Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis.

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

Inhibition of the Alternative Complement Pathway May Cause Secretion of Factor B, Enabling an Early Detection of Pancreatic Cancer.

This study aims to elucidate the cellular mechanisms behind the secretion of complement factor B (CFB), known for its dual roles as an early biomarker for pancreatic ductal adenocarcinoma (PDAC) and as the initial substrate for the alternative complement pathway (ACP). Using parallel reaction monitoring analysis, we confirmed a consistent ∼2-fold increase in CFB expression in PDAC patients compared with that in both healthy donors (HD) and chronic pancreatitis (CP) patients. Elevated ACP activity was observed in CP and other benign conditions compared with that in HD and PDAC patients, suggesting a functional link between ACP and PDAC. Protein-protein interaction analyses involving key complement proteins and their regulatory factors were conducted using blood samples from PDAC patients and cultured cell lines. Our findings revealed a complex control system governing the ACP and its regulatory factors, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, adrenomedullin (AM), and complement factor H (CFH). Particularly, AM emerged as a crucial player in CFB secretion, activating CFH and promoting its predominant binding to C3b over CFB. Mechanistically, our data suggest that the KRAS mutation stimulates AM expression, enhancing CFH activity in the fluid phase through binding. This heightened AM-CFH interaction conferred greater affinity for C3b over CFB, potentially suppressing the ACP cascade. This sequence of events likely culminated in the preferential release of ductal CFB into plasma during the early stages of PDAC. (Data set ID PXD047043.).

Common haplotypes at the CFH locus and low-frequency variants in CFHR2 and CFHR5 associate with systemic FHR concentrations and age-related macular degeneration.

Age-related macular degeneration (AMD) is the principal cause of blindness in the elderly population. A strong effect on AMD risk has been reported for genetic variants at the CFH locus, encompassing complement factor H (CFH) and the complement-factor-H-related (CFHR) genes, but the underlying mechanisms are not fully understood. We aimed to dissect the role of factor H (FH) and FH-related (FHR) proteins in AMD in a cohort of 202 controls and 216 individuals with AMD. We detected elevated systemic levels of FHR-1 (p = 1.84 × 10-6), FHR-2 (p = 1.47 × 10-4), FHR-3 (p = 1.05 × 10-5) and FHR-4A (p = 1.22 × 10-2) in AMD, whereas FH concentrations remained unchanged. Common AMD genetic variants and haplotypes at the CFH locus strongly associated with FHR protein concentrations (e.g., FH p.Tyr402His and FHR-2 concentrations, p = 3.68 × 10-17), whereas the association with FH concentrations was limited. Furthermore, in an International AMD Genomics Consortium cohort of 17,596 controls and 15,894 individuals with AMD, we found that low-frequency and rare protein-altering CFHR2 and CFHR5 variants associated with AMD independently of all previously reported genome-wide association study (GWAS) signals (p = 5.03 × 10-3 and p = 2.81 × 10-6, respectively). Low-frequency variants in CFHR2 and CFHR5 led to reduced or absent FHR-2 and FHR-5 concentrations (e.g., p.Cys72Tyr in CFHR2 and FHR-2, p = 2.46 × 10-16). Finally, we showed localization of FHR-2 and FHR-5 in the choriocapillaris and in drusen. Our study identifies FHR proteins as key proteins in the AMD disease mechanism. Consequently, therapies that modulate FHR proteins might be effective for treating or preventing progression of AMD. Such therapies could target specific individuals with AMD on the basis of their genotypes at the CFH locus.

Beyond factor H: The impact of genetic-risk variants for age-related macular degeneration on circulating factor-H-like 1 and factor-H-related protein concentrations.

