Cancer stem cells (CSC) are resistant to radiation and chemotherapy and play a significant role in cancer recurrence and metastatic disease. It is therefore important to identify alternative strategies, such as immunotherapies that can be used to control this refractory population. A CD44(+)CD24(-/low) subpopulation of cells within the B6 PyMT-MMTV transgenic mouse-derived AT-3 mammary carcinoma cell line was identified, which had CSC-like characteristics, including pluripotency and a resistance to chemo- and radiotherapy. Therefore, unlike xenograph models that require immunocompromised settings, this novel system may provide a means to study immune-mediated responses against CSC-like cells. The immunobiology of the AT-3 CSC-like cell population was studied by their surface molecule expression profile and their sensitivity to specified cell death pathways. Comparable levels of Rae-1, CD155, CD54 and higher levels of Fas and DR5 were expressed on the AT-3 CSC-like cells compared to non-CSC-like tumor cells. Expression correlated with an in vitro sensitivity to cell death by NK cells or through the ligation of the death receptors (Fas or DR5), by their ligands or anti-Fas and anti-DR5 mAbs. Indeed, compared to the rest of the AT-3 tumor cells, the CD44(+)CD24(-/low) subpopulation of cells were more sensitive to both Fas- and TRAIL-mediated cell death pathways. Therefore, despite the refractory nature of CSC to other conventional therapies, these CSC-like cells were not inherently resistant to specified forms of immune-mediated cell death. These results encourage the continued investigation into immunotherapeutic strategies as a means of controlling breast CSC, particularly through their cell death pathways.

Freshly collected chronic lymphocytic leukemia B cells (B-CLL cells) are known to be inefficient at stimulating allogeneic T cells, and to lack significant expression of B7 (CD80 and CD86) costimulatory molecules. We investigated the potential of CD40 triggering to up-regulate the expression of adhesion and costimulatory molecules on B-CLL cells, and to enhance their immunogenicity towards allogeneic T cells. B-CLL cells cocultured with human CD40 ligand-expressing mouse fibroblasts rapidly up-regulated CD54 and CD58 adhesion molecules and B7-1 (CD80) and B7-2 (CD86) costimulatory molecules, and acquired a strong stimulatory capacity towards CD4+ as well as isolated CD8+ allogeneic T cells. Costimulation by both CD80 and CD86 proved critical for allogeneic T cell proliferation and CD25 and HLA-DR expression, since these were strongly inhibited by anti-CD80 or anti-CD86 monoclonal antibodies, and completely abrogated by CTLA4-Ig fusion protein, which blocks both CD80 and CD86. B7 costimulation also proved critical for restimulation of primed B-CLL-reactive T cells. Most importantly, priming of purified CD8+ T cells with CD40-triggered allogeneic B-CLL cells resulted in cytotoxic activity against the unstimulated B-CLL cells. These findings raise the possibility that CD40 triggering of B-CLL cells might be exploited in immunotherapeutic protocols.

BACKGROUND

Preterm infants with respiratory distress syndrome (RDS) present with systemic inflammation. The role of lymphocytes in RDS is less studied. Activation of lymphocytes could mediate chronic inflammation and development of bronchopulmonary dysplasia (BPD).

OBJECTIVE

To evaluate whether T cells are activated in preterm infants with RDS and whether T cell activation is associated with the development of BPD.

METHODS

Thirty-four infants with RDS [mean gestational age 27.1 (SD 2.0) weeks, birth weight 900 (216) g] were compared with 21 infants without RDS [32.6 (1.4) weeks, 1,697 (406) g]. From blood samples taken on postnatal days 1, 3, and 7, CD4 and CD8 cell counts and their expressions of co-stimulatory molecule CD54 and adhesion molecule CD62L were determined by flow cytometry. In activated cells, expression of CD54 is increased and CD62L is decreased.

RESULTS

As compared with infants without RDS, infants with RDS had less CD4 and CD8 cells on day 3 (both p = 0.02). On day 1 and day 3, RDS was associated with increased CD54 expression on CD4 cells (p = 0.001; p = 0.03) and decreased CD62L expression on CD8 cells (both p = 0.02). Infants with RDS who developed BPD (n = 18) had higher CD54 expression on CD4 cells on day 3 (p = 0.01) and on CD8 cells on day 1 and day 3 (p = 0.01; p = 0.04) as compared with infants without BPD (n = 16).

