Complement Factor C3 Research Articles

Decreased sialylation elicits complement-related microglia response and bipolar cell loss in the mouse retina.

Sialylation plays an important role in self-recognition, as well as keeping the complement and innate immune systems in check. It is unclear whether the reduced sialylation seen during aging and in mice heterozygous for the null mutant of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (Gne+/-), an essential enzyme for sialic acid biosynthesis, contributes to retinal inflammation and degeneration. We found a reduction of polysialic acid and trisialic acid expression in several retinal layers in Gne+/- mice at 9 months of age compared to Gne+/+ wildtype (WT) mice, which was associated with a higher microglial expression of the lysosomal marker CD68. Furthermore, the total number of rod bipolar cells was reduced in 12 months old Gne+/- mice in comparison to WT mice, demonstrating loss of these retinal interneurons. Transcriptome analysis showed up-regulation of complement, inflammation, and apoptosis-related pathways in the retinas of Gne+/- mice. Particularly, increased gene transcript levels of the complement factors C3 and C4 and the pro-inflammatory cytokine Il-1β were observed by semi-quantitative real-time polymerase chain reaction (sqRT-PCR) in 9 months old Gne+/- mice compared to WT mice. The increased expression of CD68, loss of rod bipolar cells, and increased gene transcription of complement factor C4, were all prevented after crossing Gne+/- mice with complement factor C3-deficient animals. In conclusion, our data show that retinal hyposialylation in 9 and 12 months old Gne+/- mice was associated with complement-related inflammation and lysosomal microglia response, as well as rod bipolar cells loss, which was absent after genetic deletion of complement factor C3.

Effects of Aeromonas infection on the immune system, physical barriers and microflora structure in the intestine of juvenile grass carp (Ctenopharyngodon idella)

Grass carp (Ctenopharyngodon idella) is an intensively cultured and economically important herbivorous fish species in China, but its culture is often impacted by Aeromonas pathogens such as Aeromonas hydrophila and Aeromonas veronii. In this study, healthy grass carp were separately infected with A. hydrophila or A. veronii for 12, 24, 48 or 72 h. The results showed that the mRNA expression levels of intestinal inflammatory factors (tnf-α, il-1β and il-8), complement factors (c3 and c4), antimicrobial peptides (hepcidin, nk-lysin and β-defensin-1), immunoglobulins (igm and igt), and immune pathway-related signaling molecules (tlr1, tlr2, tlr4, myd88, irak4, irak1, traf6, nf-κb p65 and ap-1) were differentially upregulated in response to A. hydrophila and A. veronii challenge. Additionally, the expression levels of the intestinal pro-apoptotic genes tnfr1, tnfr2, tradd, caspase-8, caspase-3 and bax were significantly increased, whereas the expression of the inhibitory factor bcl-2 was significantly downregulated, indicating that Aeromonas infection significantly induced apoptosis in the intestine of grass carp. Moreover, the expression of intestinal tight junction proteins (occludin, zo-1, claudin b and claudin c) was significantly decreased after infection with Aeromonas. Histopathological analysis indicated the Aeromonas challenge caused severe damage to the intestinal villi with adhesions and detachment of intestinal villi accompanied by severe inflammatory cell infiltration at 12 h and 72 h. The 16S rRNA sequencing results showed that Aeromonas infection significantly altered the structure of the intestinal microflora of the grass carp at the phylum (Proteobacteria, Fusobacteria, Bacteroidetes and Firmicutes) and genus (Proteus, Cetobacterium, Bacteroides, and Aeromonas) levels. Take together, the findings of this study revealed that Aeromonas infection induces an intestinal immune response, triggers cell apoptosis, destroys physical barriers and alters microflora structure in the intestine of juvenile grass carp; the results will help to reveal the pathogenesis of intestinal bacterial diseases in grass carp.

Age-related macular degeneration: suitability of optogenetic therapy for geographic atrophy.

Age-related macular degeneration (AMD) is a growing public health concern given the aging population and it is the leading cause of blindness in developed countries, affecting individuals over the age of 55 years. AMD affects the retinal pigment epithelium (RPE) and Bruch's membrane in the macula, leading to secondary photoreceptor degeneration and eventual loss of central vision. Late AMD is divided into two forms: neovascular AMD and geographic atrophy (GA). GA accounts for around 60% of late AMD and has been the most challenging subtype to treat. Recent advances include approval of new intravitreally administered therapeutics, pegcetacoplan (Syfovre) and avacincaptad pegol (Iveric Bio), which target complement factors C3 and C5, respectively, which slow down the rate of enlargement of the area of atrophy. However, there is currently no treatment to reverse the central vision loss associated with GA. Optogenetics may provide a strategy for rescuing visual function in GA by imparting light-sensitivity to the surviving inner retina (i.e., retinal ganglion cells or bipolar cells). It takes advantage of residual inner retinal architecture to transmit visual stimuli along the visual pathway, while a wide range of photosensitive proteins are available for consideration. Herein, we review the anatomical changes in GA, discuss the suitability of optogenetic therapeutic sensors in different target cells in pre-clinical models, and consider the advantages and disadvantages of different routes of administration of therapeutic vectors.

