Very little is known about alveolar macrophage (AM) immunological function in early childhood. Using nonbronchoscopic bronchoalveolar lavage (BAL), this study sought to compare the proportion, number, and function of AM between very young and older children. BAL fluid (BALF) leukocyte parameters were determined in 63 children, and data divided into 3 age groups: group 1 (<2 yrs), group 2 >> or =2-< or =5 yrs) and group 3 >> or =6-< or =17 years). In a further subgroup of children, AM function and immune receptor expression were assessed, and data categorized into two age groups: <2 yrs and>> or =2 yrs of age. Compared to groups 2 and 3, the AM percentage in the BAL in group 1 was significantly increased (median: 98% versus 92% and 91%), as was the albumin-adjusted AM concentration. AM from children <2 yrs expressed less human leukocyte antigen (HLA)-DR (versus>> or =2 yrs of age), were less effective in reducing nitro blue tetrazolium, and released less interleukin (IL)-1 and tumour necrosis factor on lipopolysaccharide stimulation. There was no difference in release of IL-6, expression of intercellular adhesion molecule-1 (CD54), and AM stimulation of allogeneic T-cells, between children <2 yrs and>> or =2 yrs of age. It was concluded that the capacity of alveolar macrophage to stimulate T-cells is not enhanced in early childhood, and that immaturity of alveolar macrophage function may contribute to an increased susceptibility to respiratory infections in this age group.

To investigate the regulatory effects of decoy receptor 3 (DcR3) on the differentiation and function of dendritic cells (DCs), bone marrow-derived DCs (BM-DCs) from nonobese diabetic (NOD) mice were cultured with recombinant DcR3.Fc protein. Their differentiating phenotypes and T cell-stimulating functions were then evaluated. Expression of CD11c, CD40, CD54, and major histocompatibility complex I-A(g7) was reduced in cells cultured with additional DcR3.Fc, compared with DCs incubated with granulocyte macrophage-colony stimulating factor and interleukin (IL)-4, indicating that DcR3 interferes with the differentiation and maturation of BM-DCs. One of the most striking effects of DcR3.Fc on the differentiation of DCs was the up-regulation of CD86 and down-regulation of CD80, suggesting a modulatory potential to skew the T cell response toward the T helper cell type 2 (Th2) phenotype. Consistent with this, the proliferation of CD4(+) T cells cocultured with DcR3.Fc-treated DCs was significantly reduced compared with that of T cells stimulated by normal DCs. Moreover, the secretion of interferon-gamma from T cells cocultured with DcR3.Fc-treated DCs was profoundly suppressed, indicating that DcR3 exerts a Th1-suppressing effect on differentiating DCs. Furthermore, adoptive transfer experiments revealed that NOD/severe combined immunodeficiency mice received DcR3.Fc-treated DCs, and subsequently, autoreactive T cells showed delayed onset of diabetes and a decrease in diabetic severity compared with mice that received normal DCs and T cells, suggesting a future therapeutic potential in autoimmune diabetes. Data from two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight analysis show an up-regulation of some proteins-such as mitogen-activated protein kinase p38 beta, cyclin-dependent kinase 6, and signal-induced proliferation-associated gene 1-and a down-regulation of the IL-17 precursor; tumor necrosis factor-related apoptosis-inducing ligand family member-associated nuclear factor-kappaB activator-binding kinase 1; and Golgi S-nitroso-N-acetylpenicillamine in cells treated with DcR3, further demonstrating its effect on DC differentiation and function.

BACKGROUND

The capacity of inflammatory cells to adhere involves an array of adhesion molecules, and is critical to the inflammatory responses seen in childhood asthma. We aimed to determine the changes of intercellular adhesion molecule-1 (ICAM-1) and L-selectin expressed on peripheral blood (PB) T lymphocytes and natural killer (NK) cells in asthmatic children with acute exacerbation and after prednisolone therapy.

METHODS

Flow cytometric analysis was performed to determine the expression of ICAM-1 (CD54) and L-selectin (CD62L) on T (CD3+) cells and NK (CD3-/CD56+) cells of PB from children with allergic asthma with acute exacerbation and in a stable condition after prednisolone therapy. Atopic subjects without asthma and age-matched controls were also included for comparison.

