Expression of adhesion molecules on granulocytes and monocytes from patients with asthma stimulated in vitro with interleukin-8 and monocyte chemotactic protein-1.

Upregulation of adhesion molecule expression on endothelial cells (EC) and circulating leukocytes, by locally produced inflammatory mediators, may result in the enhanced infiltration of leukocytes into tissue, e.g. the airways of asthma patients. The present study investigates whether the expression of adhesion molecules on granulocytes and monocytes from asthma patients is affected by chemotactic factors, i.e. interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1). Flow cytometric analysis showed that the intrinsic expression of the various adhesion molecules on peripheral blood phagocytes from asthma patients was not different from that of healthy individuals. However, stimulation of monocytes with MCP-1 resulted only in upregulation of the expression of CD14 on monocytes from symptomatic asthma patients but not on monocytes from asymptomatic asthma patients and healthy individuals. Stimulation of granulocytes with IL-8 did not change the expression of the various beta 1- and beta 2-integrin molecules, such as VLA-4, LFA-1, CR3 and p150,95. Since earlier studies have shown that CD14 on monocytes mediates monocyte adhesion to activated vascular EC the present findings suggest that during the active phase of asthma upregulation of CD14 on monocytes by MCP-1 may lead to an increased adhesion of monocytes to vascular endothelium and their subsequent transendothelial migration into the tissue of the airways.

Effect of interleukin-8 and monocyte chemotactic protein-1 on adhesion of circulating granulocytes and monocytes from asthma patients to human venous endothelial cells.

The adhesive interactions between phagocytes and endothelial cells (EC) can be modulated by inflammatory cytokines and chemotactic proteins which are released during an inflammatory response. The aim of the present study was to investigate first whether the adhesive properties of granulocytes and monocytes from asthma patients for vascular endothelial cells differ from those of phagocytes from healthy individuals. Furthermore, we studied whether the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) can affect the binding of phagocytes to EC. No differences were observed in binding of phagocytes from asymptomatic or symptomatic asthma patients and from healthy individuals to non-stimulated or cytokine-stimulated EC. Incubation of granulocytes with IL-8 did not influence their adhesion to non-stimulated EC but inhibited the adhesion of granulocytes to IL-1-stimulated EC. Incubation of monocytes with MCP-1 did not affect their adhesion to non-stimulated or cytokine-stimulated EC. Our results indicate that adhesion of phagocytes to EC depends on the activation state of the endothelial cells but not on the origin of the phagocytes, since there were no differences in the adhesion of phagocytes from asthma patients and healthy individuals to non-stimulated or cytokine-stimulated EC.

Identification of potential HLA-A *0201 restricted CTL epitopes derived from the epithelial cell adhesion molecule (Ep-CAM) and the carcinoembryonic antigen (CEA).

The altered expression pattern of the Epithelial Cell Adhesion Molecule (Ep-CAM) and the Carcinoembryonic Antigen (CEA) on tumor cells of epithelial origin as compared to normal epithelia may permit T cells to preferentially recognize and lyse these tumor cells. The binding affinity for human leucocyte antigen A2.1 (HLA-A*0201) and the capacity to form stable peptide-major histocompatibility complex (MHC) interactions with this molecule were tested for 410 Ep-CAM-derived sequences, including an overlapping set of 9 amino-acid-long peptides, and 73 CEA-derived peptides fulfilling the HLA-A*0201 motif. Peptides with a high binding affinity and a low peptide-MHC dissociation rate were subsequently tested for their immunogenicity in HLA-A*0201Kb transgenic mice. One Ep-CAM-derived peptide and 1 CEA-derived peptide were able to reproducibly induce peptide-specific cytotoxic T cells (CTL) in these mice. This indicates that EpCAM and CEA are potential target antigens for CTL-mediated immunotherapy of epithelial cancers.

Heat shock protein 65 induces CD62e, CD106, and CD54 on cultured human endothelial cells and increases their adhesiveness for monocytes and granulocytes.

Expression of members of the heat shock protein 60 (hsp60) family in tissues has been reported to coincide with leukocyte infiltration, but it is not known whether these proteins are directly involved in the extravasation of leukocytes. Extravasation of leukocytes requires their adhesion to endothelial cells (EC) via an interaction between adhesion molecules expressed on both cell types. The aim of the present study was to investigate the effect of recombinant mycobacterial hsp65 on the adhesive characteristics of EC for monocytes and granulocytes. Incubation of EC with hsp65 induces a concentration- and time-dependent increase in adhesiveness of these EC for monocytes and granulocytes. The effect was maximal after incubation of EC with hsp65 for 4 to 6 h. In addition, incubation of EC with hsp65 induced the expression of endothelial CD62E (E-selectin), CD106 (vascular cell adhesion molecule-1) and CD54 (intercellular adhesion molecule-1). The increased adhesion of granulocytes to hsp65-stimulated EC was inhibited completely by blocking Ab against CD62E. mAb against endothelial CD62E, CD106, or CD54 or against the monocyte adhesion molecules CD14 or CD49d (very late Ag-4) did not inhibit the increased adhesion of monocytes to hsp65-stimulated EC; however, mAb against the monocyte adhesion molecule CD18 (beta2-integrin) inhibited monocyte adhesion to hsp65-stimulated EC to the same extent as monocyte adhesion to nonstimulated EC. Hsp65 did not exert its effect in an autocrine or paracrine fashion via the endogenous production of IL-1, TNF-alpha, or other factors or via contaminating LPS. Together these results indicate that hsp65 can play an important role in the adhesion of monocytes and granulocytes to EC at sites of inflammation via modulation of the adhesive characteristics of EC and thus may facilitate extravasation of these phagocytes.