Sialic acids on T cells are crucial for their maintenance and survival.

Sialic acids are found as terminal sugars on glycan structures on cellular surfaces. T cells carry these sialoglycans abundantly, and they are thought to serve multiple functions in cell adhesion, cell migration, and protection from complement attack. We studied the role of sialoglycans on T cells in a mouse model with a T cell-specific deletion of cytidine monophosphate-sialic acid synthase (CMAS), the enzyme that is crucial for the synthesis of sialoglycans. These mice showed a T-cell deficiency in peripheral lymphoid organs. Many T cells with an undeleted Cmas allele were found in the periphery, suggesting that they escaped the Cre-mediated deletion. The remaining peripheral T cells of T cell-specific Cmas KO mice had a memory-like phenotype. Additional depletion of the complement factor C3 could not rescue the phenotype, showing that the T-cell defect was not caused by a host complement activity. Cmas-deficient T cells showed a high level of activated caspase 3, indicating an ongoing apoptosis. In bone marrow chimeric cellular transfer experiments, we observed a strong competitive disadvantage of Cmas-deficient T cells compared to wild-type T cells. These results show that sialoglycans on the surface of T cells are crucial for T-cell survival and maintenance. This function has not been recognized before and is similar to the function of sialoglycans on B cells.

Early Feeding Strategy Mitigates Major Physiological Dynamics Altered by Heat Stress in Broilers.

Heat stress is one of the stressors that negatively affect broiler chickens, leading to a reduction in production efficiency and profitability. This reduction affects the economy in general, especially in hot and semi-hot countries. Therefore, improving heat tolerance of broiler chicks is a key to sustained peak performance, especially under adverse environmental heat stress conditions. The present study investigated three early feed withdrawal regimes (FWD) as a potential mitigation for thermal stress exposure. A total of 240 unsexed one-day-old Cobb-500 chicks were randomly recruited to one of four experimental groups using a completely randomized design (10 birds × 6 replicates). The experimental groups included the control group with no feed withdrawal (control), while the other three groups were subjected to early feed withdrawal for either 24 h on the 5th day of age (FWD-24), 12 h on the 3rd and 5th day of age (FWD-12), or 8 h on the 3rd, 4th, and 5th day of age (FWD-8), respectively. Production performance was monitored throughout the experiment. Meanwhile, blood and liver samples were taken at the end of the experimental period to evaluate major physiological dynamic changes. Our findings demonstrated that under chronic heat stress conditions, FWD treatments significantly improved broilers' production performance and enhanced several physiological parameters compared with the control. Serum levels of thyroid hormones were elevated, whereas leptin hormone was decreased in FWD groups compared with the control. Moreover, serum total protein, globulin, and hemoglobin levels were higher, while total cholesterol and uric acid were lower in the FWD groups. Furthermore, FWD groups showed significantly higher antioxidant marker activity with a significantly lower lipid peroxidation level. Immunoglobulin levels, lysozyme, complement factor C3, and liver heat shock protein 70 (HSP70) concentration were also elevated in FWD compared with the control. Also, serum interleukin-1β (IL-1β) and interferon-gamma (IFN-γ) significantly increased with FWD. Based on our findings, early feed withdrawal can be applied as a promising non-invasive nutritional strategy for broilers reared under chronic heat stress conditions. Such a strategy promotes the alleviation of the deleterious effects of heat stress on broiler performance, immunity, and redox status, owing to the onset of physiological adaptation and the development of thermotolerance ability.

