A Pilot Study for Legionella pneumophila Volatilome Characterization Using a Gas Sensor Array and GC/MS Techniques.

Legionellosis is a generic term describing the pneumonic (Legionnaires' disease, LD) and non-pneumonic (Pontiac fever, PF) forms of infection with bacteria belonging to the genus Legionella. Currently, the techniques used to detect Legionella spp. in water samples have certain limitations and drawbacks, and thus, there is a need to identify new tools to carry out low-cost and rapid analysis. In this regard, several studies demonstrated that a volatolomics approach rapidly detects and discriminates different species of microorganisms via their volatile signature. In this paper, the volatile organic compounds (VOCs) pattern emitted in vitro by Legionella pneumophila cultures is characterized and compared to those produced by other Legionella species and by Pseudomonas aeruginosa, using a gas sensor array and gas chromatograph mass spectrometer (GC-MS). Bacterial cultures were measured at the 3rd and 7th day after the incubation. Sensor array data analyzed via the K-nearest neighbours (k-NN) algorithm showed a sensitivity to Legionella pneumophila identification at around 89%. On the other hand, GC-MS identified a bouquet of VOCs, mainly alcohols and ketones, that enable the differentiation of Legionella pneumophila in respect to other waterborne microorganisms.

Aspergillus Species Discrimination Using a Gas Sensor Array.

The efficiency of electronic noses in detecting and identifying microorganisms has been proven by several studies. Since volatile compounds change with the growth of colonies, the identification of strains is highly dependent on the growing conditions. In this paper, the effects of growth were investigated with different species of Aspergillus, which is one of the most studied microorganisms because of its implications in environmental and food safety. For this purpose, we used an electronic nose previously utilized for volatilome detection applications and based on eight porphyrins-functionalized quartz microbalances. The volatile organic compounds (VOCs) released by cultured fungi were measured at 3, 5, and 10 days after the incubation. The signals from the sensors showed that the pattern of VOCs evolve with time. In particular, the separation between the three studied strains progressively decreases with time. The three strains could still be identified despite the influence of culture time. Linear Discriminant Analysis (LDA) showed an overall accuracy of 88% and 71% in the training and test sets, respectively. These results indicate that the presence of microorganisms is detectable with respect to background, however, the difference between the strains changes with the incubation time.

Control of Legionella Contamination and Risk of Corrosion in Hospital Water Networks following Various Disinfection Procedures.

Physical and chemical disinfection methods have been proposed with the aim of controlling Legionella water contamination. To date, the most effective procedures for reducing bacterial contamination have not yet been defined. The aim of this study was to assess the long-term effectiveness of various disinfection procedures in order to reduce both culturable and nonculturable (NC) legionellae in different hospital water networks treated with heat, chlorine dioxide, monochloramine, and hydrogen peroxide. The temperature levels and biocide concentrations that proved to give reliable results were analyzed. In order to study the possible effects on the water pipes, we verified the extent of corrosion on experimental coupons after applying each method for 6 months. The percentage of positive points was at its lowest after treatment with monochloramine, followed by chlorine dioxide, hydrogen peroxide, and hyperthermia. Different selections of Legionella spp. were observed, as networks treated with chlorine-based disinfectants were contaminated mainly by Legionella pneumophila serogroup 1, hyperthermia was associated with serogroups 2 to 14, and hydrogen peroxide treatment was associated mainly with non-pneumophila species. NC cells were detected only in heat-treated waters, and also when the temperature was approximately 60°C. The corrosion rates of the coupons were within a satisfactory limit for water networks, but the morphologies differed. We confirm here that chemical disinfection controls Legionella colonization more effectively than hyperthermia does. Monochloramine was the most effective treatment, while hydrogen peroxide may be a promising alternative to chlorine-based disinfectants due to its ability to select for other, less virulent or nonpathogenic species.

Influence of genetic polymorphisms of styrene-metabolizing enzymes on the levels of urinary biomarkers of styrene exposure.