CONCLUSIONS

In preterm infants, RDS is associated with a lower T cell count and a higher proportion of activated cells. Increased proportion of activated T cells predicts the development of BPD. Systemic T cell activation could mediate inflammation and development of BPD.

To understand the role of Epstein-Barr virus (EBV) and viral products in associated with immunophenotype and clinical outcome of primary nasopharyngeal carcinoma (NPC), the expression levels of chemokines IFN-g-induced protein 10 (IP-10, CXCL10), stromal-derived factor-1 (SDF-1, CXCL12) and its receptor CXCR4 was investigated in 56 primary NPC biopsy specimens from Chinese NPC patients in parallels with LMP1 antigen and EBER1 by immunohistochemisty (IHC) and in situ hybridization (ISH). Moreover, the expression levels of HLA class I (b-microglobulin) and II antigen (HLA-DR), and co-stimulatory molecule CD54 were also evaluated in 31 out of these 56 patients using immunohistochemisty (IHC). Our results showed that (a) the elevated expression levels of IP10, SDF-1, CXCR4, b-microglobulin, HLA-DR and CD54 in NPC lesions was 66%, 36%, 30%, 42%, 55% and 69%, respectively. (b) High expression of SDF-1 was observed in advanced NPC (N stage, p < 0.05). (c) LMP1 expression correlated with upregulation of CXCR4 and translocation of CXCR4 to the nucleus of the tumor cells. This role of LMP1 in regulating the expression of CXCR4 was confirmed in the EBV positive NPC cell line C666 by inhibition of LMP1 expression with small interfering RNA (siRNA). Our findings provide new insights on the immune status of the malignant cells which may affect the outcome of immunotherapy in NPC. The differentiated nonkeratinizing and undifferentiated types of nasopharyngeal carcinoma (NPC) are associated with Epstein-Barr virus (EBV) in South China, which has the highest incidence rate in the world. The EBV latent type II antigens include nuclear antigen 1 (EBNA1), and latent membrane protein 1 (LMP1, in approximately 50%, seen in ref. 2) and protein 2 (LMP2), in addition small non-polyadenylated viral RNAs non-coding nuclear RNAs (EBERs) and BamHI-A rightward transcripts (BARTs) expressed in NPC tumor cells. The expressions of EBV antigens in NPC tumor cells provide the targets for adoptive immunotherapy. However, the poorly differentiated NPC is always characterized by the presence of a highly cellular lymphoid stroma admixed with tumor cells. However, the role of local immunity surrounding NPC cells and the role EBV and viral products expressed in tumor cell remain unclear, which is associated with the expression of immune-related molecules including chemokines and receptors, HLA class I and II antigens, and co-stimulatory molecules and the role of EBV and viral products to alter the expression of immune-related molecules on tumor cells. It has been reported that the expression pattern of immune related-molecules on tumor cells will affect the outcome of T-cell-based adoptive immunotherapy for NPC.

We had isolated a high molecular weight polysaccharide fraction, designated as F3, and performed a comprehensive analysis of its immunomodulatory and adjuvant activities in vivo and in vitro. In vivo, F3-treated mice showed an increase in the number of dendritic cells as well as CD4, CD8, regulatory T, B, plasma, NK, and NKT cells in the spleen. F3 also elevated the levels of multiple cytokines and chemokines in the blood of mice. F3 displayed potent adjuvant activity for tetanus toxoid in the absence of alum and potentiated antibody responses to alum-containing tetanus toxoid in mice. In addition, F3 also boosted Th1 and Th2 response in vivo. In vitro, F3 induced the maturation of dendritic cells derived from human monocytes by upregulating CD40, CD54, CD80, CD83, CD86, and HLA-DR, enhanced mixed lymphocyte reaction, and stimulated the production of ten cytokines and six chemokines. In microarray analysis, expressions of 7688 genes were modulated in dendritic cells after treatment with F3, including cytokine and chemokine genes. These results provide F3 polysaccharide extract further insight into the mechanisms of action for these immunomodulatory and adjuvant activities of from Ganoderma lucidum and also offer preclinical evidence for its development as a vaccine adjuvant.