Age-related macular degeneration (AMD) is a leading cause of vision loss; there is strong genetic susceptibility at the complement factor H (CFH) locus. This locus encodes a series of complement regulators: factor H (FH), a splice variant factor-H-like 1 (FHL-1), and five factor-H-related proteins (FHR-1 to FHR-5), all involved in the regulation of complement factor C3b turnover. Little is known about how AMD-associated variants at this locus might influence FHL-1 and FHR protein concentrations. We have used a bespoke targeted mass-spectrometry assay to measure the circulating concentrations of all seven complement regulators and demonstrated elevated concentrations in 352 advanced AMD-affected individuals for all FHR proteins (FHR-1, p = 2.4 × 10-10; FHR-2, p = 6.0 × 10-10; FHR-3, p = 1.5 × 10-5; FHR-4, p = 1.3 × 10-3; FHR-5, p = 1.9 × 10-4) and FHL-1 (p = 4.9 × 10-4) when these individuals were compared to 252 controls, whereas no difference was seen for FH (p = 0.94). Genome-wide association analyses in controls revealed genome-wide-significant signals at the CFH locus for all five FHR proteins, and univariate Mendelian-randomization analyses strongly supported the association of FHR-1, FHR-2, FHR-4, and FHR-5 with AMD susceptibility. These findings provide a strong biochemical explanation for how genetically driven alterations in circulating FHR proteins could be major drivers of AMD and highlight the need for research into FHR protein modulation as a viable therapeutic avenue for AMD.

Gliosis-dependent expression of complement factor H truncated variants attenuates retinal neurodegeneration following ischemic injury.

Inherited, age-related, and acute retinal diseases are often exacerbated by an aberrant or excessive activity of the complement system. Consequently, cells not directly affected by an acute event or genetic variants may degenerate, resulting in enhanced visual impairment. The therapeutic potential of supplementation of complement factor H (FH), a key regulator of the complement cascade, is therefore particularly promising in the context of retinal diseases caused by complement activation. In this study, we engineered adeno-associated viruses (AAVs) containing sequences of two truncated human FH variants. The expression of these variants was regulated by the glial fibrillary acidic protein (GFAP) promoter, which is selectively active in gliotic Müller cells. Both FH variants consisted of FH domains 19-20, which were connected to domains 1-4 and 1-7, respectively, by a polyglycine linker. These AAVs were intravitreally injected following ischemic injury of C57BL/6J mouse retinas. We observed transgene expression in gliotic Müller cells and to some extent in astrocytes. The expression correlated directly with damage severity. Interventions resulted in decreased complement activation, accelerated normalization of microglia activity and morphological improvements. Reduced levels of C3 transcripts and C3d protein in conjunction with higher transcript levels of inhibitory regulators like Cfi and Cfh, hinted at attenuated complement activity. This study demonstrates the great potential of complement regulatory gene addition therapy. With further in vivo testing it could be applied to treat a wide range of retinal diseases where no causative therapies are available.

Eculizumab as first-line treatment for patients with severe presentation of complement factor H antibody-mediated hemolytic uremic syndrome.

BACKGROUND: Complement factor H (FH) antibody-mediated hemolytic uremic syndrome (HUS) has varying prevalence globally. Plasmapheresis and immunosuppressive drugs are the standard treatment. Recently, eculizumab has been reported as an effective alternative. This study aims to report four children with FH antibody-mediated HUS managed with eculizumab plus immunosuppression as first-line therapy. METHODS: A retrospective chart review was conducted for children aged ≤ 18 years old with complement-mediated HUS in two referral centers. Patients with FH antibody-mediated HUS treated with eculizumab as first-line therapy were included. RESULTS: Four children (aged 6-11 years old) were included. Dialysis was necessary in three patients. Eculizumab was administered 5-23 days after onset. None of them received plasmapheresis. Prednisone and mycophenolate mofetil were added after receiving positive FH antibody results. Hematological signs and kidney function improved after the second eculizumab dose. Eculizumab was discontinued in three patients after 6 months. One patient required rituximab due to persistent high FH antibody titers; discontinuation of eculizumab occurred after 15 months without recurrence. No treatment-related complications were observed. During a mean 12-month follow-up (range 6-24 months), no relapses were recorded and all patients ended with normal GFR. CONCLUSION: Our data suggest that a short course of 6 months of C5 inhibitor might be sufficient to reverse thrombotic microangiopathy symptoms and improve kidney function in patients with severe FH antibody-mediated HUS. Simultaneously, adding immunosuppressive agents might reduce the risk of relapse and allow cessation of C5 inhibition in a shorter period of time.

An unusual case of adult-onset still's disease complicated with anti-complement factor H antibodies associated atypical haemolytic uraemic syndrome.