Therapeutic effects of monoclonal antibody G250, interferons and tumor necrosis factor, in mice with renal-cell carcinoma xenografts.

Because renal-cell carcinoma (RCC) is considered relatively resistant to radio- and chemotherapy, RCC patients may benefit from new treatment modalities, e.g. immunotherapy. In vitro and in vivo studies suggest that combinations of cytokines such as interferon gamma or interferon alpha (IFN-gamma, IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) may act synergistically. In this study we tested whether a monoclonal antibody (MAb) G250, reactive with a RCC-associated antigen, showed anti-tumor effects in vivo in nude mice with established s.c. human RCC xenografts, and also whether this MAb could enhance the anti-tumor effect of combinations of IFNs and TNF-alpha. Treatment with combinations of IFN-alpha/TNF-alpha or IFN-gamma/TNF-alpha, or with MAb G250 alone, resulted in a significant inhibition of tumor growth. Treatment with MAb G250, in combination with IFN-gamma/TNF-alpha, did not result in an improve anti-tumor effect as compared to that of either treatment alone. In contrast, MAb G250 combined with IFN-alpha/TNF-alpha resulted in a significantly enhanced anti-tumor response. In one experiment, 3 out of 10 mice showed complete tumor regression, with no recurrence after 90 days. Large numbers of infiltrating macrophages were found surrounding viable and necrotic tumor tissue after treatment with G250 combined with IFN-alpha/TNF-alpha. These results suggest that combination therapy, consisting of IFN-alpha, TNF-alpha and MAbs, may have therapeutic value in the treatment of RCC.

Localization of monoclonal antibody G250 and bispecific monoclonal antibody CD3/G250 in human renal-cell carcinoma xenografts: relative effects of size and affinity.

A study was made of the relative effects of size and binding strength of various forms of the monoclonal antibody (MAb) G250, reacting with primary and metastatic human renal-cell carcinoma (RCC), on the localization in human RCC xenografts in nude mice. Preferential tumor localization was demonstrated after injection of 125I-labelled intact IgG, with increasing tumor/non-tumor ratios in time. Approximately 27.4% of the injected dose/gram (%ID/g) was localized in the xenograft 24 hr post-injection. A control MAb did not preferentially localize in xenografts. With F(ab')2 fragments, higher tumor/blood ratios were obtained, although a lower percentage of injected dose per gram was bound to the tumor, 24 and 48 hr post-injection. Using a bispecific MAb CD3/G250 F(ab')2 fragment, which reacts with CD3 on human T lymphocytes and binds monovalently to RCC, an enhanced accumulation in tumor tissue was also observed. The %ID/g tumor obtained with bispecific CD3/G250 F(ab')2 was comparable with %ID/g tumor found with G250 F(ab')2. The 10-fold lower binding affinity to RCC compared with intact IgG or F(ab')2 had only marginal effects on %ID/g tumor. These results show that MAb G250 preferentially localizes to RCC xenografts. Because injection of F(ab')2 fragments resulted in higher tumor/non-tumor ratios, G250 F(ab')2 may therefore be more suitable for diagnostic evaluation of RCC in patients. The tumor uptake is more dependent on size than on affinity. Furthermore, the data obtained with bispecific MAb CD3/G250 F(ab')2 support the hypothesis that this bispecific MAb may be able to target cytotoxic T lymphocytes to RCC in humans to mediate destruction of RCC.

Flow cytometric DNA-ploidy analysis of synchronously occurring multiple malignant tumors of the female genital tract.

In this study the authors applied flow cytometric DNA-ploidy analysis to multiple female genital tract malignant tumors in 43 patients, most of whom (n = 37) had bilateral ovarian cancer. An algorithm was developed for calculation of the likelihood ratio of the probabilities that measured DNA index differences between multiple tumor localizations within the same patient could be attributed to measurement variation or to true biologic DNA content differences. The results of this statistical analysis show that in 72% of the cases (31 of 43) this probability ratio exceeded 1. Because the probability that two independent tumors will have a near-identical aneuploid DNA content is very low, this finding supports a metastatic process rather than the occurrence of multiple primary tumors in these patients. Thus, flow cytometric DNA-ploidy analysis can be helpful in the identification of metastatic disease in patients with multiple female genital tract malignant tumors.