Current treatments for AMI centre on prompt restoration of epicardial coronary blood flow. Despite improvements, AMI is still associated with significant morbidity and mortality. Novel approaches are therefore keenly sought. Intercellular adhesion molecule-1 (ICAM-1, CD54) is a member of the immunoglobulin superfamily. It is implicated in neutrophil and monocyte-endothelial cell adhesion, processes contributing to myocardial neutrophil infiltration and microvascular coronary slow flow, both viewed as important to the pathophysiologic responses in AMI. ICAM-1 would therefore appear an important potential therapeutic target in this context, and is the subject of this review.

In this study, the impact of the beta-adrenergic antagonist propranolol on resting and acute psychological- and physical-stress-induced circulating leukocyte numbers and the density of cellular adhesion molecules was investigated. In a randomized double-blind crossover design, 45 healthy volunteers performed a 15-min public speaking task and 21 subjects performed a 16-min bicycle exercise after 5 days of ingesting a placebo and after 5 days of ingesting 100 mg/day propranolol. One week of ingesting propranolol modestly elevated the numbers of CD62L+ (P<0.019) but not CD62L- T-lymphocytes. Moreover, propranolol preferentially blunted-psychological stress-induced increases in naïve T-helper (CD4+CD62L+; P<0.049) and naïve T-cytotoxic lymphocytes (CD8+CD62L+; P<0.003), as well as activated T-cytotoxic lymphocytes (CD8+CD29+; P<0.005). However, exercise-induced increases in leukocyte numbers were enhanced following propranolol treatment (P<0.04). In contrast to the effect on the numbers of adhesion-molecule-bearing cells, there was only a modest effect of propranolol on stress-induced alterations of the density of CD62L, CD54 and CD11a. In this study, propranolol treatment interfered with the adrenergic regulation of circulating leukocyte numbers by blunting psychological stress effects but enhancing exercise effects. Propranolol affected the cell activation status to a lesser extent, as reflected by the density of adhesion molecules.

Polyinosinic:polycytidylic acid (poly I:C) is a synthetic double-stranded polyribonucleotide that elicits immune responses analogous to those observed during viral infection. It is also known to modulate the expression of certain autoimmune disorders including diabetes mellitus in the BB rat and NOD mouse. The mechanism underlying these immunomodulatory effects is not known, but it could involve activation of vascular endothelium. We now report that parenteral poly I:C induces rat pancreatic endothelium to hyperexpress intercellular adhesion molecule 1 (CD54). This is accompanied by a perivascular recruitment of mononuclear cells to the exocrine pancreas. Corollary in vitro studies demonstrated that poly I:C is a potent activator of both rat and human endothelial cells in culture. It upregulates endothelial expression of several leukocyte adhesion molecules, stimulates the release of interleukin-6 and interleukin-8, and antagonizes interferon-gamma induction of major histocompatibility complex class II expression. We conclude that poly I:C activates endothelial cells to express surface molecules and cytokines in a pattern classically associated with leukocyte recruitment. These effects may in part contribute to the immunomodulatory effects of poly I:C in animal models of autoimmunity.

BACKGROUND

Adhesion is crucial in the metastatic process of malignant tumours. Recently, the expression of certain selectins on intratumoral vessels has been shown to be associated with the clinical outcome of melanoma patients.

OBJECTIVE

For the first time, this study examines the serum concentrations of circulating soluble vascular cellular adhesion molecule 1 (CD 106), endothelial leucocyte adhesion molecule (CD62E), sP-selectin (CD62P) and sCD44v5 in comparison to soluble intercellular adhesion molecule 1 (sICAM-1, CD54) in a series of 34 melanoma patients at different clinical stages and 11 normal donors using ELISA:

RESULTS

sICAM-1 and sP-selectin levels were statistically elevated in all subgroups of melanoma patients compared to controls (p < 0.01). Circulating ICAM-1 as well as sP-selectin might be valuable additional serological tumour markers which correspond to tumour load and correlate with progression of malignant melanoma.

CONCLUSIONS

Measurement of sICAM-1 and sP-selectin serum levels might therefore provide a sensitive tool for monitoring the clinical course of melanoma.

