Multiplex pathogen detection based on spatially addressable microarrays of barcoded resins.

Suspension microsphere immunoassays are rapidly gaining recognition in antigen identification and infectious disease biodetection due to their simplicity, versatility and high-throughput multiplex screening. We demonstrate a multiplex assay based on antibody-functionalized barcoded resins (BCRs) to identify pathogen antigens in complex biological fluids. The binding event of a particular antibody on given bead (fluorescence) and the identification of the specific pathogen agent (vibrational fingerprint of the bead) can be achieved in a dispersive Raman system by exciting the sample with two different laser lines. Anthrax protective antigen, Franciscella tularensis lipopolysaccharide and CD14 antigens were accurately identified and quantified in tetraplex assays with a detection limit of 1 ng/mL. The rapid, versatile and simple analysis enabled by the BCRs demonstrates their potential for multiplex antigen detection and identification in a reconfigurable microarray format.

CD14 and TLR4 are expressed early in tammar (Macropus eugenii) neonate development.

Marsupials are born in a relatively underdeveloped state and develop during a period of intensive maturation in the postnatal period. During this period, the young marsupial lacks a competent immune system, but manages to survive despite the potential of exposure to environmental pathogens. Passive immune transfer via the milk is one well-recognised strategy to compensate the neonate, but there also may be innate immune mechanisms in place. In this study, CD14 and Toll-like receptor 4 (TLR4), integral molecular components of pathogen recognition, were identified and characterised for the first time in a marsupial, the tammar wallaby (Macropus eugenii). Functional motifs of tammar CD14 and the toll/interleukin receptor (TIR) domain of TLR4 were highly conserved. The lipopolysaccharide (LPS) binding residues and the TLR4 interaction site of CD14 were conserved in all marsupials. The TIR signalling domain had 84% identity within marsupials and 77% with eutherians. Stimulation of adult tammar leukocytes resulted in the induction of a biphasic pattern of CD14 and TLR4 expression, and coincided with increased production of the pro-inflammatory cytokine TNF-alpha. Differential patterns of expression of CD14 and TLR4 were observed in tammar pouch young early in development, suggesting that early maturation of the innate immune system in these animals may have developed as an immune survival strategy to protect the marsupial neonate from exposure to microbial pathogens.

Purification of soluble CD14 fusion proteins and use in an electrochemiluminescent assay for lipopolysaccharide binding.

CD14, a 55kDa lipopolysaccharide binding glycoprotein, is a key element in both LPS-mediated activation of cells and endotoxin detoxification. A gene fragment containing residues 1-348 of the human LPS receptor CD14, representing the extracellular form of the molecule, was fused to the CH(2)-CH(3) portion of the human IgG heavy chain or to a 6x His tag and transfected into CHO cells. Stable cell lines of each were grown to produce recombinant protein in unsupplemented serum free media and CD14His was purified by ion-exchange chromatography. After passive immobilization onto a carbon surface both forms of the CD14 fusion proteins bound LPS-biotin in a dose-dependent manner in an electrochemiluminescent assay. Binding was inhibited by the anti-CD14 antibody S39 as well as by unlabeled LPS. This report describes an efficient method of purifying CD14 and a novel assay to detect bioactive lipopolysaccharide.

Identification of lipopolysaccharide-binding proteins in porcine milk.

Septicemia and endotoxemia initiated by bacterial lipopolysaccharide (LPS) are relatively common in suckling and weaned piglets. Maternal milk is a source of both nutrition and immune protection for piglets. Passive transfer of colostral antibodies is necessary for protection of neonatal piglets against diseases, but the concentration of immunoglobulins in milk rapidly declines during the 1st wk of lactation in all mammals. We hypothesized, therefore, that nonimmunoglobulin substances in milk contribute to the innate protection of neonates against septicemia during the suckling period. Using LPS-affinity chromatography for isolation of LPS-binding proteins and liquid chromatography-mass spectrometry for their identification, we identified in porcine milk the following proteins with LPS-binding capacity: lactoferrin, soluble CD14, serum amyloid A, alpha-S1 casein, beta-casein, and kappa-casein. For lactoferrin, alpha-S1 casein, and kappa-casein, in vitro pepsin digestion did not inhibit LPS-binding activity, whereas combined digestion with pepsin and pancreatin abolished it. The biologic functions of these LPS-binding proteins and peptides were not determined.

Tuberculosis bacilli still posing a threat. Polymorphism of genes regulating anti-mycobacterial properties of macrophages.

