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International audience; T cell exclusion causes resistance to cancer immunotherapies via immune checkpoint blockade (ICB). Myeloid cells contribute to resistance by expressing signal regulatory protein-α (SIRPα), an inhibitory membrane receptor that interacts with ubiquitous receptor CD47 to control macrophage phagocytosis in the tumor microenvironment. Although CD47/SIRPα-targeting drugs have been assessed in preclinical models, the therapeutic benefit of selectively blocking SIRPα, and not SIRPγ/CD47, in humans remains unknown. We report a potent synergy between selective SIRPα blockade and ICB in increasing memory T cell responses and reverting exclusion in syngeneic and orthotopic tumor models. Selective SIRPα blockade stimulated tumor nest T cell recruitment by restoring murine and human macrophage chemokine secretion and increased anti-tumor T cell responses by promoting tumor-antigen crosspresentation by dendritic cells. However, nonselective SIRPα/SIRPγ blockade targeting CD47 impaired human T cell activation, proliferation, and endothelial transmigration. Selective SIRPα inhibition opens an attractive avenue to overcoming ICB resistance in patients with elevated myeloid cell infiltration in solid tumors.

Background Identifying biomarkers to predict kidney transplant failure and to define new therapeutic targets requires more comprehensive understanding of the immune response to chronic allogeneic stimulation. Methods We investigated the frequency and function of CD8+ T cell subsets-including effector memory (EM) and terminally differentiated EM (TEMRA) CD8+ T cells-in blood samples from 284 kidney transplant recipients recruited 1 year post-transplant and followed for a median of 8.3 years. We also analyzed CD8+ T cell reactivity to donor-specific PBMCs in 24 patients who had received living-donor kidney transplants. Results Increased frequency of circulating TEMRA CD8+ T cells at 1 year post-transplant associated with increased risk of graft failure during follow-up. This association remained after adjustment for a previously reported composite of eight clinical variables, the Kidney Transplant Failure Score. In contrast, increased frequency of EM CD8+ T cells associated with reduced risk of graft failure. A distinct TEMRA CD8+ T cell subpopulation was identified that was characterized by expression of FcγRIIIA (CD16) and by high levels of proinflammatory cytokine secretion and cytotoxic activity. Although donor-specific stimulation induced a similar rapid, early response in EM and TEMRA CD8+ T cells, CD16 engagement resulted in selective activation of TEMRA CD8+ T cells, which mediated antibody-dependent cytotoxicity. Conclusions At 1 year post-transplant, the composition of memory CD8+ T cell subsets in blood improved prediction of 8-year kidney transplant failure compared with a clinical-variables score alone. A subpopulation of TEMRA CD8+ T cells displays a novel dual mechanism of activation mediated by engagement of the T-cell receptor or of CD16. These findings suggest that TEMRA CD8+ T cells play a pivotal role in humoral and cellular rejection and reveal the potential value of memory CD8+ T cell monitoring for predicting risk of kidney transplant failure.

International audience; Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature hematopoietic precursors known to suppress immune responses. Interaction of SIRP alpha (SIRPα), expressed by myeloid cells, with the ubiquitous receptor CD47 is an important immune checkpoint of the innate response regulating macrophages and dendritic cells functions. We previously described that MDSC expressing SIRPα accumulated after transplantation and maintained kidney allograft tolerance. However, the role of the SIRPα/CD47 axis on MDSC function remained unknown. Here, we found that blocking SIRPα or CD47 with monoclonal antibodies (mAbs) induced differentiation of MDSC into myeloid cells overexpressing MHC class II, CD86 costimulatory molecule and increased secretion of macrophage-recruiting chemokines (eg, MCP-1). Using a model of long-term kidney allograft tolerance sustained by MDSC, we observed that administration of blocking anti-SIRPα or CD47 mAbs induced graft dysfunction and rejection. Loss of tolerance came along with significant decrease of MDSC and increase in MCP-1 concentration in the periphery. Graft histological and transcriptomic analyses revealed an inflammatory (M1) macrophagic signature at rejection associated with overexpression of MCP-1 mRNA and protein in the graft. These findings indicate that the SIRPα-CD47 axis regulates the immature phenotype and chemokine secretion of MDSC and contributes to the induction and the active maintenance of peripheral acquired immune tolerance.