Immunological Significance of Meconium-Stained Amniotic Fluid with Complement Factors C3 and CD4 Levels for Pregnancy Health

Background: Meconium stained liquor (MSL) occurs when the fetal anal sphincter muscles relax in utero, tinting the amniotic fluid green, yellow, or brown. It is less common in premature fetuses. Method: Complement factors (C3 and CD4) were assessed in maternal serum and amniotic fluid from 30 women with MSL undergoing diagnostic fetoscopy at gestational week. Serum levels were consistently higher than control levels, which were higher than levels in amniotic fluid. Results: In the MSL group versus the control group, mean C3 levels in serum and amniotic fluid were (200.3913±37.6689 vs. 155.1321.8257) and (100.912±22.1869 vs. 98.0027±30.0989 pg/ml), respectively, showing statistically significant differences (p<0.01). Significant differences (p<0.01) were also observed between amniotic fluid and serum samples (155.1321 vs. 200.3913 pg/ml) in the MSL group, but not in the control group (100.912 vs. 98.0027 pg/ml). Mean CD4 levels in serum and amniotic fluid in the MSL group versus the control group were (13.1072 ± 4.2871 vs. 7.8651±2.5954) and (4.0372 ± 1.766 vs. 4.2328 ± 1.3943 pg/ml), respectively, with statistically significant differences (p<0.01). Significant differences (p<0.01) were noted between amniotic fluid and serum samples (7.8651 vs. 13.1072 pg/ml) in the MSL group, but not in the control group (4.2328 vs. 4.0372 pg/ml). Conclusion: Elevated levels of C3 and CD4 in serum and meconium-stained amniotic fluid indicate potential issues in pregnancy, suggesting their utility for early detection of this pathological condition to safeguard fetal health.

A longitudinal prospective study of active tuberculosis in a Western Europe setting: insights and findings.

This study investigates the potential of inflammatory parameters (IP), symptoms, and patient-related outcome measurements as biomarkers of severity and their ability to predict tuberculosis (TB) evolution. People with TB were included prospectively in the Stage-TB study conducted at five clinical sites in Barcelona (Spain) between April 2018 and December 2021. Data on demographics, epidemiology, clinical features, microbiology, and Sanit George Respiratory Questionnaire (SGRQ) and Kessler-10 as Health-Related Quality of Life (HRQoL) were collected at three time points during treatment. C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), neutrophil/lymphocyte, and monocyte/lymphocyte ratios (NLR and MLR), complement factors C3, C4, and cH50, clinical and microbiological data, and HRQoL questionnaires were assessed at baseline, 2months, and 6months. Their ability to predict sputum culture conversion (SCC) and symptom presence after 2months of treatment was also analysed. The study included 81 adults and 13 children with TB. The CRP, ESR, NLR, and MLR values, as well as the presence of symptoms, decreased significantly over time in both groups. Higher IP levels at baseline were associated with greater bacillary load and persistent symptoms. Clinical severity at baseline predicted a delayed SCC. Kessler-10 improved during follow-up, but self-reported lung impairment (SGRQ) persisted in all individuals after 6months. IP levels may indicate disease severity, and sustained high levels are linked to lower treatment efficacy. Baseline clinical severity is the best predictor of SCC. Implementing health strategies to evaluate lung function and mental health throughout the disease process may be crucial for individuals with TB.

Amiloride Reduces Urokinase/Plasminogen-Driven Intratubular Complement Activation in Glomerular Proteinuria.

Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic activity might be involved. The urokinase-type plasminogen activator (uPA)-plasminogen cascade activates complement and generates C3a and C5a in vitro / ex vivo in urine from healthy persons when exogenous, inactive, plasminogen, and complement factors are added. Amiloride inhibits uPA and attenuates complement activation in vitro and in vivo . In conditional podocin knockout (KO) mice with severe proteinuria, blocking of uPA with monoclonal antibodies significantly reduces the urine excretion of C3a and C5a and lowers tissue NLRP3-inflammasome protein without major changes in early fibrosis markers. This mechanism provides a link to proinflammatory signaling in proteinuria with possible long-term consequences for kidney function. Persistent proteinuria is associated with tubular interstitial inflammation and predicts progressive kidney injury. In proteinuria, plasminogen is aberrantly filtered and activated by urokinase-type plasminogen activator (uPA), which promotes kidney fibrosis. We hypothesized that plasmin activates filtered complement factors C3 and C5 directly in tubular fluid, generating anaphylatoxins, and that this is attenuated by amiloride, an off-target uPA inhibitor. Purified C3, C5, plasminogen, urokinase, and urine from healthy humans were used for in vitro / ex vivo studies. Complement activation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and ELISA. Urine and plasma from patients with diabetic nephropathy treated with high-dose amiloride and from mice with proteinuria (podocin knockout [KO]) treated with amiloride or inhibitory anti-uPA antibodies were analyzed. The combination of uPA and plasminogen generated anaphylatoxins C3a and C5a from intact C3 and C5 and was inhibited by amiloride. Addition of exogenous plasminogen was sufficient for urine from healthy humans to activate complement. Conditional podocin KO in mice led to severe proteinuria and C3a and C5a urine excretion, which was attenuated reversibly by amiloride treatment for 4 days and reduced by >50% by inhibitory anti-uPA antibodies without altering proteinuria. NOD-, LRR- and pyrin domain-containing protein 3-inflammasome protein was reduced with no concomitant effect on fibrosis. In patients with diabetic nephropathy, amiloride reduced urinary excretion of C3dg and sC5b-9 significantly. In conditions with proteinuria, uPA-plasmin generates anaphylatoxins in tubular fluid and promotes downstream complement activation sensitive to amiloride. This mechanism links proteinuria to intratubular proinflammatory signaling. In perspective, amiloride could exert reno-protective effects beyond natriuresis and BP reduction. Increased Activity of a Renal Salt Transporter (ENaC) in Diabetic Kidney Disease, NCT01918488 and Increased Activity of ENaC in Proteinuric Kidney Transplant Recipients, NCT03036748 .

