Gertie J. Oostingh

PS3, A Semisynthetic β-1,3-Glucan Sulfate, Diminishes Contact Hypersensitivity Responses Through Inhibition of L-and P-Selectin Functions

Leukocyte extravasation is initiated by an interaction of selectin adhesion molecules and appropriate carbohydrate ligands. Targeting those interactions seems a promising approach to treat chronic inflammation. We developed a beta-1, 3-glucan sulfate (PS3) with inhibitory activity toward L and P-selectins under static conditions. Here, detailed investigation showed inhibition of P- and L-selectins, but not E-selectin under flow conditions (relative reduction of interaction with appropriate ligands to 34.4+/-16.6, 8.5+/-3.6, or 99.5+/-9.9%, respectively, by PS3 for P-, L- or E-selectin). Intravital microscopy revealed reduction of leukocyte rolling in skin microvasculature from 22.7+/-5.0 to 12.6+/-4.0% after injection of PS3. In the next experiments, mice were sensitized with 2,4,-dinitrofluorobenzene (DNFB), and lymphocytes were transferred into syngeneic recipients, which were challenged by DNFB. Inflammatory responses were reduced when immunity was generated in mice treated with PS3 or in L-selectin-deficient mice. No effect was observed when L-selectin-deficient donor mice were treated with PS3, further suggesting that PS3 acted primarily through inhibition of L-selectin. Elicitation of a contact hypersensitivity response was reduced in P-selectin-deficient and in PS3-treated mice. Again, PS3 had no effect in P-selectin-deficient mice. PS3 is a potent P- and L-selectin inhibitor that may add to the therapy of inflammatory diseases.

Sensitisation to swine leukocyte antigens in patients with broadly reactive HLA specific antibodies.

We have previously shown that IgG HLA specific antibodies in the sera of highly sensitised patients awaiting renal transplantation can cross‐react with swine leukocyte antigens (SLA). In this study we determined the frequency of patient serum IgG HLA specific antibody binding to a porcine lymphocyte panel and the likelihood of locating a cross‐match negative pig donor for sensitised patients. Serum samples (n = 82) were obtained from 35 sensitised [current IgG panel reactive antibodies (PRA) > 10%] and seven nonsensitised patients awaiting renal transplantation at Addenbrookes Hospital, Cambridge, UK. Fifty sera had IgG HLA specific PRA of 11–84%, 20 had IgG PRA of > 84% and 12 had 0% PRA (negative controls). Sera were absorbed with porcine erythrocytes to remove xenoreactive natural antibodies and tested for cross‐reactive IgG HLA specific antibody binding by flow cytometry against a panel of porcine lymphocytes obtained from 23 human decay accelerating factor (hDAF) transgenic pigs. A total of 1884 cross‐match combinations were tested and 369 (20%) gave a positive porcine lymphocyte cross‐match. For sera from sensitised patients with IgG PRA (11–64%), only 6 of 805 (0.75%) cross‐match tests were positive. In contrast, for sera from patients with high IgG PRA (> 64%), 363 of 805 (45%) cross‐match tests were positive (p < 0.0001). There was no difference in the frequency of positive cross‐matches between patient sera with IgG PRA 65–84% and highly sensitised patient sera with IgG PRA 85–100% [156/345 (45%) vs. 207/460 (45%)]. This study demonstrates that only patient sera with broadly reactive IgG HLA specific PRA (> 64%) cross‐react with porcine lymphocytes. If future clinical trials of xenotransplantation are undertaken, it may be of value to select a cross‐match‐negative pig organ donor for such patients.

PECAM-1 Polymorphism Affects Monocyte Adhesion to Endothelial Cells

Background. Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) plays an important role in leukocyte-endothelial cell adhesion and transmigration. Single nucleotide polymorphisms of PECAM-1 encoding amino acid substitutions at positions 98 leucine/valine (L/V), 536 serine/asparagine (S/N), and 643 arginine/glycine (R/G) occur in strong genetic linkage resulting in two common haplotypes (LSR and VNG). These PECAM-1 polymorphisms are associated with graft-versus-host disease after hematopoietic stem cell transplantation and with cardiovascular disease, but whether they influence PECAM-1 function is unknown. Methods. We examined the effect of homozygous and heterozygous expression of the PECAM-1 LSR and VNG genotypes on the adhesive interactions of peripheral blood monocytes and activated endothelial cell monolayers under shear stress in a flow-based cell adhesion assay. Results. There was no difference in monocyte adhesion between the two homozygous genotypes of PECAM-1 but when monocytes expressed both alleles in heterozygous form, firm adhesion of monocytes to endothelial cells was markedly increased. PECAM-1 polymorphism expressed in homozygous or heterozygous form by endothelial cells did not influence monocyte adhesion. Conclusions. This is, to our knowledge, the first demonstration that PECAM-1 genotype can alter the level of monocyte binding to endothelial cells and a demonstration that heterozygous expression of a polymorphic protein may lead to altered function.