Mast cells (MC) are important cellular components of the immune network in diverse organs. The skin MC has likewise been implicated in IgE- and complement-mediated cutaneous reactions. Such reactions supposedly involve specific cell surface membrane receptors. In this study, the cell surface marker profile of human skin MC was established using monoclonal antibodies (MoAb) against defined CD antigens. MC were isolated from juvenile foreskin (n = 55) and adult mammary skin (n = 5). The reactivity of MC with MoAb was assessed by a combined toluidine blue/immunofluorescence staining technique. Confirming our previous analyses on lung MC, foreskin MC reacted with MoAb against CD9, CD29, CD33, CD43, CD44, CD45, CD46, CD51, CD54, CD55, CD58, CD59, CD61, and CD117 (c-kit). Foreskin MC were also recognized by MoAb to CD47, CD48, CD49d, CD53, CD60, CD63, CD81, CD82, CD84, CD87, CD92, CD97, CD98, and CD99. Recently clustered CD antigens detectable on foreskin MC were CD147 (neurothelin), CD149 (MEM133), CD151 (PETA-3), and CD157 (BST-1). In contrast to lung MC and MC from adult skin, foreskin MC were found to express CD88 (C5aR). Also, cutaneous MC (from both juvenile foreskin and adult mammary skin), but not lung MC, were found to bind the CD32 MoAb IV.3, 2E1, and FLI8.26 (Fc gammaRII). The CD50 antigen (ICAM-3) was detectable on lung MC, but not on foreskin MC or MC of adult mammary skin. In summary, our data show that cutaneous MC and lung MC express an almost identical phenotype; however, in contrast to lung MC, cutaneous MC appear to express substantial amounts of CD32 and to lack CD50. In addition, foreskin MC, unlike MC from adult skin or lung, express CD88.

HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45⁺ microvesicles (MV) which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1BaL or matched non-depleted preparations, the presence of CD45⁺ MVs was shown to enhance DC maturation and ICAM-1 (CD54) expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24⁺) MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates. Recombinant HSP90α and β and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1 infected subjects.

During early neonatal life, important changes occur in the gut. The intestine is challenged by both milk and a microbial flora. Later on, at weaning, the diet of mice changes from milk to pelleted food leading to changes in microbial contents. This period seems essential for a complete development of the mucosal immune system. We investigated the development of both intraepithelial (IEL) and lamina propria lymphocytes (LPL), from day 5, and every 5 days, up to day 30 after birth. IEL and LPL were isolated from the small intestine and the phenotype was assessed by FACS analyses, using antibodies for detection of T-cell markers CD3, TCR alpha beta, TCR gamma delta, CD4, CD8 alpha, CD8 beta, CD5, CD18, CD54, and CD49d. Our data show a clear increase in the number of LPL just before weaning, while the number of IEL increased after day 15. A more mature pattern of membrane antigen expression of both IEL and LPL was observed at weaning. The adhesion molecules CD18, CD54, and CD49d, essential for cellular communication of lymphocytes, showed an expression peak at weaning. In conclusion, the mouse mucosal immune system develops during the first 3 weeks of neonatal life leading to the formation of a more mature immune system at weaning.

Human MM is a haematologic disorder characterized by the accumulation of malignant plasma cells (PC), primarily in the bone marrow (BM). Although these cells characteristically home to the BM, in recent years several groups have detected the presence of related malignant B cells in the peripheral blood (PB) which could be implicated in the progression and spread of the disease. However, the proportion and origin of these clonotypic circulating B cells is still controversial. In this study, using a triple-staining flow cytometric procedure and a whole blood lysis method, PB B lineage cells could be divided into two populations according to their distinct repertoires of cell adhesion molecules and B cell antigens in untreated MM patients. The results show that: (i) the percentage and the absolute number of PB CD19+ B cells were decreased in MM patients compared with controls; (ii) the quantity and percentage of B cell antigens (CD20, CD22, CD24, DR, CD138) and adhesion molecules (beta1- and beta2-integrins, CD44, CD54, CD56, CD61 and CD62L) expressed by these PB CD19+ cells of MM patients and healthy subjects were similar and all of them were virtually polyclonal cells; (iii) a very minor circulating CD19-CD38++CD45-/dim subset was also detected which expressed CD138 (B-B4) (high intensity), monoclonal cytoplasmic immunoglobulin (cIg), and was negative for pan-B antigens (CD19, CD20, CD24, DR), surface immunoglobulin (sIg) and several adhesion molecules such as CD62L, CD18 and CD11a; this CD19-CD38++CD45-/dim CD138++ subset was not found in normal blood and exhibited a phenotypic profile which was closely related to that of malignant BM plasma cells, with the exception of the CD56 antigen. Polymerase chain reaction (PCR) analysis of IgH clonotypic rearrangements confirmed these results. We postulate that, in MM patients, circulating B lineage cells may be divided into two different categories: polyclonal CD19+ B cells and a very minor proportion of clonal CD138++ PC that escape from the BM.