Septic shock is a severe disease state characterised by the body's life threatening response to infection. Complex interactions between endothelial cells and circulating monocytes are responsible for microvasculature dysfunction contributing to the pathogenesis of this syndrome. Here, we intended to determine whether microparticles derived from activated monocytes contribute towards inflammatory processes and notably vascular permeability. We found that endotoxin stimulation of human monocytes enhances the release of microparticles of varying phenotypes and mRNA contents. Elevated numbers of LPS-induced monocytic microparticles (mMP) expressed CD54 and contained higher levels of transcripts for pro-inflammatory cytokines such as TNF, IL-6 and IL-8. Using a prothrombin time assay, a greater reduction in plasma coagulation time was observed with LPS-induced mMP than with non-stimulated mMP. Co-incubation of mMP with the human brain endothelial cell line hCMEC/D3 triggered their time-dependent uptake and significantly enhanced endothelial microparticle release. Unexpectedly, mMP also modified signalling pathways by diminishing pSrc (tyr416) expression and promoted endothelial monolayer tightness, as demonstrated by endothelial impedance and permeability assays. Altogether, these data strongly suggest that LPS-induced mMP have contrasting effects on the intercellular communication network and display a dual potential: enhanced pro-inflammatory and procoagulant properties, together with protective function of the endothelium.

Multiple myeloma (MM) cells express idiotypic proteins and other tumor-associated antigens which make them ideal targets for novel immunotherapeutic approaches. However, recent reports show the presence of Kaposi's sarcoma herpesvirus (KSHV) gene sequences in bone marrow dendritic cells (BMDCs) in MM, raising concerns regarding their antigen-presenting cell (APC) function. In the present study, we sought to identify the ideal source of DCs from MM patients for use in vaccination approaches. We compared the relative frequency, phenotype, and function of BMDCs or peripheral blood dendritic cells (PBDCs) from MM patients versus normal donors. DCs were derived by culture of mononuclear cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The yield as well as the pattern and intensity of Ag (HLA-DR, CD40, CD54, CD80, and CD86) expression were equivalent on DCs from BM or PB of MM patients versus normal donors. Comparison of PBDCs versus BMDCs showed higher surface expression of HLA-DR (P =.01), CD86 (P =. 0003), and CD14 (P =.04) on PBDCs. APC function, assessed using an allogeneic mixed lymphocyte reaction (MLR), demonstrated equivalent T-cell proliferation triggered by MM versus normal DCs. Moreover, no differences in APC function were noted in BMDCs compared with PBDCs. Polymerase chain reaction (PCR) analysis of genomic DNA from both MM patient and normal donor DCs for the 233-bp KSHV gene sequence (KS330233) was negative, but nested PCR to yield a final product of 186 bp internal to KS330233 was positive in 16 of 18 (88.8%) MM BMDCs, 3 of 8 (37.5%) normal BMDCs, 1 of 5 (20%) MM PBDCs, and 2 of 6 (33.3%) normal donor PBDCs. Sequencing of 4 MM patient PCR products showed 96% to 98% homology to the published KSHV gene sequence, with patient specific mutations ruling out PCR artifacts or contamination. In addition, KHSV-specific viral cyclin D (open reading frame [ORF] 72) was amplified in 2 of 5 MM BMDCs, with sequencing of the ORF 72 amplicon revealing 91% and 92% homology to the KSHV viral cyclin D sequence. These sequences again demonstrated patient specific mutations, ruling out contamination. Therefore, our studies show that PB appears to be the preferred source of DCs for use in vaccination strategies due to the ready accessibility and phenotypic profile of PBDCs, as well as the comparable APC function and lower detection rate of KSHV gene sequences compared with BMDCs. Whether active KSHV infection is present and important in the pathophysiology of MM remains unclear; however, our study shows that MMDCs remain functional despite the detection of KSHV gene sequences.

The use of immunotherapy to treat patients with plasma cell dyscrasias (PCD) such as multiple myeloma (MM) and Waldenström's macroglobulinemia (WM) has gained enormous interest in recent years, with considerable efforts being mounted by many investigators. These efforts have included the use of serotherapy (antibody-mediated immunotherapy), vaccination strategies aimed at inducing allogeneic as well as autologous anti-MM immunity, and the use of donor lymphocyte infusions (DLIs). A number of cell surface antigens on malignant plasma cells and/or B cells in MM and/or WM patients have been proposed for use in tumor cell-targeted serotherapy, including immunoglobulin idiotype, CD19, CD20, CD38, CD54, CD138, HM1.24, and MUC1 core protein. Ongoing clinical trials are examining serotherapy targeting CD20 (in MM and WM) and CD38 (in MM), with early reports of responses to the anti-CD20 monoclonal antibody (mAb) Rituximab (Genentech, South San Francisco, CA) in patients with WM and certain patients with MM. The use of agents to induce MM- and WM-selective antigens for targeting in serotherapy has been proposed based on studies demonstrating the upregulation of CD20 by interferon-gamma (IFN-gamma), and of MUC1 core protein by dexamethasone (DEX) on malignant plasma cells. Strategies to induce allogeneic anti-MM immunity have included immunization of the marrow donor to idiotypic protein, as well as DLI. In addition, proposed immunization strategies aimed at inducing autologous immunity include vaccination with dendritic cells pulsed with MM antigens, MM cell-dendritic cell fusions, carrier-linked idiotype protein, catalytic subunit of telomerase, or DNA encoding for single-chain variable fragments (scFv) linked to a carrier protein gene. Whole tumor vaccination strategies are also being examined and include the use of MM cells transfected and/or stimulated with cytokines, costimulatory molecules, or CD40 ligand. Finally, potential obstacles to the use of immunotherapy, including the presence of resistance antigens on MM and WM tumor cells, are discussed.