Acute phase proteins in patients with recurrent pulmonary tuberculosis

Objective. Study of initial levels of acute phase proteins in patients with pulmonary tuberculosis with and without relapse as predictors of unfavorable disease progression. Materials and methods. 2 groups of patients were examined: the base group - 55 patients with pulmonary tuberculosis with relapse and the control group - 55 patients with pulmonary tuberculosis without relapse. Acute phase protein content was studied in patients before and after the intensive phase of treatment. The content of complement factors C3 and C4, haptoglobin, ceruloplasmin, properdin, C-reactive protein, IL-6, VSH, leukocyte content was studied. Results. The obtained results reveal that acute phase proteins can be considered basic predictors of ineffective antituberculosis treatment: the high indices of leukocyte, C-reactive protein, IL-6, ESR, and haptoglobin.

Eculizumab Utilization in Hemolytic Uremic Syndrome (HUS) Presenting with Acute Hemolysis

Background: Hemolytic uremic syndrome (HUS) is thrombotic microangiopathy in which microthrombi are formed, consisting mainly of platelets, which will occlude the arterioles and capillaries leading to a triad of intravascular hemolysis, thrombocytopenia, and acute kidney failure. Case Presentation: A 2-year-old boy presented to our outpatient clinic with persistent diarrhea streaked with blood, vomiting, signs of dehydration, and an ingested throat. The complete blood count (CBC) revealed acute hemolysis with mild normocytic hypochromic anemia, anisopoikilocytosis, fragmented red blood cells, and moderate thrombocytopenia. The patient experienced a drastic course which was dramatically improved after the administration of eculizumab. Conclusion: Eculizumab is a recombinant humanized monoclonal antibody against complement component C5; that effectively blocks the cleavage of complement factor C5 and, subsequently, the complement membrane attacks complex C5B-9.78. While on eculizumab, our patient's kidney function improved significantly after the first dose with a creatinine of 0.56 mg/dl.

Locus coeruleus MRI contrast is associated with Complement factor C3b in cerebrospinal fluid

AbstractBackgroundDegeneration of the Locus coeruleus (LC) is associated with Alzheimer’s disease (AD) pathology and cognitive decline. Recent evidence from animal models indicates that this relationship may be mediated by neuroinflammation. Here we examined how LC MRI contrast is related to CSF biomarkers of neuroinflammation in humans.MethodThe LC as well as the Substantia nigra (SN) were visualized and segmented using neuromelanin‐sensitive T1‐weighted FLASH‐MRI images acquired at 3T and MRI contrast values were calculated as previously described (Betts et al., 2019b). A cohort of 65 participants with MRI contrast and CSF data from the DZNE longitudinal cognitive impairment and dementia (DELCODE) study (20 HC, 22 SCD, 16 MCI, 7 AD dementia; 53.8% female) were included in the analyses. We performed correlational analyses to test for associations between LC and SN contrast with a panel of 4 Principal Components (PC) created from a Principial Component Analysis on 16 individual inflammation‐related CSF biomarkers (Fig. 1). Both bivariate and partial correlation analyses were performed between PCs and MRI contrast values using sex, age, site and CSF levels of AD pathology, namely Aß42/40‐ratio and phosphorylated tau, as covariates (see Fig. 2).ResultWe found a significant positive association between LC MRI contrast and the inflammatory component PC4 including the proinflammatory complement factors C3 and C3b across the entire sample (r = 0.365, p = 0.003). Additional post‐hoc correlational analyses showed this association with LC MRI contrast was mainly due to a correlation with C3b (r = 0.349, p = 0.004). A significant positive association was still observed (r = 0.347, p = 0.008) in a subgroup of individuals comprising healthy older adults and individuals with a risk of AD dementia (HC, SCD and MCI) as shown in Fig. 2. In contrast, no significant associations between SN MRI contrast and any neurodegenerative or inflammatory measure were observed indicating effects were specific to the LC.ConclusionThe results demonstrate LC MRI contrast is associated with complement factors in CSF, in particular complement factor C3b in healthy older adults and individuals with a risk of AD dementia. Further analyses are required to determine if elevated complement factors may predict degeneration of the LC and subsequent cognitive decline.

