C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation

Complement C1q is the activator of the classical pathway. However, it is now recognized that C1q can exert functions unrelated to complement activation. Here we show that C1q, but not C4, is expressed in the stroma and vascular endothelium of several human malignant tumours. Compared with wild-type (WT) or C3- or C5-deficient mice, C1q-deficient (C1qa(-/-)) mice bearing a syngeneic B16 melanoma exhibit a slower tumour growth and prolonged survival. This effect is not attributable to differences in the tumour-infiltrating immune cells. Tumours developing in WT mice display early deposition of C1q, higher vascular density and an increase in the number of lung metastases compared with C1qa(-/-) mice. Bone marrow (BM) chimeras between C1qa(-/-) and WT mice identify non-BM-derived cells as the main local source of C1q that can promote cancer cell adhesion, migration and proliferation. Together these findings support a role for locally synthesized C1q in promoting tumour growth

the neutrophil activating protein of helicobacter pylori crosses endothelia to promote neutrophil adhesion in vivo

Helicobacter pylori induces an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by infiltration of neutrophils/polymorphonuclear cells (PMNs) and mononuclear cells. The H. pylori neutrophil-activating protein (HP-NAP) activates PMNs, monocytes, and mast cells, and promotes PMN adherence to the endothelium in vitro. By using intravital microscopy analysis of rat mesenteric venules exposed to HP-NAP, we demonstrated, for the first time in vivo, that HP-NAP efficiently crosses the endothelium and promotes a rapid PMN adhesion. This HP-NAP-induced adhesion depends on the acquisition of a high affinity state of β2 integrin on the plasma membrane of PMNs, and this conformational change requires a functional p38 MAPK. We also show that HP-NAP stimulates human PMNs to synthesize and release a number of chemokines, including CXCL8, CCL3, and CCL4. Collectively, these data strongly support a central role for HP-NAP in the inflammation process in vivo: indeed, HP-NAP not only recruits leukocytes from the vascular lumen, but also stimulates them to produce messengers that may contribute to the maintenance of the flogosis associated with the H. pylori infection

Critical role and therapeutic control of the lectin pathway of complement activation in an abortion-prone mouse mating

The abortion-prone mating combination CBA/J 7 DBA/2 has been recognized as a model of preeclampsia, and complement activation has been implicated in the high rate of pregnancy loss observed in CBA/J mice. We have analyzed the implantation sites collected from DBA/2-mated CBA/J mice for the deposition of the complement recognition molecules using CBA/J mated with BALB/c mice as a control group. MBL-A was observed in the implantation sites of CBA/J 7 DBA/2 combination in the absence of MBL-C and was undetectable in BALB/c-mated CBA/J mice. Conversely, C1q was present in both mating combinations. Searching for other complement components localized at the implantation sites of CBA/J 7 DBA/2, we found C4 and C3, but we failed to reveal C1r. These data suggest that complement is activated through the lectin pathway and proceeds to completion of the activation sequence as revealed by C9 deposition. MBL-A was detected as early as 3.5 d of pregnancy, and MBL-A deficiency prevented pregnancy loss in the abortion-prone mating combination. The contribution of the terminal complex to miscarriage was supported by the finding that pregnancy failure was largely inhibited by the administration of neutralizing Ab to C5. Treatment of DBA/2-mated CBA/J mice with Polyman2 that binds to MBL-A with high affinity proved to be highly effective in controlling the activation of the lectin pathway and in preventing fetal loss

New insight into antiphospholipid syndrome: antibodies to \u3b22glycoprotein I-domain 5 fail to induce thrombi in rats

Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of \u3b22glycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analysed the in vivo pro-coagulant effect of two groups of 5 serum IgG each reacting selectively with domain 1 or domain 5 in LPS-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy and vascular deposition of \u3b22glycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-\u3b22glycoproteinI antibodies served as controls. All the anti-domain 1 positive IgG exhibited potent pro-coagulant activity while the anti-domain 5 positive and the negative control IgG failed to promote blood clot and vessels occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound \u3b22glycoprotein I while able to interact with fluid-phase \u3b22glycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit in vivo thrombogenic effect and the interaction of these antibodies with circulating \u3b22glycoproteinI suggest their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining really at risk patients for more appropriate treatments to avoid recurrences and disability