International audience; The critical role of neutrophils in pathological inflammation, notably in various autoimmune disorders, is currently the focus of renewed interest. Here, we demonstrate for the first time that activation of neutrophils with various inflammatory stimuli induces the release of extracellular vesicles (EVs) that are internalized by endothelial cells (ECs), thus leading to the transfer of miR-223, miR-142–3p and miR-451 and subsequent endothelial damage. Indeed, while miR-223 has little effect on EC responses, we show that the induced expression of miR-142–3p and miR-451 in ECs results in profound cell damage, especially in inflammatory conditions, characterized by a dramatic increase in cell apoptosis, impaired angiogenic repair responses, and the induction of IL-6, IL-8, CXCL10 and CXCL11 expression. We show that the strong deleterious effect of miR-142–3p may be due in part to its ability to block the activation of ERK1/2 and eNOS-mediated signals in ECs. miR-142–3p also inhibits the expression of RAC1, ROCK2 and CLIC4, three genes that are critical for EC migration and angiogenic responses. Importantly, miR-223, miR-142–3p and miR-451 are markedly increased in kidney biopsies from patients with active ANCA-associated vasculitis, a severe autoimmune disease that is prototypical of a neutrophil-induced microvascular damage. Taken together, our results suggest that miR-142–3p and miR-451 released in EVs by activated neutrophils can target EC to trigger an inflammatory cascade and induce direct vascular damage, and that therapeutic strategies based on the inhibition of these miRNAs in ECs will have implications for neutrophil-mediated inflammatory diseases.

BACKGROUND: Identifying biomarkers to predict kidney transplant failure and to define new therapeutic targets requires more comprehensive understanding of the immune response to chronic allogeneic stimulation. METHODS: We investigated the frequency and function of CD8(+) T cell subsets—including effector memory (EM) and terminally differentiated EM (TEMRA) CD8(+) T cells—in blood samples from 284 kidney transplant recipients recruited 1 year post-transplant and followed for a median of 8.3 years. We also analyzed CD8(+) T cell reactivity to donor-specific PBMCs in 24 patients who had received living-donor kidney transplants. RESULTS: Increased frequency of circulating TEMRA CD8(+) T cells at 1 year post-transplant associated with increased risk of graft failure during follow-up. This association remained after adjustment for a previously reported composite of eight clinical variables, the Kidney Transplant Failure Score. In contrast, increased frequency of EM CD8(+) T cells associated with reduced risk of graft failure. A distinct TEMRA CD8(+) T cell subpopulation was identified that was characterized by expression of FcγRIIIA (CD16) and by high levels of proinflammatory cytokine secretion and cytotoxic activity. Although donor-specific stimulation induced a similar rapid, early response in EM and TEMRA CD8(+) T cells, CD16 engagement resulted in selective activation of TEMRA CD8(+) T cells, which mediated antibody-dependent cytotoxicity. CONCLUSIONS: At 1 year post-transplant, the composition of memory CD8(+) T cell subsets in blood improved prediction of 8-year kidney transplant failure compared with a clinical-variables score alone. A subpopulation of TEMRA CD8(+) T cells displays a novel dual mechanism of activation mediated by engagement of the T-cell receptor or of CD16. These findings suggest that TEMRA CD8(+) T cells play a pivotal role in humoral and cellular rejection and reveal the potential value of memory CD8(+) T cell monitoring for predicting risk of kidney transplant failure.

Rat and human CD4(+) and CD8(+) Tregs expressing low levels of CD45RC have strong immunoregulatory properties. We describe here that human CD45 isoforms are nonredundant and identify distinct subsets of cells. We show that CD45RC is not expressed by CD4(+) and CD8(+) Foxp3(+) Tregs, while CD45RA/RB/RO are. Transient administration of a monoclonal antibody (mAb) targeting CD45RC in a rat cardiac allotransplantation model induced transplant tolerance associated with inhibition of allogeneic humoral responses but maintained primary and memory responses against cognate antigens. Anti-CD45RC mAb induced rapid death of CD45RC(high) T cells through intrinsic cell signaling but preserved and potentiated CD4(+) and CD8(+) CD45RC(low/-) Tregs, which are able to adoptively transfer donor-specific tolerance to grafted recipients. Anti-CD45RC treatment results in distinct transcriptional signature of CD4(+) and CD8(+) CD45RC(low/-) Tregs. Finally, we demonstrate that anti-human CD45RC treatment inhibited graft-versus-host disease (GVHD) in immune-humanized NSG mice. Thus, short-term anti-CD45RC is a potent therapeutic candidate to induce transplantation tolerance in human.