CD19-Targeted CAR-T Cells in Refractory Systemic Autoimmune Diseases: A Monocentric Experience from the First Fifteen Patients

Background: CD19-targeting chimeric antigen receptor (CAR) T cells are highly efficient in B cell malignancies. However, CD19 CAR T cells also target autoreactive B cells that trigger autoimmune diseases (AID) including systemic lupus erythematosus (SLE), idiopahtic inflammatory myositis (IIM) and systemic sclerosis (SSc). Achieving long-term remission in AID with standard therapy remains challenging and usually necessitates long-term treatment. Aim: To test whether CD19 CAR T cells achieve a deeper reset of B cells, eradicate autoimmunity, and achieve lasting drug-free remission in autoantibody-dependent AID. Methods: Patients with treatment-refractory SLE, IIM and SSc were eligible if they had signs of active organ involvement, failure of multiple immunomodulatory therapies, and serious or immediately life-threatening disease. CAR T cell therapy was offered via an expanded access program. Autologous CD19 CAR T cells MB-CART19.1 were produced with the Miltenyi prodigy platform. After leukapheresis on day -13, manufacturing commenced in house and lymphodepletion was started with fludarabine 25 mg/m2/d intravenously (i.v.) on days -5 to -3 and cyclophosphamide 1000 mg/m2/d i.v. on day -3. On day 1, all patients received 1x106 CAR T cells / kg body weight. Results: 15 patients (8 SLE, 4 SSc, and 3 IIM) were apheresed and were treated with a single infusion of CD19 CAR T cells. Median disease duration before CAR T was 3 years [1-20], median follow-up after CAR T was 12 months [2-28]. All patients failed on a median of 5 [2-14] previous treatments. Except for a maximum of 10 mg prednisolone daily, all immunomodulatory drugs were stopped prior to leukapheresis. After infusion, CAR T cells rapidly expanded till day 8.6 ± 0.8. CD19+ B cells were rapidly eliminated from peripheral blood after a mean of 5.9 ± 2.2 days. In 12/15 patients, B cells reoccurred at day 111±50. The last three patients with 77, 51 and 16 days follow-up are still B cell aplastic. All 8 SLE patient reached a complete remission after 3 months and maintain a SLE Disease Activity Index (SLEDAI) of 0 since. 3/3 IIM patients experienced major improvement and normalization of CK after 3 months which is ongoing. Of 4 SSc patients, 3 patients with >3 months follow-up show a decreased disease activity by EULAR AI by -4.3 [-4.3; -3.6]. All 15 patients entirely stopped immunosuppressive drugs. Overall safety of CAR T Cell therapy in AID was very favourable with cytokine-release syndrome (CRS) of grade 0 in 4, grade 1 in 10, and grade 2 in 1 patient. 6/15 patients received tocilizumab. One grade 1 immune-effector cell associated neurotoxicity syndrome (ICANS) presented as vertigo two weeks after CAR T infusion. No prolonged (>28 days) bone marrow suppression occurred. The Five SLE patients with a follow-up of 14-24 months stayed in remission despite reconstitution of B cells. In line, complement factor C3 stayed normal, proteinuria remained absent or at very low levels, and autoantibody seroconversion persisted. IgG responses to standard vaccines remained stable up to month 24 and were boosted by re-vaccination. Total B cell numbers gradually increased to a median of 230 cells/µl [183; 886] at month 12 and were mostly of a naïve phenotype. The early drop of CD19+CD27+ memory B cells increased again at month 12 indicating maturation of the B cells emerging after CAR T treatment. Conclusion: CD19 CAR T cells induce persistent, drug-free remission in three distinct autoantibody dependent AID with very good tolerability. Though hypothetical cure appears possible, longer follow up is needed. This unprecedented drug-free remission is remarkable as B cells functionally reconstitute. An investigator-initiated trial is currently expanding our experience of efficacy and safety.