Styrene exposure is still present in different occupational settings including manufacture of synthetic rubber, resins, polyesters and plastic. The aim of this work was to investigate the effects of polymorphic genes CYP2E1, EPHX1, GSTT1, and GSTM1 on the urinary concentrations of the styrene metabolites mandelic acid (MA), phenylglyoxylic acid (PGA) and on the concentration ratios between (MA+PGA) and urinary styrene (U-Sty) and airborne styrene (A-Sty), in 30 workers from two fiberglass-reinforced plastic manufacturing plants and 26 unexposed controls. Personal air sampling and biological monitoring results revealed that sometimes exposure levels exceeded both the threshold limit value (TLV) and the biological exposure index (BEI) suggested by the American Conference of Governmental Industrial Hygienists. A significantly reduced excretion of styrene metabolites (MA+PGA) in individuals carrying the CYP2E1*5B and CYP2E1*6 heterozygote alleles, with respect to the homozygote wild type, was observed only in the exposed group. A reduction was also detected, in the same group, in subjects carrying the slow allele EPHX1 (codon 113), through the lowering of (MA+PGA)/urinary styrene concentration ratio. In addition, the ratio between MA+PGA and the personal airborne styrene concentration appeared to be modulated by the predicted mEH activity, in the exposed group, as evidenced by univariate linear regression analysis. Our results confirm some previous hypotheses about the role of the polymorphism of genes coding for enzymes involved in the styrene detoxification pathway: this may significantly reduce the levels of excreted metabolites and therefore it must be taken into account in the interpretation of the biological monitoring results for occupational exposure.

Exposure to airborne culturable microorganisms and endotoxin in two Italian poultry slaughterhouses.

Even if slaughterhouses' workers handle large amounts of organic material and are potentially exposed to a wide range of biological agents, relatively little and not recent data are available. The main objective of this study was to characterize indoor concentrations of airborne bacteria, fungi, and endotoxin mod = Im (endotoxin∼Gram-negative*plant*filter) in two Italian poultry slaughterhouses. Air samples near air handling units inlets were also collected. Since there are not standardized protocols for endotoxin sampling and extraction procedures, an additional aim of the study was to compare the extraction efficiency of three different filter.. The study was also aimed at determining the correlation between concentrations of Gram-negative bacteria and endotoxin. In Plant A bacterial levels ranged from 17.5 to 2.6×10(3) CFU/m3. The highest concentrations were observed in evisceration area of chickens, between the automatic detachment of the neck and washing offal, and near birds coupling before hair-chilling. The highest mean value of Gram-negative (266.5 CFU/m3) was found near the washing offal of turkeys. In Plant B bacterial concentration ranged from 35 to 8×10(3) CFU/m3. The highest concentration. with the highest value of Gram-negative (248 CFU/m3), was found after defeathering. Fungal concentrations were overall lower than those found for bacteria (range: 0-205 CFU/m3 in Plant A and 0-146.2 CFU/m3 in Plant B). The microbial flora was dominated by Gram-negative and coagulase-negative staphylococci for bacteria and by species belonging to Cladosporium, Penicillium and Aspergillus genera for molds. The highest endotoxin concentrations were measured in washing offal for Plant A (range: 122.7-165.9 EU/m3) and after defeathering for Plant B (range: 0.83-38.85 EU/m3). In this study airborne microorganisms concentrations were lower than those found in similar occupational settings and below the occupational limits proposed by some authors. However, these microorganisms may exert adverse effects on exposed workers, in particular for those engaged in the early slaughtering stages, as evidenced by the presence of pathogenic species. The detection of pathogenic bacteria near AHU inlet may constitute a risk to public health and environmental pollution.

Aire regulates the transfer of antigen from mTECs to dendritic cells for induction of thymic tolerance.

To investigate the role of Aire in thymic selection, we examined the cellular requirements for generation of ovalbumin (OVA)-specific CD4 and CD8 T cells in mice expressing OVA under the control of the rat insulin promoter. Aire deficiency reduced the number of mature single-positive OVA-specific CD4(+) or CD8(+) T cells in the thymus, independent of OVA expression. Importantly, it also contributed in 2 ways to OVA-dependent negative selection depending on the T-cell type. Aire-dependent negative selection of OVA-specific CD8 T cells correlated with Aire-regulated expression of OVA. By contrast, for OVA-specific CD4 T cells, Aire affected tolerance induction by a mechanism that operated independent of the level of OVA expression, controlling access of antigen presenting cells to medullary thymic epithelial cell (mTEC)-expressed OVA. This study supports the view that one mechanism by which Aire controls thymic negative selection is by regulating the indirect presentation of mTEC-derived antigens by thymic dendritic cells. It also indicates that mTECs can mediate tolerance by direct presentation of Aire-regulated antigens to both CD4 and CD8 T cells.