International audience; Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example α1-3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV) infection. For this purpose, a double knock-out pig lacking α1-3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking α1-3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection.

Background: In heart allograft in the rat, a sustained costimulation blockade with CTLA4Ig prevents alloreactive T-cell activation and promotes a long-term graft survival through the action of tolerogeneic dendritic cells. It is unclear whether similar mechanisms might occur after xenotransplantation. To test that hypothesis, we have analyzed the action of CTLA4Ig in a model of CD4+T cell-mediated xenograft rejection. Methods: Hamster hearts were transplanted into LEW.1A rats receiving an accommodation-inducing treatment consisting of a short course administration of LF15-0195 and a daily administration of cyclosporine A (CSA). To achieve long-term delivery of CTLA4Ig, an intravenous administration of an adenovirus vector coding for mouse CTLA4Ig (Ad-CTLA4Ig) was added to the accommodation induction protocol. On day 40 post-transplantation, rejection was induced by CSA withdrawal. In other xenograft recipients, CD28/B7 costimulation was inhibited at that time only by injections of CTLA4Ig or anti-CD28 antibodies. Graft survival, immunohistology, as well as development of antibodies and regulatory cells were examined. Results: Xenografts survived 6 days after CSA withdrawal in controls and were rejected, as previously described, through the action of CD4+ xenoreactive T cells. Interfering with CD28/B7 costimulation inhibited this xenoreactive T cell response and delayed rejection to day 10. In recipients that had received Ad-CTLA4Ig, survival was prolonged to day 19 and this was accompanied by the appearance of regulatory cells exhibiting non-donor-specific suppressive activity dependent on IL-2, NO, and IDO. These regulatory cells were different from those previously identified after Ad-CTLA4Ig administration in heart allograft in the rat. In these recipients, rejection occurred as a consequence of an evoked anti-donor IgM response and complement activation and not of a cellular rejection as complement inhibition with cobra venom factor further prolonged xenograft survival. Conclusion: CD28/B7 blockade delays CD4+ T cell-mediated rejection after CSA withdrawal in accommodated recipients of hamster heart xenografts. In addition, a sustained expression of CTLA4Ig has the potential of inducing cellular regulatory mechanisms. However, such treatment does not prevent the development of xenoreactive IgM antibodies that participate in vascular rejection processes in a complement-dependent manner.

In transplantation tolerance, numerous regulatory populations have the capacity to inhibit allograft rejection; however, their compensatory capacities have never been clearly evidenced. We have previously demonstrated that the tolerogenic effect mediated by CD8+CD45RClow regulatory T cells (Tregs) in a model of organ transplantation with CD40Ig could be abrogated by permanent depletion of CD8+ cells that resulted in allograft rejection in half of the recipients. This result demonstrated that CD8+ Tregs were essential, but also that half of the recipients still survived indefinitely. We also demonstrated that no other regulatory populations, besides CD8+ Tregs, could induce and maintain allograft tolerance in CD40Ig-treated tolerant animals. In the current study, we analyzed the mechanisms that arose following CD8+ Treg depletion and allowed establishment of networks of new regulatory cells to maintain allograft survival. We identified regulatory B cells (Bregs) and regulatory myeloid cells (RegMCs) as being responsible of the maintenance of the long-term allograft survival. We demonstrated that both regulatory cell subsets efficiently inhibited antidonor immune responses in adoptively transferred recipients. Although Bregs were induced, they were not essential for the maintenance of the graft as demonstrated in IgM-deficient recipients. In addition, we showed that RegMCs were the most suppressive and acted alone, whereas Bregs activity was associated with increased suppressive activity of other subsets in adoptively transferred recipients. Altogether, to our knowledge, we demonstrated in this study for the first time the emergence of both Bregs and RegMCs following Tregs depletion and highlighted the importance of regulatory cell networks and their synergistic potential in transplantation.