BACKGROUND: Atypical haemolytic uremic syndrome (aHUS) is an uncommon form of thrombotic microangiopathy (TMA). However, it remains difficult to diagnose the disease early, given its non-specific and overlapping presentation to other conditions such as thrombotic thrombocytopenic purpura and typical HUS. It is also important to identify the underlying causes and to distinguish between primary (due to a genetic abnormality leading to a dysregulated alternative complement pathway) and secondary (often attributed by severe infection or inflammation) forms of the disease, as there is now effective treatment such as monoclonal antibodies against C5 for primary aHUS. However, primary aHUS with severe inflammation are often mistaken as a secondary HUS. We presented an unusual case of adult-onset Still's disease (AOSD) with macrophage activation syndrome (MAS), which is in fact associated with anti-complement factor H (anti-CFH) antibodies related aHUS. Although the aHUS may be triggered by the severe inflammation from the AOSD, the presence of anti-CFH antibodies suggests an underlying genetic defect in the alternative complement pathway, predisposing to primary aHUS. One should note that anti-CFH antibodies associated aHUS may not always associate with genetic predisposition to complement dysregulation and can be an autoimmune form of aHUS, highlighting the importance of genetic testing. CASE PRESENTATION: A 42 years old man was admitted with suspected adult-onset Still's disease. Intravenous methylprednisolone was started but patient was complicated with acute encephalopathy and low platelet. ADAMTS13 test returned to be normal and concurrent aHUS was eventually suspected, 26 days after the initial thrombocytopenia was presented. Plasma exchange was started and patient eventually had 2 doses of eculizumab after funding was approved. Concurrent tocilizumab was also used to treat the adult-onset Still's disease with MAS. The patient was eventually stabilised and long-term tocilizumab maintenance treatment was planned instead of eculizumab following haematology review. Although the aHUS may be a secondary event to MAS according to haematology opinion and the genetic test came back negative for the five major aHUS gene, high titre of anti-CFH antibodies was detected (1242 AU/ml). CONCLUSION: Our case highlighted the importance of prompt anti-CFH antibodies test and genetic testing for aHUS in patients with severe AOSD and features of TMA. Our case also emphasized testing for structural variants within the CFH and CFH-related proteins gene region, as part of the routine genetic analysis in patients with anti-CFH antibodies associated aHUS to improve diagnostic approaches.

Anti-C5 monoclonal antibody treatment showing pathological resolution of complement-mediated atypical hemolytic uremic syndrome: a case report.

BACKGROUND: No reports have shown histological changes before and after anti-C5 monoclonal antibody treatment in patients with atypical hemolytic uremic syndrome (aHUS). Here, we report a rare case of complement-mediated aHUS with a complement factor H (CFH) mutation and anti-CFH antibodies who underwent multiple kidney biopsies. CASE PRESENTATION: A 53-year-old woman developed aHUS with CFH gene mutation [c.3572C > T (p. Ser1191 Leu)] and anti-CFH antibodies. Her father had succumbed to acute kidney injury (AKI) in his 30 s. She exhibited AKI, thrombocytopenia, and hemolytic anemia with schistocytes. After improving the platelet count with one session of plasma exchange, a kidney biopsy was performed one month after the onset of symptoms. Blood vessel thrombosis, obvious endothelial swelling, endocapillary hypercellularity, and subendothelial exudative lesions in the glomeruli and arterioles were detected. Anti-C5 monoclonal antibody treatment with eculizumab immediately improved disease activity. A second biopsy 3 months later revealed marked improvement of endothelial injuries with residual membrane double contours and exudative lesions. A third biopsy at 17 months after gradual improvement of kidney function showed a further decrease of double contours along with alterations of the exudative lesions to fibrous intimal thickening. CONCLUSIONS: This is the first report showing the pathophysiology of aHUS in the kidneys and the efficacy of anti-C5 monoclonal antibody treatment by presenting serial kidney pathological features before and after anti-C5 monoclonal antibody treatment. Since her CFH mutation was considered the most important pathological condition, treatment centered on eculizumab was administered, resulting in a good long-term prognosis. In addition, kidney pathological resolution in aHUS occurred over 1 year after anti-C5 monoclonal antibody treatment.

A complement factor H homolog, heparan sulfation, and syndecan maintain inversin compartment boundaries in C. elegans cilia.

Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. Canonical disease models suggest that defective interactions between complement factor H (CFH) and cell surface heparan sulfate (HS) result in increased alternative complement pathway activity, cytolytic damage, and tissue inflammation in the retina. Although these factors are thought to contribute to increased disease risk, multiple studies indicate that noncanonical mechanisms that result from defective CFH and HS interaction may contribute to the progression of AMD as well. A total of 60 ciliated sensory neurons in the nematode Caenorhabditis elegans detect chemical, olfactory, mechanical, and thermal cues in the environment. Here, we find that a C. elegans CFH homolog localizes on CEP mechanosensory neuron cilia where it has noncanonical roles in maintaining inversin/NPHP-2 within its namesake proximal compartment and preventing inversin/NPHP-2 accumulation in distal cilia compartments in aging adults. CFH localization and maintenance of inversin/NPHP-2 compartment integrity depend on the HS 3-O sulfotransferase HST-3.1 and the transmembrane proteoglycan syndecan/SDN-1. Defective inversin/NPHP-2 localization in mouse and human photoreceptors with CFH mutations indicates that these functions and interactions may be conserved in vertebrate sensory neurons, suggesting that previously unappreciated defects in cilia structure may contribute to the progressive photoreceptor dysfunction associated with CFH loss-of-function mutations in some AMD patients.

Complement factor H attenuates TNF-α-induced inflammation by upregulating EIF3C in rheumatoid arthritis.

OBJECTIVE: To explore the role and underlying mechanism of Complement Factor H (CFH) in the peripheral and joint inflammation of RA patients. METHODS: The levels of CFH in the serum and synovial fluid were determined by ELISA. The pyroptosis of monocytes was determined by western blotting and flow cytometry. The inflammation cytokine release was tested by ELISA. The cell migration and invasion ability of fibroblast-like synoviocytes (FLS) were tested by Wound healing Assay and transwell assay, respectively. The potential target of CFH was identified by RNA sequencing. RESULTS: CFH levels were significantly elevated in the serum and synovial fluid from RA and associated with high sensitivity C-reactive protein (hs-CRP), erythrocyte sedimentation rate (ESR), and disease activity score 28 (DAS28). TNF-α could inhibit CFH expression, and CFH combined with TNF-α significantly decreased cell death, cleaved-caspase 3, gasdermin E N-terminal (GSDME-N), and inflammatory cytokines release (IL-1ß and IL-6) of RA-derived monocytes. Stimulated with TNF-α increased CFH levels in RA FLS and CFH inhibits the migration, invasion, and TNF-α-induced production of inflammatory mediators, including proinflammatory cytokines (IL-6, IL-8) as well as matrix metalloproteinases (MMPs, MMP1 and MMP3) of RA FLSs. The RNA-seq results showed that CFH treatment induced upregulation of eukaryotic translation initiation factor 3 (EIF3C) in both RA monocytes and FLS. The migration of RA FLSs was promoted and the expressions of IL-6, IL-8, and MMP-3 were enhanced upon EIF3C knockdown under the stimulation of CFH combined with TNF-α. CONCLUSION: In conclusion, we have unfolded the anti-inflammatory roles of CFH in the peripheral and joints of RA, which might provide a potential therapeutic target for RA patients.

A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material.

BACKGROUND: Komagataella phaffii (Pichia pastoris) is a methylotrophic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being explored in this organism to facilitate cost-effective biomanufacturing. Small, stable artificial chromosomes in K. phaffii could offer unique advantages by accommodating multiple integrations of extraneous genes and their promoters without accumulating perturbations of native chromosomes or exhausting the availability of selection markers. RESULTS: Here, we describe a linear "nano"chromosome (of 15-25 kb) that, according to whole-genome sequencing, persists in K. phaffii over many generations with a copy number per cell of one, provided non-homologous end joining is compromised (by KU70-knockout). The nanochromosome includes a copy of the centromere from K. phaffii chromosome 3, a K. phaffii-derived autonomously replicating sequence on either side of the centromere, and a pair of K. phaffii-like telomeres. It contains, within its q arm, a landing zone in which genes of interest alternate with long (approx. 1-kb) non-coding DNA chosen to facilitate homologous recombination and serve as spacers. The landing zone can be extended along the nanochromosome, in an inch-worming mode of sequential gene integrations, accompanied by recycling of just two antibiotic-resistance markers. The nanochromosome was used to express PDI, a gene encoding protein disulfide isomerase. Co-expression with PDI allowed the production, from a genomically integrated gene, of secreted murine complement factor H, a plasma protein containing 40 disulfide bonds. As further proof-of-principle, we co-expressed, from a nanochromosome, both PDI and a gene for GFP-tagged human complement factor H under the control of PAOX1 and demonstrated that the secreted protein was active as a regulator of the complement system. CONCLUSIONS: We have added K. phaffii to the list of organisms that can produce human proteins from genes carried on a stable, linear, artificial chromosome. We envisage using nanochromosomes as repositories for numerous extraneous genes, allowing intensive engineering of K. phaffii without compromising its genome or weakening the resulting strain.