Human dendritic cell subsets from spleen and blood are similar in phenotype and function but modified by donor health status.

Mouse dendritic cells (DC) have been extensively studied in various tissues, especially spleen, and they comprise subsets with distinct developmental origins, surface phenotypes, and functions. Considerably less is known about human DC due to their rarity in blood and inaccessibility of other human tissues. The study of DC in human blood has revealed four subsets distinct in phenotype and function. In this study, we describe four equivalent DC subsets in human spleen obtained from deceased organ donors. We identify three conventional DC subsets characterized by surface expression of CD1b/c, CD141, and CD16, and one plasmacytoid DC subset characterized by CD304 expression. Human DC subsets in spleen were very similar to those in human blood with respect to surface phenotype, TLR and transcription factor expression, capacity to stimulate T cells, cytokine secretion, and cross-presentation of exogenous Ag. However, organ donor health status, in particular treatment with corticosteroid methylprednisolone and brain death, may affect DC phenotype and function. DC T cell stimulatory capacity was reduced but DC were qualitatively unchanged in methylprednisolone-treated deceased organ donor spleen compared with healthy donor blood. Overall, our findings indicate that human blood DC closely resemble human spleen DC. Furthermore, we confirm parallels between human and mouse DC subsets in phenotype and function, but also identify differences in transcription factor and TLR expression as well as functional properties. In particular, the hallmark functions of mouse CD8α(+) DC subsets, that is, IL-12p70 secretion and cross-presentation, are not confined to the equivalent human CD141(+) DC but are shared by CD1b/c(+) and CD16(+) DC subsets.

IL-10 controls cystatin C synthesis and blood concentration in response to inflammation through regulation of IFN regulatory factor 8 expression.

Cystatin C (CstC) is a cysteine protease inhibitor of major clinical importance. Low concentration of serum CstC is linked to atherosclerosis. CstC can prevent formation of amyloid ß associated with Alzheimer's disease and can itself form toxic aggregates. CstC regulates NO secretion by macrophages and is a TGF-ß antagonist. Finally, the serum concentration of CstC is an indicator of kidney function. Yet, little is known about the regulation of CstC expression in vivo. In this study, we demonstrate that the transcription factor IFN regulatory factor 8 (IRF-8) is critical for CstC expression in primary dendritic cells. Only those cells with IRF-8 bound to the CstC gene promoter expressed high levels of the inhibitor. Secretion of IL-10 in response to inflammatory stimuli downregulated IRF-8 expression and consequently CstC synthesis in vivo. Furthermore, the serum concentration of CstC decreased in an IL-10-dependent manner in mice treated with the TLR9 agonist CpG. CstC synthesis is therefore more tightly regulated than hitherto recognized. The mechanisms involved in this regulation might be targeted to alter CstC production, with potential therapeutic value. Our results also indicate that caution should be exerted when using the concentration of serum CstC as an indicator of kidney function in conditions in which inflammation may alter CstC production.

Cutting edge: priming of CD8 T cell immunity to herpes simplex virus type 1 requires cognate TLR3 expression in vivo.

Despite its potential for involvement in viral immunity, little evidence links TLR3 to adaptive antiviral responses. Here we show that TLR3 is required for the generation of CD8 T cell immunity to HSV-1. The magnitude of the gB-specific CD8 T cell response after flank infection by HSV-1 was significantly reduced in mice lacking TIR domain-containing adaptor-inducing IFN-beta or TLR3, but not MyD88. Impaired CTL induction was evident in chimeric mice lacking TLR3 in bone marrow (BM)-derived cells. Among the dendritic cell subsets, TLR3 was expressed by CD8alpha(+) dendritic cells, known to be involved in priming HSV-1-specific CD8 T cells. Use of mixed BM chimeras revealed that TLR3 and the MHC class I-restriction element must be expressed by the same BM-derived cell for effective priming. These data imply that a cognate linkage between TLR3 and MHC class I is required for efficient CTL priming to HSV-1.