Normal and malignant plasma cells were investigated for the expression of seven cellular adhesion molecules by immunofluorescence microscopy. The antigens investigated were CD2 and its ligand, LFA-3 (CD58). LFA-1 alpha (CD11a) and LFA-1 beta (CD18) and their ligand ICAM-1 (CD54), H-CAM (lymphocyte homing receptor; CD44) and N-CAM (CD56). Marrow from 18 patients with myeloma, two with plasma cell leukaemia (PCL), four with monoclonal gammopathy of uncertain significance (MGUS) and 10 normal allogeneic bone marrow donors was studied. All plasma cells from normals and multiple myeloma patients were negative for CD2, CD11a and CD18. All normal and myeloma marrow plasma cells were positive for ICAM-1. 16/18 myeloma cases tested, and all other samples (normal, MGUS and PCL), contained plasma cells positive for H-CAM. Only one normal, but 12/16 myelomas tested were positive for N-CAM (P less than 0.02). One of four MGUS cases was moderately positive and one other weakly positive for N-CAM. Both PCLs were N-CAM negative. 12/18 myelomas were positive for LFA-3, but only two normals (P less than 0.05). All MGUS cases were negative for LFA-3, as was one PCL, the other being weakly positive. Three cases were negative for both adhesion molecules, three cases expressed only N-CAM or LFA-3 and 10 cases expressed both. LFA-3 and N-CAM are expressed significantly in myeloma rather than normal plasma cells. Cases of MGUS may express N-CAM but not, in this small series, LFA-3. Plasma cells in the peripheral blood (PCL) and plasma cell lines express little or no LFA-3 or N-CAM.

OBJECTIVE

Previous studies have shown that hepatic natural killer (NK) cells, also called pit cells, have a higher cytotoxicity against certain tumor cells and have a higher expression of the cell adhesion molecule CD11a as compared with blood NK cells. We further investigated the involvement of the adhesion molecules, reported to be involved in target cell killing by blood NK cells, in pit cell-mediated colon carcinoma cell killing.

METHODS

51Cr-release and DNA fragmentation were used to quantify target cell lysis and apoptosis, respectively. Adhesion of pit cells to CC531s monolayers was quantitated.

RESULTS

Flow cytometric analysis showed that pit cells expressed CD2, CD11a, CD18 and CD54. CC531s cells expressed only CD54. Treatment of freshly isolated pit cells with monoclonal antibodies (mAbs) to CD11a and CD18 inhibited not only the pit cell-mediated CC531s cytolysis but also the pit cell-induced apoptosis of CC531s cells. The combination of mAbs to CD11a, CD18 and CD54 further increased the inhibition of pit cell-mediated CC531s cytolysis and apoptosis. Anti-CD2 mAb did not affect these processes. The binding of pit cells to CC531s cells was also inhibited by anti-CD11a, and CD18 mAbs, but not by anti-CD2 mAb. Anti-CD54 mAb reduced the target cell killing and the binding only slightly.

CONCLUSIONS

These results indicate that CD11a/CD18 (LFA-1) present on pit cells plays an important role in pit cell-mediated target cell adhesion, lysis and apoptosis. This finding might explain why pit cells, which have a higher expression of LFA-1 as compared to blood NK cells, are more cytotoxic against tumor cells as compared to blood NK cells.

We previously demonstrated that mucosal immunization with SHIV virus-like particles (VLPs) was able to induce strong humoral and cellular immune responses against HIV envelope protein (Env). To understand the mechanism for such enhanced immune responses, we studied the interaction between VLPs and dendritic cells (DCs) in initiating immune responses. We found that about 50% of DCs were bound by octadecyle rhodamine B (R18) labeled SHIV VLPs. The bound SHIV VLPs were internalized by DCs when cultured at 37 degrees C. Incubation of immature human PBMC-derived DCs with SHIV VLPs for 48 h resulted in the significant up-regulation of CD40, CD80, CD83, CD54, CD86, HLA-A, B, C and HLA-DR, DP, DQ molecules on activated DC CD11c+ subpopulations. SHIV VLPs efficiently stimulated DCs to release IL-12, IFN-gamma and TNF-alpha. Furthermore, SHIV VLPs-activated DCs were fully functional in inducing allogeneic T cell proliferation. We conclude that DCs can interact and process SHIV VLPs efficiently and may be critical in initiation of SHIV VLPs-induced immune responses. Thus, interaction between VLPs and DCs may play an important role in the enhancement of immune responses in VLPs-based vaccination.

