Effect of protein C gene mutation on coagulation and inflammation in hemorrhagic shock.

INTRODUCTION: Trauma patients are at high risk of complications and death from coagulopathy and inflammatory organ failure. Recent evidence implicates protein C (PC) as a key mediator of this process. We hypothesized that a mutation in the PC gene would ameliorate the inflammatory and coagulopathic response to hemorrhagic shock (HS) and resuscitation. METHODS: FHH wild type and PC mutant rats underwent controlled hemorrhage for 120 min with 70% of blood volume removed. Rats were resuscitated with Ringers lactate (2x shed blood volume) and shed blood. Animals were sacrificed 4 h post-HS. Controls were untreated naïve rats. RESULTS: AST and NFkB lung protein levels were elevated similarly in both WT and mutants compared with naïve rats. Plasma fibrinogen levels decreased significantly with progression of HS compared with baseline (BL) levels and returned towards normal 4 h after resuscitation. PC activity was similar in both groups at BL (0.5 ± 0.08 versus 0.6 ± 0.14; P = 0.14) and decreased from BL by 53% ± 24% in WT (P =0.08), by 67% ± 11% in mutants (P = 0.03) at sacrifice, and was not different between groups (P = 0.29). CONCLUSIONS: Our model of HS and resuscitation produced a hypocoaguable, hyperinflammatory state with increased levels of NFkB and decreased levels of fibrinogen and PC levels. The mutated PC did not appear to alter these responses in our model of HS and resuscitation.

Soluble donor-like MHC class I proteins induce CD4(+)CD25(+)CD8(-)FoxP3(+) cells with potential to ameliorate graft chronic injury.

Conventional immunosuppressive therapies failed to prevent allograft chronic rejection. New approaches to modulate recipient immune response are needed. Donor-like MHC class I soluble proteins demonstrated therapeutic potential to suppress chronic rejection. The present study was designed to clarify the ability of MHC class I soluble proteins to induce T regulatory cells with true regulatory potential in a fully allogeneic rat cardiac transplant model. Donor-like MHC class I proteins upregulate small population of splenic CD8(-) negative CD4(+)CD25(+)FoxP3(+) positive cells. CD4(+) splenocytes after MHC therapy suppress lymphocyte proliferation against donor antigens in vitro. ACI recipients of WF hearts treated with CD4(+) cells, induced with donor-like MHC class I proteins (CD4-MHC), demonstrated stable survival of the transplanted organ (MST >120 days; n=17). Histology revealed that grafts of recipients treated with CD4-MHC had 23.6% vessels affected 100 days postgrafting. On the contrary, hearts obtained from long-term surviving hosts treated with CD4(+) cells induced with high-dose CsA (CD4-CsA) had 50-70% of affected vessels. CD4-MHC class I treated transplants were mostly CD3(-) negative, had low level of mast and FoxP3(+) cell infiltration compared to CD4-CsA treated hearts. Intragraft CD4(+) cells were close to mast cells in morphology. The same graft tissues had similar number of CD4(+) positive cells and mast cells suggesting existence of CD4(+) positive mast cells. On the other hand, a negligible number of FoxP3(+) positive cells in the grafts after CD4-MHC treatment supports the idea of CD4(+) positive FoxP3(+) negative mast cells population. We demonstrate that donor-like MHC class I protein therapy induces population of CD4(+)CD25(+)CD8(-)FoxP3(+) cells with potential to ameliorate development of transplant vascular disease and evoke CD4(+) positive FoxP3 negative mast cells in the secondary hosts.

Intensity of transplant chronic rejection correlates with level of graft-infiltrating regulatory cells.