To investigate the mechanism of synovial pannus formation in rheumatoid arthritis, immunohistochemical and immunoelectron microscopic studies with monoclonal antibodies against the adhesion molecules, CD54 (ICAM-1), CD11a (LFA-1), CDw49a (VLA-1), CDw49b (VLA-2), CDw49c (VLA-3), Cdw49d (VLA-4) and CDw49e (VLA-5), were carried out to determine the pattern of distribution of these molecules at the rheumatoid synovial cartilage junction. Treatment with anti-ICAM-1 resulted in membrane staining of most of the macrophages and fibroblasts infiltrating the synovial tissue and bordering and pannus-cartilage junction, suggesting the possibility that ICAM-1 may function to facilitate the adhesion of synovial type A cells bearing ICAM-1 to type B cells of the pannus. ICAM-1 positive macrophages and fibroblasts were often found to be in contact with lymphoid cells, suggesting also that a cellular immune reaction occurs in the formation of the pannus. In addition, VLA-3, VLA-4 and, particularly, VLA-5 were the predominant beta 1 integrins expressed by rheumatoid synovial pannus. Sine these three integrins all function as fibronectin receptors, it is possible that the fibronectin-rich environment of the rheumatoid cartilage surface effectively traps pannus cells expressing high levels of these molecules. The VLA-5 molecule was found in a pericellular and interterritorial matrix distribution in the present study, strongly suggesting that a receptor-ligand interaction between VLA-5 and cartilage matrix may occur at the early stage of pannus formation. Furthermore, an increase in beta 1 integrin may be necessary for the growth of the pannus and also for the upregulation of the VLA molecules, leading secondarily to increased attachment.

We examined the role of intracellular adhesion molecule 1 (ICAM-1 or CD54) in the development of pulmonary edema in rabbits after pulmonary artery occlusion and reperfusion using a monoclonal antibody (MAb) RR1/1 directed against ICAM-1, a ligand for the CD18 leukocyte adhesion glycoprotein complex. A vascular clamp was placed around the left pulmonary artery for 24 h and then released to allow reperfusion for 2 h. Lungs subjected to 24 h of unilateral pulmonary artery occlusion showed increased binding of 125I-labeled RR1/1 and immunocytochemical evidence of ICAM-1 expression in pulmonary vascular endothelial cells compared with the contralateral lung. MAbs RR1/1 (0.5 mg/kg) or IB4 (1.0 mg/kg) (MAb directed against an epitope on the CD18 adhesion glycoprotein) was infused 45-60 min before the start of reperfusion to assess the roles of ICAM-1 and CD18 in the response. After reperfusion, the lungs were removed, suspended from one end of a weighing balance, and perfused with Ringer-albumin (0.5 g/100 ml), and the changes in lung weight were monitored for 60 min. Lung tissue myeloperoxidase (MPO) content (a marker of neutrophil sequestration) was determined after reperfusion. The increases in lung weight gain in the RR1/1- and IB4-treated groups of 960 +/- 100 and 865 +/- 110 mg, respectively, were less (P less than 0.05) than in untreated controls (3,550 +/- 725 mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Salmonellae have evolved several mechanisms to evade host clearance. Here, we describe the influence on bacterial immune escape of the effector protein SopB, which is translocated into the cytosol through a type III secretion system. Wild-type bacteria, as well as the sseC and aroA attenuated mutants exerted a stronger cytotoxic effect on dendritic cells (DC) than their SopB-deficient derivatives. Cells infected with the double sseC sopB, phoP sopB and aroA sopB mutants also exhibited higher expression of MHC, CD80, CD86 and CD54 molecules, and showed a stronger capacity to process and present an I-E(d)-restricted epitope from the influenza hemagglutinin (HA) to CD4+ cells from TCR-HA transgenic mice in vitro. The incorporation of an additional mutation into the sopB locus of the attenuated sseC, phoP and aroA mutants resulted in the stimulation of improved humoral and cellular immune responses following oral vaccination. The obtained results define a new potential immune escape strategy of this important pathogen, and also demonstrate that this mechanism can be subverted to optimize the immune responses elicited using Salmonella as a live vaccine carrier.

The Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disorders that arise in immunosuppressed individuals are considered to resemble EBV-transformed in vitro lymphoblastoid cell lines (LCLs) with a mature activated B-cell phenotype. In this study of human lymphoproliferative disorders in the severe combined immunodeficiency mouse model, however, we demonstrate that EBV-infected tumor cells are not LCL-like but are predominantly plasmacytoid and that this phenotype correlates with reduced expression of EBV latent genes. B-cell tumors developed within 3-6 weeks after injection of LCLs into severe combined immunodeficiency mice. The tumors and the injected LCLs were analyzed by flow cytofluorometry for B-cell differentiation and activation markers and by ribonuclease protection assay for cellular and viral gene expression. No differences in the expression of CD19 and CD21 were observed. However, a decrease in CD23, CD11a (lymphocyte function-associated antigen LFA-1), and CD58 (LFA-3) expression and an increase in CD38 (a plasma-cell-associated antigen), CD54 (intracellular adhesion molecule ICAM-1), and HLA class I in the tumor cells relative to the LCLs was observed. Two-color flow cytofluorometric analysis showed that the predominant population >> 80%) in LCLs was CD23hi/CD38lo and that the major population in LCL-derived tumors was CD23lo/CD38hi. Cell cycle analysis showed that, in contrast to actively cycling LCLs, the majority of tumor cells had exited the cell cycle and were restricted to G0/G1 phase. Finally, and most important, a reduction in mRNA for the EBV latent genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein (LMP1) was observed in the tumors.

OBJECTIVE

Changes in extracellular matrix glycosaminoglycans (GAGs) of the intestinal mucosa have been associated with inflammatory bowel disease. The aim of the present study was to follow the changes in GAGs metabolism during the progression from non-inflamed to inflamed intestinal colon of patients with Crohn's disease (CD), using direct biochemical analysis and specific immunohistochemistry against chondroitin/dermatan sulfate and heparan sulfate.

METHODS

The content of GAGs from inflamed and non-inflamed colon of eight patients with active CD was estimated by uronic acid per dry weight of tissue and analyzed by agarose gel electrophoresis and ion-exchange chromatography. Intestinal sections were stained using antibodies against dermatan sulfate/chondroitin 4-sulfate (DS/CS), heparan sulfate (HS), and ICAM-1 (CD54), and analyzed by confocal microscopy.

RESULTS

There was a reduction in the amount of GAGs in the non-inflamed colon of patients with CD. In the inflamed colon, HS, CS and DS showed increased concentrations compared with the non-inflamed colon. GAGs showed a diffuse distribution in the lamina propria and in the basement membrane of both inflamed and non-inflamed mucosa of patients with CD.

CONCLUSIONS

We observed a marked reduction in GAGs with altered patterns of distribution in the non-inflamed colon of patients with CD. The increase in the synthesis of GAGs observed in the inflamed colon may be a compensatory mechanism for the restoration of the integrity of the intestinal mucosa.