Trafficking of antigen-specific T cells into the central nervous system (CNS) is an important initiating step in inflammation in the brain. In spite of the extensive knowledge about the role of adhesion molecules in T cell migration across peripheral vessels, the mechanism of the entry of antigen-specific T cells into the CNS is not known. This work was designed to study the regulatory roles of adhesion molecules in antigen-specific T cell migration into the CNS. Antigen-specific T cells were tracked in an in vivo migration assay using T cell receptor (TCR) transgenic mice having 95% of T cells specific for a defined antigen. pigeon cytochrome c (PCC). TCR transgenic mice were cannulated intraventricularly (IVT) for PCC antigen infusion and cerebrospinal fluid (CSF) sampling. Upon PCC infusion into the CNS, the number of alpha/beta TCR+ Vbeta3+ Mac1- cells in the CSF was characterized in the presence or absence of anti-adhesion molecule reagents. We found that antibodies against VCAM-1 (CD106), VLA-4 (CD49d/CD29), ICAM-1 (CD54), and LFA-1 (CD11a/CD18) did not influence the increased number of antigen-specific T cells in the CSF However, upon intravenous (i.v.) injection, anti-PECAM-1 (CD31) antibody or PECAM-Ig chimeric molecule inhibited the trafficking of alpha/beta TCR+ Vbeta3+ Mac1- cells into the CNS. The expression of PECAM-1 (CD31) was also up-regulated on antigen-specific T cells in a time-dependent manner in vitro upon antigenic stimulation. The antigen-induced activation of T cells in vivo was measured by CD44 and LFA-1 expression and found to be comparable between mPECAMIg-treated mice and wild-type serum control-treated groups. This indicates that CD31 inhibition of antigen-specific T cell accumulation in the CNS is probably not due to a functional inhibition of these cells. Finally, adoptive transfer of CFSE-labeled AND transgenic cells into naïve animals resulted in the accumulation of these cells in the CNS upon PCC IVT immunization that was also inhibited by mPECAMIg treatment. Hence, PECAM-1 (CD31) might play an important role in regulating antigen-specific T cells trafficking in CNS inflammatory diseases.

Adipose tissue is a source of adult multipotent stem cells that can differentiate along mesenchymal lineage. When mature fat cells obtained from human subcutaneous adipose tissue were maintained with attachment to the ceiling surface of culture flasks filled with medium, two fibroblastic cell populations appeared at the ceiling and the bottom surface. Both populations were positive to CD13, CD90, and CD105, moderately positive to CD9, CD166, and CD54, negative to CD31. CD34, CD66b, CD106, and CD117, exhibited potential of unlimited proliferation, and differentiated along mesenchymal lineage to produce adipocytes, osteoblasts, and chondrocytes. The population that appeared at the ceiling surface showed higher potential of adipogenic differentiation. These observations showed that the cells tightly attached to mature fat cells can generate two fibroblastic cell populations with multiple but distinct potential of differentiation. Since enough number of both populations for clinical transplantation can be easily obtained by maintaining fat cells from a small amount of subcutaneous adipose tissue, this method has an advantage in preparing autologous cells for patients needing repair of damaged tissues by reconstructive therapy.

We have investigated the in vitro uptake, toxicity, phenotypic consequences and transfection efficiency of a stealth NGR/PEG/PDBA-coupled-SHA-PEI/pDNA targeting polyplex loaded with PLGA-PEG-PLGA tri-block copolymer in human monocyte-derived dendritic cells (DCs). Modification with PEG effectively shielded and reduced non-specific phagocytosis by immature DCs to approximately 20%. Coupling the NGR cell-specific peptide to the PEGylated polyplex (NGR/PEG/PDBA-SHA-PEI/pDNA) however resulted in specific and enhanced phagocytosis in DCs without any observable toxicity at the optimum concentration of 0.25% of the copolymer. DNase treatment had no effect on DNA integrity in the encapsulated polyplex. Confocal microscopy confirmed intracellular localization of the targeting NGR/PEG/PDBA-SHA-PEI/pDNA microparticles, resulting in more enhanced uptake of the radiolabeled plasmid DNA and approximately 5- and 10-fold increase over the control tri-block Pluronic F68 copolymer and the non-targeting polyplex, respectively. More importantly, phagocytosis of the targeting microparticles neither altered the functionality of immature DCs nor the phenotypic expression of DC-specific cell surface molecules, CD80, CD86, CD40 and CD54 (ICAM-1), suggesting that uptake of the targeting microparticles by themselves did not induce DC maturation. Taken together, these results suggest that PLGA-PEG-PLGA encapsulation of this stealth targeting polyplex has no negative effects on key properties of immature DCs and should pave the way for targeting DCs for vaccination purposes.