CD8+, CD8-, and plasmacytoid dendritic cell generation in vitro using flt3 ligand.

The generation of dendritic cells (DCs) from monocytes and early progenitors in GM-CSF cultures has been the gold standard for in vitro generation of DCs for three decades. However, the most recent evidence suggests that these cultures represent the migratory and inflammatory DC subtypes and not the DC subtypes found in the steady state. By contrast a different culture method was described where mouse bone marrow is cultured with flt3 ligand for 9 days. Here, we describe this method in detail for the generation of the phenotypic, functional, and developmental equivalents of CD8(+), CD8(-), and plasmacytoid DCs. This includes growth and purification of recombinant flt3 ligand from Chinese hamster ovary cells, isolation of bone marrow cells, and phenotypic characterization of the subsets. This simple method allows generation of large numbers of DCs (60-100 million from one mouse) compared to splenic DC isolation (5 million per mouse).

c-Rel is required for the development of thymic Foxp3+ CD4 regulatory T cells.

During thymopoiesis, a unique program of gene expression promotes the development of CD4 regulatory T (T reg) cells. Although Foxp3 maintains a pattern of gene expression necessary for T reg cell function, other transcription factors are emerging as important determinants of T reg cell development. We show that the NF-kappaB transcription factor c-Rel is highly expressed in thymic T reg cells and that in c-rel(-/-) mice, thymic T reg cell numbers are markedly reduced as a result of a T cell-intrinsic defect that is manifest during thymocyte development. Although c-Rel is not essential for TGF-beta conversion of peripheral CD4(+)CD25(-) T cells into CD4(+)Foxp3(+) cells, it is required for optimal homeostatic expansion of peripheral T reg cells. Despite a lower number of peripheral T reg cells in c-rel(-/-) mice, the residual peripheral c-rel(-/-) T reg cells express normal levels of Foxp3, display a pattern of cell surface markers and gene expression similar to those of wild-type T reg cells, and effectively suppress effector T cell function in culture and in vivo. Collectively, our results indicate that c-Rel is important for both the thymic development and peripheral homeostatic proliferation of T reg cells.

The C-type lectin Clec12A present on mouse and human dendritic cells can serve as a target for antigen delivery and enhancement of antibody responses.

We have cloned the mouse and human C-type lectin Clec12A, expressed both, and produced mAb recognizing both. Mouse Clec12A is highly expressed on splenic CD8(+) dendritic cells (DC) and plasmacytoid DC. A proportion of CD8(-)DC also expresses lower levels of Clec12A, as do monocytes, macrophages, and B cells. Human CLEC12A, like the mouse counterpart, is expressed on blood monocytes and DC, including pDC and BDCA-3(+)DC, the proposed equivalent of mouse CD8(+)DC. To determine whether Ag targeted to Clec12A could induce immune responses, mice were injected with a rat mAb recognizing Clec12A, or a control rat mAb, then production of anti-rat Ig was measured. Anti-Clec12A mAb alone produced only moderate responses, but these were amplified by coinjecting only small amounts of LPS as a DC activation agent. Furthermore, when OVA was conjugated to anti-Clec12A mAb, OVA-specific T cells were induced to proliferate. This Ag presentation to naive T cells was due to targeting conventional DC, because their ablation eliminated T cell activation. The potent Ab responses induced using microgram amounts of anti-Clec12A and minimal amounts of adjuvant demonstrate that this molecule can be used as an Ag-delivery target to enhance Ab responses to vaccines.

Monoclonal antibodies generated by DNA immunization recognize CD2 from a broad range of primates.