CD7 is a 40-kDa protein found primarily on T, NK, and pre-B cells; the function of the CD7 protein in the immune system is largely unknown. The K12 (SECTM1) protein was originally identified by its location just upstream of the CD7 locus. The K12 gene encodes a transmembrane protein of unknown function. In order to clone a K12-binding protein, we generated a soluble version of the human K12 protein by fusing its extracellular domain to the Fc portion of human IgG(1). Flow cytometry experiments showed that the K12-Fc fusion protein bound at high levels to both human T and NK cells. Precipitation experiments using K12-Fc on (35)S-radiolabeled NK cells lysates indicated that the K12 cognate was an approximately 40-kDa protein. A human peripheral blood T cell cDNA expression library was screened with the K12-Fc protein, and two independent, positive cDNA clones were identified and sequenced. Both cDNAs encoded the same protein, which was CD7. Thus, K12 and CD7 are cognate proteins that are located next to each other on human chromosome 17q25. Additionally, we have cloned the gene encoding the mouse homologue of K12, shown that it maps near the mouse CD7 gene on chromosome 11, and established that the mouse K12 protein binds to mouse, but not human, CD7. Mouse K12-Fc inhibited in a dose-dependent manner concanavalin A-induced proliferation, but not anti-TcRalpha/beta induced proliferation, of mouse lymph node T cells. Human K12-Fc stimulated the up-regulation of CD25, CD54, and CD69 on human NK cells in vitro.

BACKGROUND

Type II alveolar epithelial cells (AECII) are well known for their role in the innate immune system. More recently, it was proposed that they could play a role in the antigen presentation to T lymphocytes but contradictory results have been published both concerning their surface expressed molecules and the T lymphocyte responses in mixed lymphocyte cultures. The use of either AECII cell line or fresh cells could explain the observed discrepancies. Thus, this study aimed at defining the most relevant model of accessory antigen presenting cells by carefully comparing the two models for their expression of surface molecules necessary for efficient antigen presentation.

METHODS

We have compared by flow cytometry the surface expression of the major markers involved in the immunological synapse on the A549 cell line, the most popular model of type II alveolar epithelial cells, and freshly isolated cells. HLA-DR, CD80, CD86, ICOS-L, CD54, CD58 surface expression were studied in resting conditions as well as after IFN-γ/TNF-α treatment, two inflammatory cytokines, known to modulate some of these markers.

RESULTS

The major difference found between the two cells types was the very low surface expression of HLA-DR on the A549 cell line compared to its constitutive expression on freshly isolated AECII. The surface expression of co-stimulatory molecules from the B7 family was very low for the CD86 (B7-2) and ICOS-L (B7-H2) and absent for CD80 (B7-1) on both freshly isolated cells and A549 cell line. Neither IFN-γ nor TNF-α could increase the expression of these classical co-stimulatory molecules. However CD54 (ICAM-1) and CD58 (LFA-3) adhesion molecules, known to be implicated in B7 independent co-stimulatory signals, were well expressed on the two cell types.

CONCLUSIONS

Constitutive expression of MHC class I and II molecules as well as alternative co-stimulatory molecules by freshly isolated AECII render these cells a good model to study antigen presentation.

OBJECTIVE

We attempted to evaluate endothelial dysfunction and the role of AECAs in systemic lupus (SL) with low disease activity. We quantified endothelial microparticles (EMps) and attempted to find the best flow cytometry method for that purpose.

METHODS

CD105, CD144 and CD146 were tested, individually or in combination, on EMp-enriched plasma. Twenty-three healthy blood donors and 27 SL patients were evaluated. SL patients with a SLEDAI <10 (median 2.6) were evaluated in our outpatient clinic. For each patient, EMps (CD105-CD146(+), CD45(-)) and AECAs were quantified and characterized.

RESULTS

The monochrome composite marker CD105-CD146 appeared to be the most efficient in detecting EMps. SL patients had more circulating EMps than healthy donors: respective median values were 2575 and 130 EMps/microl (P < 0.001). SL patients had more CD54(+) and CD54(-) EMps than healthy donors (496 vs 34 EMps CD54(+)/microl, P < 0.0001; 1875 vs 89 EMps CD54(-)/microl, P < 0.0001). The ratio CD54(+) EMps/total EMps was lower for lupus patients than for healthy individuals (20.3 vs 33.7%, P = 0.03). Twenty-four patients (89%) were positive for AECAs. EMp counts were not significantly higher for patients with AECAs.

CONCLUSIONS

Monochrome composite marker is efficient in detecting the whole population of EMps using flow cytometry. SL patients with low disease activity have a marked endothelial dysfunction. EMps released from the endothelium originate from activated and non-activated cells. AECAs do not seem to be the main cause of endothelial dysfunction in this population.