RESULTS

Percentages of PB non-CD3, CD56+ NK cells, but not CD3+ T cells, increased in asthmatic children with acute exacerbation, compared to those assessed in a stable condition after a course of prednisolone. However, significant decrease of ICAM-1 (P = 0.01) and L-selectin (P = 0.01) expression on PB NK cells, but not on T cells, were found in children with acute asthma compared to those in a stable condition. NK cells in children with acute asthma showed minimal expression of CD69 and CD25.

CONCLUSIONS

Results suggests that either NK cells expressing ICAM-1 and L-selectin selectively migrated into inflamed lung tissues, or subsets of NK cells not expressing ICAM-1/L-selectin were expanded during acute exacerbation of childhood asthma.

In patients with the acquired immunodeficiency syndrome, the incidence of non-Hodgkin's lymphoma is increased. Two major subgroups of AIDS-related NHL (ARL) have been defined: Burkitt-type NHL (BL) and polymorphic centroblastic/immunoblast-rich large cell lymphomas (CB/IB LCL). These subgroups differ in their association with the Epstein-Barr virus (EBV) and thus possibly in their pathogenesis. We studied the expression of EBER (EBV small RNA's), and EBV latent antigens LMP-1 and EBNA-2 in 43 cases of ARL and related this to histology and immune status (CD4-cell count). In addition, in 19 cases the expression of adhesion molecules (LFA-1 (CD18), ICAM-1 (CD54), alpha4beta1 integrin (CD49d/CD29), L-selectin (CD62L) and CD44) was studied. We found major differences between the two subgroups. Patients with BL had significantly higher CD4-cell counts; only 40% of their lymphomas were EBV-positive, and when EBV-positive, were of the type I latency phenotype. Expression of adhesion molecules important for immune recognition was absent or low in all BL. In contrast, the majority of CB/IB LCL were EBER-positive (79%). 58% of EBV-positive LCL (particularly those in patients with CD4-cell counts below 0.2 x 10(9)/1) had a type II or III latency phenotype. Most LCL showed expression of LFA-1, ICAM-1 and alpha4beta1 integrin. CD44s expression was restricted to CB/IB LCL, in whom high expression of the metastasis-associated exon v6-containing CD44 variant was also observed. The observed EBV-latency types and full expression of adhesion molecules suggest that defective Epstein-Barr virus immunity is important in the pathogenesis of CB/IB large cell lymphomas.

Osteoclast-like giant-cell neoplasms of the urinary tract are rare. They are composed of ovoid or spindle-shaped mononuclear cells with evenly spaced osteoclast-like giant cells. Terminology, histogenesis, and biologic behavior of these tumors remain controversial. Six cases of osteoclast-like giant-cell neoplasms of the urinary tract were identified from the consultation files of two of the authors. Patients were all male and elderly (range 65-82), with the exception of one 39-year-old male. In all, 3/6 tumors developed in the bladder and 3/6 in the renal pelvis. Size ranged from 5 to 11 cm. One bladder and three renal pelvis tumors were high stage (pT3) at time of presentation. Adjacent to the osteoclast-like giant-cell neoplasm in the same specimen, all patients had urothelial carcinoma in situ and/or high-grade papillary urothelial carcinoma. Multinucleated cells had identical morphological and immunohistochemical properties of osteoclasts; positive for CD-68, LCA, CD51 and CD54, and negative for cytokeratins and EMA. Varying percentages of mononuclear cells expressed alpha-smooth muscle actin (4/6), desmin (1/6), S-100 (4/6), LCA (2/6) and CD68 (6/6). However, mononuclear cells were also positive for epithelial markers in 4/6 tumors (cytokeratins AE-1/AE-3, Cam 5.2, CK7 and/or EMA). p53 stained mononuclear tumor cells in three cases, paralleling the staining on the accompanying urothelial carcinoma. Ki-67 stained mononuclear tumor cells, but not osteoclast-like giant cells. Follow-up data were available in five cases. One patient developed recurrence of noninvasive urothelial carcinoma and is still alive. Four patients were dead due to disease within 15 months, three with distant metastases. The intimate association of these tumors with urothelial carcinoma along with their immunohistochemical profile supports an epithelial origin for the mononuclear cells and non-neoplastic reactive histiocytic lineage for the osteoclast-like giant cells.