Using heterologous prime-boost (DNA immunization followed by immunization with transfected cells), we have generated depleting mouse anti-baboon CD2 monoclonal antibodies (mAb). These anti-CD2 mAb recognized a diverse range of primate CD2 from New World monkeys and Old World monkeys to humans and have potent immunosuppressive activity for human allo-MLR responses and anti-tetanus-toxoid recall responses. There was no upregulation of activation markers or release of cytokines when the mAb were incubated with human peripheral blood mononuclear cells. Using chimeric NOD-SCID IL2rgamma(null) mice, the mAb were shown to deplete human and cynomolgus monkey T cells in vivo. These anti-CD2 mAb may therefore be important immunological tools in allo- and xenotransplantation.

The impact of circulating dendritic cells on the development and differentiation of thymocytes.

Central tolerance is established through the negative selection of self-reactive thymocytes and the induction of T-regulatory cells (T-regs). A role for thymic epithelial cells in mediating both negative selection and T-reg induction has been clearly shown. The role of thymic dendritic cells (DCs) in these processes has not been clearly determined but has been the focus of recent studies. Thymic DCs include two conventional DC (cDC) subtypes, CD8(lo)Sirpalpha(hi/+) (CD8(lo)Sirpalpha(+) herein) and CD8(hi)Sirpalpha(lo/-) (CD8(hi)Sirpalpha(-) herein). It has been shown that these DC subsets have distinct developmental origins, the CD8(hi)Sirpalpha(-) cDCs developing intra-thymically and the CD8(lo)Sirpalpha(+) migrating into the thymus from the periphery. Furthermore, an important role for thymic DCs in the induction of T-regs has been shown. In this review, the role of DCs in the development and education of T cells in the thymus will be reviewed, with emphasis on the role of circulatory DCs in mediating these processes.

Dendritic cells in the thymus contribute to T-regulatory cell induction.

Central tolerance is established through negative selection of self-reactive thymocytes and the induction of T-regulatory cells (T(R)s). The role of thymic dendritic cells (TDCs) in these processes has not been clearly determined. In this study, we demonstrate that in vivo, TDCs not only play a role in negative selection but in the induction of T(R)s. TDCs include two conventional dendritic cell (DC) subtypes, CD8(lo)Sirpalpha(hi/+) (CD8(lo)Sirpalpha(+)) and CD8(hi)Sirpalpha(lo/-) (CD8(hi)Sirpalpha(-)) [corrected] which have different origins. We found that the CD8(hi)Sirpalpha(+) DCs represent a conventional DC subset that originates from the blood and migrates into the thymus. Moreover, we show that the CD8(lo)Sirpalpha(+) DCs demonstrate a superior capacity to induce T(R)s in vitro. Finally, using a thymic transplantation system, we demonstrate that the DCs in the periphery can migrate into the thymus, where they efficiently induce T(R) generation and negative selection.

Differential MHC class II synthesis and ubiquitination confers distinct antigen-presenting properties on conventional and plasmacytoid dendritic cells.

The importance of conventional dendritic cells (cDCs) in the processing and presentation of antigen is well established, but the contribution of plasmacytoid dendritic cells (pDCs) to these processes, and hence to T cell immunity, remains unclear. Here we showed that unlike cDCs, pDCs continued to synthesize major histocompatibility complex (MHC) class II molecules and the MHC class II ubiquitin ligase MARCH1 long after activation. Sustained MHC class II-peptide complex formation, ubiquitination and turnover rendered pDCs inefficient in the presentation of exogenous antigens but enabled pDCs to continuously present endogenous viral antigens in their activated state. As the antigen-presenting abilities of cDCs and pDCs are fundamentally distinct, these two cell types may activate largely nonoverlapping repertoires of CD4(+) T cells.

Distinct functional capacities of mouse thymic and splenic dendritic cell populations.

Dendritic cells (DC) are antigen-presenting cells that activate naive T cells. Murine DC are a heterogeneous population and can be subdivided into distinct subsets with different immune regulatory functions, namely the conventional DC (cDC), which include the CD8(+)Sirpalpha(-) and CD8(-)Sirpalpha(+) DC, and the plasmacytoid DC (pDC). In this study, the phenotype and function of DC subsets in both the thymus and spleen were compared. Significant differences between the thymic and splenic DC were observed in the expression of genes encoding chemokine receptors (CCRs), toll-like receptors (TLRs) and chemokines. Thymic DC expressed high levels of genes encoding a unique set of chemokines (CCL17 and CCL22) known to be important for T-cell development. Moreover, the capacity of the DC from the two organs to produce IL-6, IFN-alpha and IL-12p70 in response to the TLR 9 agonist CpG differed markedly, indicating intrinsic functional differences between subsets with similar surface phenotype. These results indicate that the microenvironment is an important factor that contributes to the functional specification of DC subsets in different lymphoid tissues.