Recent studies suggest that particular dendritic cells (DC) subpopulations may be tolerogenic. To test the capacity of different DC subpopulations to modulate allograft rejection, we generated two distinct populations of rat bone marrow-derived DCs (BMDC) with low doses of GM-CSF and IL-4. The non-adherent population (nBMDC), which are the 'classical' DCs was able to stimulate naive allogeneic T cells and could be induced to completely mature using various stimuli. In contrast, the adherent population (aBMDC), which displayed an immature phenotype, was unable to stimulate T cells and was more resistant to maturation. We found that syngeneic aBMDCs, injected one day before transplantation, induced significant prolongation of heart allograft survival and decreased anti-donor humoral and cellular responses. Similarly, syngeneic aBMDCs inhibited T-cell responses to KLH in the spleen but not in lymph node in a KLH immunization model without graft. This effect was not antigen specific and could be reversed using an inhibitor of inducible nitric oxide synthase. This compartmentalized inhibition could be in part explained by the fact that the majority of syngeneic adherent cells administered intravenously were found in the spleen with some of them reaching the T-cell areas. These data suggest that syngeneic aBMDCs can modulate immune responses.

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International audience; T cell depletion is commonly used in organ transplantation for immunosuppression; however, a restoration of T cell homeostasis following depletion leads to increased memory T cells, which may promote transplant rejection. The cytokine IL-7 is important for controlling lymphopoiesis under both normal and lymphopenic conditions. Here, we investigated whether blocking IL-7 signaling with a mAb that targets IL-7 receptor α (IL-7Rα) alone or following T cell depletion confers an advantage for allograft survival in murine transplant models. We found that IL-7R blockade alone induced indefinite pancreatic islet allograft survival if anti-IL-7R treatment was started 3 weeks before graft. IL-7R blockade following anti-CD4- and anti-CD8-mediated T cell depletion markedly prolonged skin allograft survival. Furthermore, IL-7 inhibition in combination with T cell depletion synergized with either CTLA-4Ig administration or suboptimal doses of tacrolimus to induce long-term skin graft acceptance in this stringent transplant model. Together, these therapies inhibited T cell reconstitution, decreased memory T cell numbers, increased the relative frequency of Tregs, and abrogated both cellular and humoral alloimmune responses. Our data suggest that IL-7R blockade following T cell depletion has potential as a robust, immunosuppressive therapy in transplantation.

Material, methods and results In rat recipients of kidney allografts where tolerance was induced by costimulation blockade, we observed a 2-fold decrease of plasma CCL5 levels as compared to control syngeneic grafted animals (0.60 ± 0.17 vs. 1.17 ± 0.48 ng/ml; p

Background Antagonist antibodies targeting CD28 have been proposed as an alternative to the use of CD80/86 antagonists to modulate T cell responses in autoimmunity and transplantation. Advantages would be the blockade of CD28mediated co-stimulatory signals without impeding co-inhibitory signals depending on CD80 interactions with CTLA-4 and PD-L1 that are important for the control of immune responses and for the function of regulatory T cells. Anti-CD28 antibodies are candidate antagonists only if they prevent access to the CD80/86 ligands without simultaneously stimulating CD28 itself, a process that is believed to depend on receptor multimerization.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells that are believed to inhibit immune responses in the contexts of cancer and organ transplantation, in association with regulatory T cells (Treg). However, the way in which MDSC cooperate with Treg remains elusive. In this study, we used DNA microarrays to analyze gene expression in blood-derived MDSC from rat recipients of kidney allografts. We found CCL5 (Rantes), a chemotactic C-C motif 5 chemokine, to be strongly downregulated after treatment with a tolerizing regimen. The amount of CCL5 protein was also lower in the plasma of tolerant recipients, whereas intragraft CCL5 was unchanged. Because CCL5 is chemotactic for Treg, we hypothesized that a gradient of CCL5 between the graft and peripheral blood might contribute to the intragraft localization of Treg in tolerant animals. To test this hypothesis, we treated tolerant rat recipients of kidney allografts with recombinant rat CCL5 to restore normal plasma concentrations. This led to a strong reduction in intragraft Treg monitored by immunohistofluorescence and by quantitative real-time PCR measurement of Foxp3 mRNA. Ultimately, this treatment led to an increase in serum creatinine concentrations and to kidney graft rejection after about a month. The kidney function of syngeneic grafts was not affected by a similar administration of CCL5. These data highlight the contribution of MDSC to the establishment of a graft-to-periphery CCL5 gradient in tolerant kidney allograft recipients, which controls recruitment of Treg to the graft where they likely contribute to maintaining tolerance.