Glycophorin A is a protein with an abundant glycosylation (60% carbohydrate by weight), and studies have suggested that resistance of target cells to natural killing may be correlated with the level of glycophorin A expression. To assess the role of glycophorin A and of its carbohydrates in sensitivity to lysis by natural killer (NK) cells, the glycoprotein was inserted into the membrane of K562 target cells using electropulsation. Peripheral blood lymphocytes were used as effector cells. When glycophorin A was inserted into the membrane, the level of resistance to NK cell attack increased with the number of glycophorin A molecules electroinserted. The resistance to lysis was not due to a defect in target cell-effector cell binding. Electroassociation of glycophorin A did not cause a decrease in the expression of either "positive signals" for NK cells (such as CD71, CD15, and CD32 antigens) or cellular adhesion molecules (CD18, CD29, CD54, and CD58). Furthermore, electroinsertion of glycophorin A did not trigger any "negative signals," such as class I HLA antigen expression. Finally, it was shown that the sialic acid and O-linked oligosaccharides of glycophorin A did not play any role in its effect against NK cells. Conversely, the unique N-linked oligosaccharide was shown to be essential for resistance to occur.

Adult stem cells that reside in adult tissues have been deemed to possess great potential for clinical application because of their capabilities of self-renewal and differentiation. However, the limitations such as infection risks and low isolation rate make the search for appropriate source to be continued. Here, we demonstrate isolation of progenitors from human foreskin tissue samples, which have fibroblast-like morphology and could be easily propagated for more than 50 passages. These foreskin-derived fibroblast-like stromal cells (FDSCs) expressed CD90, CD105, CD166, CD73, SH3, and SH4, which is similar to the immunophenotypes of human bone marrow-derived mesenchymal stem cells. In comparison with Hs68, the human fibroblast cell line, FDSCs are positive for CD105 and absent for CD54 expression. Further, FDSCs could be induced to differentiate into osteocytes, adipocytes, neural cells, smooth muscle cells, Schwann-like cells, and hepatocyte-like cells. Interestingly, when cultured in Dulbecco's modified Eagle's medium/F12 medium, FDSCs can form spheres with increased expression levels of fibronectin and vimentin. In conclusion, foreskin can serve as a valuable source of multipotent cells with the capabilities for endodermal, mesodermal, and ectodermal cells. Coupled with the advantages of their easy access, isolation, and propagation, these foreskin-derived stromal cells might be of potential use in future studies in stem cell differentiation and therapeutic application.

In a clinical phase I/II trial, pediatric patients with high-risk malignancies were treated with ex vivo IL-2-stimulated donor natural killer (NK) cells after transplantation with haploidentical stem cells. To evaluate the potential negative effects of the immunosuppressive drug mycophenolate mofetil (MMF) used for immunotherapy, the functionality and signaling of ex vivo NK cells was investigated. Our results show that during NK cell expansion, long-term (9 days) incubation with mycophenolic acid (MPA), the active metabolite of MMF, in therapeutically relevant concentrations led to the severe inhibition of NK cell proliferation. This correlated with a significantly reduced cytokine/chemokine secretion and the inhibited acquisition of surface receptors regarding cytotoxicity (e.g., NKp30, NKp44, NKp46, NKG2D), adhesion/migration (e.g., ICAM-1/CD54, LFA-1/CD11a, CD62L, CXCR3) and activation (e.g., CD25). Moreover, MPA prevented phosphorylation of the central signaling molecules STAT-3/-4/-5, AKT and ERK1/2. In contrast, short-term (24 h) MPA incubation of IL-2-stimulated NK cells had no or only marginal effects on the activated NK cell phenotype, including receptor expression, cytokine/chemokine secretion and intracellular signaling. Further, short-term MPA incubation only moderately affected the highly cytotoxic activity of previously IL-2-stimulated NK cells. In conclusion, while long-term MPA incubation significantly compromised ex vivo NK cell functionality, previously IL-2-activated NK cells seemed to be rather resistant to short-term MPA treatment. This finding supports the use of IL-2-activated NK cells as immunotherapy, especially for patients treated with MMF after haploidentical stem cell transplantation.