Intraluminal assessment of inflammatory factors in patients with intracranial aneurysms.

The formation, growth, and rupture of intracranial aneurysms (IA) are due to several pathophysiological mechanisms, including focal hemodynamic injury and inflammation of the arterial wall. We investigated the differences between venous, parent artery, and intra-aneurysmal blood by measuring inflammatory factors and antibodies in patients with ruptured (rIA) or unruptured intracranial aneurysms (uIA). A prospective study was performed in patients who presented with IA and required endovascular treatment. Blood was drawn from the lumen of the aneurysm sac, the parent artery, and the peripheral veins, to determine the serum concentrations of complement factors C3, C4, IgG, IgM, IgA antibodies, and C-reactive protein (CRP). Thirty-six patients (15 with uIA and 21 with rIA) were enrolled in the study. In both groups, C3, C4, IgM, IgG, and IgA showed a gradual decrease from venous to intra-aneurysmal samples, but only IgG in the parent artery and intra-aneurysmal samples reached a significant decrease in uIA compared with venous samples. Accordingly, C3 and IgG concentrations in the intra-aneurysmal samples showed a significant decrease in rIA compared with venous samples. A significant increase in CRP concentrations was observed in parent artery and intra-aneurysmal samples from patients with rIA compared with patients with uIA; a significant increase in C3 concentrations was observed in parent artery samples from patients with rIA compared with patients with uIA, and a significant decrease in IgM concentrations was observed in venous, parent artery, and intra-aneurysmal samples from patients with rIA compared with patients with uIA. A decrease in C3 and IgG in the aneurysm sac indicates activation of the complement system in the arterial wall. CRP in the aneurysm sac and lumen of the parent artery was significantly increased in ruptured compared with unruptured aneurysms, whereas venous, parent artery, and intra-aneurysmal IgM were decreased in ruptured compared with unruptured aneurysms. These results argue for the role of an ongoing inflammatory process in aneurysms leading to their growth and rupture.

Extracellular Matrix Remodeling in Atopic Dermatitis Harnesses the Onset of an Asthmatic Phenotype and Is a Potential Contributor to the Atopic March

The development of atopic dermatitis in infancy, and subsequent allergies, such as asthma in later childhood, is known as the atopic march. The mechanism is largely unknown, however the course of disease indicates an inter-epithelial crosstalk, through the onset of inflammation in the skin and progression to other mucosal epithelia. In this study, we investigated if and how skin-lung epithelial crosstalk contributes to the development of the atopic march. First, we emulated inter-epithelial crosstalk through indirect coculture of bioengineered atopic-like skin disease models and three-dimensional bronchial epithelial models triggering an asthma-like phenotype in the latter. A subsequent secretome analysis identified thrombospondin-1, CD44, complement factor C3, fibronectin, and syndecan-4 as potentially relevant skin-derived mediators. Because these mediators are extracellular matrix-related proteins, we then studied the involvement of the extracellular matrix, unveiling distinct proteomic, transcriptomic, and ultrastructural differences in atopic samples. The latter indicated extracellular matrix remodeling triggering the release of the above-mentioned mediators. Invivo mouse data showed that exposure to these mediators dysregulated activated circadian clock genes which are increasingly discussed in the context of atopic diseases and asthma development. Our data point toward the existence of a skin-lung axis that could contribute to the atopic march driven by skin extracellular matrix remodeling.

Short-term and long-term efficacies of combined use of irbesartan and calcitriol for the treatment of IgA nephropathy

Purpose: To determine the efficacy and safety of irbesartan plus calcitriol in the treatment of IgA nephropathy, and its effect on inflammatory injury and complement systemMethods: Differences in renal function, inflammatory response, immune function, complement factor levels, clinical efficacy, and incidence of adverse reactions between IgA nephropathy patients treated with irbesartan (control, n = 50) and those treated with irbesartan + calcitriol (study group, n = 50) after 2 and 12 months, were retrospectively analyzed. As treatment progressed, protein in 24-h urine, and creatinine and BUN in both groups were gradually reduced.Results: The serum levels of complement factors C1q and C3 in both groups gradually increased, while C4 level gradually decreased. Relative to pre-treatment, at 2- and 12-months post-treatment, serum levels of C1q and C3 in both groups were raised, while C4 level decreased (p < 0.05). Relative to control, serum C1q and C3 in the study group were raised, while C4 level was decreased (p < 0.05). Relative to the control group, total treatment effectiveness in the study group increased at 2- and 12- months post-treatment (p < 0.05). There was no significant difference in the incidence of adverse reactions between the control and study groups during the treatment.Conclusion: Treatment with irbesartan + calcitriol significantly improves renal function in patients with IgA nephropathy, reduces inflammatory response, and improves immune function and clinical effectiveness with high safety profile. More clinical trials should be carried out to validate the findings of this study.