One third of the earth's population is infected with Mycobacterium tuberculosis, but only 5-10% of the infected individuals will develop active disease over their lifetime. To identify the genes responsible for the variation in the human susceptibility/resistance to tuberculosis (TB) we determined the polymorphisms of three genes crucial for the function of macrophages, in TB patients and healthy controls with no past history of TB. We found no association between the polymorphisms of the NRAMP-INT4, MBL (codons 52, 54, 57) and CD14-159 genes and TB in a Caucasian Polish population. However, we have suggested a possible involvement of CD14 and MBL molecules in the host-mycobacteria interactions on the basis of the significant increase in the serum CD14 and MBL in TB patients compared to healthy controls.

Purification and characterization of human soluble CD14 expressed in Pichia pastoris.

CD14 is a protein that mediates lipopolysaccharide (LPS)-induced biological responses such as activation of a transcriptional factor, nuclear factor (NF)-kappaB. It exists as a soluble form (sCD14) in serum and mediates LPS responses of epithelial and endothelial cells as well as a membrane-bound form (mCD14) on monocytes and macrophages. To obtain sCD14 in large quantity for its structural and functional characterization, we expressed the full-length form of human recombinant sCD14 (rsCD14) in a methylotrophic yeast, Pichia pastoris. The recombinant protein was expressed as a major protein in the culture supernatant and purified by ammonium sulfate precipitation, followed by three steps of ion exchange chromatographies. Finally, 1.6 mg of the protein was obtained in high purity from 2L of the supernatant and its identity to sCD14 was confirmed by NH(2)-terminal amino acid sequence analysis. The purified protein was found to have N-linked sugars by an analysis of enzymatic deglycosylation. A native PAGE analysis revealed that the protein was able to form complexes with LPS. In addition, the rsCD14 protein could mediate the LPS-mediated activation of NF-kappaB in human embryonic kidney 293 cells transfected with Toll-like receptor 4 and MD-2, indicating that the purified protein is biologically active. Thus, the rsCD14 protein expressed in P. pastoris and highly purified in a large amount is useful for its structural and functional studies.

Bacterial fimbriae and their peptides activate human gingival epithelial cells through Toll-like receptor 2.

Gingival epithelial cells are a central component of the barrier between oral microflora and internal tissues. Host responses to periodontopathic bacteria and surface components containing fimbriae are thought to be important in the development and progression of periodontal diseases. To elucidate this mechanism, we established immortalized human gingival epithelial cells (HGEC) that were transfected with human papillomavirus. HGEC predominantly expressed Toll-like receptor (TLR) 2, but not TLR4 or CD14. They also induced interleukin-8 (IL-8) production when stimulated with Porphyromonas gingivalis fimbriae and Staphylococcus aureus peptidoglycan, but not Escherichia coli-type synthetic lipid A. Furthermore, an active synthetic peptide composed of residues 69 to 73 (ALTTE) of the fimbrial subunit protein, derived from P. gingivalis and similar to a common component of cell wall peptidoglycans in parasitic bacteria, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), significantly induced IL-8 production and NF-kappaB activation in HGEC, and these cytokine-producing activities were augmented by a complex of soluble CD14 and lipopolysaccharide-binding protein (LBP). IL-8 production in HGEC stimulated with these bacterial components was clearly inhibited by mouse monoclonal antibody to human TLR2. These findings suggest that P. gingivalis fimbrial protein and its active peptide are capable of activating HGEC through TLR2.

The lipopolysaccharide co-receptor CD14 is present and functional in seminal plasma and expressed on spermatozoa.

CD14 is a 54 000-molecular weight (MW) glycolipid-anchored membrane glycoprotein, expressed on myeloid cells, which functions as a member of the lipopolysaccharide (LPS) receptor complex. Soluble forms of CD14 have been reported in plasma, cerebrospinal fluid, amniotic fluid and breast milk. In plasma and breast milk, soluble CD14 has been implicated as a regulator of T- and B-cell activation and function. Expression of CD14 in the male reproductive system has not previously been investigated. We here show that soluble CD14 is present in seminal plasma at levels comparable to those in serum. Spermatozoa expressed CD14 on their membranes, as demonstrated by fluorescence microscopy and flow cytometry. Post-vasectomy, the levels of seminal plasma CD14 (spCD14) were much reduced, implying an origin distal to the point of transection of the vas deferens. Ultracentrifugation analyses demonstrated that spCD14 was not associated with lipid complexes, indicating that it lacks the glycolipid anchor. Purified spCD14 mediated activation by LPS of CD14-negative cells. These findings suggest that CD14 may play a hitherto unexplored role in immune defence and cell activation in the male reproductive tract.