Recruitment of neutrophils into alveolar air spaces is an early event in the pathogenesis of pneumonia due to Streptococcus pneumoniae. This results from chemokines released by activated endothelial and epithelial cells and alveolar macrophages. Culture supernatants of 6 wild-type strains of S. pneumoniae, shown to contain choline-binding protein A (CbpA; clades A and B), induced release of chemokine CXCL8 from the human alveolar epithelial cell line A549, whereas a CbpA deletion mutant elicited significantly reduced CXCL8 release, compared with that of its isogenic parent (P<.01). Recombinant CbpA up-regulated expression of messenger RNA of CXCL8 and CCL2 but not of XCL1, CXCL10, CCL1, CCL3, CCL4, or CCL5 in A549 cells and induced increased secretion of CXCL8, CCL2, CXCL1, and CXCL5 in a dose- and time-dependent manner. CbpA also increased the expression of intercellular adhesion molecule 1 (CD54) by A549 cells. Thus, CbpA of S. pneumoniae induces the transcription and release of proinflammatory molecules by human alveolar epithelial cells.

Epidemiological data indicate that there is a strong association between intake of trans-18 : 2 fatty acids (TFA) and sudden cardiac death. There is little known about the mechanisms by which TFA exert harmful effects on the cardiovascular system. The present in vitro study is the first to demonstrate the effects of membrane-incorporated C18 : 2 TFA on human aortic endothelial cell (HAEC) function. Trans-18 : 2 fatty acids were incorporated to a greater extent (2-fold) in the phospholipid fraction of endothelial cells than that of cis-18 : 2; furthermore, these fatty acids were enriched to a similar extent in the TAG fraction. Flow cytometric analysis indicated that TFA treatment of HAEC significantly increased the expression of endothelial adhesion molecules, including intercellular adhesion molecule-1 (CD54) and vitronectin receptor (CD51/CD61). Incorporation of TFA into membranes increased HAEC adhesion to fibronectin- or vitronectin-coated plates by 1.5- to 2-fold, respectively. Neutrophil and monocyte adhesion to HAEC monolayers was nearly proportional to adhesion molecule expression. TFA treatment also induced the release of monocyte chemoattractant protein-1 by nearly 3-fold in non-stimulated HAEC. Furthermore, we examined the role of TFA on in vitro angiogenic assays. Chemotactic migration of TFA-treated HAEC toward sphingosine-1-phosphate (SPP) was significantly increased compared with controls. Conversely, capillary morphogenesis of TFA-treated HAEC was significantly inhibited in response to SPP, suggesting that TFA incorporation suppresses endothelial cell differentiation. In conclusion, these in vitro studies demonstrated that TFA play a role in the induction of pro-inflammatory responses and endothelial cell dysfunction.

Polyploidy and binuclearity are characteristics of the mammalian liver. Increasing polyploidisation occurs with age and after administration of various drugs and chemicals. This study was designed to examine the function of ploidy by addressing several questions: (1) Does the increase in size of polyploid hepatocytes have any physiological function by altering surface receptor expression such as intercellular adhesion molecule-1 (ICAM-1, CD54) or IFNgammaR? and (2) Do polyploid cells respond differently to inflammatory cytokines such as interferon gamma (IFNgamma)? We have developed a method to accurately measure the volume of live isolated hepatocytes using confocal microscopy and image analysis. Using flow cytometry, we have shown that the expression of ICAM-1 increases with increasing DNA content and IFNgammaR is not detectable on isolated mouse hepatocytes. Diploid (2n), tetraploid (4n) and octoploid (8n) hepatocytes were found to be equally susceptible to IFNgamma-induced apoptosis in vitro. Although the function of polyploidy remains unanswered, we have described some of the characteristics of polyploidy in isolated hepatocytes and in vitro.

Interactions of alveolar macrophages with respiratory epithelium may play a key role in hyperoxia-induced lung inflammation. We studied the effect of cell-cell contact with epithelial cells in hyperoxia on macrophages' secretion of interleukin-8 (IL-8). A549 pulmonary epithelial cells and THP-1 monocyte/macrophage cells were cultured either singly, in contact coculture, or prevented from contact by a porous membrane, and exposed to oxygen or room air. Phorbol-12-myristate-13-acetate-(PMA)-treated THP-1 cells were exposed to the same conditions. Neither cell line cultured alone produced appreciable amounts of IL-8 in hyperoxia. Contact cocultures exposed to hyperoxia produced increased IL-8, while in the noncontact coculture it was attenuated. Both cell-cell contact and PMA increased THP-1 cell CD54 expression. Intracellular IL-8 production was increased in contact cocultured, hyperoxia exposed THP-1 cells, while the A549 sample showed no change. Increased IL-8 mRNA expression was not demonstrated in cocultured, hyperoxic exposed THP-1 cells, suggesting nontranscriptional regulation of IL-8 protein levels. Contact with epithelial cells appears to potentiate macrophage responses to hyperoxia.

