RESUMEN

Background: C4d deposits are present in a substantial proportion of patients with IgA nephropathy (IgAN), indicating the activation of the lectin pathway (LP) of the complement system. It seems that patients with activated LP have worse renal prognosis. The aim of this study was to investigate the prevalence and prognostic significance of C4d in our cohort of patients with primary IgA nephropathy (pIgAN). Methods: Patients with pIgAN were recruited from a hospital register of kidney biopsies of the Department of Nephrology and Dialysis, Dubrava University Hospital, Zagreb. Additional immunohistochemistry staining for C4d was performed on paraffin-embedded kidney tissue, and patients were stratified into being C4d positive or C4d negative. The clinical and histologic features of patients were analyzed and compared regarding C4d positivity. The primary outcome was defined as kidney failure (KF), and predictor variables of KF and renal survival were analyzed. Results: Of a total of 95 patients with pIgAN included in the study, C4d was present in 43 (45.3%). C4d-positive patients had a higher value of systolic (p = 0.039) and diastolic (p = 0.006) blood pressure at diagnosis as well as higher 24 h proteinuria (p = 0.018), serum urate (p = 0.033), and lower eGFR (p < 0.001). C4d-positive patients had worse renal survival (p < 0.001), higher rates of disease progression to KF (p < 0.001), and higher proteinuria (p < 0.001) and lower eGFR (p < 0.001) at the last follow-up. Glomerular C4d was an independent predictor of disease progression to KF (HR = 5.87 [0.95 CI 1.06-32.44], p = 0.032). Conclusions: C4d is an independent predictor of disease progression in patients with pIgAN. C4d may be used as an additional marker of progressive disease course in IgAN. The therapeutic implications of C4d status in IgAN, particularly in terms of complement inhibitors application, are not yet known.

RESUMEN

Terminal complement complex (TCC) deposition was identified in human degenerated discs. To clarify the role of terminal complement activation in disc degeneration (DD), we investigated respective activating mechanisms and cellular effects in annulus fibrosus (AF) cells. Isolated cells from human AF, nucleus pulposus (NP), and endplate (EP) were stimulated with human serum alone or with zymosan and treated with either the C3 inhibitor Cp40 or the C5 antibody eculizumab. Complement activation was determined via anaphylatoxin generation and TCC deposition detection. Thereby, induced catabolic effects were evaluated in cultured AF cells. Moreover, C5 cleavage under degenerative conditions in the presence of AF cells was assessed. Zymosan-induced anaphylatoxin generation and TCC deposition was significantly suppressed by both complement inhibitors. Zymosan induced gene expression of ADAMTS4, MMP1, and COX2. Whereas the C3 blockade attenuated the expression of ADAMTS4, the C5 blockade reduced the expression of ADAMTS4, MMP1, and COX2. Direct C5 cleavage was significantly enhanced by EP conditioned medium from DD patients and CTSD. These results indicate that terminal complement activation might be functionally involved in the progression of DD. Moreover, we found evidence that soluble factors secreted by degenerated EP tissue can mediate direct C5 cleavage, thereby contributing to complement activation in degenerated discs.

Asunto(s)

RESUMEN

Background: Colorectal cancer (CRC) is a common global malignancy associated with high invasiveness, high metastasis, and poor prognosis. CRC commonly metastasizes to the liver, where the treatment of metastasis is both difficult and an important topic in current CRC management. Methods: Microarrays data of human CRC with liver metastasis (CRCLM) were downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database to identify potential key genes. Differentially expressed (DE) genes (DEGs) and DEmiRNAs of primary CRC tumor tissues and metastatic liver tissues were identified. Microenvironment Cell Populations (MCP)-counter was used to estimate the abundance of immune cells in the tumor micro-environment (TME), and weighted gene correlation network analysis (WGCNA) was used to construct the co-expression network analysis. Gene Ontology and Kyoto Encyclopaedia of Gene and Genome (KEGG) pathway enrichment analyses were conducted, and the protein-protein interaction (PPI) network for the DEGs were constructed and gene modules were screened. Results: Thirty-five pairs of matched colorectal primary cancer and liver metastatic gene expression profiles were screened, and 610 DEGs (265 up-regulated and 345 down-regulated) and 284 DEmiRNAs were identified. The DEGs were mainly enriched in the complement and coagulation cascade pathways and renin secretion. Immune infiltrating cells including neutrophils, monocytic lineage, and cancer-associated fibroblasts (CAFs) differed significantly between primary tumor tissues and metastatic liver tissues. WGCN analysis obtained 12 modules and identified 62 genes with significant interactions which were mainly related to complement and coagulation cascade and the focal adhesion pathway. The best subset regression analysis and backward stepwise regression analysis were performed, and eight genes were determined, including F10, FGG, KNG1, MBL2, PROC, SERPINA1, CAV1, and SPP1. Further analysis showed four genes, including FGG, KNG1, CAV1, and SPP1 were significantly associated with CRCLM. Conclusions: Our study implies complement and coagulation cascade and the focal adhesion pathway play a significant role in the development and progression of CRCLM, and FGG, KNG1, CAV1, and SPP1 may be metastatic markers for its early diagnosis.