Expression of IL-18 mRNA and secretion of IL-18 are reduced in monocytes from patients with atopic dermatitis.

BACKGROUND: IL-18 has been found to be an IFN-gamma-inducing factor that plays an important role in T(H)1 cell activation. Recently, IL-18 has also been found to enhance a T(H)2 cellular response in a specific setting. OBJECTIVE: The aim of this study was to elucidate the role of monocytes and soluble factors, with special focus on IL-18, in the pathogenesis of atopic dermatitis (AD). METHODS: The release of cytokines from PBMCs and purified monocytes was measured through use of ELISA; mRNA expression was evaluated by RT-PCR. The results from patients with AD were compared with those from healthy controls. RESULTS: IL-18 secretion was reduced in both unstimulated and lipopolysaccharide-stimulated monocytes from patients with AD. The mRNA expression of IL-18 and IL-1 beta-converting enzyme was significantly reduced in unstimulated monocytes from patients with AD (P <.03 and P <.01, respectively). Patients with AD had an elevated secretion of prostaglandin E(2) (PGE(2)) from unstimulated PBMCs (P <.001). The anti-PGE(2) antibody reversed the suppressive effect of PGE(2) on IL-18 secretion in unstimulated PBMCs from patients with AD. CONCLUSIONS: Decreased IL-18 production, together with a significantly reduced IL-18 and ICE mRNA expression in unstimulated monocytes and elevated PGE(2) secretion from PBMCs, was associated with the pathogenesis of AD.

Soluble CD14 in human breast milk and its role in innate immune responses.

Immune factors secreted in milk are important for health in the neonatal gut. We have detected the bacterial pattern recognition receptor, soluble CD14 (sCD14) in human breast milk at different times during lactation. The molecule occurs in a single form in milk, in contrast to human serum, in which there are two isoforms. Produced by mammary epithelial cells, milk sCD14 mediates secretion of innate immune response molecules such as interleukin-8, tumor necrosis factor-alpha, and epithelial neutrophil activator-78 by CD14-negative intestinal epithelial cells exposed to lipopolysaccharide (LPS) or bacteria. Although present at low concentrations in milk, LPS-binding protein may be implicated in the biological effects observed. Our findings support the premise that milk sCD14 acts as a 'sentinel' molecule and immune modulator in homeostasis and in the defense of the neonatal intestine. In so doing, it may prevent the immune and inflammatory conditions of the gut to which non-breastfed infants are predisposed.

[Clone and expression of human soluble CD14 and study of its function].

Human soluble CD14(sCD14) cDNA fragment was amplified using total RNA extracted from U937 cells by RT-PCR of sCD14 gene, and the recombinant expression plasmid pEF1/HisC/sCD14 348aa was constructed. Then the expression in eukaryotic cell was carry out by liposome transfection method. It demonstrated that the expression level was relatively high by scanning map identification. The expressed product was purified by immunoaffinity chromatography and the purity was above 90%. The changes of CD14 brought by LPS stimulating U937 cell proved the product had the function of combine with LPS.

A CD14-independent LPS receptor cluster.

Bacterial lipopolysaccharide (LPS), the major structural component of the outer wall of Gram-negative bacteria, is a potent initiator of an inflammatory response and serves as an indicator of bacterial infection. Although CD14 has been identified as the main LPS receptor, accumulating evidence has suggested the possible existence of other functional receptor(s). In this study, using affinity chromatography, peptide mass fingerprinting and fluorescence resonance energy transfer, we have identified four new proteins that form an activation cluster after LPS ligation and are involved in LPS signal transduction. Here we present evidence that implicates heat shock proteins 70 and 90, chemokine receptor 4 and growth differentiation factor 5 as the main mediators of activation by bacterial lipopolysaccharide.

Soluble CD14 enriched in colostrum and milk induces B cell growth and differentiation.