Targeted delivery of neutralizing anti-C5 antibody to renal endothelium prevents complement- dependent tissue damage

Complement activation is largely implicated in the pathogenesis of several clinical conditions and its therapeutic neutralization has proven effective in preventing tissue and organ damage. A problem that still needs to be solved in the therapeutic control of complement-mediated diseases is how to avoid side effects associated with chronic neutralization of the complement system, in particular, the increased risk of infections. We addressed this issue developing a strategy based on the preferential delivery of a C5 complement inhibitor to the organ involved in the pathologic process. To this end, we generated Ergidina, a neutralizing recombinant anti-C5 human antibody coupled with a cyclic-RGD peptide, with a distinctive homing property for ischemic endothelial cells and effective in controlling tissue damage in a rat model of renal ischemia/reperfusion injury (IRI). As a result of its preferential localization on renal endothelium, the molecule induced complete inhibition of complement activation at tissue level, and local protection from complement-mediated tissue damage without affecting circulating C5. The ex vivo binding of Ergidina to surgically removed kidney exposed to cold ischemia supports its therapeutic use to prevent posttransplant IRI leading to delay of graft function. Moreover, the finding that the ex vivo binding of Ergidina was not restricted to the kidney, but was also seen on ischemic heart, suggests that this RGD-targeted anti-C5 antibody may represent a useful tool to treat organs prior to transplantation. Based on this evidence, we propose preliminary data showing that Ergidina is a novel targeted drug to prevent complement activation on the endothelium of ischemic kidney

Targeting CD34(+) cells of the inflamed synovial endothelium by guided nanoparticles for the treatment of rheumatoid arthritis

Despite the advances in the treatment of rheumatoid arthritis (RA) achieved in the last few years, several patients are diagnosed late, do not respond to or have to stop therapy because of inefficacy and/or toxicity, leaving still a huge unmet need. Tissue-specific strategies have the potential to address some of these issues. The aim of the study is the development of a safe nanotechnology approach for tissue-specific delivery of drugs and diagnostic probes. CD34 + endothelial precursors were addressed in inflamed synovium using targeted biodegradable nanoparticles (tBNPs). These nanostructures were made of poly-lactic acid, poly-caprolactone, and PEG and then coated with a synovial homing peptide. Immunofluorescence analysis clearly demonstrated their capacity to selectively address CD34 + endothelial cells in synovial tissue obtained from human, mouse, and rat. Biodistribution studies in two different animal models of rheumatoid arthritis (antigen-induced arthritis/AIA and collagen-induced arthritis/CIA) confirmed the selective accumulation in inflamed joints but also evidenced the capacity of tBNP to detect early phases of the disease and the preferential liver elimination. The therapeutic effect of methotrexate (MTX)-loaded tBNPs were studied in comparison with conventional MTX doses. MTX-loaded tBNPs prevented and treated CIA and AIA at a lower dose and reduced administration frequency than MTX. Moreover, MTX-loaded tBNP showed a novel mechanism of action, in which the particles target and kill CD34 + endothelial progenitors, preventing neo-angiogenesis and, consequently, synovial inflammation. tBNPs represent a stable and safe platform to develop highly-sensitive imaging and therapeutic approaches in RA targeting specifically synovial neo-angiogenesis to reduce local inflammation

Plasminogen activator-coated nanobubbles targeting cell-bound β2-glycoprotein I as a novel thrombus-specific thrombolytic strategy

