Antimicrobial susceptibility testing of Gram-positive and -negative bacterial isolates directly from spiked blood culture media with Raman spectroscopy.

Patients suffering from bacterial bloodstream infections have an increased risk of developing systematic inflammatory response syndrome (SIRS), which can result in rapid deterioration of the patients' health. Diagnostic methods for bacterial identification and antimicrobial susceptibility tests are time-consuming. The aim of this study was to investigate whether Raman spectroscopy would be able to rapidly provide an antimicrobial susceptibility profile from bacteria isolated directly from positive blood cultures. First, bacterial strains (n = 133) were inoculated in tryptic soy broth and incubated in the presence or absence of antibiotics for 5 h. Antimicrobial susceptibility profiles were analyzed by Raman spectroscopy. Subsequently, a selection of strains was isolated from blood cultures and analyzed similarly. VITEK®2 technology and broth dilution were used as the reference methods. Raman spectra from 67 antibiotic-susceptible strains showed discriminatory spectra in the absence or at low concentrations of antibiotics as compared to high antibiotic concentrations. For 66 antibiotic-resistant strains, no antimicrobial effect was observed on the bacterial Raman spectra. Full concordance with VITEK®2 data and broth dilution was obtained for the antibiotic-susceptible strains, 68 % and 98 %, respectively, for the resistant strains. Discriminative antimicrobial susceptibility testing (AST) profiles were obtained for all bacterial strains isolated from blood cultures, resulting in full concordance with the VITEK®2 data. It can be concluded that Raman spectroscopy is able to detect the antimicrobial susceptibility of bacterial species isolated from a positive blood culture bottle within 5 h. Although Raman spectroscopy is cheap and rapid, further optimization is required, to fulfill a great promise for future AST profiling technology development.

A novel lysosome-associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment.

We have identified a novel lysosome-associated membrane glycoprotein localized on chromosome 3q26.3-q27, DC-LAMP, which is homologous to CD68. DC-LAMP mRNA is present only in lymphoid organs and DC. A specific MAb detects the protein exclusively in interdigitating dendritic cells. Expression of DC-LAMP increases progressively during in vitro DC differentiation, but sharply upon activation with LPS, TNFalpha, or CD40L. Confocal microscopy confirmed the lysosomal distribution of the protein. Furthermore, DC-LAMP was found in the MHC class II compartment immediately before the translocation of MHC class II molecules to the cell surface, after which it concentrates into perinuclear lysosomes. This suggests that DC-LAMP might change the lysosome function after the transfer of peptide-MHC class II molecules to the surface of DC.

The cytokine profile expressed by human dendritic cells is dependent on cell subtype and mode of activation.

In the present study, we have analyzed the pattern of cytokines expressed by two independent dendritic cell (DC) subpopulations generated in vitro from human cord blood CD34+ progenitors cultured with granulocyte-macrophage CSF and TNF-alpha. Molecularly, we confirmed the phenotypic differences discriminating the two subsets: E-cadherin mRNA was only detected in CD1a+-derived DC, whereas CD68 and factor XIIIa mRNAs were observed exclusively in CD14+-derived DC. Semiquantitative reverse-transcriptase PCR analysis revealed that both DC subpopulations spontaneously expressed IL-1alpha, IL-1beta, IL-6, IL-7, IL-12 (p35 and p40), IL-15, IL-18, TNF-alpha, TGF-beta, macrophage CSF, and granulocyte-macrophage CSF, but not IL-2, IL-3, IL-4, IL-5, IL-9, and IFN-gamma transcripts. Both subpopulations were shown to secrete IL-12 after CD40 triggering. Interestingly, only the CD14+-derived DC secreted IL-10 after CD40 activation, strengthening the notion that the two DC subpopulations indeed represent two independent pathways of DC development. Furthermore, both DC subpopulations expressed IL-13 mRNA and protein following activation with PMA-ionomycin, but not with CD40 ligand, in contrast to IL-12 and IL-10, revealing the existence of different pathways for DC activation. Finally, we confirmed the expression of IL-7, IL-10, and IL-13 mRNA by CD4+ CD11c+ CD3- DC isolated ex vivo from tonsillar germinal centers. Thus, CD14+-derived DC expressing IL-10 and factor XIIIa seemed more closely related to germinal center dendritic cellsGCDC than to Langerhans cells.

CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3alpha and is highly expressed in human dendritic cells.

Dendritic cells initiate immune responses by ferrying antigen from the tissues to the lymphoid organs for presentation to lymphocytes. Little is known about the molecular mechanisms underlying this migratory behavior. We have identified a chemokine receptor which appears to be selectively expressed in human dendritic cells derived from CD34+ cord blood precursors, but not in dendritic cells derived from peripheral blood monocytes. When stably expressed as a recombinant protein in a variety of host cell backgrounds, the receptor shows a strong interaction with only one chemokine among 25 tested: the recently reported CC chemokine macrophage inflammatory protein 3alpha. Thus, we have designated this receptor as the CC chemokine receptor 6. The cloning and characterization of a dendritic cell CC chemokine receptor suggests a role for chemokines in the control of the migration of dendritic cells and the regulation of dendritic cell function in immunity and infection.

Follicular dendritic cells specifically express the long CR2/CD21 isoform.

This paper describes an antibody (mAb 7D6) that specifically recognizes human follicular dendritic cells (FDCs). By expression cloning, a cDNA clone encoding for the long human CR2/ CD21 isoform (CD21L) that contains an additional exon (10a) was isolated. We demonstrated that FDCs selectively express CD21L, while B cells selectively express the short CR2/CD21 lacking exon 10a (CD21S). By screening mouse Ltk- cells transfected with the CD21L cDNA, we further showed that the other two anti-human FDC mAbs DRC-1 and KiM4 also recognize CD21L. Thus, CD21L represents the first characterized human FDC-specific molecule, which may confer unique functions of FDCs in germinal center development.

CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF alpha.

Human dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34+ hematopoietic progenitors in presence of GM-CSF+TNF alpha for 12 d. The present study demonstrates that cord blood CD34+ HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CD1a and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CD80, CD83, CD86, CD58, high HLA class II). CD1a+ precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14+ progenitors mature into CD1a+ DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIIIa described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA+ naive T cells. Interestingly, the CD14+ precursors, but not the CD1a+ precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T cells. Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14(+)-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.

CD40 and B cell antigen receptor dual triggering of resting B lymphocytes turns on a partial germinal center phenotype.

Phenotypic alterations occur when resting human B lymphocytes become germinal center (GC) cells. These include the induction of surface CD38, CD95 (FAS/APO-1), and carboxy-peptidase-M (CPM), a recently described GC marker. However, the factors that govern the in vivo induction of these surface molecules on B cells remain unknown. Here, we purified resting (CD38-) human B lymphocytes from tonsils in an attempt to establish culture conditions resulting in the induction of these three GC markers. We show that interferon (IFN) alpha or IFN-gamma, as well as antibodies against the B cell antigen receptor (BCR), could induce CD38 on resting B lymphocytes, a phenomenon further enhanced by CD40 stimulation. Concomitantly, CD95 was upregulated by CD40 ligation and, to a lesser extent, by IFN-gamma. By contrast, CPM expression could be upregulated only through BCR triggering. This CPM induction was specifically enhanced by CD19 or CD40 ligation. CD40 + BCR stimulation of resting B cells with CD40 ligand-transfected fibroblastic cells in the presence of cross-linked anti-BCR monoclonal antibodies resulted in the coexpression of CD38, CD95, and CPM. As GC cells, these cells also expressed CD71, CD80 (B7.1), and CD86 (B7.2), but not CD24. However, CD10+ or CD44- B cells could not be detected in these culture conditions, suggesting that yet other signals are required for the induction of these GC markers. Consistent with a GC phenotype, CD40 + BCR-stimulated cells exhibited reduced viability when cultured for 20 h in the absence of stimulus. These results first demonstrate that cotriggering of resting B cells through BCR and CD40 induces both phenotypic and functional GC features. They also show that IFN and CD19 triggering of resting B cells specifically modulate the expression of GC markers.