Transplantation is the treatment of choice for patients with end-stage organ failure. Its success is limited by side effects of immunosuppressive drugs, such as inhibitors of the calcineurin pathway that prevent rejection by reducing synthesis of interleukin-2 by T cells. Moreover, none of the existing drugs efficiently prevent the eventual rejection of the organ. Blocking the CD28-mediated T cell costimulation pathway is a nontoxic alternative immunosuppression strategy that is now achieved by blockade of CD80/86, the receptor for CD28 on antigen-presenting cells. However, interaction of CD80/86 with cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is required for immune regulation. Therefore, CD28 blockade, instead of CD80/86 blockade, might preserve regulatory signals mediated by CTLA-4 and preserve immune regulation. By using monovalent antibodies, we identified true CD28 antagonists that induced CTLA-4-dependent decreased T cell function compatible with regulatory T (Treg) cell suppression. In transplantation experiments in primates, blocking CD28 augmented intragraft and peripheral blood Treg cells, induced molecular signatures of immune regulation, and prevented graft rejection and vasculopathy in synergy with calcineurin inhibition. These findings suggest that targeting costimulation blockade at CD28 preserves CTLA-4-dependent immune regulation and promotes allograft survival.

The immune tolerance to rat kidney allografts induced by a perioperative treatment with anti-CD28 Abs is associated with a severe unresponsiveness of peripheral blood cells to donor Ags. In this model, we identified an accumulation in the blood of CD3−class II−CD11b+CD80/86+ plastic-adherent cells that additionally expressed CD172a as well as other myeloid markers. These cells were able to inhibit proliferation, but not activation, of effector T cells and to induce apoptosis in a contact-dependent manner. Their suppressive action was found to be under the control of inducible NO synthase, an enzyme also up-regulated in tolerated allografts. Based on these features, these cells can be defined as myeloid-derived suppressor cells (MDSC). Interestingly, CD4+CD25highFoxP3+ regulatory T cells were insensitive in vitro to MDSC-mediated suppression. Although the adoptive transfer of MDSC failed to induce kidney allograft tolerance in recently transplanted recipients, the maintenance of tolerance after administration of anti-CD28 Abs was found to be dependent on the action of inducible NO synthase. These results suggest that increased numbers of MDSC can inhibit alloreactive T cell proliferation in vivo and that these cells may participate in the NO-dependent maintenance phase of tolerance.

Background . Preliminary studies showed that heat treatment of glutaraldehyde preserved valvular bioprostheses mitigates calcification. This study was carried out to define the physicochemical characteristics of the heat-treated tissues to elucidate the mechanism involved in the mitigation. Methods . Glut bovine pericardium or porcine valve samples were treated at 50°C in a 0.625% glutaraldehyde solution for 2 months. Some samples underwent assay for shrinkage temperature, moisture content, ninhydrin test, and acid hydrolysis, and other samples were incubated in human serum for 3 days and then analyzed by electrophoresis to study protein adsorption. Results . Heat treatment mitigated calcification without adversely affecting shrinkage temperature (84.81°C versus 83.95°C) and moisture content (78.68% versus 78.71%). A significant reduction in free amino groups (0.15 versus 0.37 mol NH 2 /mol collagen) and a significant increase in resistance to acid hydrolysis were observed. Total protein content was similar, but significant differences were found for four proteins adsorbed in the tissues (167, 45, 11.6, and 10 kDa). Conclusions . The anticalcification effect of heat treatment may be attributed to structural changes, lipid extraction, increased resistance, and modifications of the type and concentration of the proteins adsorbed in the tissue.