Comparison of allogeneic and xenogeneic in vitro T-cell proliferative responses in sensitized patients awaiting kidney transplantation.

Abstract:  Highly sensitized patients awaiting kidney transplantation may be potential candidates for future clinical trials using pig organ donors. Because of crossreactivity between human leucocyte antigens (HLA) and swine leucocyte antigens (SLA), such patients might have heightened T‐cell responses to porcine xenoantigens. We determined whether lymphocytes from allo‐sensitized patients displayed secondary (cyclosporine resistant) T‐cell proliferative responses against porcine xenoantigens. Lymphocytes from six non‐sensitized, seven sensitized [immunoglobulin G (IgG) panel reactive antibodies (PRA) 11 to 84%], 14 highly sensitized patients (IgG PRA > 84%) and 12 healthy individuals were tested [in the presence and absence of Cyclosporin A (CsA)] to determine their proliferative response to human (allogeneic) and to porcine (xenogeneic) stimulator cells. Lymphocytes from all study groups showed a strong proliferative response to allogeneic and xenogeneic stimulator cells with no significant difference between non‐sensitized and sensitized individuals. Addition of CsA (100 and 500 ng/ml) inhibited (>90%) proliferation of lymphocytes from all non‐sensitized patients to both allogeneic and xenogeneic stimulators. CsA was less effective at inhibiting proliferation of lymphocytes from sensitized patients and highly sensitized patients to allogeneic stimulators [29% (n = 21) and 50% (n = 42) respectively were resistant to CsA inhibition (100 ng/ml)]. In contrast, cyclosporine inhibited proliferation of lymphocytes from the majority of sensitized and highly sensitized patients to xenogeneic stimulator cells [14% (n = 21) and 14% (n = 42) respectively were resistant to CsA inhibition (100 ng/ml)]. HLA sensitized patients awaiting renal transplantation display cyclosporine resistant proliferative T‐cell responses to allogeneic stimulators but proliferative responses to xenogeneic stimulators are more amenable to suppression by CsA. This finding suggests that humoral sensitisation to HLA antigens is not necessarily indicative of a heightened in vitro T‐cell response to SLA antigens.

Autoreactive T cell responses in pemphigus and pemphigoid

Pemphigus and pemphigoid are cutaneous autoimmune diseases characterised by autoantibodies directed against specific adhesion proteins of the epidermis and dermal-epidermal junction. These proteins are usually associated with desmosomes or hemidesmosomes. Binding of antibodies to their targets leads to the loss of cell-cell or cell-matrix adhesion and subsequently to blister formation. The humoral aspects of the autoimmune responses in pemphigus and pemphigoid have been extensively studied in the past. More recently, the cellular interactions resulting in the formation of autoantibodies and the involvement of autoreactive T cells in these diseases have attracted increased interest. In this review, the current knowledge on T cell involvement in pemphigus and pemphigoid is summarised.

Potential implications of ABO blood group for vascular rejection in pig to human kidney xenotransplantation.

Abstract:

Computer-aided analysis of cell interactions under dynamic flow conditions

Abstract:  Experimentally, initial steps of leucocyte extravasation, including tethering and rolling, are analysed in endothelial cell flow chambers. Given the complexity and speed of endothelial‐immune cell interaction, computer‐aided advances of this analysis are highly desirable. Herein, we compared two established methods, hand counting and tracking software, with novel analysis software using defined movies recorded at standard conditions of endothelial‐leucocyte interactions. As a first validation, cell counts and velocity parameters determined by seven experienced experts revealed no statistic differences to both semi‐automated tracking and fully computerized analyses. Nevertheless, interindividual variations were substantial for hand counting. In additional experiments, velocity distributions between 1 and 800 μm/s picked up by the fully computerized analysis matched well with the tracking software as indicated by speed vector histograms. With respect to the time consumed for a defined set of movies, hand counting took 3.6 ± 1.6 h, tracking software 4.5 ± 1.2 h, whereas fully automated analysis consumed less than 15 min, reaching real‐time mode. Thus, a validated and fully computerized method yielded functional flow chamber data unbiased, independent from an examiner, and reaching high‐throughput level, which in turn will allow a substantial progress in understanding this process central for skin inflammation.