Distribution of carboxypeptidase M on lymphoid and myeloid cells parallels the other zinc-dependent proteases CD10 and CD13.

Monoclonal antibody (MoAb) M27 was generated after immunization of mice with the human B-lineage acute lymphoblastic leukemia cell line Pre-ALP. Under reducing conditions, MoAb M27 precipitated a 60-kD surface-membrane molecule from Pre-ALP cells. Expression cloning of Pre-ALP cDNA showed that M27 recognizes carboxypeptidase M (CPM), a cell-surface, zinc-dependent protease known to cleave off basic C-terminal amino acids from peptide hormones. Using M27 antibody, CPM was detected only at discrete B lymphocyte developmental stages, namely on committed precursors and on germinal center cells. CPM was also expressed on mature T cells, mainly after activation. These results provide the first description of a carboxy-peptidase on lymphoid cells. In addition, CPM was found on granulocytes and monocytes, but not on their progenitors. Strikingly, CPM was present only on CD38+ cells, irrespective of lineage affiliation. Of interest, CPM displayed a largely overlapping distribution with the CD10 and CD13 peptidases, with which it shares common substrates (enkephalins, bradykinin). Collectively, the present data show a previously unrecognized distribution pattern of CPM on lymphoid and myeloid cells and suggest that CPM may cooperate with CD10 and CD13 to regulate biologic activity of peptide hormones on leukocytes.

Growth inhibitory and agonistic signals of interleukin-7 (IL-7) can be mediated through the CDw127 IL-7 receptor.

The present study was aimed at identifying surface-membrane molecules involved in the regulation of human B-cell ontogeny. For this purpose, murine monoclonal antibodies (MoAbs) were generated against Pre-Alp, a pre-B acute lymphoblastic leukemia (ALL) cell line, and MoAb R34.34 was selected for further characterization. R34.34 recognized a molecule expressed on normal B-cell precursors (BCP) but not on mature B cells. The antibody also reacted with T lymphocytes, a subpopulation of monocytes from peripheral blood, and a subset of CD34+ cells. Immunoprecipitation analysis indicated that R34.34 recognizes an 80-kD molecular weight antigen. Antibody R34.34 was further found to be directed against an epitope interfering with binding of interleukin-7 (IL-7) to Pre-Alp cells. Expression cloning from a Pre-Alp cDNA library showed that R34.34 antigen is CDw127, the 75- to 80-kD IL-7 receptor. Proliferation of the B-lineage ALL cell lines Reh and Mieliki was inhibited by IL-7, and this effect was specifically reverted by MoAb R34.34. In addition, antibody R34.34 specifically inhibited IL-7-dependent proliferation of normal BCP, Pre-Alp cells, and peripheral T cells. These results imply that both inhibitory and proliferative effects of IL-7 can be mediated through the same receptor on various lineages. R34.34 antibody should be important for the analysis of signal transduction through CDw127.

Expression of a 32-kDa ligand for the CD40 antigen on activated human T lymphocytes.

To identify the ligand(s) of the human CD40 antigen, a cDNA encoding the extracellular domain of the CD40 antigen was fused to a cDNA encoding the constant region (Fc) of human IgG1. The CD40-Fc fusion protein was able to specifically bind to CD4+ and various CD8+ T cell clones activated with immobilized anti-CD3. The 125I-labeled CD40-Fc fusion protein bound anti-CD3 activated CD4+ T cell clone (MT9) with an equilibrium dissociation constant (Kd) of 10-20 nM. The human CD40-binding protein expressed on the cell surface of activated T lymphocytes is a monomeric protein of approximately 32 kDa. Minor components of 29 kDa and 17 kDa were also detected. A small proportion of CD4+ and CD8+ blood mononuclear T cells activated by anti-CD3 expressed the CD40 ligand but its detection was best observed following depletion of B cells. Addition of B cells to purified T cells abolished the binding of CD40-Fc obtained after anti-CD3 activation.