RESUMEN

Thrombotic microangiopathies (TMA) constitute a group of different disorders that have a common underlying mechanism: the endothelial damage. These disorders may exhibit different mechanisms of endothelial injury depending on the pathological trigger. However, over the last decades, the potential role of the complement system (CS) has gained prominence in their pathogenesis. This is partly due to the great efficacy of complement-inhibitors in atypical hemolytic syndrome (aHUS), a TMA form where the primary defect is an alternative complement pathway dysregulation over endothelial cells (genetic and/or adquired). Complement involvement has also been demonstrated in other forms of TMA, such as thrombotic thrombocytopenic purpura (TTP) and in Shiga toxin-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS), as well as in secondary TMAs, in which complement activation occurs in the context of other diseases. However, at present, there is scarce evidence about the efficacy of complement-targeted therapies in these entities. The relationship between complement dysregulation and endothelial damage as the main causes of TMA will be reviewed here. Moreover, the different clinical trials evaluating the use of complement-inhibitors for the treatment of patients suffering from different TMA-associated disorders are summarized, as a clear example of the entry into a new era of personalized medicine in its management.

RESUMEN

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett's Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.

Asunto(s)

RESUMEN

For last months, humanity has faced a formidable unknown enemy, which is presented as a new coronavirus infection. Despite the fact that the causative agents of new diseases appear at a certain frequency and that the virus SARS-CoV-2 has certain common properties with its predecessors, at the moment we are dealing with a new unknown pathogenesis of the development of severe complications in patients with risk factors. A final understanding of pathological process mechanisms is the goal of the scientific community. Summarizing research data from different countries, it became obvious that in severe cases of viral infection, we are dealing with a combination of the systemic inflammatory response syndrome, disseminated intravascular coagulation and thrombotic microangiopathy (TMA). Thrombotic microangiopathy is represented by a group of different conditions in which thrombocytopenia, hemolytic anemia, and multiple organ failure occur. The article reflects the main types of TMA, pathogenesis and principles of therapy. The main participants in the process are described in detail, including the von Willebrand factor and ADAMTS-13. Based on the knowledge available, as well as new data obtained from patients with COVID-19, we proposed possible models for the implementation of conditions such as sepsis, TMA, and DIC in patients with severe new coronavirus infection. Through a deeper understanding of pathogenesis, it will be possible to develop more effective diagnosis and therapy.

Asunto(s)

RESUMEN

Systemic inflammation is thought to underlie many of the metabolic manifestations of cachexia in cancer patients. The complement system is an important component of innate immunity that has been shown to contribute to metabolic inflammation. We hypothesized that systemic inflammation in patients with cancer cachexia was associated with complement activation. Systemic C3a levels were higher in cachectic patients with inflammation (n = 23, C-reactive protein (CRP) ≥ 10 mg/L) as compared to patients without inflammation (n = 26, CRP < 10 mg/L) or without cachexia (n = 13) (medians 102.4 (IQR 89.4-158.0) vs. 81.4 (IQR 47.9-124.0) vs. 61.6 (IQR 46.8-86.8) ng/mL, respectively, p = 0.0186). Accordingly, terminal complement complex (TCC) concentrations gradually increased in these patient groups (medians 2298 (IQR 2022-3058) vs. 1939 (IQR 1725-2311) vs. 1805 (IQR 1552-2569) mAU/mL, respectively, p = 0.0511). C3a and TCC concentrations were strongly correlated (rs = 0.468, p = 0.0005). Although concentrations of C1q and mannose-binding lectin did not differ between groups, C1q levels were correlated with both C3a and TCC concentrations (rs = 0.394, p = 0.0042 and rs = 0.300, p = 0.0188, respectively). In conclusion, systemic inflammation in patients with cancer cachexia is associated with the activation of key effector complement factors. The correlations between C1q and C3a/TCC suggest that the classical complement pathway could play a role in complement activation in patients with pancreatic cancer.