The Noncanonical Role of Complement Factor H in Retinal Pigment Epithelium (RPE) Cells and Implications for Age-Related Macular Degeneration (AMD).

Age-related macular degeneration (AMD) is a complex degenerative disease of the retina. Dysfunction of the retinal pigment epithelium (RPE) occurs in early stages of AMD, and progressive RPE atrophy leads to photoreceptor death and visual impairments that ultimately manifest as geographic atrophy (GA), one of the late-stage forms of AMD. AMD is caused by a combination of risk factors including aging, lifestyle choices, and genetic predisposition. A gene variant in the complement factor H gene (CFH) that leads to the Y402H polymorphism in the factor H protein (FH) confers the second highest risk for the development and progression of AMD. FH is a major negative regulator of the alternative pathway of the complement system, and the FH Y402H variant leads to increased complement activation, which is detectable in AMD patients. For this reason, various therapeutic approaches targeting the complement system have been developed, however, so far with very limited or no efficacy. Interestingly, recent studies suggest roles for FH beyond complement regulation. Here, we will discuss the noncanonical functions of FH in RPE cells and highlight the potential implications of those functions for future therapeutic approaches.

A genome-wide association study identifies key modulators of complement factor H binding to malondialdehyde-epitopes.

Genetic variants within complement factor H (CFH), a major alternative complement pathway regulator, are associated with the development of age-related macular degeneration (AMD) and other complementopathies. This is explained with the reduced binding of CFH or its splice variant factor H-like protein 1 (FHL-1) to self-ligands or altered self-ligands (e.g., malondialdehyde [MDA]-modified molecules) involved in homeostasis, thereby causing impaired complement regulation. Considering the critical role of CFH in inhibiting alternative pathway activation on MDA-modified surfaces, we performed an unbiased genome-wide search for genetic variants that modify the ability of plasma CFH to bind MDA in 1,830 individuals and characterized the mechanistic basis and the functional consequences of this. In a cohort of healthy individuals, we identified rs1061170 in CFH and the deletion of CFHR3 and CFHR1 as dominant genetic variants that modify CFH/FHL-1 binding to MDA. We further demonstrated that FHR1 and FHR3 compete with CFH for binding to MDA-epitopes and that FHR1 displays the highest affinity toward MDA-epitopes compared to CFH and FHR3. Moreover, FHR1 bound to MDA-rich areas on necrotic cells and prevented CFH from mediating its cofactor activity on MDA-modified surfaces, resulting in enhanced complement activation. These findings provide a mechanistic explanation as to why the deletion of CFHR3 and CFHR1 is protective in AMD and highlight the importance of genetic variants within the CFH/CFHR3/CFHR1 locus in the recognition of altered-self in tissue homeostasis.

The Reduction of Uromodulin, Complement Factor H, and Their Interaction Is Associated with Acute Kidney Injury to Chronic Kidney Disease Transition in a Four-Time Cisplatin-Injected Rat Model.

Uromodulin is recognized as a protective factor during AKI-to-CKD progression, but the mechanism remains unclear. We previously reported that uromodulin interacts with complement factor H (CFH) in vitro, and currently aimed to study the expression and interaction evolution of uromodulin and CFH during AKI-to-CKD transition. We successfully established a rat model of AKI-to-CKD transition induced by a four-time cisplatin treatment. The blood levels of BUN, SCR, KIM-1 and NGAL increased significantly during the acute injury phase and exhibited an uptrend in chronic progression. PAS staining showed the nephrotoxic effects of four-time cisplatin injection on renal tubules, and Sirius red highlighted the increasing collagen fiber. Protein and mRNA levels of uromodulin decreased while urine levels increased in acute renal injury on chronic background. An extremely diminished level of uromodulin correlated with severe renal fibrosis. RNA sequencing revealed an upregulation of the alternative pathway in the acute stage. Renal CFH gene expression showed an upward tendency, while blood CFH localized less, decreasing the abundance of CFH in kidney and following sustained C3 deposition. A co-IP assay detected the linkage between uromodulin and CFH. In the model of AKI-to-CKD transition, the levels of uromodulin and CFH decreased, which correlated with kidney dysfunction and fibrosis. The interaction between uromodulin and CFH might participate in AKI-to-CKD transition.