Elevated complement C3 and increased CD8 and type 1 helper lymphocyte T populations in patients with post-COVID-19 condition

BackgroundBiological markers associated to post-COVID-19 condition (PCC) have not been clearly identified. MethodsEighty-two patients attending our post-COVID-19 outpatient clinic were recruited and classified as fully recovered (40.2%) or presenting with PCC (59.8%). Clinical and radiological data, laboratory markers, cytokines, and lymphocyte populations were analyzed. ResultsMedian number of days after hospitalization was 78.5 [p25-p75: 60–93] days. PCC was significantly more frequent in women, in patients with a previously critical COVID-19, and in those with two or more comorbidities. No differences were found in lymphocyte counts, ferritin, C-reactive protein, D-dimer or sCD25, IL-1β, IL-1Ra, IL-6, CXCL8, IL-17A, IL-18, IL-22, IFN-γ, TNF-α, and IL-10 cytokines levels. PCC patients showed significantly higher levels of complement factor C3 than fully recovered patients: median C3 128 mg/dL [p25-p75:107–135] vs 111 mg/dL [p25-p75: 100–125] (p =.005), respectively. In the flow cytometry assessment of peripheral blood lymphocyte subpopulations, PCC patients showed significantly increased CD8 populations compared to fully recovered patients: median CD8: 529 [p25-p75: 384–683] vs 370/mm3 [p25-p75:280–523], p =.007. When type 1, 2, 17/22, and 17.1 helper and follicular T lymphocyte subpopulations were analyzed, the frequency of Th1 was significantly higher in PCC patients compared to fully recovered patients (30% vs 38.5%, p =.028). ConclusionPatients with a post-COVID-19 condition showed significantly increased immunological parameters of inflammation (complement factor C3 and CD8 and Th1 T lymphocyte populations) compared to fully recovered patients. These parameters could be used as biological markers of this condition.

Low levels of complement factor C3 at diagnosis can predict outcome in antineutrophil antibody associated vasculitis.

Experimental data support the involvement of complement in the pathogenesis of antineutrophil antibody associated vasculitis, and clinical studies describe a more severe disease phenotype in patients with antineutrophil antibody associated vasculitis and complement activation. In the present study, we looked for an association between circulating serum complement factor 3 levels at diagnosis and outcomes. One hundred sixty-four patients with antineutrophil antibody associated vasculitis who underwent kidney biopsy at our center during the last 15years were retrospectively reviewed. Patients were categorized according to their serum complement factor 3 level at diagnosis. Patient and renal survival were compared between those above and below the median serum complement factor 3 at diagnosis. During the first year, 6 patients died and 53 reached end-stage renal disease. Death or end-stage renal disease at one-year were significantly more common in the low serum complement factor 3 group (44 vs. 29%, p = 0.037). In the multivariable analysis, serum complement factor 3 was the strongest negative outcomepredictor (HR, 95%CI 0.118, (0.021-0.670)). The lower the serum complement factor 3 level at baseline, the higher the risk of dialysis and death. The risk was particularly high for both endpoints if the serum complement factor 3 concentration was below 0.9g/l at baseline. Complement activation at diagnosis may identify a distinct subgroup of patients with antineutrophil antibody associated vasculitis and higher risk for poor outcomes. However, it remains to be proven whether inhibition of serum complement factor 3 is beneficial and safein the clinical setting.

Microangiopathy in multiple myeloma: a case of carfilzomib-induced secondary thrombotic microangiopathy successfully treated with plasma exchange and complement inhibition

BackgroundThrombotic microangiopathy (TMA) is a potentially organ and life-threatening condition affecting patients with multiple myeloma (MM). Cases of proteasome inhibitor-induced TMA and specifically carfilzomib-induced TMA have been rarely reported and standards for diagnostic workup and treatment are not available.Case presentationWe describe a case of a male MM patient under salvage therapy including proteasome inhibitor carfilzomib following chemotherapy and autologous stem cell transplantation. The patient then developed acute kidney injury with clinical and laboratory signs of TMA. Hemodialysis became necessary and treatment with plasma exchange was initiated followed by therapy with C5 complement inhibitor eculizumab which led to amelioration of kidney function and hemolysis parameters.ConclusionWe report a patient with suspected proteasome inhibitor-induced secondary thrombotic microangiopathy that has been successfully treated with plasma exchange and eculizumab, a monoclonal antibody targeting complement factor C5.