Bronchiolitis is characterized histologically by epithelial necrosis and peribronchial infiltration of leukocytes, with a high percentage of neutrophils in the airways. We investigated the expression of adhesion molecules (CD11a, CD11b, CD18, CD31, CD54, and CD62L) on neutrophils from nasopharyngeal aspirates (NPAs) and peripheral blood (PB) of infants with respiratory syncytial virus (RSV)-induced bronchiolitis. The expression of CD31 and CD62L on neutrophils from NPAs is decreased and the expression of CD11b, CD18, and CD54 on neutrophils from NPAs is increased compared with cells from PB of RSV-infected infants. The expression of CD18 and CD54 on neutrophils from PB of RSV-infected infants is also increased compared with cells from PB of control infants. Shedding of CD31 and CD62L on neutrophils in RSV infection may contribute to the neutrophil emigration from blood to airways; the upregulation of Mac-1 (CD11b/CD18) and CD54 on neutrophils may help explain the high percentage of neutrophils in the airways of RSV bronchiolitis; and the upregulation of Mac-1 may be involved in the increased neutrophil-airway epithelial adhesion in RSV infection.

Conventional aldehyde based fixatives produce good morphological preservation. However, owing to their cross-linking mechanism of action, epitope loss may occur during fixation compromising the tissue for subsequent immunohistochemical (IHC) analysis. IHC is an important tool for characterizing antigen, cytokine and cytomorphological markers. The increasing use of mouse models for study of pathogenesis has highlighted the need to investigate alternative fixatives. In the study reported here, tissue samples from RIII mice with immune mediated lesions, Mycobacterium bovis infected mice, and uninfected control mice were fixed in either zinc salt fixative or buffered formalin, then tested for IHC using a panel of antibodies (CD3, CD4, CD8, CD45, CD54, F4/80, Interferon-gamma and MIP2). Zinc salt fixation preserved processing-sensitive murine cell markers (CD4, CD8 and CD54) and improved the intensity of immunolabeling for CD45, F4/80 and CD3. Buffered formalin failed to preserve any of the processing-sensitive murine epitopes for demonstration by subsequent IHC.

Lymphocyte activation induces or increases the expression of several surface structures, none of which is characteristic of an activated cell subset. In particular, structures such as CD45RO, CD25, CD26, CD49b, CD54, CD71 are expressed by the vast majority of lymphocytes at various times following in vitro activation. CD39 molecules were originally identified on activated B lymphocytes and have recently been described on activated T cell clones. In the present report, we have characterized phenotypically and functionally defined cell subsets generated during an in vitro allostimulation. Results indicated that the percentage of CD39+ cells reached a maximum at day 6 and remained stable thereafter. We demonstrate that CD39 expression allows the identification within the allosensitized CD8+ cytotoxic cells of distinct subsets of cells mediating allo cytotoxic T lymphocyte or natural killer (NK)-like reactivity. More precisely, CD8+CD39+ alloactivated cells mainly mediate specific killer activity, whereas CD8+CD39- alloactivated cells predominantly exhibit NK-like reactivity. Further, we show a high functional correlation associated with the lack of CD39 expression on NK-like alloactivated CD8+ cells, while there is no association with CD56 or CD57 NK-associated structures.

