Inhibition of the lectin pathway of complement activation reduces LPS-induced acute respiratory distress syndrome in mice

Acute respiratory distress syndrome (ARDS) is a life-threatening disorder with a high rate of mortality. Complement activation in ARDS initiates a robust inflammatory reaction that can cause progressive endothelial injury in the lung. Here, we tested whether inhibition of the lectin pathway of complement could reduce the pathology and improve the outcomes in a murine model of LPS-induced lung injury that closely mimics ARDS in human. In vitro, LPS binds to murine and human collectin 11, human MBL and murine MBL-A, but not to C1q, the recognition subcomponent of the classical pathway. This binding initiates deposition of the complement activation products C3b, C4b and C5b-9 on LPS via the lectin pathway. HG-4, a monoclonal antibody that targets MASP-2, a key enzyme in the lectin pathway, inhibited lectin pathway functional activity in vitro, with an IC50 of circa 10nM. Administration of HG4 (5mg/kg) in mice led to almost complete inhibition of the lectin pathway activation for 48hrs, and 50% inhibition at 60hrs post administration. Inhibition of the lectin pathway in mice prior to LPS-induced lung injury improved all pathological markers tested. HG4 reduces the protein concentration in bronchoalveolar lavage fluid (p<0.0001) and levels of myeloid peroxide (p<0.0001), LDH (p<0.0001), TNFα and IL6 (both p<0.0001). Lung injury was significantly reduced (p<0.001) and the survival time of the mice increased (p<0.01). From the previous findings we concluded that inhibition of the lectin pathway has the potential to prevent ARDS pathology

Chromatin Structure Regulates Gene Conversion

Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vλ pseudogene array is characterized by histone modifications associated with active chromatin. We directly demonstrated the importance of chromatin structure for gene conversion, using a regulatable experimental system in which the heterochromatin protein HP1 (Drosophila melanogaster Su[var]205), expressed as a fusion to Escherichia coli lactose repressor, is tethered to polymerized lactose operators integrated within the pseudo-Vλ donor array. Tethered HP1 diminished histone acetylation within the pseudo-Vλ array, and altered the outcome of Vλ diversification, so that nontemplated mutations rather than templated mutations predominated. Thus, chromatin structure regulates homology-directed repair. These results suggest that histone modifications may contribute to maintaining genomic stability by preventing recombination between repetitive sequences

Activation of caspase-3-like protease by digitonin-treated lysosomes

AbstractApoptosis, a naturally occurring programmed cell death or cell `suicide', has been paid much attention as one of the critical mechanisms for morphogenesis and tissue remodeling. Activation of cysteine aspartases (caspases) is one of the critical steps leading to apoptosis. Although a mitochondria-mediated pathway has been postulated to be one of the activation mechanism of caspase-3, another subcellular compartment might be involved in the activation of the enzyme. The present study shows that the supernatant fraction of digitonin-treated lysosomes strongly activates Ac-DEVD-CHO inhibitable caspase-3-like protease. Activation of caspase-3-like protease by digitonin-treated lysosomal fractions was specifically suppressed by leupeptin and E-64, inhibitors of cysteine protease. These results indicate that leakage of lysosomal cysteine protease(s) into the cytosolic compartment might be involved in the activation of caspase-3-like protease

Inhibition of the lectin pathway of complement activation reduces LPS-induced acute respiratory distress syndrome in mice.

Peer reviewed: TrueAcute respiratory distress syndrome (ARDS) is a life-threatening disorder with a high rate of mortality. Complement activation in ARDS initiates a robust inflammatory reaction that can cause progressive endothelial injury in the lung. Here, we tested whether inhibition of the lectin pathway of complement could reduce the pathology and improve the outcomes in a murine model of LPS-induced lung injury that closely mimics ARDS in human. In vitro, LPS binds to murine and human collectin 11, human MBL and murine MBL-A, but not to C1q, the recognition subcomponent of the classical pathway. This binding initiates deposition of the complement activation products C3b, C4b and C5b-9 on LPS via the lectin pathway. HG-4, a monoclonal antibody that targets MASP-2, a key enzyme in the lectin pathway, inhibited lectin pathway functional activity in vitro, with an IC50 of circa 10nM. Administration of HG4 (5mg/kg) in mice led to almost complete inhibition of the lectin pathway activation for 48hrs, and 50% inhibition at 60hrs post administration. Inhibition of the lectin pathway in mice prior to LPS-induced lung injury improved all pathological markers tested. HG4 reduces the protein concentration in bronchoalveolar lavage fluid (p<0.0001) and levels of myeloid peroxide (p<0.0001), LDH (p<0.0001), TNFα and IL6 (both p<0.0001). Lung injury was significantly reduced (p<0.001) and the survival time of the mice increased (p<0.01). From the previous findings we concluded that inhibition of the lectin pathway has the potential to prevent ARDS pathology

Secondary Complement Deficiency Impairs Anti-Microbial Immunity to Klebsiella pneumoniae and Staphylococcus aureus During Severe Acute COVID-19.

A high incidence of secondary Klebsiella pneumoniae and Staphylococcus aureus infection were observed in patients with severe COVID-19. The cause of this predisposition to infection is unclear. Our data demonstrate consumption of complement in acute COVID-19 patients reflected by low levels of C3, C4, and loss of haemolytic activity. Given that the elimination of Gram-negative bacteria depends in part on complement-mediated lysis, we hypothesised that secondary hypocomplementaemia is rendering the antibody-dependent classical pathway activation inactive and compromises serum bactericidal activity (SBA). 217 patients with severe COVID-19 were studied. 142 patients suffered secondary bacterial infections. Klebsiella species were the most common Gram-negative organism, found in 58 patients, while S. aureus was the dominant Gram-positive organism found in 22 patients. Hypocomplementaemia was observed in patients with acute severe COVID-19 but not in convalescent survivors three months after discharge. Sera from patients with acute COVID-19 were unable to opsonise either K. pneumoniae or S. aureus and had impaired complement-mediated killing of Klebsiella. We conclude that hyperactivation of complement during acute COVID-19 leads to secondary hypocomplementaemia and predisposes to opportunistic infections

High affinity mAbs selected from DTLacO cells.

<p>(A) Above, binding profiles of successive DTLacO LacI-HP1 populations selected for recognition of cell surface receptors, VEGFR2, TIE2 and TROP2. Rounds of selection designated above peaks (S0–S8). Below, saturation binding kinetics, indicating apparent k_D. (B) Specificity of selected DTLacO populations. FACS analysis of binding of cell populations selected for high affinity recognition of VEGFR2, TIE2 or TROP2 to recombinant VEGFR2, TIE2, TROP2, SAv or ovalbumin (OVA). Solid peaks represent the negative reference control (secondary antibody alone), and green lines represent staining with antigen. (C) Schematic alignment of V_H and V_λ regions of mAbs selected for binding to VEGFR2, TIE2 and TROP2. Thin horizontal blue lines represent chicken framework regions, thicker horizontal lavender lines against background shading identify CDRs, vertical bars indicate single residue differences relative to the most common DTLacO sequence, and triangle indicates insertion.</p