BACKGROUND: The understanding of chronic rejection (transplant vascular sclerosis, or TVS) mechanisms is a major goal of transplantation. In this study we tested a cardiac transplant model for TVS development in connection with emerging T-regulatory cells (T-regs). We used 40-mer peptides derived from the donor MHC Class I alpha1 helix of the alpha1-domain to make recipients tolerant. METHODS: ACI recipients were transplanted with either RT1.A(u) (WF), RT1.A(l) (LEW), RT1.A(c) (PVG), or RT1.A(b) (BUF) cardiac grafts. The grafts were analyzed 120 days later for TVS and development of T-regs. RESULTS: Donor MHC peptides were injected through the portal vein (0.1 mg) into ACI recipients of WF hearts in addition to sub-therapeutic cyclosporine (CsA, 10 mg/kg for 3 days post-operatively). Peptide treatment specifically prolonged graft survival for >100 days (n = 31). ACI recipients of WF or LEW hearts treated with PVG peptides promptly rejected the transplanted grafts (15 +/- 4 and 20 +/- 1 days, respectively). Presence of T-regs in tolerant recipients was confirmed by the adoptive transfer of T cells into a new cohort of syngeneic recipients (mean survival time [MST] >100 days, n = 3). CD4(+) and FoxP3(+) cells were detected in 70% of the chronically rejected grafts vs 38% (CD4) and 22% (FoxP3) in the well-preserved transplants. IgG and IgM deposits were found in only half of surviving cardiac grafts with a high level of TVS. Blood vessels in grafts with attenuated TVS were 80% IgG and IgM positive. Interleukin (IL)-4 and IL-2 were markedly down-regulated in the hearts with high TVS compared with well-preserved grafts. Long-term-surviving hearts demonstrated increased IL-10 expression. Interferon-gamma (IFN-gamma) was more evident in the grafts with a high TVS. CONCLUSIONS: Donor MHC Class I peptides can specifically prolong transplant survival and generate T-regs. The level of intragraft T-regs correlates with severity of TVS and IL-2/IL-4 down-regulation.

Class I MHC allochimeric presentation of composite immunogenic and self epitopes induces tolerance to genetically diverse rat strains.

Functional topography of rat class I major histocompatibility complex (MHC) molecule was studied. The alpha1-helical sequences that are shared by class I RT1.A(l) and RT1.A(u) were substituted in the RT1.A(a) molecule to produce the composite [alpha(1h)(l/u)]-RT1.A(a) MHC class I allochimeric molecule. Dominant immunogenic epitopes that induce accelerated rejection were identified within the hypervariable regions of the alpha1 domain of RT1.A(a), RT1.A(l), and RT1.A(u). Peri-transplant portal venous delivery of MHC class I allochimeric proteins, that included composite alpha1 helical immunodominant epitopes of RT1.A(u) and RT1.A(l), induced donor-specific tolerance to RT1(u) (Wistar Furth, WF) and RT1(l) Lewis, LEW) disparate cardiac allografts in ACI (RT1(a)) hosts. Allochimeric generated tolerance was characterized by absence of T cell deletion or anergy. Donor specific IgM allo-Abs was not detected, while IgG alloresponse was markedly attenuated in sera of tolerant hosts. Further, long-term allografts in allochimeric-conditioned hosts exhibited moderate B cell infiltration when compared to rejecting controls. Analysis of intragraft cytokines revealed selective upregulation of IL-10 and marked inhibition of IL-2, IFN-gamma, and IL-4. Our findings indicate the emergence of a peripherally induced tolerant state, afforded by the novel approach of soluble class I allochimeric conditioning that presents donor immunogenic epitopes in the context of recipient class I determinants.

Inhibition of chronic rejection by antibody induced vascular accommodation in fully allogeneic heart allografts.

BACKGROUND: The potential role of altered antibody responses as an effector protective mechanism to induce graft accommodation has been widely investigated in xenogeneic responses. Here we investigate the protective effects of antibody binding to vascular endothelium in a fully mismatched allogeneic model of heart transplantation. METHODS: ACI recipients of WF cardiac grafts were treated either with allochimeric [alpha1h ]-RT1.A class I major histocompatibility complex (MHC) extracts (1 mg/rat, p.v. day 0) or high dose of CsA (10 mg/kg/day, p.o., day 0-6). Cardiac allografts were evaluated at 100 days posttransplant by immunohistology for evidence of chronic rejection and/or vascular accommodation. Activation of apoptotic or antiapoptotic mechanisms was verified by DNA fragmentation (TUNEL) analysis. RESULTS: Allochimeric therapy resulted in inhibition of chronic rejection, absence of neointimal formation and induction of vascular accommodation of fully allogeneic WF hearts in ACI hosts. Such accommodation was evident by IgG and IgM vascular endothelial binding and marked reduction of DNA fragmentation. In contrast, CsA therapy resulted in marked neointimal proliferation, without evidence of vascular accommodation. Immunohistochemical analysis failed to demonstrate vascular endothelial antibody binding. Further, severe chronic rejection following CsA treatment was accompanied by marked DNA fragmentation. CONCLUSION: Alteration of humoral immunity induces vascular accommodation in allogeneic transplantation. Vascular accommodation is the underlying mechanism for inhibition allograft vasculopathy following allochimeric MHC class I therapy.