Accessory functions of cellular adhesion molecules (CAM) in activation, adhesion, migration, and cytotoxicity of natural killer (NK) cells are partly dependent on activation by cytokines. We studied effects of interleukin 12 (IL12) on expression and function of adhesion molecules on human NK cells and compared them to the effects mediated by IL2. Target binding of NK cells was significantly increased by IL12, leading to an increased level of conjugate formation with K562 target cells as well as enhanced binding to tumor monolayers. IL12 also induced significant levels of cytotoxicity against fresh tumor cell targets in purified human NK cells. IL12 significantly enhanced adhesion and subsequent migration of NK cells through 3-microns-pore-size polycarbonate filters. However, IL2 was a more potent activator of these functions, which have been shown to be partly mediated by CD2, CD58, beta 2 integrins, and ICAM-1. As assessed by flow cytometry, IL12 also induced significant up-regulation in the proportion or mean fluorescence intensity of NK cells positive for the following activation markers and adhesion molecules: CD25, HLA-DR, CD69, CD71, CD56, CD2, and CD54. Among the beta 2 integrins, IL12 selectively increased expression of CD11a on NK cells, although to a significantly lower level than that induced by IL2. IL12 had different in vitro effects than IL2 on expression and function of the beta 1 integrins. Whereas IL2 induced marked up-regulation in expression of the beta 1 integrins, CD49b, -c, -d, and -e, IL12 had no demonstrable effect over a wide range of concentrations. In addition, while IL2-activated NK cells showed significantly increased integrin-dependent adhesion to fibronectin- or laminin-coated plates, IL12-activated cells were less adherent to fibronectin and were unchanged in their adherence to laminin. Our data demonstrate that IL12 is involved in interactions of NK cells with fresh or cultured tumor cell targets, biologic substrates, or extracellular matrix molecules. Although the magnitude of its in vitro effects on adhesion-dependent functions of NK cells was significantly smaller than that of IL2, lower doses of IL12 were required to up-regulate functions of CAM, and this may be an attractive feature of IL12 as a potential therapeutic cytokine.

We studied the potential of multidimensional flow cytometry to evaluate the frequency and maturation/activation status of dendritic cells in minimally manipulated peripheral blood mononuclear cell preparations (i.e., only separated on Ficoll-Hypaque) of normal donors and cancer patients. A rare subset of HLA-DR+ leukocytes (less than 1% mononuclear cells) was detected in blood of normal donors that displayed all the features of dendritic cells: these cells had high forward-light-scatter characteristics and coexpressed CD4, CD86 and CD54 surface antigens, but lacked the lineage-associated surface markers of T cells, B cells, monocytes, granulocytes or NK i.e. they were CD3-, CD19-, CD20-, CD14-, CD11b-, CD16-, CD56-). These physical and phenotypic properties were virtually identical to those of immunomagnetically sorted leukocytes characterized as dendritic-cells on the basis of morphology, phenotype and high stimulatory activity in allogeneic mixed-lymphocyte cultures. Using this flow-cytometric approach we observed that the frequency of dendritic cell-like cells in peripheral blood mononuclear cell specimens of cancer patients receiving chemotherapy alone or those recovering from stem cell transplantation was significantly lower than that of normal individuals (mean +/- SE: 0.36 +/- 0.05%, 0.14 +/- 0.06%, and 0.75 +/- 0.04% respectively). Multidimensional flow-cytometric analysis of dendritic cells might represent an important new tool for assessing immunocompetence, and for monitoring the effects of therapeutic regimens on the immune system.

Four human breast cancer cell lines (T47D, ZR-75-1, MCF7D and HS578T) were examined for the effects of cytokines on expression of cell surface antigens. Interferon (IFN)gamma up-regulated the expression of ICAM-I (CD54) in all the cell lines and coordinately up-regulated both CD54 and CD40 expression in T47D. Tumour necrosis factor (TNF)alpha, interleukin (IL)1 alpha, IL1 beta and IL6 also up-regulated the expression of CD54 in all the cell lines but CD40 was unaffected. Levels of expression of CD11a, CD18, CD49b, CD58 and CD71 were unaltered by these cytokines. Conditioned medium (CM) generated from human fibroblasts, and in particular from foetal cells, was highly effective in up-regulating expression of ICAM-1 but not of CD40 in the breast cancer cell lines. ICAM-1 induction correlated with IL6 bioactivity in these CMs. Combinations of IL6 with other cytokines, such as IL1, resulted in further increases in ICAM-1 expression. Our observations suggest that IL6 is involved in intercellular signalling between mesenchyme and breast cancer epithelium.