Background . Several techniques have been proposed to mitigate calcification of glutaraldehyde-preserved bioprostheses. None has been fully satisfactory. Knowing that heat induces significant molecular changes, we investigated the potential benefit of the heat treatment of glutaraldehyde-fixed tissues. Methods . Samples of porcine valves and bovine pericardium treated in 0.625% glutaraldehyde were submitted to temperatures from 37° to 70°C for 2 to 12 weeks and then implanted subcutaneously in newborn rats for 3 months. In a second protocol, nine heat-treated porcine valve bioprostheses and seven control porcine valve bioprostheses were implanted in the mitral position in sheep for 20 weeks. Results . Spectrophotometry and x-ray analysis comparing heat-treated versus non–heat-treated tissues showed the following: porcine valve: 6.7 ± 2.3 μg Ca/mg (n = 8) versus 239.9 ± 2.9 μg Ca/mg (n = 8); bovine pericardium: 19.5 ± 8 μg Ca/mg (n = 6) versus 108 ± 10.3 μg Ca/mg (n = 8); and porcine valve bioprostheses: 0 versus ++. Conclusions . Heat treatment of glutaraldehyde-treated bioprostheses significantly mitigated calcification in both subcutaneous and mitral position.

Background . The methods currently used to analyze the process of calcification of bioprostheses give only global information on calcium deposition. We investigated the potential advantage of infrared spectroscopy, which makes it possible to analyze the various components of the calcification process, ie, lipids, proteins, and calcium deposits. Methods . Sixty porcine aortic leaflets were fixed in 0.6% glutaraldehyde and then subsequently implanted in 10-day-old Wistar rats. The valve leaflets were removed 2, 7, 14, 21, 35, and 56 days after implantation. Results . Before implantation infrared spectroscopic analysis revealed the presence of proteins only. On day 2 after implantation, all valves showed minor lipid deposits. On day 7, amorphous calcium phosphate was detected. Between days 7 and 14, crystalline forms of calcium phosphate appeared and amorphous calcium phosphate progressively changed into carbapatite over the 56-day period. Conclusions . Infrared spectroscopy yields valuable additional information on the nature and kinetics of the various components of glutaraldehyde-treated tissues after implantation. It may prove to be important in the evaluation of new techniques of calcium mitigation.

Calcification of non-osseous tissues such as heart valves or vessels is a major concern in clinical practice. The exact mechanism is still unknown. Numerous studies have shown that mineral deposits of crystalline hydroxyapatite within these tissues were associated with increased non-collagenous protein content. More recently osteopontin was found to be associated with calcification in living tissues such as vessels and native human aortic valves. The aim of this study was to determine whether or not non-collagenous proteins can also be found in non-living tissues such as glutaraldehyde-pretreated porcine valves after implantation in humans. Thirty-eight glutaraldehyde pretreated porcine bioprostheses were studied: 16 not implanted and 22 after 11 years of implantation in the aortic and mitral valve position in humans. In areas of calcification vizualized by Von Kossa staining and microradiography, immunostaining using polyclonal antibodies against calcium-binding proteins showed osteopontin positive staining and no staining for osteocalcin, bone sialoprotein or osteonectin. In uncalcified areas and in non-implanted values, staining for osteopontin or other calcium-binding proteins was negative. Western blot analysis of macroscopically calcified and uncalcified areas showed that several proteins were adsorbed in implanted values and confirmed the presence of osteopontin in the calcified areas, while no immunolabelling was found in non-calcified areas, in uncalcified valves and in non-implanted valves. Thus the presence of osteopontin in the calcified areas of bioprosthetic heart valves implanted in human indicates that this protein is associated with bioprosthetic valvular calcification. Since these values are made of non-living connective tissue, and no cell immunostained for osteopontin was found around the calcified area, this suggests that a non-cellular mediated mechanism involving protein adsorption may play a role in bioprosthetic valvular calcification.