RESUMEN

Acute kidney injury (AKI) is a common and severe complication of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) causing progressive chronic kidney disease (CKD), end-stage renal disease (ESRD) or death. Pathogenic ANCAs, in particular proteinase 3 (PR3) and myeloperoxidase (MPO), trigger a deleterious immune response resulting in pauci-immune necrotizing and crescentic glomerulonephritis (GN), a common manifestation of glomerular injury in AAV. However, there is growing evidence that activation of the complement pathway contributes to the pathogenesis and progression of AAV. We here aimed to compare glomerular and tubulointerstitial lesions in ANCA GN and extrarenal manifestation of AAV in association with levels of circulating complement components C3c and C4. METHODS: Plasma levels of C3c and C4 in a total number of 53 kidney biopsies with ANCA GN were retrospectively included between 2015 and 2020. Glomerular and tubulointerstitial lesions were evaluated according to established scoring systems for ANCA GN and analogous to the Banff classification. RESULTS: We here show that circulating levels of C3c and C4 in ANCA GN were comparable to the majority of other renal pathologies. Furthermore, hypocomplementemia was only detectable in a minor subset of ANCA GN and not correlated with renal or extrarenal AAV manifestations. However, low levels of circulating C3c correlated with AKI severity in ANCA GN independent of systemic disease activity or extrarenal AAV manifestation. By systematic scoring of glomerular and tubulointerstitial lesions, we provide evidence that low levels of circulating C3c and C4 correlated with vasculitis manifestations to distinct renal compartments in ANCA GN. CONCLUSIONS: We here expand our current knowledge about distinct complement components in association with vasculitis manifestations to different renal compartments in ANCA GN. While low levels of C4 correlated with glomerulitis, our observation that low levels of circulating complement component C3c is associated with interstitial vasculitis manifestation reflected by intimal arteritis implicates that C3c contributes to tubulointerstitial injury in ANCA GN.

Asunto(s)

RESUMEN

PURPOSE: Formation of terminal complement complex (TCC), a downstream complement system activation product inducing inflammatory processes and cell lysis, has been identified in degenerated discs. However, it remains unclear which molecular factors regulate complement activation during disc degeneration (DD). This study investigated a possible involvement of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) and the lysosomal protease cathepsin D (CTSD). METHODS: Disc biopsies were collected from patients suffering from DD (n = 43) and adolescent idiopathic scoliosis (AIS, n = 13). Standardized tissue punches and isolated cells from nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) were stimulated with 5% human serum (HS) alone or in combination with IL-1ß, CTSD or zymosan. TCC formation and modulation by the complement regulatory proteins CD46, CD55 and CD59 were analysed. RESULTS: In DD tissue cultures, IL-1ß stimulation decreased the percentage of TCC + cells in AF and EP (P < 0.05), whereas CTSD stimulation significantly increased TCC deposition in NP (P < 0.01) and zymosan in EP (P < 0.05). Overall, the expression of CD46, CD55 and CD59 significantly increased in all isolated cells during culture (P < 0.05). Moreover, cellular TCC deposition was HS concentration dependent but unaffected by IL-1ß, CTSD or zymosan. CONCLUSION: These results suggest a functional relevance of IL-1ß and CTSD in modulating TCC formation in DD, with differences between tissue regions. Although strong TCC deposition may represent a degeneration-associated event, IL-1ß may inhibit it. In contrast, TCC formation was shown to be triggered by CTSD, indicating a multifunctional involvement in disc pathophysiology.

Asunto(s)

RESUMEN

Thrombotic microangiopathy is a rare but serious complication that affects kidney transplant recipients. It appears in 0.8-14% of transplanted patients and negatively affects graft and patient survival. It can appear in a systemic form, with hemolytic microangiopathic anemia, thrombocytopenia, and renal failure, or in a localized form, with progressive renal failure, proteinuria, or arterial hypertension. Post-transplant thrombotic microangiopathy is classified as recurrent atypical hemolytic uremic syndrome or de novo thrombotic microangiopathy. De novo thrombotic microangiopathy accounts for the majority of cases. Distinguishing between the 2 conditions can be difficult, given there is an overlap between them. Complement overactivation is the cornerstone of all post-transplant thrombotic microangiopathies, and has been demonstrated in the context of organ procurement, ischemia-reperfusion phenomena, immunosuppressive drugs, antibody-mediated rejection, viral infections, and post-transplant relapse of antiphospholipid antibody syndrome. Although treatment of the causative agents is usually the first line of treatment, this approach might not be sufficient. Plasma exchange typically resolves hematologic abnormalities but does not improve renal function. Complement blockade with eculizumab has been shown to be an effective therapy in post-transplant thrombotic microangiopathy, but it is necessary to define which patients can benefit from this therapy and when and how eculizumab should be used.