It has been reported that human immunodeficiency virus type 1 (HIV-1) bound to follicular dendritic cells (FDCs) remains highly infectious to CD4(+) T cells even when it forms immune complexes with neutralizing antibody (HIV-1/IC). To elucidate the role of FDCs in HIV-1 transmission to CD4(+) T cells in lymph nodes, we have isolated and purified FDCs from human tonsils and examined whether the HIV-1/IC trapped on their surface is infectious to CD4(+) T cells. To our surprise, not the HIV-1/IC but the antibody-free HIV-1 on FDCs could be transmitted to CD4(+) T cells. Furthermore, in contrast to previous studies showing that FDCs are productively infected with HIV-1, the present study clearly demonstrated that FDCs were not the target cells for HIV-1 infection. FDCs could capture the viral particles on their surface; however, the binding of HIV-1 to FDCs was strongly inhibited by the presence of anti-CD54 (ICAM-1) monoclonal antibody (MAb) and anti-CD11a (LFA-1) MAb, suggesting that the adhesion molecules play an important role in the interaction between HIV-1 and FDCs.

OBJECTIVE

Activation of cannabinoid CB(2) receptors protects against various forms of ischaemia-reperfusion (I/R) injury. Δ(8) -Tetrahydrocannabivarin (Δ(8) -THCV) is a synthetic analogue of the plant cannabinoid Δ(9) -tetrahydrocannabivarin, which exhibits anti-inflammatory effects in rodents involving activation of CB(2) receptors. Here, we assessed effects of Δ(8) -THCV and its metabolite 11-OH-Δ(8) -THCV on CB(2) receptors and against hepatic I/R injury.

METHODS

Effects in vitro were measured with human CB(2) receptors expressed in CHO cells. Hepatic I/R injury was assessed in mice with 1h ischaemia and 2, 6 or 24h reperfusion in vivo.

RESULTS

Displacement of [(3) H]CP55940 by Δ(8) -THCV or 11-OH-Δ(8) -THCV from specific binding sites in CHO cell membranes transfected with human CB(2) receptors (hCB(2) ) yielded K(i) values of 68.4 and 59.95 nM respectively. Δ(8) -THCV or 11-OH-Δ(8) -THCV inhibited forskolin-stimulated cAMP production by hCB(2) CHO cells (EC(50) = 12.95 and 14.3 nM respectively). Δ(8) -THCV, given before induction of I/R, attenuated hepatic injury (measured by serum alanine aminotransferase and aspartate aminotransferase levels), decreased tissue protein carbonyl adducts, 4-hydroxy-2-nonenal, the chemokines CCL3 and CXCL2,TNF-α, intercellular adhesion molecule 1 (CD54) mRNA levels, tissue neutrophil infiltration, caspase 3/7 activity and DNA fragmentation. Protective effects of Δ(8) -THCV against liver damage were still present when the compound was given at the beginning of reperfusion. Pretreatment with a CB(2) receptor antagonist attenuated the protective effects of Δ(8) -THCV, while a CB(1) antagonist tended to enhance it.

CONCLUSIONS

Δ(8) -THCV activated CB(2) receptors in vitro, and decreased tissue injury and inflammation in vivo, associated with I/R partly via CB(2) receptor activation.

BACKGROUND

This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

This study investigated the expression of intercellular adhesion molecule-1 (ICAM-1; CD54) by cells of the central nervous system (CNS) during acute experimental allergic encephalomyelitis (EAE) and chronic relapsing EAE (CREAE). In the CNS of normal guinea pigs, only a few endothelial cells expressed detectable levels of ICAM-1, whereas during the active phases of the disease ICAM-1 was present on cells of the perivascular infiltrate and the endothelia of both lesion- and non-lesion-associated blood vessels. In addition, cultured cerebrovascular endothelia maintained in 'standard' culture medium did not express ICAM-1, but they could be induced to express this antigen on incubation in a lymphocyte-conditioned medium. These findings suggest that the induction of ICAM-1 on CNS endothelia may be important in antigen presentation or in promoting lymphocyte extravasation across the blood-brain barrier in inflammatory disorders of the CNS.

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