Upregulation of HIF-1α contributes to complement activation in transplantation-associated thrombotic microangiopathy.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a severe complication of haematopoietic stem cell transplantation (HSCT). Complement activation is involved in the development of TA-TMA. However, the underlying mechanism is unclear. Therefore, 21 samples of TA-TMA and 1:1 matched controls were measured for hypoxia-inducible factor-1α (HIF-1α) and complement protein. The mechanism was investigated both in vitro and in vivo. In this study, we found that levels of HIF-1α were significantly higher in TA-TMA patients than that in non-TA-TMA controls. Upregulation of HIF-1α induced an increase in membrane-bound complement C3 and dysfunction of human umbilical vein endothelial cells (HUVECs) in vitro. Increasing HIF-1α in vivo led to C3 and C5b-9 deposition in the glomerular endothelial capillary complex, thrombocytopenia, anaemia, and increased serum lactate dehydrogenase (LDH) levels in wild-type (WT) but not in C3-/- mice subjected to HSCT. High platelet aggregation in peripheral blood and CD41-positive microthrombi in the kidney were also found in dimethyloxallyl glycine (DMOG)-treated mice, recapitulating the TA-TMA phenotype seen in patients. Comprehensive analysis, including DNA array, luciferase reporter assay, chromatin immunoprecipitation (ChIP)-seq, and quantitative polymerase chain reaction (PCR), revealed that HIF-1α interacted with the promoter of complement factor H (CFH) to inhibit its transcription. Decreased CFH led to complement activation in endothelial cells.

Functional characterization of complement factor H in host defense against bacterial pathogen in Nile tilapia (Oreochromis niloticus).

Complement factor H (CFH), a multifunctional soluble complement regulatory protein, can bind to a variety of pathogens and play a crucial role in host innate immune defense. To explore the functional characteristics of CFH (OnCFH) in Nile tilapia (Oreochromis niloticus), we cloned and characterized the open reading frame (ORF) of OnCFH in this study. The full-length of OnCFH ORF is 1359 bp, encoding 452 aa for a 48.85 kDa peptide, and its predicted structure containing six short complement-like repeats (SCRs). The analysis of tissue distribution showed that OnCFH was constitutively expressed in all tested tissues, with the highest in the liver. Upon Streptococcus agalactiae and Aeromonas hydrophila stimuli in vivo and in vitro, OnCFH mRNA transcript was significantly upregulated in head kidney tissue as well as head kidney monocytes/macrophages. Further, the recombinant OnCFH protein ((r)OnCFH) could bind to pathogenic bacteria in a dose-dependent. Moreover, it got involved in the regulation of inflammation as well as phagocytosis of monocytes/macrophages. The knockdown of OnCFH remarkably decreased the amount of bacteria in the head kidney. In summary, our data demonstrated that OnCFH could participate in the immune response of Nile tilapia against bacterial infection.

Anti-factor H antibody associated hemolytic uremic syndrome following SARS-CoV-2 infection.

BACKGROUND: The pathogenesis of autoantibody generation in anti-factor H (FH) antibody associated atypical hemolytic uremic syndrome (aHUS) is unknown and is perhaps triggered by an infectious or environmental agent. We observed an unusual increase of patients with anti-FH antibody associated aHUS coinciding with the second pandemic wave in New Delhi and suspected that SARS-CoV-2 infection might be a potential trigger. METHODS: We screened for SARS-CoV-2 infection using reverse transcriptase polymerase chain reaction (RT-PCR) and serology in 13 consecutive patients with anti-FH antibody associated aHUS during the past year in New Delhi. RESULTS: We report 5 patients, 4-13 years old, who presented with a febrile illness without respiratory symptoms during the second pandemic wave. Of these, 3 patients presented with a relapse 25-85 months following the initial episode of aHUS. SARS-CoV-2 was detected by RT-PCR in 1 patient and by serology in 4 patients (median titer 47.1 cut-off index). Patients had high titers of anti-FH antibodies (median 2,300 AU/ml). Genetic studies, done in 3 of the 5 patients, showed homozygous CFHR1 deletion without other significant genetic abnormalities. Specific management comprised plasma exchanges and oral prednisolone, combined with either cyclophosphamide or mycophenolate mofetil. At median follow-up of 3.3 months, the estimated glomerular filtration rate in 4 patients ranged from 62 to 110 ml/min/1.73 m2; one patient was dialysis-dependent. CONCLUSION: Increased vigilance is required during the pandemic, especially in patients with anti-FH associated aHUS, who might relapse despite quiescent disease for a prolonged period. A higher resolution version of the Graphical abstract is available as Supplementary information.

