstrating that HLA-DR transmits signals in the APC. In this study, we have addressed the question of the outcome of HLA-DR signals on APC of the monocyte/DC lineages throughout their differentiation from immature to mature APC. DC were generated from both monocytes and CD34+ cells of the same individual, macrophages were differentiated from monocytes. Immunophenotypical analysis clearly distinguished these populations. HLA-DR-mediated signals led to marked apoptosis in mature DC of either CD34 or monocytic origin. Significantly less apoptosis was observed in immature DC of either origin. Nonetheless, even immature DC were more susceptible to HLA-DR-mediated apoptosis than macrophages, whereas monocytes were resistant to HLA-DR-mediated apoptosis. The mechanism of HLA-DR-mediated apoptosis was independent of caspase activation. Taken together, these data lead to the notion that signals generated via HLA-DR lead to the demise of mature professional APC, thereby providing a means of limiting the immune response.

HLA-DR inhibits granulocytic differentiation without inducing apoptosis of CD34 cells.

Hematopoietic progenitors express HLA-DR molecules. However the significance of HLA-class II molecules on CD34+ cells remains unknown. The primary role of HLA-class-II molecules is antigen presentation although a second role, that of signal transduction, has been established in B cells. The role of HLA-DR in hematopoiesis was examined by determining the ability of CD34+ progenitor cells to differentiate to "Colony Forming Unit Granulocyte-Macrophage" (CFU-GM) and "Burst Forming Unit Erythrocyte" (BFU-E) in the presence of anti-HLA-DR monoclonal antibody. We observed a reduction in the number of CFU-GM which was due in part to down regulation of granulocyte rather than monocyte differentiation. These observations suggest that HLA-DR signals can regulate myelopoiesis. We point out especially the role of the HLA-DR molecule in the switch of CFU-GM between granulocyte or monocyte lineages. Although HLA-DR mediated apoptosis has been described in mature B lymphocytes apoptosis of CD34+ cells was excluded as a mechanism.

Differential expression of major histocompatibility complex class Ia, Ib, and II molecules on monocytes-derived dendritic and macrophagic cells.

Blood monocyte derived antigen presenting cells (APC) such as dendritic cells and macrophages are considered as major promising tools for antitumoral immunotherapy. In order to contribute to their phenotype characterization, we have precisely investigated their levels of expression of MHC class Ia, Ib (HLA-G) and II molecules using mainly flow cytometry quantification assays. APC were generated from monocytes cultured for 7 days in the presence of GM-CSF and IL-4 or M-CSF. These cells, which exhibited known morphological and immunological features of dendritic cells and macrophages respectively, were evidenced to display high expression of MHC class Ia and class II antigens in comparison to that found in monocytes. Dendritic cells and macrophages thus expressed 2-fold more and 4-fold more MHC class Ia molecules and 5-fold and 3-fold more MHC class II DR molecules than parental monocytes. In addition, expression of MHC class II DP and DQ molecules, not or only barely detected in monocytes, was clearly demonstrated in the two kinds of APC. In contrast, monocytes, dendritic cells and macrophages failed to express MHC class Ib HLA-G antigen. The up-regulation in monocyte-derived APC of MHC class Ia and II molecules mediating the presentation of antigen peptides to lymphocytes fully supports the interest of such APC in antitumoral immunotherapy.

CpG methylation patterns in the 5' part of the nonclassical HLA-G gene in peripheral blood CD34+ cells and CD2+ lymphocytes.

A dominant goal of research focused on the nonclassical human leukocyte antigen G (HLA-G) gene is to understand the molecular mechanism involved in its limited expression. In the present report, we examined DNA methylation as a potential regulatory mechanism of HLA-G transcription in two cell types of the adult lymphomyeloid lineage: CD2+ lymphocytes express several mRNA isoforms while transcripts are undetectable in CD34+ hematopoietic cells. The methylation status of 63 CpG sites in the promoter and in the 5' CpG island was established using bisulfite-treated genomic DNA sequencing. Methylation was first analyzed by the direct sequencing of bisulfite-treated and amplified products. The general patterns of CpG methylation in the 5' part of the gene were found to be similar for CD34+ cells and CD2+ lymphocytes: the distribution of methylation was not uniform across the 63 CpG sites. In the promoter region, both CpG dinucleotides were partially or fully methylated whereas in the CpG island, several CpG sites were totally demethylated. Unexpectedly, in HLA-G positive CD2+ lymphocytes, a great number of CpG dinucleotides displayed a higher frequency of methylation relative to that found in CD34+ cells. However, the sequence analysis of cloned products revealed that the molecules have different methylation patterns which suggests that the HLA-G gene is differentially expressed in CD2+ cells. Our results suggest that methylation is not the sole mechanism that achieves the repression of HLA-G transcription in immature CD34+ cells.