RESUMEN

Upon traumatic injuries or carious lesions, the elimination of bacteria infiltrating the pulp is recognized as a prerequisite for initiating the regeneration process. Complement is a major system involved in initiating the inflammatory reaction and the subsequent bacteria elimination. This plasma system of above 35 proteins is synthesized by the liver and some immune cells. It is activated by 3 pathways: the classical, alternative, and lectin pathways that can be triggered by physical injuries, infection, and biomaterials. Recent data have shown that the pulp fibroblast represents a unique nonimmune cell type able to synthesize Complement proteins. Indeed, after physical injuries/bacteria stimulation, the pulp fibroblast has been shown to synthesize and to activate the complement system leading to the production of biologically active molecules such as C5a, C3b, and the membrane attack complex. This local secretion represents a rapid and efficient mechanism for eliminating bacteria invading the pulp, thus supporting complement activation from the plasma. Pulp fibroblast-secreted Complement proteins allow cariogenic bacteria direct lysis via membrane attack complex formation on their surface, phagocytic cell recruitment by producing C5a and cariogenic bacteria opsonization by C3b fixation on their surface, stimulating cariogenic bacteria phagocytosis. Overall, this review highlights that, in addition to initiating the inflammatory reaction, pulp fibroblasts also provide a powerful control of this inflammation via local Complement activation. The pathogen elimination capacity by fibroblast-produced complement demonstrates that this system is a strong local actor in arresting bacterial progression into the dental pulp.

Asunto(s)

RESUMEN

Deposits of complement components have been documented in several human tumors suggesting a potential involvement of the complement system in tumor immune surveillance. In vitro and in vivo studies have revealed a double role played by this system in tumor progression. Complement activation in the cancer microenvironment has been shown to promote cancer growth through the release of the chemotactic peptide C5a recruiting myeloid suppressor cells. There is also evidence that tumor progression can be controlled by complement activated on the surface of cancer cells through one of the three pathways of complement activation. The aim of this review is to discuss the protective role of complement in cancer with special focus on the beneficial effect of complement-fixing antibodies that are efficient activators of the classical pathway and contribute to inhibit tumor expansion as a result of MAC-mediated cancer cell killing and complement-mediated inflammatory process. Cancer cells are heterogeneous in their susceptibility to complement-induced killing that generally depends on stable and relatively high expression of the antigen and the ability of therapeutic antibodies to activate complement. A new generation of monoclonal antibodies are being developed with structural modification leading to hexamer formation and enhanced complement activation. An important progress in cancer immunotherapy has been made with the generation of bispecific antibodies targeting tumor antigens and able to neutralize complement regulators overexpressed on cancer cells. A great effort is being devoted to implementing combined therapy of traditional approaches based on surgery, chemotherapy and radiotherapy and complement-fixing therapeutic antibodies. An effective control of tumor growth by complement is likely to be obtained on residual cancer cells following conventional therapy to reduce the tumor mass, prevent recurrences and avoid disabilities.

Asunto(s)

RESUMEN

Envenomation by Loxosceles spiders can result in severe systemic and local reactions, which are mainly triggered by Sphingomyelinase D (SMase D), a toxic component of Loxosceles venom. SMase D induces a systemic inflammatory condition similar to the reaction observed during an endotoxic shock. Considering the potent pro-inflammatory potential of Loxosceles venom and the SMase D, in this study we have used the whole human blood model to study the endotoxic-like shock triggered by SMase D. Recombinant purified SMase D from L. intermedia venom, similarly to LPS, induced activation of blood leukocytes, as observed by the increase in the expression of CD11b and TLR4, production of reactive oxygen and nitrogen species (superoxide anion and peroxynitrite) and release of TNF-α. Complement consumption in the plasma was also detected, and complement inhibition by compstatin decreased the SMase D and LPS-induced leukocyte activation, as demonstrated by a reduction in the expression of CD11b and TLR4 and superoxide anion production. Similar results were found for the L. intermedia venom, except for the production of TNF-α. These findings indicate that SMase D present in Loxosceles venom is able to activate leukocytes in a partially complement-dependent manner, which can contribute to the systemic inflammation that follows envenomation by this spider. Thus, future therapeutic management of systemic Loxosceles envenomation could include the use of complement inhibitors as adjunct therapy.