We describe a subset of peripheral CD14+ cells, coexpressing the CD34 progenitor marker and able to migrate across endothelial cell monolayers. On culture with granulocyte-macrophage-CSF, this population differentiated into dendritic cells expressing CD83, CD80, HLA-DR(bright), CD86, and CD54. These dendritic cells were immunostimulatory, in that they induced proliferation of allogenic and tetanus toxoid-specific T lymphocytes. The CD14+ CD34+ population expressed higher levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and alpha4beta1 integrin than the CD14+ CD34- counterpart, being dull positive for other integrins. Using stably transfected PECAM-1+, VCAM-1+, or ICAM-1+ cells, we found that PECAM-1 and, to a lesser extent, VCAM-1, could support transmigration of CD14+ CD34+ cells, whereas the alphaL-ICAM-1 interaction was involved in cell adhesion. PECAM-1-driven transmigration was conceivably dependent on a haptotactic gradient, as it was reduced by 80% across NIH3T3 cells transfected with the PECAM-1-delta cyto deletion mutant. This mutant lacks the cytoplasmic tail and displays a reduced tendency to localize at the intercellular junctions, thus failing to form a molecular junctional gradient. Once differentiated, dendritic cells derived from CD14+ CD34+ precursors retained their transendothelial migratory capability, using both PECAM-1 and ICAM-1 for transmigration. We suggest that a subset of CD14+ CD34+ circulating leukocytes can localize to peripheral tissues and differentiate into functional dendritic cells, thus representing a functional reservoir of potential APC. PECAM-1, constitutively expressed on vascular endothelium, is likely to play a relevant role in the egress of this population from the bloodstream.

Understanding why human immunodeficiency virus (HIV) preferentially infects some CD4(+) CD45RO(+) memory T cells has implications for antiviral immunity and pathogenesis. We report that differential expression of a novel secreted factor, ps20, previously implicated in tissue remodeling, may underlie why some CD4 T cells are preferentially targeted. We show that (i) there is a significant positive correlation between endogenous ps20 mRNA in diverse CD4 T-cell populations and in vitro infection, (ii) a ps20(+) permissive cell can be made less permissive by antibody blockade- or small-interference RNA-mediated knockdown of endogenous ps20, and (iii) conversely, a ps20(low) cell can be more permissive by adding ps20 exogenously or engineering stable ps20 expression by retroviral transduction. ps20 expression is normally detectable in CD4 T cells after in vitro activation and interleukin-2 expansion, and such oligoclonal populations comprise ps20(positive) and ps20(low/negative) isogenic clones at an early differentiation stage (CD45RO(+)/CD25(+)/CD28(+)/CD57(-)). This pattern is altered in chronic HIV infection, where ex vivo CD4(+) CD45RO(+) T cells express elevated ps20. ps20 promoted HIV entry via fusion and augmented CD54 integrin expression; both of these effects were reversed by anti-ps20 antibody. We therefore propose ps20 to be a novel signature of HIV-permissive CD4 T cells that promotes infection in an autocrine and paracrine manner and that HIV has coopted a fundamental role of ps20 in promoting cell adhesion for its benefit. Disrupting the ps20 pathway may therefore provide a novel anti-HIV strategy.