The potential of human adipose stem cells (ASCs) for regenerative medicine has received recognition owing to their ease of isolation and their multilineage differentiation capacity. Additionally, low immunogenicity and immunosuppressive properties make them a relevant cell source when considering immunomodulation therapies and allogeneic stem cell treatments. In the current study, immunogenicity and immunosuppression of ASCs were determined through mixed lymphocyte reactions. The immunogenic response was analyzed after cell isolation and expansion in fetal bovine serum (FBS), human serum (HS)-supplemented medium, and xeno-free and serum-free (XF/SF) conditions. Additionally, the immunophenotype and the secretion of CXC chemokine ligand 8 (CXCL8), CXCL9, CXCL10, C-C chemokine ligand 2 (CCL2), CCL5, interleukin 2 (IL-2), IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor-α, interferon-γ, transforming growth factor-β1, indoleamine 2,3-deoxygenase, Galectin-1, and Galectin-3 were analyzed. The results showed that ASCs were weakly immunogenic when expanded in any of the three conditions. The significantly strongest suppression was observed with cells expanded in FBS conditions, whereas higher ASC numbers were required to display suppression in HS or XF/SF conditions. In addition, statistically significant differences in protein secretion were observed between direct versus indirect cocultures and between different culture conditions. The characteristic immunophenotype of ASCs was maintained in all conditions. However, in XF/SF conditions, a significantly lower expression of CD54 (intercellular adhesion molecule 1) and a higher expression of CD45 (lymphocyte common antigen) was observed at a low passage number. Although culture conditions have an effect on the immunogenicity, immunosuppression, and protein secretion profile of ASCs, our findings demonstrated that ASCs have low immunogenicity and promising immunosuppressive potential whether cultured in FBS, HS, or XF/SF conditions.

The lymphocyte function-associated antigen-1 (LFA-1) (also known as CD11a/CD18 and αLβ₂), is just one of many integrins in the human body, but its significance is derived from its exclusive presence in leukocytes. In this review, we summarize the studies relating LFA-1 and its major ligand ICAM-1 (or CD54) with cancer, through the function of lymphocytes and myeloid cells on tumor cells. We consider how LFA-1 mediates the interaction of leukocytes with tumors and the role of ICAM-1 in tumor dynamics, which can be independent of its interaction with LFA-1. We also offer a more detailed examination of the role of LFA-1 within B-cell chronic lymphocytic leukemia. Finally, we discuss the role that exosomes harboring LFA-1 play in tumor growth and metastasis.

OBJECTIVE

Gastric cancer is the most common gastrointestinal malignancy and the leading cause of cancer-related deaths in East Asia. Increasing evidence has revealed that autophagy is closely associated with tumor initiation and progression. The present work aimed to investigate the role of autophagy in adjuvant chemotherapy for gastric cancer.

METHODS

Gastric cancer stem cells (CSCs) were isolated from gastric cancer cell lines using the cell surface markers CD44 and CD54 and cultured in a three-dimensional cell culture system. Western blotting was used to detect their protein expression levels in gastric CSCs. In addition, the cells were treated with inhibitors to investigate the underlying mechanisms of autophagy.

RESULTS

After isolation of gastric CSCs expressing CD44 and CD54, Western blot analysis showed that the levels of the autophagic marker LC3II were markedly enhanced in CD44+CD54+ gastric CSCs. Moreover, the ratio of LC3II/LC3I protein levels was higher in CD44+CD54+ gastric CSCs than in non-CSCs. By contrast, both a chemotherapeutic agent (5-fluorouracil) and autophagy inhibitor (chloroquine) exhibited an inhibitory effect on the cell viability of gastric CSCs, and their combination further enhanced such inhibitory effects. Mechanistically, the addition of Notch inhibitor decreased the cell viability of gastric CSCs treated with 5-fluorouracil and chloroquine. In addition, 5-fluorouracil and chloroquine both increased the expression of Notch1 in gastric CSCs.

CONCLUSIONS

These findings show that autophagy regulated drug sensitivity of gastric cancer cells through the Notch signaling pathway.

