Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature.

Neutrophil extracellular traps (NETs) composed of DNA decorated with histones and proteases trap and kill bacteria but also injure host tissue. Here we show that during a bloodstream infection with methicillin-resistant Staphylococcus aureus, the majority of bacteria are sequestered immediately by hepatic Kupffer cells, resulting in transient increases in liver enzymes, focal ischaemic areas and a robust neutrophil infiltration into the liver. The neutrophils release NETs into the liver vasculature, which remain anchored to the vascular wall via von Willebrand factor and reveal significant neutrophil elastase (NE) proteolytic activity. Importantly, DNase although very effective at DNA removal, and somewhat effective at inhibiting NE proteolytic activity, fails to remove the majority of histones from the vessel wall and only partly reduces injury. By contrast, inhibition of NET production as modelled by PAD4-deficiency, or prevention of NET formation and proteolytic activity as modelled in NE(-/-) mice prevent collateral host tissue damage.

Binding affinity of anti-xylitol antibodies to canine hepatic vessels.

Xylitol is used as a sugar substitute in food products. Dogs have been reported to experience lethal liver injury after accidental ingestion of xylitol. Because liver injury may be a serious consequence of canine immune-mediated reactions, antibodies produced against xylitol may attack the liver. Therefore, in the present study, we evaluated whether binding sites for xylitol antibodies are located at the liver or not. Anti-xylitol antibodies were generated by immunization of rabbits with a xylose-bovine serum albumin conjugate. Immunohistological examination showed that binding sites for the anti-xylitol antibodies were located in the hepatic arteries and the portal veins. Western blotting analyses by using a canine liver homogenate showed 4 protein bands with different molecular weights which reacted with anti-xylitol antibodies. Therefore, binding of anti-xylitol antibodies to the vessels may be the first step in an immune-mediated pathogenic response in xylitol toxicity. Further studies are necessary to determine the effects of anti-xylitol antibodies on the liver in the pathogenesis of xylitol toxicity.

Bacille Calmette Guérin (BCG) can induce Kawasaki disease-like features in programmed death-1 (PD-1) gene knockout mice.

OBJECTIVES: Various genetic variants of inhibitory immune signals have been suspected as feasible causes of Kawasaki disease (KD). We investigated the associative role of programmed death-1 (PD-1) gene in the pathogenesis of KD by injecting bacilli Calmette Guérin (BCG) to PD-1 gene knockout (PD-1KO) mice. METHODS: In order to induce KD-like clinical manifestations in young PD-1KO mice, intradermal injection of the bacilli Calmette Guérin (BCG) was performed twice on the abdominal skin with a 4-week interval. For defining the role of BCG, heat shock protein (HSP) 65 was challenged. In addition, Staphylococcus aureus was adopted as a microorganism that does not contain HSP65 structure. One month after the second injection, heart, liver, and kidneys were removed and examined. RESULTS: PD-1KO mice showed KD-like features including prolonged fever for more than 5 days, erythematous swelling on soles, tail skin desquamation, and gallbladder (GB) hydrops. Inflammatory cell aggregation and intimal proliferation in at least more than one coronary artery was found in all PD-1KO mice whereas scanty coronary lesion was found in wild type (WT) mice. When the PD-1KO mice were injected twice with HSP65, coronary arterial lesions similar to those seen after BCG injection were observed. Inflammatory reactions in other organs including hepatic arteries, renal arteries, and biliary arteries were also observed in PD-1KO mice. CONCLUSIONS: Our data suggest that PD-1 gene may be one of the genetic predispositions of KD and antigens containing HSP65 structure could be a triggering factor of KD by our animal model of KD.

Cytomegalovirus and its association with hepatic artery thrombosis after liver transplantation.

BACKGROUND: Hepatic artery thrombosis (HAT) is a cause of morbidity and graft loss in approximately 7% of patients after an orthotopic liver transplantation (OLT). Although technical problems are often thought to be the cause of HAT, in general the etiology remains unclear. Because cytomegalovirus (CMV) can infect endothelial cells in vitro and lead to a rapid procoagulant response, it can be hypothesized that, in the absence of CMV antibodies, latent CMV in an allograft may become activated and promote or contribute to vascular thrombosis. Therefore, the purpose of this study was to examine the relationship between CMV serology of the donor and recipient with the development of HAT after OLT. METHODS: Between July 1988 and November 1995 (University of Wisconsin era), 490 OLTs were performed in 413 patients. Subsequently, four patients were excluded in whom the CMV serology results of the donor were not available. Sixteen of the 409 patients developed HAT within 30 days after liver transplantation. The control group consisted of the other 393 patients. RESULTS: The incidence of HAT was 12.5% in 64 CMV D+R- patients and 0% in 52 CMV D-R- patients. However, in the other combinations (D+R+ and D-R+), the incidence was only 2.8% (P = 0.005). Eight of the 16 patients with HAT belonged to the CMV D+R- group. CONCLUSIONS: We conclude that CMV-seronegative patients receiving a seropositive allograft may be at risk for early HAT. Seropositivity of the donor alone and of the recipient alone was not significantly related to the incidence of HAT. Prophylactic treatment with ganciclovir and/or anticoagulation should be evaluated to prevent this complication.