Background . The incidence of calcification of porcine valve bioprostheses shows important, and as yet unexplained, variations. Previous studies by others showed that osteopontin and osteocalcin are expressed in calcified porcine valve bioprostheses. However, no study has yet explored other proteins that could also be involved. Methods . Twelve porcine valve bioprostheses were retrieved from 12 patients and were separated into two groups. Group 1 (n = 6) had early calcification after 4 to 9 years (mean, 6 ± 2.3 years). The mean age of the patients at the time of implantation was 46 ± 9 years. Group 2 (n = 6) had no calcification after 8 to 14 years (mean, 12 ± 2.8 years). The mean age was 47 ± 13.4 years. These valves were analyzed by electrophore-sis, and the bands were quantified by densitometry. Results . A 14-kd protein showed a 50% increase in the calcified group. A 31-kd protein found in the calcified group was not detected in the noncalcified group. Three other proteins (45, 39, and 28 kd) showed reduced adsorption in the calcified group. Conclusions . Important differences were found in the proteins adsorbed in calcified and noncalcified bioprostheses after implantation in patients. Besides osteopontin and osteocalcin, several other proteins may play a role in the process of calcification of valvular bioprostheses.

Background . Proteins adsorbed by bioprosthetic tissues after implantation play a major role in the process of calcification. We investigated whether there are differences in protein adsorption between bovine pericardial and porcine valvular tissues that could correlate with the differences observed clinically. Methods . Glutaraldehyde-treated bovine pericardial and porcine valve samples were implanted subcutaneously in rats and retrieved 1 month after implantation. Total protein content was assessed by Lowry's method. Qualitative analysis was performed by polyacrylamide gel electrophoresis. Quantitative analysis was performed by densitometry. Results . Total protein content showed a higher protein concentration in porcine valve tissue than in pericardialtissue: 149 ± 22.6 μg/mg dry tissue versus 108 ± 12.7 μg/mg dry tissue (38% increase). In pericardial tissue, four protein bands (17, 16, 15.5, and 13.5 kd) showed decreased concentration when compared with porcine valve tissue, whereas one band (11 kd) showed increased concentration. Conclusions . Significant differences were found in protein content between bovine pericardial and porcine valve tissues. Correlations with clinical findings may lead to a better understanding of the mechanism involved in the process of calcification, particularly the role played by the structure of the tissues.

Having recalled a few essential definitions in the theory of internal friction in solids, the authors, in a first chapter, study the propagation of longitudinal waves in a rod; they start from a simple law relating locally stress, strain and their first derivatives with respect to time. This law which is more general thanHooke's law has been proposed first byZener to explain the anelastic behaviour of a standard linear solid. The authors calculate the internal friction resulting from energy dissipation when the vibration is forced then free. The second chapter deals with problems arising when one tries to measure the internal friction in flexure of metallic specimens having the shape of rods or circular plates. The authors develop the basic flexure equations applied to the two types of specimen; then they determine the fundamental characteristics, which appear in internal friction measurement purposes.

Antagonist antibodies targeting CD28 have been proposed as an alternative to the use of CD80/86 antagonists to modulate T cell responses in autoimmunity and transplantation. Advantages would be the blockade of CD28-mediated co-stimulatory signals without impeding the co-inhibitory signals dependent on CD80 interactions with CTLA-4 and PD-L1 that are important for the control of immune responses and for the function of regulatory T cells. Anti-CD28 antibodies are candidate antagonists only if they prevent access to the CD80/86 ligands without simultaneously stimulating CD28 itself, a process that is believed to depend on receptor multimerization. In this study, we evaluated the impact of different formats of a potentially antagonist anti-human CD28 antibody on T cell activation. In particular, we examined the role of valency and of the presence of an Fc domain, two components that might affect receptor multimerization either directly or in the presence of accessory cells expressing Fc receptors. Among monovalent (Fab’, scFv), divalent (Fab’2), monovalent-Fc (Fv-Fc) and divalent-Fc (IgG) formats, only the monovalent formats showed consistent absence of induced CD28 multimerization and absence of associated activation of phosphoinositol-3-kinase, and clear antagonist properties in T cell stimulation assays. In contrast, divalent antibodies showed agonist properties that resulted in cell proliferation and cytokine release in an Fc-independent manner. Conjugation of monovalent antibodies with polyethylene glycol, α-1-antitrypsin or an Fc domain significantly extended their in vivo half-life without modifying their antagonist properties. In conclusion, these data indicate that monovalency is mandatory for maintaining the antagonistic activity of anti-CD28 monoclonal antibodies.