The effect of RGD-sequence-containing pentapeptides and monoclonal antibodies (MAbs) against the adhesion molecules CD11a-c/CD18, ICAM-1 (CD54) and CD2 on the binding and cytotoxicity of endogenous (freshly purified) and IL-2-stimulated CD3-negative NK cells has been studied. Antibody to CD18 exerted the most significant inhibition of adhesion and cytotoxicity of endogenous NK cells to MOLT-4 lymphoma cells, followed by antibodies to CD2 and CD54. Antibodies to either CD11a, CD11b or CD11c did not inhibit adhesion when used separately, whereas as a mixture their inhibitory capacity was as strong as that of anti-CD18. Antibodies against CD18, CD54 and CD2 exerted an additive effect on the inhibition of adhesion. Their combination eradicated the binding of endogenous NK cells. The RGD-containing peptide did not inhibit the binding or cytotoxicity of freshly purified NK cells to MOLT-4, whereas some inhibition was detected against the adenocarcinoma cell line COLO-205. According to FACS analysis, IL-2 increases the expression of CD2 and CD54 on NK cells. However, the relative contribution of the adhesion molecules to the NK cell binding did not change as a result of the stimulation with IL-2. The RGD-peptide substantially inhibited the binding of IL-2-stimulated killer cells to COLO, and the combination of this peptide with MAbs to CD18, CD54 and CD2 practically blocked the adhesion. Our results indicate that both CD11a-c/CD18- (involving the 3 heterodimers) and CD2-dependent adhesion pathways are used by LGL in endogenous and IL-2 stimulated natural killing. In addition, RGD-binding receptors are involved in adhesion to some target cells.

OBJECTIVE

We previously reported a correlation between levels of micro particles carrying CD31 (PMP(CD31+)) and disease activity in MS. However, the effects of long term (12 month) treatment with high dose, high frequency interferon-β1a (Rebif™) on plasma levels of PMP(CD31+), PMP(CD146+), and PMP(CD54+) and MRI measures of disease activity have not yet been assessed.

METHODS

During this prospective 1-year study, we used flow cytometry to measure changes in plasma micro particles (PMP) bearing CD31 (PMP(CD31+)), CD146 (PMP(CD146+)), and CD54/ICAM-1 (PMP(CD54+)) in 16 consecutive patients with relapsing-remitting MS (RRMS) before and after 3, 6, and 12 months of subcutaneous therapy with interferon-beta1a (44 micrograms, 3X weekly). At each visit, clinical exams and expanded disability status scale (EDSS) scores were recorded.

RESULTS

Plasma levels of PMP(CD31+), and PMP(CD54+) were significantly reduced by treatment with IFN-β1a. PMP(CD146+) appeared to decrease only at 3 months and did not persist at 6 and 12 months (p = 0.0511). In addition, the decrease in plasma levels of PMP(CD31+) and PMP(CD54+) levels at 12 months were associated with a significant decrease in the number and volume of contrast enhancing T1-weigthed lesions.

CONCLUSIONS

Our data suggest that serial measurement of plasma micro particles (PMP), particularly in the initial stages of MS (when neuro-inflammatory cascades are more intense), may serve as reliable and reproducible surrogate markers of response to IFN-β1a therapy for MS. In addition, the progressive decline in plasma levels of PMP(CD31+) and PMP(CD54+) further supports the concept that IFN-β1a exerts stabilizing effect on the cerebral endothelial cells during pathogenesis of MS.

Giant cell tumor (GCT) of bone is a primary osteolytic tumor that is characterized by the formation of osteoclast-like giant cells. In addition to GCT of bone, extraskeletal GCT are known to be formed in several soft tissues. Giant cells in GCT of bone were suggested to be identical to osteoclasts, but the characterization of giant cells in extraskeletal GCT remains incomplete. In this study, a case of sarcomatoid hepatocellular carcinoma with osteoclast-like giant cells was analyzed. Immunohistochemistry revealed the expression of almost all markers of osteoclasts: tartrate-resistant acid phosphatase, CD68, CD51, CD54 and matrix metalloprotease-9, in osteoclast-like giant cells in the tumor. In situ hybridization revealed the expression of receptor activator of nuclear factor-kappa B (RANK) in the giant cells and receptor activator of nuclear factor-kappa B ligand (RANKL) in the tumor cells. The hepatic origin of the sarcomatoid hepatocellular carcinoma cells was confirmed by the expression of albumin. This is the first report suggesting that hepatocyte-derived cells possess the potential for osteoclastogenesis. In addition, these findings suggest that osteoclast-like cells in the hepatocellular carcinoma were formed by the same mechanism as osteoclastogenesis in bone.