We studied the effects of a C60 water suspension at 4 microg/mL (nC60) and the water soluble fullerenol C60(OH)24 at final concentrations of 1-100 microg/mL on human umbilical vein endothelial cells (HUVECs) in culture. We found that a 24 hr treatment of HUVECs with C60(OH)24 at 100 microg/mL significantly increased cell surface expression of ICAM-1(<em>CD54</em>) (67 +/- 4% <em>CD54</em>+ cells vs. 19 +/- 2 % <em>CD54</em>0 cells in control; p < 0.001). In addition, this treatment induced the expression of tissue factor (CD142) on HUVECs (54 +/- 20% CD142+ cells vs 4 +/- 2% CD142+ cells in control; p = 0.008) and increased exposure of phosphatidylserine (PS) (29 +/- 2% PS+ cells vs. 12 +/- 5% PS+ cells in control; p < 0.001). Analysis of cell cycle and DNA fragmentation (TUNEL) showed that both nC60 and C60(OH)24 caused G1 arrest of HUVECs and C60(OH)24 induced significant apoptosis (21 +/- 2% TUNEL+ cells at 100 microg/mL of C60(OH)24 vs. 4 +/- 2% TUNEL+ cells in control; p < 0.001). We also demonstrated that both nC60 and C60(OH)24 induced a rapid concentration dependent elevation of intracellular calcium [Ca2+]i. This could be inhibited by EGTA, suggesting that the source of [Ca2+]i in fullerene stimulated calcium flux is predominantly from the extracellular environment. In conclusion, fullerenol C60(OH)24 had both pro-inflammatory and pro-apoptotic effects on HUVECs, indicating possible adverse effects of fullerenes on the endothelium.

OBJECTIVE

The authors have previously shown that a monoclonal antibody (mAb) that recognizes intercellular adhesion molecule-1 (ICAM-1), also known as CD54, when administered systemically inhibits experimental vasospasm in a rat femoral artery model, suggesting that ICAM-1 and leukocyte-endothelial adhesion play a crucial role in the molecular chain of events leading to posthemorrhagic vasospasm. In this report the authors confirm this hypothesis with mAbs directed against lymphocyte function-associated antigen-1 ([LFA-1] CD11a/CD18), the molecule on the surface of leukocytes that interacts with ICAM-1.

METHODS

Femoral arteries in 38 Sprague-Dawley rats were isolated and exposed to autologous blood. Twenty-nine animals were then randomized into three groups and received intraperitoneal injections of anti-LFA-1 mAb (10 rats), anti-ICAM-1 mAb (10 rats), or an isotype-matched control mAb (nine rats). Injections were administered at 3 hours and 3, 6, and 9 days after surgery. Before their deaths, six animals underwent spleen harvest, and splenocytes were used in fluorescence-activated cell sorter (FACS) analysis to verify saturation of appropriate binding sites. Animals were killed at 12 days and vessels were harvested for histological study and measurement of the luminal cross-sectional area. Nine animals were randomized as earlier, killed 24 hours after a single injection of mAb, and evaluated for periadventitial infiltration of granulocytes and macrophages. Results of FACS analysis demonstrated saturation of both LFA-1 and ICAM-1 binding sites in animals treated with the respective mAb. The mean ratios of blood-exposed to saline-exposed luminal cross-sectional areas (expressed as the percentage of lumen patency) were 90.1 +/- 5.8% (mean +/- standard error of the mean) for animals treated with the anti-LFA-1 mAb (p = 0.0218), 94.2 +/- 3.3% for animals treated with the anti-ICAM-1 mAb (p = 0.0067), and 62 +/- 7.4% for animals treated with the isotype-matched control mAb. Macrophage and granulocyte counts in the periadventitial region were 39.5 +/- 3.2/hpf for animals treated with anti-LFA-1 mAb (p = 0.001), 42 +/- 3.7/hpf for animals treated with anti-ICAM-1 mAb (p = 0.003), and 72.2 +/- 6.2/hpf for control animals.

CONCLUSIONS

The systemic administration of anti-LFA-1 or anti-ICAM-1 mAb initiated 3 hours after exposure to autologous blood inhibits the development of delayed chronic vasospasm at 12 days in a rat femoral artery model and leads to a significant reduction in periadventitial inflammatory cells at 24 hours. The authors conclude that blocking the migration of inflammatory cells across the endothelial surface of an artery after adventitial exposure to blood prevents the initiation of biological cascades necessary for the subsequent development of chronic vasospasm.

Environmental hyperthermia and exercise produce extensive changes in gene expression in human blood cells, but it is unknown whether this also happens during febrile-range hyperthermia. We tested the hypothesis that heat shock protein (HSP) and immunomodulatory stress gene expression correlate with fever in intensive care unit patients. Whole blood messenger RNA was obtained over consecutive days from 100 hospitalized patients suffering from sepsis or noninfectious systemic inflammatory response syndrome (SIRS) as defined by conventional criteria. The most abnormal body temperature in the preceding 24 h was recorded for each sample. Expression analysis was performed using the Affymetrix U133 chip. ANCOVA followed by correlation analysis was performed on a subset of 278 prospectively identified sequences of interest. Temperature affected expression of 60 sequences, either independently or as a function of clinical diagnosis. Forty-eight of these (representing 38 genes) were affected by temperature only, including several HSPs, transcription factors heat shock factor (HSF)-1 and HSF-4, cellular adhesion molecules such as ICAM1/CD54 and JAM3, toll receptors TLR-6 and TLR-7, ribosomal proteins, and a number of molecules involved in inflammatory pathways. Twelve sequences demonstrated temperature-dependent responses that differed significantly between patients with sepsis and noninfectious SIRS: CXCL-13; heat shock proteins DNAJB12 and DNAJC4; the F11 receptor; folate hydrolase 1; HSF-2; HSP 70 proteins HSPA1A, HSPA1B, and HSPA1L; interleukin 8; lipopolysaccharide binding protein; and prostaglandin E synthase. Febrile-range temperatures achieved during sepsis and noninfectious SIRS correlate with detectable changes in stress gene expression in vivo, suggesting that fever can activate HSP gene expression and modify innate immune responses. For some genes, it appears that clinical condition can alter temperature-sensitive gene expression. Collectively, these data underscore the potential importance of body temperature in shaping the immune response to infection and injury.