Asunto(s)

RESUMEN

Envenomation by Loxosceles spiders can result in severe systemic and local reactions, which are mainly triggered by Sphingomyelinase D (SMase D), a toxic component of Loxosceles venom. SMase D induces a systemic inflammatory condition similar to the reaction observed during an endotoxic shock. Considering the potent pro inflammatory potential of Loxosceles venom and the SMase D, in this study we have used the whole human blood model to study the endotoxic-like shock triggered by SMase D. Recombinant purified SMase D from L. intermedia venom, similarly to LPS, induced activation of blood leukocytes, as observed by the increase in the expression of CD11b and TLR4, production of reactive oxygen and nitrogen species (superoxide anion and peroxynitrite) and release of TNF-alpha. Complement consumption in the plasma was also detected, and complement inhibition by compstatin decreased the SMase D and LPS-induced leukocyte activation, as demonstrated by a reduction in the expression of CD11b and TLR4 and superoxide anion production. Similar results were found for the L. intermedia venom, except for the production of TNF-alpha. These findings indicate that SMase D present in Loxosceles venom is able to activate leukocytes in a partially complement-dependent manner, which can contribute to the systemic inflammation that follows envenomation by this spider. Thus, future therapeutic management of systemic Loxosceles envenomation could include the use of complement inhibitors as adjunct therapy.

RESUMEN

OBJECTIVE: To investigate the expression of complement system's activation factors in plasma of patients with severe preeclampsia and their correlations with anti-angiogenesis factors. METHODS: A case-control study was performed. The study group consisted of 30 cases of early-onset severe preeclampsia (EOSPE) and 30 cases of late-onset severe preeclampsia (LOSPE). Thirty cases were selected as the early-onset control group (E-control) and 30 as the late-onset control group (L-control), with the weeks of gestation matched. Enzyme-linked immunosorbent assay (ELISA) was used to test C3a, C5a, MAC, sEng, and sflt-1 in the maternal peripheral circulation. RESULTS: The complement system's activation factors C3a, C5a, and MAC were increased significantly in EOSPE and LOSPE (all P < 0.01) compared with E/L-control. Plasma levels of C3a correlated inversely with plasma sEng (r = -0.454, P < 0.001) and sflt-1 (r = -0.326, P = 0.011) in preeclampsia patients, while MAC correlated with soluble endoglin (sEng; r = 0.343, P = 0.007) and soluble fms-like tyrosine kinase-1 (sflt-1; r = 0.318, P = 0.013). There were no significant correlations between complement system's activation-related factors and the anti-angiogenesis factors in healthy control group. CONCLUSIONS: Abnormal activation of the complement system exists in the maternal circulation of patients with E/L-onset severe preeclampsia. There were correlations between the abnormal activation of the complement system and the abnormal expression of anti-angiogenesis factors in patients with severe preeclampsia, but the correlation was not strong.

Asunto(s)

RESUMEN

Peptide-based nanoparticles have emerged as promising drug delivery systems for targeted cancer therapy. Yet, the biocompatibility of these nanoparticles has not been elucidated. Here, the in vitro biocompatibility and toxicity and in vivo immunocompatibility and bioactivity of the self/coassembling peptide AC8 in its nanoparticle form are evaluated. AC8 showed minimal hemolytic activity (5%) and did not cause aggregation of red blood cells. The in vitro assay revealed that AC8 did not activate the complement system via the classical or alternative pathway but did activate the lectin pathway to a small extent. However, AC8 showed no C3a and C5a anaphylotoxin activation suggesting that complement activation did not proceed to the later, inflammatory, stages. The in vivo immune response assay showed that administration of AC8 to BALB/c mice had no effect on the weight of immune organs or body weight of mice at doses less than 0.1 mg/kg. This peptide also did not have any effect on the expression of CD3+ T-cells and natural killer (NK) cells, the ratio of CD4+/CD8+ T-cell, and the proliferation of B-cells. These results suggest that AC8 can be a potential carrier candidate for drug delivery.

Asunto(s)

RESUMEN

Antivenoms have been widely used for more than a century for treating snakebites and other accidents with poisonous animais. Despite their efficacy, the use of heterologous antivenoms involves the possibility of adverse reactions due to activation of the immune system. In this paper, alternatives for antivenom production already in use were evaluated in light of their ability to minimize the occurrence of adverse reactions. These effects were classified according to their molecular mechanism as: anaphylactic reactions mediated by IgE, anaphylactoid reactions: aused by complement system activation, and pyrogenic reactions produced mainly by the presence of endotoxins in the final product. ln the future, antivenoms may be replaced by humanized antibodies, specific neutralizing compounds or vaccination. Meanwhile, improvements in antivenom quality will be focused on the obtainment of more purified and specific product in compliance with good manufacturing practices and at an affordable cost

Asunto(s)