The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement.

A novel dendritic cell (DC)-restricted molecule, Clec9A, was identified by gene expression profiling of mouse DC subtypes. Based on sequence similarity, a human ortholog was identified. Clec9A encodes a type II membrane protein with a single extracellular C-type lectin domain. Both the mouse Clec9A and human CLEC9A were cloned and expressed, and monoclonal antibodies (mAbs) against each were generated. Surface staining revealed that Clec9A was selective for mouse DCs and was restricted to the CD8(+) conventional DC and plasmacytoid DC subtypes. A subset of human blood DCs also expressed CLEC9A. A single injection of mice with a mAb against Clec9A, which targets antigens (Ags) to the DCs, produced a striking enhancement of antibody responses in the absence of added adjuvants or danger signals, even in mice lacking Toll-like receptor signaling pathways. Such targeting also enhanced CD4 and CD8 T-cell responses. Thus, Clec9A serves as a new marker to distinguish subtypes of both mouse and human DCs. Furthermore, targeting Ags to DCs with antibodies to Clec9A is a promising strategy to enhance the efficiency of vaccines, even in the absence of adjuvants.

Selective suicide of cross-presenting CD8+ dendritic cells by cytochrome c injection shows functional heterogeneity within this subset.

Cross-presentation as a fundamental pathway of activating CD8(+) T cells has been well established. So far the application of this concept in vivo is limited, and the mechanisms that specialize CD8(+) dendritic cells (DCs) for this task are not fully understood. Here we take advantage of the specific cytosolic export feature of cross-presenting DCs together with the property of cytosolic cytochrome c (cyt c) in initiating Apaf-1-dependent apoptosis selectively in cross-presenting DCs. A single i.v. injection of cyt c in B6 mice produced a 2- to 3-fold reduction in splenic CD8(+) DCs but not in Apaf-1-deficient mice. Functional studies both in vivo and in vitro showed that cyt c profoundly abrogated OVA-specific CD8(+) T cell proliferation through its apoptosis-inducing effect on cross-presenting DCs. More importantly, in vivo injection of cyt c abolished the induction of cytotoxic T lymphocytes to exogenous antigen and reduced subsequent immunity to tumor challenge. In addition, only a proportion of CD8(+) DCs that express abundant IL-12 and Toll-like receptor 3 were efficient cross-presenters. Our data support the hypothesis that cross-presentation in vivo requires cytosolic diversion of endocytosed proteins, conferring cross-presentation specialization to a proportion of CD8(+) DCs. We propose that DCs incapable of such transfer, even within the CD8(+) DC subset, are unable to cross-present. Our model opens an avenue to specifically target cross-presenting DCs in vivo for manipulating cytotoxic T lymphocyte responses toward infections, tumors, and transplants.

Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo.

The development of functionally specialized subtypes of dendritic cells (DCs) can be modeled through the culture of bone marrow with the ligand for the cytokine receptor Flt3. Such cultures produce DCs resembling spleen plasmacytoid DCs (pDCs), CD8(+) conventional DCs (cDCs) and CD8(-) cDCs. Here we isolated two sequential DC-committed precursor cells from such cultures: dividing 'pro-DCs', which gave rise to transitional 'pre-DCs' en route to differentiating into the three distinct DC subtypes (pDCs, CD8(+) cDCs and CD8(-) cDCs). We also isolated an in vivo equivalent of the DC-committed pro-DC precursor cell, which also gave rise to the three DC subtypes. Clonal analysis of the progeny of individual pro-DC precursors demonstrated that some pro-DC precursors gave rise to all three DC subtypes, some produced cDCs but not pDCs, and some were fully committed to a single DC subtype. Thus, commitment to particular DC subtypes begins mainly at this pro-DC stage.