High prevalence of CFHR deletions in Indian women with pregnancy-associated hemolytic uremic syndrome.

AIM: Pregnancy-associated hemolytic uremic syndrome (P-aHUS) is an important cause of peripartum acute kidney injury. Studies from Europe have described mutations in complement regulator genes, and data in Indian patients is scarce. Hence this study used multiplex ligation-dependent probe amplification (MLPA) to identify variants in complement genes in P-aHUS patients. METHODS: We present 17 patients of P-aHUS who were investigated for complement protein levels and genetic analysis with MLPA for complement genes. Plasma exchange therapy was offered to all patients presenting in acute phase. RESULTS: Mean age 26.74 (3.36) years with 15/17 delivered by caesarean section. Eleven patients received early (within 7 days) plasma exchange, three were dialysis-dependent at 3 months and seven were dialysis-free. Only one of the three patients receiving late (after 7 days) plasma exchange was dialysis-free. MLPA showed that 11 patients had heterozygous deletions of exons 3, 5, 6 of CFHR1 and upstream region of exons 1, 2, 3, 6 and intron 4 of CFHR3 gene while four patients had homozygous deletions at the same loci. Two patients had no MLPA-detectable variations. CONCLUSION: This study reports a high proportion of deletions of exons of CFHR1 & CFHR3 genes in Indian P-aHUS patients detectable by MLPA by copy number variations. This needs confirmation in large multicentre studies. Plasma exchange can be an effective therapy in the non-availability of Eculizumab.

Early graft loss due to acute thrombotic microangiopathy accompanied by complement gene variants in living-related kidney transplantation: case series report.

BACKGROUND: Recently, early graft loss has become very rare in living-related kidney transplantation (LKT) as a result of decreased risk of hyperacute rejection and improvements in immunosuppressive regimens. Post-transplant acute thrombotic microangiopathy (TMA) is a rare, multi-factorial disease that often occurs shortly after kidney transplantation and is usually resistant to treatment with dismal renal outcomes. The complement genetic variants may accelerate the development of TMA. However, the complement genetic test was seldom performed in unknown native kidney disease recipients scheduled for LKT. CASE PRESENTATION: We reported three cases of unknown native kidney diseases who had fulminant TMA in the allograft shortly after LKT. Both the donors and the recipients were noted to carry complement genetic variants, which were identified by genetic testing after transplantation. However, all recipients were refractory to treatment and had allograft loss within 3 months after LKT. CONCLUSION: This case series highlights the suggestion to screen complement gene variants in both the donors and the recipients with unknown native kidney diseases scheduled for LKT.

An Infant Case of Streptococcus Pneumoniae-Associated Thrombotic Microangiopathy with Heterozygous CFI Mutation and CFHR3-CFHR1 Deletion.

Thrombotic microangiopathy (TMA) is a disease that causes organ damage due to microvascular hemolytic anemia, thrombocytopenia, and microvascular platelet thrombosis. Streptococcus pneumoniae-associated TMA (spTMA) is a rare complication of invasive pneumococcal infection. In addition, atypical hemolytic uremic syndrome (aHUS) is TMA associated with congenital or acquired dysregulation of complement activation. We report the case of a nine-month-old boy with refractory nephrotic syndrome complicated by spTMA in the setting of heterozygous complement factor-I (CFI) gene mutation and CFHR3-CFHR1 deletion. He repeatedly developed thrombocytopenia, anemia with schistocytes, hypocomplementemia, and abnormal coagulation triggered by infection, which manifested clinically with convulsions and an intraperitoneal hematoma. Eculizumab (a monoclonal humanized anti-C5 antibody) provided transient symptomatic benefit including improvement in thrombocytopenia; however, he developed unexplained cardiac arrest and was declared brain dead a few days later. In this report, we highlight the diagnostic challenges of this case and the causal relationship between spTMA and complement abnormalities and consider the contribution of heterozygous mutation of CFI and CFHR3-CFHR1 deletion.