Heat-shock protein 65 and activated gamma/delta T cells in injured arteries.

Because immune mechanisms are implicated in atherogenesis, we investigated the T-lymphocyte subset and factors related to its activation after acute arterial ligation (22 ligated and 13 non-ligated specimens). Ligated arteries produced heat-shock protein 65 (hsp65) and were infiltrated with activated T cells (mostly dendritic, CD3+, CD4-, CD8-, and gamma/delta T-cell-receptor bearing). The protein was found with dendritic T cells, with immunogold-labelled hsp65 beside the dendritic processes. Thus, the immune reaction after acute arterial injury may be associated with binding and recognition of in-situ hsp65 by dendritic gamma/delta T-cells.

Induction of HLA class II antigen and interleukin 2 receptor expression in acute vascular rejection of human kidney allografts.

The induction of HLA class II antigens on graft tubular cells and IL-2 receptor expression on the infiltrating lymphoid cells was studied in 245 prospective aspiration biopsies taken during the first posttransplant month from 20 human renal allografts with histologically verified acute vascular rejection (AVR). Based on the histological findings, the specimens were categorized into two main groups: biopsies from grafts with features of AVR only, and biopsies with a combination of AVR and acute cellular rejection (ACR). Also in the second group the AVR findings were predominant. Biopsies were further divided into two subgroups, depending on whether the rejection was reversible or irreversible. Evaluation of class II and IL-2R expression was done by indirect immunoperoxidase staining using monoclonal antibodies. The initial posttransplant tubular cell class II expression was low in all 20 grafts, with 5-15% positive tubular cells, and IL-2R expression was negative. All 13 grafts with a combination of AVR and ACR displayed class II induction, closely correlating to the blast response, with 50% positive tubular cells on days 2-7 after the onset of rejection, and declining thereafter back to prerejection level in grafts with reversible rejection. In grafts with irreversible rejection, tubular cell class II expression remained elevated. A similar pattern was observed with regard to IL-2R expression: the IL-2R positive cells disappeared from the grafts with reversible rejection, but they persisted in the irreversible rejections. The same pattern of class II and IL-2R expression was observed in grafts with pure AVR and reversible rejection. Instead, completely different findings were seen in grafts with pure AVR and irreversible rejection: there was neither class II induction on tubular cells nor IL-2R expression on lymphoid cells. The persistant inflammation was dominated by mononuclear phagocytes, and no blast response could be detected during the entire follow-up. These findings demonstrate a close relationship between IL-2R expression and tubular cell class II induction also in AVR, in the majority of cases. On the other hand, the findings in grafts with pure AVR in histology and irreversible rejection suggest that AVR is a heterogenous group of rejections, where different cellular and molecular mechanisms are operating.

Detection of factor VIII/coagulant antigen in human liver tissue.

Factor VIII, a high molecular weight glycoprotein complex which has an important role in haemostasis, consists of two immunologically as well as functionally discernible moieties that can be isolated separately. These are factor VIII/von Willebrand factor (FVIII/vWF) which is associated with the factor VIII-related antigen (FVIIIRAg), and the factor VIII/procoagulant activity (FVIII/C) which is associated with the factor VIII/procoagulant antigen (FVIII/CAg). The FVIII/C activity is decreased or absent in patients with haemophilia A (for a review of the structure and function of the factor VIII complex, see refs 1 and 2). Immunological techniques, combined with cell culture, have demonstrated that FVIIIRAg is present in and synthesized by endothelial cells and megakaryocytes. However, the organ and/or cell type responsible for the production of FVIII/C has not been established. Indirect evidence derived from organ transplantation in experimental animals suggests that the liver is the most likely organ for FVIII/C production. Here we have used a monoclonal antibody against FVIII/CAg in combination with a sensitive immunostaining technique to demonstrate the presence of FVIII/CAg in hepatic sinusoidal endothelial cells.