Protective role of unconjugated bilirubin on complement-mediated hepatocytolysis.

Hyperbilirubinemia and complement-mediated immune attack on hepatocyte membrane are common features of certain hepatic diseases. To assess whether unconjugated bilirubin (UB) counteracts complement-mediated hepatocytolysis, we first generated a rabbit polyclonal antibody (Ab) against rat hepatocyte plasma membrane (RHPM). An assay performed with isolated rat hepatocytes in the presence of the polyclonal Ab and rat serum as complement donor demonstrated that UB inhibits cell lysis, as lactate dehydrogenase release into the medium was inhibited by the pigment in a dose-dependent manner. Immunofluorescence microscopy studies showed that UB significantly attenuates the binding of C3 to the hepatocyte-Ab complex. Further enzyme immunoassay studies showed that UB interferes the binding of C1q to purified anti-RHPM IgG, also in a dose-dependent manner. A dot-blot assay showed that [14C]-UB binds to C1q and human serum albumin (HSA) to a similar extent. A differential spectrum analysis of UB in the presence of C1q further confirmed that the pigment interacts with this protein. In conclusion, we demonstrated an inhibitory action of UB on complement-mediated Ab-induced hepatocytolysis, this action being evidenced at pathophysiological pigment concentrations (171 microM and higher). A direct binding of the pigment to C1q is likely involved.

Antiphospholipid and antioangiogenic activity in females with recurrent miscarriage and antiphospholipid syndrome.

Background Antiphospholipid syndrome is an autoimmune disease characterized by thrombosis, fetal losses and thrombocytopenia associated to antiphospholipid antibodies. They are directed to phospholipids, such as cardiolipins (anticardiolipin) and lupus anticoagulant or to complexes formed by phospholipids and protein cofactors, such as ß2 glycoprotein 1 (a-ß2GP1) and annexin V (a-annexin V). These auto-antibodies may be considered as a family of antibodies involved in thrombotic events and antiphospholipid activity. On the other hand, some proangiogenic factors are involved in the normal development of placental vasculature, such as the vascular endothelial growth factor. Overexpression of vascular endothelial growth factor receptor in its soluble form (sVEGFR-1) has been associated to a higher antiangiogenic activity. Our aim was to analyse the association between anticardiolipin, lupus anticoagulant, a-ß2GP1, a-annexin V and sVEGFR-1 with recurrent miscarriage before week 10 of gestation in females with antiphospholipid syndrome. Methods We studied 24 females (primary or secondary antiphospholipid syndrome), who were divided into two groups: females with recurrent miscarriage before week 10 of gestation (M; n = 12) and females with no history of fetal loss (NM; n = 12). Anticardiolipin, a-ß2GP1, a-annexin V and sVEGF-R1 concentrations were assessed by ELISA, while lupus anticoagulant was assessed by screening and confirmatory tests. Results A significant association was observed between the number of positive biomarkers and the belonging group ( P < 0.05). Besides, a positive result for lupus anticoagulant and a-ß2GP1 was found to be significantly associated to the M group ( P < 0.05). Conclusions Lupus anticoagulant and a-ß2GP1 may be implicated in pregnancies complicated by recurrent miscarriage in females with antiphospholipid syndrome.