Endothelial cells (ECs) are involved in the process of angiogenesis, the outgrowth of new vessels from preexisting blood vessels. If available in sufficiently large numbers, ECs could be used therapeutically to establish blood flow through in vitro engineered tissues and tissues suffering from severe ischemia. Adipose tissue (AT) is an easily available source of large number of autologous ECs. Here we describe the isolation, in vitro expansion, and characterization of human AT derived ECs (AT-ECs). AT-ECs proliferated rapidly through 15-20 population doublings. The cultured cells showed cobblestone morphology and expressed EC markers including CD31, CD144, eNOS, CD309, CD105, von Willebrand factor, CD146, CD54, and CD102. They bound Ulex europaeus agglutinin I lectin and took up DiI-Ac-LDL. The AT-ECs formed capillary-like tubes in Matrigel in vitro and formed functional blood vessels in Matrigel following subcutaneous injection into immunodeficient mice. In conclusion, AT-ECs reach clinically significant cell numbers after few population doublings and are easily accessible from autologous AT, which also contains mesenchymal stem cells/pericytes. Thus, AT yields two cell populations that may be used together in the treatment of tissue ischemia and in clinical applications of tissue engineering.

BACKGROUND

The present study was aimed at determining whether routine prenatal measurements of circulating soluble intercellular adhesion molecule (sICAM-1; CD54) and soluble vascular cell adhesion molecule (sVCAM-1; CD106) in midgestation have predictive value for the identification of pregnant women destined to develop preeclampsia or other complications of pregnancy during late gestation.

METHODS

Plasma sICAM-1 and sVCAM-1 were analyzed between weeks 22 and 29 of gestation in 1543 pregnant women and related to the outcome of pregnancy in a prospective longitudinal study.

RESULTS

Plasma sICAM-1 and sVCAM-1 in uncomplicated pregnancies were normally distributed and varied over a small range (sICAM-1, SD = 22.5%; sVCAM-1, SD = 25.5%). Of all analyzed uncomplicated pregnancies, 54 (3.95%) were identified with concentrations of sICAM-1 or sVCAM-1 above the mean + 2 SD. In contrast, of 177 pregnancies with complications (prevalence, 11.5%), 97 (55%) had sICAM-1 or sVCAM-1 concentrations above the same cutoffs weeks before the onset of disease. The sensitivities of sICAM-1 and sVCAM-1 measurements were 66% for preeclampsia and hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome), 42% for gestational hypertension, 50% for fetal retardation, 46% for preterm labor, 50% for gestational diabetes mellitus, 67% for gestational proteinuria, and 70% for infections during pregnancy. Taken together, routine prenatal sICAM-1 and sVCAM-1 measurements had an overall predictive value of 64%.

CONCLUSIONS

Midgestation measurements of circulating sICAM-1 and sVCAM-1 have a high predictive value (area under the curve of combined sICAM-1 and sVCAM-1 measurements determined by ROC analysis, 0.85) and may identify up to 55% of pregnant women who will later develop a severe pregnancy-related complication.

