Factor H (fH) and properdin both modulate complement; however, fH inhibits activation, and properdin promotes activation of the alternative pathway of complement. Mutations in fH associate with several human kidney diseases, but whether inhibiting properdin would be beneficial in these diseases is unknown. Here, we found that either genetic or pharmacological blockade of properdin, which we expected to be therapeutic, converted the mild C3 GN of an fH-mutant mouse to a lethal C3 GN with features of human dense deposit disease. We attributed this phenotypic change to a differential effect of properdin on the dynamics of alternative pathway complement activation in the fluid phase and the cell surface in the fHmutant mice. Thus, in fH mutation-related C3 glomerulopathy, additional factors that impact the activation of the alternative pathway of complement critically determine the nature and severity of kidney pathology. These results show that therapeutic manipulation of the complement system requires rigorous diseasespecific target validation.

Complement plays a key role in host defense, but its dysregulation can cause autologous tissue injury. Complement activation is normally controlled by regulatory proteins, including factor H (FH) in plasma and membrane cofactor protein (MCP) on the cell surface. Mutations in FH and MCP are linked to atypical hemolytic uremic syndrome, a type of thrombotic microangiopathy (TMA) that causes renal failure. We describe here that disruption of FH function on the cell surface can also lead to disseminated complement-dependent macrovascular thrombosis. By gene targeting, we introduced a point mutation (W1206R) into murine FH that impaired its interaction with host cells but did not affect its plasma complement-regulating activity. Homozygous mutant mice carrying this mutation developed renal TMA as well as systemic thrombophilia involving large blood vessels in multiple organs, including liver, lung, spleen, and kidney. Approximately 30% of mutant mice displayed symptoms of stroke and ischemic retinopathy, and 48% died prematurely. Genetic deficiency of complement C3 and factor D prevented both the systemic thrombophilia and renal TMA phenotypes. These results demonstrate a causal relationship between complement dysregulation and systemic angiopathy and suggest that complement activation may contribute to various human thrombotic disorders involving both the micro- and macrovasculature.

Objective— Inflammation contributes to hypertension-induced cardiac damage and fibrotic remodeling. Complement activation produces anaphylatoxins, which are major inflammatory effectors. Here, we investigated the role of complement anaphylatoxins in angiotensin II (Ang II)–induced cardiac remodeling. Approach and Results— We measured human plasma levels of complement anaphylatoxins in hypertensive individuals and controls and studied the role of complement activation in a mouse model of Ang II–induced hypertension and cardiac injury. We found that complement 5a (C5a) concentration was more elevated in hypertensive individuals than in controls. Infusion of Ang II in mice for 7 days led to increased anaphylatoxin concentration in plasma and perivascular C3b deposition in the heart. C5a receptor (C5aR)–deficient but not C3a receptor–deficient mice exhibited markedly reduced cardiac remodeling and inflammation after Ang II infusion. Pharmacological inhibition of C5a production by an anti-C5 monoclonal antibody produced similar effects to C5aR deficiency. Bone marrow chimera experiments revealed that C5aR expression on bone marrow–derived cells was critical in mediating Ang II–induced cardiac injury and remodeling. The C5aR pathway regulated the expression of adhesion molecules on peripheral monocytes, as well as infiltration and cytokine production of macrophage in the heart. Conclusions— Complement is activated in hypertensive hearts, and the C5aR signaling pathway on blood monocytes/macrophages plays a pathological role in Ang II–induced cardiac inflammation and remodeling. Therapeutic inhibition of complement may protect patients from hypertension-related heart injury.

The design, synthesis, and properties of a new pyrene excimer-forming probe of DNA have been described. 2,2-(Aminomethyl)propanediol was converted by the reaction with 1-pyrenebutylic acid to bis-pyrene-modified propanediol as a fluorescent non-nucleosidic linker. The bis-pyrene-modified linker can be incorporated via phosphoramidite chemistry into the 5'-terminal or internal positions of oligonucleotides (ODNs). The terminally modified ODNs showed almost similar affinity for complementary DNA when compared with the corresponding unmodified ODNs. The duplexes containing the bis-pyrene in the main chain exhibited higher melting temperatures relative to the corresponding duplexes containing propanediol linker at the same position. The UV and CD spectral studies indicate that the stacking interactions between the pyrene and DNA bases occur in the internally modified duplex and do not in the terminally modified duplex. The bis-pyrene modified linker itself displays excimer (E at 480 nm) and monomer (M at 380 nm) emission in a quantum yield (QY) of 0.17 and the E/M intensity ratio of 15. Incorporation of this linker into the terminal or internal positions of ODNs reduced the QY (0.003-0.009) and the E/M ratio (0.3-0.8). While small changes in the QY and E/M ratio was obtained in binding of the internally labeled ODNs to DNA, up to 27-fold increase in the QY and 17-fold increase in the E/M ratio was observed upon hybridization of the terminally labeled ODNs with DNA. The excimer and monomer fluorescence changes were found to be sensitive to a mismatch base present in the target DNA. The bis-pyrene-modified ODNs thus provide a sequence-sepcific fluorescent probe of DNA.

Complement is implicated in the pathogenesis of ischemia reperfusion injury (IRI). The activation pathway(s) and effectors(s) of complement in IRI may be organ-specific and remain to be fully characterized. We previously developed a renal IRI model in decay-accelerating factor (DAF) and CD59 double knockout (DAF−/−CD59−/−) mice. Here we used this model to dissect the pathway(s) by which complement is activated in renal IRI and to evaluate whether C3a or C5a receptor (C3aR, C5aR)-mediated inflammation or the membrane attack complex (MAC) was pathogenic. We crossed DAF−/−CD59−/− mice with mice deficient in various complement components or receptors including C3, C4, factor B (fB), factor properdin (fP), mannose-binding lectin (MBL), C3aR and C5aR or immunoglobulin (Ig) and assessed renal IRI in the resulting mutant strains. We found that deletion of C3, fB, fP, C3aR or C5aR significantly ameliorated renal IRI in DAF−/−CD59−/− mice, whereas deficiency of C4, Ig, or MBL had no effect. Treatment of DAF−/−CD59−/− mice with an anti-C5 mAb reduced renal IRI to a greater degree than C5aR deficiency. We also generated and tested a function-blocking anti-mouse fP mAb and showed it to ameliorate renal IRI when given to DAF−/−CD59−/− mice 24 hr before, but not 4 or 8 hrs after, ischemia/reperfusion. These results suggest that complement is activated via the alternative pathway during the early phase of reperfusion and both anaphylatoxin-mediated inflammation and the MAC contribute to tissue injury. Further, they demonstrate a critical role of properdin and support its therapeutic targeting in renal IRI.

The results suggest a correlation between expression levels of TLRs and cytokines, and chronic HCV infection. TLR3 recognizes double-stranded RNA and induces type 1 interferon synthesis. Collectively, suppressed expression of TLR3 in cells transfected with entire HCV may be responsible for continuous HCV infection, although a part of the HCV gene enhances its expression.

A pyrene-excimer-forming probe allowed the easy and sensitive detection of a single base mismatch in target DNA. This was due to the faster strand exchange rate compared to a fully-matched target.

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