Neutrophil inhibitory factor (NIF) is a recently cloned 41-kDa protein from the canine hookworm that binds CD11b/CD18 and inhibits CD11b/CD18-dependent neutrophil adhesion. We evaluated NIF's effects on neutrophil-dependent lung injury in guinea pigs. Pulmonary vascular endothelial CD54 (ICAM-1) was induced in buffer-perfused lungs by 90-min exposure to 1000 U/ml TNF-alpha. Human neutrophils (2 x 10(7)) were added to the perfusate and activated by 5 x 10(-9) PMA; in some lungs, the neutrophils were pretreated with NIF (100 nM) before their addition to the perfusate. Lung injury was assessed by wet:dry weight ratio, and neutrophil uptake by lung myeloperoxidase (MPO) activity. HUVEC exposed to TNF-alpha for 90 min were assayed for neutrophil adhesion, and we compared PMA-stimulated neutrophil adhesion to endothelial cells and fibrinogen-coated plates. PMA-induced pulmonary edema (lung wet:dry ratio increased from 8.8 +/- 0.7 to 18.8 +/- 4.4) was inhibited by NIF (10.0 +/- 1.0). Lung MPO activity concomitantly decreased from 17.1 +/- 6.1 to 8.7 +/- 1.8 U/mg dry lung tissue in the NIF-treated group, similar to controls (6.9 +/- 2.0). Endothelial monolayer experiments confirmed that NIF reduced neutrophil adherence (basal adhesion of 11 +/- 3% increased to 30 +/- 5% with TNF-alpha pretreatment of endothelial cells, an increase that was reduced to 10 +/- 4% with NIF). Moreover, NIF prevented PMA-induced neutrophil adhesion to fibrinogen, a CD11b/CD18-dependent event, but produced a smaller decrease in adherence to endothelial cells, which also involves CD11a/CD18 integrins. These studies indicate that NIF prevents neutrophil-dependent lung vascular injury by inhibiting neutrophil adhesion to the TNF-alpha-activated endothelium.

The receptors and the array of cell adhesion molecules regulating MHC-unrestricted cytotoxic activity of NK cells toward tumor targets have not completely characterized. Antibody inhibition studies suggest roles for a number of cell adhesion molecules (CAMs). Recent studies suggest that CAMs can function to stabilize cell-to-cell interactions and/or to provide costimulatory signals that are crucial for T cell activation. It has been difficult to experimentally demonstrate that adhesion molecules also function as costimulators in NK cell-mediated cytotoxicity. We have developed an experimental system using cells transfected with genes encoding huICAM-1 and/or LFA-3 to investigate the function of adhesion molecules. Here we report that neither the expression of transfected ICAM-1 or LFA-3 alone nor the expression of both ICAM-1 and LFA-3, in the absence of MHC class I molecules, converts a murine cell line that is resistant to NK cell-mediated lysis into a susceptible one. We next tested the ability of ICAM-1 or LFA-3-mediated interactions to provide costimulation of NK cell cytolytic activity using a "three cell" experimental system comprising human NK cells, 51C-labeled target cells, and transfected mouse cells as a source of costimulation. The ability of NK cells to lyse K562 cells or anti-CD16-coated target cells was significantly enhanced by the addition of ICAM-1-transfected cells, whereas the addition of cells transfected with LFA-3 or irrelevant genes did not enhance lytic activity. Since the transfected huICAM-1 interacts with NK cells at sites spatially separate from the NK cell-target cell interactions, our data suggest that LFA-1-ICAM-1 or MAC-1-ICAM-1 interactions can provide remote costimulation, via signaling events, to induce cytotoxic activity in NK cells.

Human intestinal T lymphocytes are constantly exposed to a large number of foreign antigens without developing a systemic immune response. One crucial mechanisms leading to this intestinal hyporesponsiveness is based on impaired signal transduction through the T cell receptor/CD3 complex in lamina propria T lymphocytes (LP-T). In this study, we addressed the question whether a lack of co-stimulatory/progression signals might also contribute to LP-T hyporesponsiveness. To this end, isolated human monocyte populations from the intestinal lamina propria were obtained and their phenotypes as well as their capacity to promote T cell activation studied. Here, we demonstrate that lamina propria macrophages (LP-MO), in contrast to peripheral blood monocytes (PB-MO), do not support proliferation of either LP-T or PB-T. This may be due to the low expression of ligands (CD54, CD58, CD80) for the T cell accessory receptors CD11/18, CD2 and CD28/CTLA-4 on mucosal macrophages. Thus, down-regulation of both recognition/competence and co-stimulatory/progression signals contribute to intestinal hypo- or unresponsiveness.