All-trans retinoic acid (ATRA) is used to treat patients with acute promyelocytic leukaemia (APL), inducing APL cells to differentiate into abnormal neutrophils. To investigate the possible relationship between the chromosome translocation t(15;17) found in APL and ATRA treatment, the human myeloid leukaemia cell lines HL60 and NB4, that are PML-RARalpha negative and positive, respectively, were treated with ATRA and immunophenotyped using a CD antibody microarray. For HL60 cells, ATRA induced major increases in descending order of CD38, CD11b, CD45RO, CD11c, CD54 and CD36 with repression of CD117 and CD44. For NB4 cells, ATRA induced major increases in descending order of CD11c, CD54, CD11a, CD11b, CD53, CD65, CD138, CD66c and T-cell receptor alpha/beta (TCRalpha/beta), with repression of CD38 and CD9. The induction of a number of these CD antigens is consistent with the known differentiation of these leukaemias to abnormal neutrophils. Approximately half of the antigens up-regulated by ATRA on NB4 cells were adhesion molecules, including CD11a, CD11b, CD11c, CD54, CD66c and CD138, consistent with the increased adhesiveness of leukaemia cells observed for APL patients treated with ATRA. On HL60 cells, ATRA induced expression of CD38, CD43 and CD45RO and repressed CD117, while the converse was true on NB4 cells that contain chimeric PML-RARalpha. For NB4 cells, ATRA induced some remarkable increases in CD antigens not seen for HL60: CD14 (16.6-fold), CD32 (27.8), CD53 (20.5), CD65 (139), CD66c (79.7), CD126 (15.1), and CD138 (57.6). The expression of these antigens may be regulated by PML-RARalpha in the presence of ATRA. Such CD antigens could be targets for synergistic treatment of APL with therapeutic antibodies following ATRA treatment.

Bone marrow-derived dendritic cells (DC) of the rat have not been as well characterized as those from the mouse. Here, large quantities of bone marrow-derived rat DC were generated when Flt-3 ligand (FL) was used as an adjunct to granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). These cells displayed a typical DC phenotype, expressing MHC class II, CD54, CD80, CD86, and CD11b/c. These DC also uniformly expressed low levels of CD161 and expressed OX62 in a bimodal distribution. Few cells were recovered from cultures grown without FL, and they failed to express OX62 or CD161. The DC generated with FL were more potent antigen-presenting cells in mixed lymphocyte cultures than cells grown without FL, and among FL-derived cells, the OX62+ cells were slightly more stimulatory than OX62- cells. Thus, FL is a useful cytokine for obtaining large quantities of functional rat DC subsets in vitro.

1alpha,25-Dihydroxyvitamin D3 (1,25D3) induces HL60 cells to acquire a monocyte-like phenotype, while cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) resemble macrophages. Using a microarray of 82 CD antibodies, 24 cluster of differentiation (CD) antigens were detected on HL60 cells. 1,25D3 induced the following antigens in decreasing order of the change: CD14, CD11c, CD11b, CD54, CD86, CD38 and CD66c, with repression of CD117, CD71, CD95, CD45 and CD64. TPA induced the following antigens in decreasing order of the change: CD11c, CD9, CD11b, CD54, CD38, CD45RO and CD66c, with repression of CD4, CD117, CD95, CD71 and CD64. The results presented provide a basis for monitoring differentiation therapy of myeloid leukaemias in patients.

To determine whether cell cultures maintain the cellular heterogeneity of primary tissues and may therefore be used for in vitro modeling of breast cancer subtypes, we evaluated the expression of a cell surface marker panel in breast cancer cell cultures derived from various subtypes of human breast carcinoma. We used a four-color flow cytometry strategy to immunophenotype seven human breast cancer cell cultures and four reference breast cancer cell lines. We analyzed 28 surface markers selected based on their potential to distinguish epithelial or mesenchymal lineage, to identify stem cell populations, and to mediate cell adhesion and migration. We determined their ability to form mammospheres and analyzed luminal cytokeratins CK18, CK19, and myoepithelial/basal CK5, SMA (alpha-smooth muscle actin), and vimentin expression by western blot. All cell surface markers showed a unimodal profile. Ten/28 markers were homogenously expressed. Four (CD66b, CD66c, CD165, CD324) displayed negative/low expression. Six (CD29, CD55, CD59, CD81, CD151, CD166) displayed homogenous high expression. Eighteen (CD9, CD10, CD24, CD26, CD44, CD47, CD49b, CD49f, CD54, CD61, CD90, CD105, CD133, CD164, CD184, CD200, CD227, CD326) were heterogeneously expressed. Spearman's rank test demonstrated a significant correlation (p< 0.001) between mesenchymal phenotype and breast cancer cell cultures. Breast cancer cell cultures, all CD44+, displayed concomitant high expression of only three antigens (CD10, CD54, CD90), and low expression of CD326; cell cultures formed mammospheres and expressed CK5, SMA and vimentin, and were weakly CK19-positive. We demonstrate that breast cancer cell cultures preserve inter-tumor heterogeneity and express stem/progenitor markers that can be identified, quantified and categorized by flow cytometry. Therefore, cell cultures can be used for in vitro modeling of breast cancer subtypes; immunophenotyping may mirror breast cancer heterogeneity and reveal molecular characteristics of individual tumors useful for testing target therapy.