Induction of resting B cell growth and differentiation requires a complex series of temporally coordinated signals that are initiated on contact with activated helper T cells. These signals complement one another, each rendering the B cell susceptible to factors supporting progressive activation. Here, we demonstrate that soluble CD14 (sCD14) bypasses the physiological sequelae of events that limit B cell activation. B cell growth and differentiation in vitro is induced by both native and recombinant forms of sCD14 at nanomolar concentrations. sCD14-mediated cellular activation does not require membrane CD14 expression, depends on a region of CD14 that is not involved in lipopolysaccharide binding, and requires functional Toll-like receptor 4. Consistent with biological activity of sCD14 in vitro, its administration to neonatal mice enhances Ig secretion. The results presented establish sCD14 as a naturally occurring soluble B cell mitogen of mammalian origin.

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.

Expression and refolding of functional fragments of the human lipopolysaccharide receptor CD14 in Escherichia coli and Pichia pastoris.

CD14 is a high-affinity cellular receptor specific for bacterial lipopolysaccharides (LPS), present in the bacterial cell wall. Binding of LPS to CD14 initiates the innate component of immune response and triggers a response that can lead to septic shock. In order to provide recombinant protein for the study of LPS-CD14 molecular interactions we have expressed human CD14 in Escherichia coli and Pichia pastoris. In bacteria, the protein was produced in high yield in the form of inclusion bodies. We have optimized the procedure for its refolding and generated correctly folded protein. A procedure to monitor the refolding efficiency by using conformation-specific anti-human CD14 monoclonal antibody has been established. A fragment of 152 amino acids of CD14 which retains the ability to bind LPS has been produced in a methylotrophic yeast, P. pastoris, expression system. The recombinant protein from yeast is glycosylated and secreted into the medium. The CD14 fragment was purified to homogeneity by immunoaffinity chromatography. Recombinant CD14 from both bacteria and yeast bind to LPS.

Functional and phenotypic analysis of thymic CD34+CD1a- progenitor-derived dendritic cells: predominance of CD1a+ differentiation pathway.

Whether thymic dendritic cells (DC) are phenotypically and functionally distinct from the monocyte lineage DC is an important question. Human thymic progenitors differentiate into T, NK, and DC. The latter induce clonal deletion of autoreactive thymocytes and therefore might be different from their monocyte-derived counterparts. The cytokines needed for the differentiation of DC from thymic progenitors were also questioned, particularly the need for GM-CSF. We show that various cytokine combinations with or without GM-CSF generated DC from CD34+CD1a- but not from CD34+CD1a+ thymocytes. CD34+ thymic cells generated far fewer DC than their counterparts from the cord blood. The requirement for IL-7 was strict whereas GM-CSF was dispensable but nonetheless improved the yield of DC. CD14+ monocytic intermediates were not detected in these cultures unless macrophage-CSF (M-CSF) was added. Cultures in M-CSF generated CD14-CD1a+ DC precursors but also CD14+CD1a- cells. When sorted and recultured in GM-CSF, CD14+ cells down-regulated CD14 and up-regulated CD1a. TNF-alpha accelerated the differentiation of progenitors into DC and augmented MHC class II transport to the membrane, resulting in improved capacity to induce MLR. The trafficking of MHC class II molecules was studied by metabolic labeling and immunoprecipitation. MHC class II molecules were transported to the membrane in association with invariant chain isoforms in CD14+ (monocyte)-derived and in CD1a+ thymic-derived DC but not in monocytes. Thus, thymic progenitors can differentiate into DC along a preferential CD1a+ pathway but have conserved a CD14+ maturation capacity under M-CSF. Finally, CD1a+-derived thymic DC and monocyte-derived DC share very close Ag-processing machinery.

CD14 transgenic mice expressing membrane and soluble forms: comparisons of levels of cytokines and lethalities in response to lipopolysaccharide between transgenic and non-transgenic mice.

Two different metallothionein promoter-mouse CD14 fusion genes were constructed. The membrane form of the CD14 fusion gene, designated M14M, contained the full-length CD14 cDNA sequence, whereas the soluble form of the fusion gene, designated M14S, was truncated to lack the sequence for the phosphatidylinositol-anchoring site. Expression of transgenic RNA in M14M and M14S mice on the basal diet was abundant in the liver. After maintenance with water containing ZnSO4 (50 mM) for 4 days, expression of transgenic RNA in M14M and M14S mice was strong in the small intestine. Immunohistochemical analysis demonstrated CD14 expression in these organs in M14S and M14M mice. Levels of CD14 in sera from M14S mice after zinc administration were significantly higher than these animals maintained with normal water, M14M mice after zinc administration and non-transgenic mice. Sera from M14S and M14M mice after stimulation with lipopolysaccharide LPS (LPS) demonstrated significantly lower levels of tumor necrosis factor-alpha and IL-6 than those from non-transgenic mice. Lethality in endotoxin shock produced by i.p. injection of 30-40 microg/g body wt LPS was not different between M14S, M14M and non-transgenic mice. However, survival rates in the lethal Shwartzman reaction induced by priming and challenge injections of LPS were significantly higher in M14M and M14S mice than in non-transgenic mice.