Beta2-glycoprotein I (β2-GPI) is a serum protein widely recognized as the main target of antibodies present in patients with anti-phospholipid syndrome (APS). β2-GPI binds to activated endothelial cells, platelets and leukocytes, key players in thrombus formation. We developed a new targeted thrombolytic agent consisting of nanobubbles (NBs) coated with recombinant tissue plasminogen activator (rtPA) and recombinant antibody specific for cell-bound β2-GPI. The therapeutic efficacy of targeted nanobubbles was evaluated in vitro, using platelet-rich blood clots, and in vivo in three different animal models: 1) thrombosis developed in a rat model of APS; 2) ferric chloride-induced mesenteric thrombosis in rats, and 3) thrombotic microangiopathy in a mouse model of atypical hemolytic uremic syndrome (C3-gain-of-function mice). Targeted nanobubbles bound preferentially to platelets and leukocytes within thrombi and to endothelial cells through β2-GPI expressed on activated cells. In vitro, rtPA-targeted NBs (rtPA-tNBs) induced greater lysis of platelet-rich blood clots than untargeted NBs. In a rat model of APS, administration of rtPA-tNBs caused rapid dissolution of thrombi and, unlike soluble rtPA that induced transient thrombolysis, prevented new thrombus formation. In a rat model of ferric chloride triggered thrombosis, rtPA-tNBs, but not untargeted NBs and free rtPA, induced rapid and persistent recanalization of occluded vessels. Finally, treatment of C3-gain-of-function mice with rtPA-tNBs, that target β2-GPI deposited in kidney glomeruli, decreased fibrin deposition, and improved urinalysis data with a greater efficiency than untargeted NBs. Our findings suggest that targeting cell-bound β2-GPI may represent an efficient and thrombus-specific thrombolytic strategy in both APS-related and APSunrelated thrombotic conditions

Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice.

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4 to 25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, PMN and NK cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications

C5aR and C5L2 act in concert to balance immunometabolism in adipose tissue

Recent studies suggested that the immunometabolic receptors; C5aR and C5L2, constitutively self-associate into homo-/heterodimers and that acylation stimulating protein (ASP/C3adesArg) or C5a treatment of adipocytes increased their colocalization. The present study evaluates the C5aR contribution in adipocytes to the metabolic and immune responses elicited by ligand stimulation.The effects of C5a, ASP, and insulin on cytokine production, triglyceride synthesis (TGS), and key signaling pathways were evaluated in isolated primary adipocytes and cultured 3T3-L1 differentiated adipocytes. In addition, mRNA expression of IRS1 and PGC1α was compared in adipose tissue samples from WT vs. C5aRKO mice.Both C5a and ASP directly increased MCP-1 (238. ±. 4%;

Multiple-Organ Complement Deposition on Vascular Endothelium in COVID-19 Patients

Increased levels of circulating complement activation products have been reported in COVID\u201019 patients, but only limited information is available on complement involvement at the tissue level. The mechanisms and pathways of local complement activation remain unclear. The aim of this study was to investigate the deposition of complement components in the lungs, kidneys, and liver in patients with COVID\u201019 patients and to determine the pathway/s of complement activation. We performed immunofluorescence analyses of autopsy specimens of lungs, kidney, and liver from 12 COVID\u201019 patients who died of acute respiratory failure. Snap\u2010frozen samples embedded in OCT were stained with antibodies against complement components and activation products, IgG, and spike protein of SARS\u2010CoV\u20102. Lung deposits of C1q, C4, C3, and C5b\u20109 were localized in the capillaries of the interalveolar septa and on alveolar cells. IgG displayed a similar even distribution, suggesting classical pathway activation. The spike protein is a potential target of IgG, but its uneven distribution suggests that other viral and tissue molecules may be targeted by IgG. FB deposits were also seen in COVID\u201019 lungs and are consistent with activation of the alternative pathway, whereas MBL and MASP\u20102 were hardly detectable. Analysis of kidney and liver specimens mirrored findings observed in the lung. Complement deposits were seen on tubules and vessels of the kidney with only mild C5b\u20109 staining in glomeruli, and on the hepatic artery and portal vein of the liver. Complement deposits in different organs of deceased COVID\u201019 patients caused by activation of the classical and alternative pathways support the multi\u2010organ nature of the disease and the contribution of the complement system to inflammation and tissue damage