#5990 COMPLEMENT GENES PROFILE OF PATIENTS WITH SEVERE HYPERTENSION AND RENAL THROMBOTIC MICROANGIOPATHY

Abstract Background and Aims Hereditary or acquired complement system dysregulation found to be driving cause of endothelial damage in atypical haemolytic uremic syndrome (aHUS) – ultrarare life-threatening disease, that always manifests with renal thrombotic microangiopathy (TMA), resulting in poor outcome in absence of appropriate treatment. Nevertheless, several studies show high incidence of rare genetic variants of complement system in other TMA’s, including hypertension-induced TMA. Here we report results of complement genes screening in patients (pts) with histologically identified renal TMA and severe hypertension in Russia, at first diagnosed as hypertension-induced TMA. Method A total of 28 Caucasian pts diagnosed with renal TMA (confirmed by kidney biopsy in all cases) and severe hypertension enrolled in the study. The main characteristic of the clinical picture was the absence of microangiopathic hemolysis and thrombocytopenia in prevailing of the pts, thus most of the primary and secondary causes of TMA clinically excluded. Examination of DNA samples was performed in all pts using next generation sequencing (NGS) technology. For statistical analysis continuous variables were presented as Mean (±Standart deviation) or Median (interquartile range) as appropriate. Results Baseline demographic and clinical characteristics of group are listed in Table 1. 13 pts (46%) had malignant form of hypertension (MHT) confirmed by eye examination. Mean systolic arterial pressure (SAP) observed at the time of presentation was 210±29 mmHg, mean diastolic arterial pressure (DAP) was 122±19 mmHg. Kidney morphology revealed chronic TMA lesions in 12 pts (43%), acute and chronic alterations simultaneously detected in 13 pts (46%). Predominantly acute lesions with glomerular and extraglomerular thrombi found in 3 (11%) pts only. Complement genes abnormalities revealed in 7 (25%) out of 28 pts. Complement factor I (CFI) rare variants were found in 2 pts (CFI c. 772<A, CFI c.1217G>A), one of this pts also had additional variant in complement factor H related protein 4 (CFHR4 c.766G>A). Complement factor C3 rare variant was observed in 1 pt (C3 c.2203C>T), membrane cofactor protein (MCP or CD46) rare variant in 1 pt (CD46 c. 198A>T), complement factor H related protein 5 (CFHR5) variant detected in 1 pt (CFHR5 c.136C>T), homozygous deletion of CFHR1 and CFHR3 detected in 1 pt and heterozygous deletion of the same complement factor H related protein genes found in another pt. Conclusion A quarter of pts with TMA and severe hypertension may have genetic defects in the complement system. These results confirmed that complement-mediated HUS may be misdiagnosed as hypertension-induced TMA. Lack of convincing hematological signs of TMA is common in this group, making aHUS diagnosis more sophisticated.

#5524 THE ROLE OF C5AR VERSUS C3 IN THROMBOTIC MICROANGIOPATHY, ISCHEMIC KIDNEY NECROSIS, AND ACUTE KIDNEY INJURY INDUCED BY CHOLESTEROL CRYSTAL EMBOLISM

Abstract Background and Aims Certain thrombotic microangiopathies (TMA) respond to inhibition of complement factor C5, which abrogates formation of the membrane attack complex and hence impairs host defense. Inhibition of C5a receptor (C5aR) avoids this safety problem and still controls autoimmune systemic vasculitis. We speculated that targeting C5aR could be sufficient also in TMA, e.g., related to cholesterol crystal embolism (CCE). Method We induced experimental TMA in C3-/-, C5aR-/- or wildtype mice by injecting CC into the left kidney artery of mice and analyzed thrombotic angiopathy, drop in measured glomerular filtration rate (GFR), and ischemic necrosis 24 hours after CCE. Results In wildtype mice, CC injection caused diffuse TMA followed by a consistent drop of GFR compared to baseline and ischemic kidney necrosis. Genetic deficiency in C3 convertase profoundly attenuated TMA and hence GFR drop and ischemic kidney necrosis indicating a key role of the complement system in CCE-related TMA. Genetic deficiency of C5aR attenuated TMA, GFR drop and ischemic necrosis to the same extent indicating that C5aR provides the main contribution of the complement system to the pathogenesis of TMA. Conclusion We conclude that targeting the C5a/C5aR axis could be sufficient to attenuate also TMA for which a better safety profile would be expected as compared to C5 inhibition.