Surface phenotype analysis of CD16+ monocytes from leukapheresis collections for peripheral blood progenitors.

In peripheral blood progenitor cell (PBPC) collections from patients with solid tumour or haematological malignancy, monocytes were separated into two subpopulations. The majority of monocytes expressed CD14 at a high density without CD16 antigen (the CD14+CD16- monocytes). The remaining monocytes co-expressed CD14 and CD16 (the CD14+CD16+ monocytes). These CD14+CD16+ monocytes amounted to 20.6 +/- 15.8%, while those in peripheral blood (PB) obtained from healthy volunteers were 7.3 +/- 3.1% (P < 0.05). When subdividing the CD14+CD16+ monocytes into CD14brightCD16dim and CD14dimCD16bright cells, both populations were found to be increased in PBPC collections. Since typical CD14+CD16+ monocytes are the CD14dimCD16bright population, we compared the additional surface antigens on CD14dimCD16bright monocytes with those of CD14+CD16- monocytes. In PBPC collections, the CD14dimCD16bright monocytes exhibited lower levels of CD11b, CD15, CD33 and CD38 expression and higher levels of CD4, CD11a, CD11c and MHC class II, and also revealed a higher percentage of CD4+ cells and a lower percentage of CD15+ cells and CD38+ cells, compared with the CD14+CD16- monocytes. When compared with the CD14dimCD16bright monocytes in PB, those in PBPC collections exhibited higher expression of CD4 and lower expression of CD11b, and also showed higher percentages of CD4+ cells and CD38+ cells and a lower percentage of CD11b+ cells. These results suggest that PBPC collections may be rich in the CD14+CD16+ monocytes in which the proportion of the immature population is increased. It is likely that these monocytes participate in the haematological and immune recovery after PBPC transplantation.

Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide.

Septic shock induced by lipopolysaccharide (LPS) triggering of cytokine production from monocytes/macrophages is a major cause of morbidity and mortality. The major monocyte/macrophage LPS receptor is the glycosylphosphatidylinositol (GPI)-anchored glycoprotein CD14. Here we demonstrate that CD14 coimmunoprecipitates with Gi/Go heterotrimeric G proteins. Furthermore, we demonstrate that heterotrimeric G proteins specifically regulate CD14-mediated, LPS-induced mitogen-activated protein kinase (MAPK) activation and cytokine production in normal human monocytes and cultured cells. We report here that a G protein binding peptide protects rats from LPS-induced mortality, suggesting a functional linkage between a GPI-anchored receptor and the intracellular signaling molecules with which it is physically associated.

Quantitation of surface CD14 on human monocytes and neutrophils.

The absolute number of membrane-expressed CD14, the most important endotoxin receptor, on human monocytes and neutrophils shows remarkable variation in the literature. To quantify these numbers two fluorescence methods using fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (mAb) were applied. A commercially available set of standard beads was used in flow cytometry to quantitate CD14 with eight different mAbs. Independent from their isotype the various mAbs showed minor differences and indicated that peripheral blood monocytes expressed 99,500-134,600 (115,400 +/- 10,600) and neutrophils 1,900-4,400 (3,300 +/- 800) CD14 receptors. There was no significant difference in CD14 expression on leukocytes in unprocessed freshly obtained whole blood and after a Ficoll isolation procedure. However, a short temperature shift resulted in a 1.3- to 1.6-fold up-regulation of CD14. The results obtained with the reference beads were verified with fluorescence Scatchard analysis and spectrofluorometry using mAb 26ic-FITC and showed 109,500 CD14 per monocyte and 6,700 CD14 per neutrophil. For comparison the number of CD14 on the monocytic THP-1 cells and Fc gamma-receptors on human leukocytes were determined using the reference beads and flow cytometry and gave results comparable to published data. Our data indicate that resting human monocytes express roughly 110,000 CD14 molecules on their surface using a simple fluorometric assay. Correct determination of the number of CD14 and other cell surface receptors is of importance in the monitoring of septic patients.