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Since 2008, there have been several recalls due to insufficient thiamine levels in canned cat food. Cats have a high requirement of thiamine, and deficiencies can lead to death within a month if not treated. Limited studies have been published regarding the impact of processing on thiamine loss. Therefore, it was our objective to determine the effect of container size and type on thiamine retention during processing of cat food. A model canned cat food was produced and placed in two container sizes (small: 89-104 mL vs medium: 163-207 mL) and three container types (can, pouch, and tray). Within each replicate batch (64 containers per replicate, two replicates), thermocouple probes were inserted into 14 separate containers. The retort time was determined by thermocouple heat penetration to meet Fo = 8 min at 121°C and 21 psi. The three sampling points for vitamin analysis were 1) batter without vitamins, 2) batter with vitamins, and 3) post retort loaf product. Samples from 1 and 2 were stored in the freezer (-20°C) and from 3 were stored at room temperature. Composite samples were analyzed for proximates (moisture, crude protein, crude fat, ash), pH, and B vitamin (thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, folic acid, cobalamin) concentrations. Results were analyzed using the GLM procedure in SAS (v. 9.4) with means and interactions separated using Fischer LSD method by significant F values and an a of 5%. The proximate composition and pH were similar (P > 0.10) among treatments. Neither container size nor container type had an effect on riboflavin, pyridoxine, or cobalamin concentrations (average 87.0, 179.0, 0.41 mg/kg, respectively). Small containers retained more (P < 0.05) thiamine than medium (3,209 and 2,513 mg/kg), niacin (909 and 861 mg/kg), and folic acid (22 vs 15 mg/kg), while pantothenic acid retention in small containers was lower (P < 0.05) than medium (273 and 324 mg/kg, respectively). The main effect means for container type only influenced thiamine concentration, wherein retention was greater for pouches than for trays with cans intermediate to both (2,540, 2,359, and 2,274 mg/kg). This work suggests that thermal processing of B vitamins can be influenced by container size and type, which likely relate to the controls over the heating and cooling cycle. [ABSTRACT FROM AUTHOR]

Feline grooming habits can lead to accumulation in the stomach and formation of masses (trichobezoars), hairballs, that are regurgitated. Some pet food companies have created diets intended to force the concretion into the intestines, primarily by supplementing large quantities of insoluble fiber like cellulose. Miscanthus grass (M) possesses a similar content of insoluble fiber and may be an alternative for this purpose. Therefore, the objectives of this study were to determine the effects of M on hairball passage in cats. Experimental diets were composed of 90% basal ration and 10% of either M or rice flour (C). Cats (12 American Shorthair) were individually fed one of the two extruded diets for 16 days of adaptation; then feces were collected for 5 days. Hair masses were washed from feces and counted, and total hair weight and fecal dry weight were quantified. Hair masses were sorted by their length and diameter (extra small: <10.0 mm × <5.0 mm, small: 10.0-20.0 mm × 3.5-6.5 mm, medium: 20.0-30.0 mm × 4.0-7.0 mm, large: 30.0-40.0 mm × 4.5-8.5 mm, and extra-large >40.0 mm × >5.0 mm). Treatments were fed as a switchback design, and data were analyzed using the GLM procedure in SAS (v. 9.4) with means separated by significant alpha (5%). Total dry fecal weight was greater for cats fed M than C (P < 0.0001; 112.99 vs. 66.71, respectively). There was a tendency for less total fecal hair mass per gram of dry feces (P = 0.0712) and fewer fecal hairball counts per gram of dry feces (P = 0.1082) for cats fed M. However, hairball size (P = 0.2431) total fecal hairball weight (P = 0.2541), and total fecal hair weight (P = 0.3027) did not differ between treatments. The addition of M lead to some improvement in gastrointestinal management of hair in short-hair cats not otherwise predisposed to hairball. Use of predisposed animals, long-hair cats, a larger number of animals, and (or) larger fiber particle size may increase differences among the treatments. In conclusion, M decreased hairball size and total fecal hair mass per gram of feces, which could be an indication of increased ability of cats to eliminate hair masses through fecal excretion and reduce their regurgitation in a home setting. [ABSTRACT FROM AUTHOR]

Wet pet foods make up 18% of the market. Most studies report thermal inactivation of pathogens, but data reporting the heat penetration in containers other than cans are scarce. Therefore, the objective of this work was to determine heat penetration in various container types and sizes of pet food. The experiment was conducted as a 2 × 3 factorial arrangement of treatments with two general container sizes (small: 89-104 mL vs medium; 163-207 mL) and three container types (can, pouch, and tray). A model cat formula was produced for all six experimental treatments, and each was produced in duplicate over six days in commercial scale equipment. The containers were cooked in a retort (cans: SJ Reid Retort, Bellingham, WA; trays and pouches: FMC retort, Madera, CA) with thermocouples attached to the center of representative cans, pouches, or trays (n = 14) in each batch. Software (Calsoft Systems, v. 5.0.5) was used to record the internal temperatures and lethality during processing. Cumulative lethality values were determined for both the cooking and cooling processes. Data were analyzed using the GLM procedure of SAS (v. 9.4), and main effect means and interactions were separated by significant F test. There was an interaction (P < 0.05) between container size and type for time to reach the F08, wherein the medium can and tray had the longest time (45.5 and 46.3 min, respectively), the small can and tray and medium pouch were intermediate (35.4, 36.0, and 32.0 min, respectively), and the small pouch had the shortest time (36.0 min). There was no difference for either main effect of container type or size on heating lethality values (each main effect average Fo = 10.3). Container size did not affect cooling lethality values (average Fo = 4.6), but pouches and trays had a higher (P < 0.05) cooling lethality than cans (Fo = 4.5, 5.9 vs 3.3, respectively). Total lethality values were not affected by container size (average Fo = 14.9), but there was a trend (P = 0.0569) for pouches and trays to have a higher total lethality than cans. These heating differences may have an impact on the retention of heat labile nutrients like thiamine. [ABSTRACT FROM AUTHOR]

To assist weight management, some pet foods are produced with higher fiber content to increase bulking and dilute the energy content of the diet. Miscanthus grass (M) is a novel fibrous ingredient that might have similar functionality to standard fibers used by the pet food industry like cellulose (C) and beet pulp (B). The objective of this study was to determine the effects of dried ground M, relative to C, and B on extrusion processing of dog and cat foods. Both dog and cat diets were made with 10% of each M, C, or B and 90% basal ration. Each diet was split into 3 batches and mixed separately prior to production in a commercial scale single screw extruder (E525, ExtruTech, Sabetha, KS). During production, preconditioner water (PW) and steam (PS) and extruder water (EW) and steam (ES) injections and SME were recorded every 20 minutes. At each time point, 5 kibbles were randomly sampled for diameter and length out of the extruder and exiting the dryer. From the kibble and die diameters, sectional expansion ratio index (SEI) was calculated (kD2/dD2). Data were analyzed using the GLIMIX procedure of SAS (v. 9.4), and means were separated by significant alpha (5%). For both dog and cat foods, extruder parameters were similar among treatments (cat diets: PW 39.0 kg*h-1, PS 28.6 kg*h-1, EW 0 kg*h-1, ES 18.8 kg*h-1, SME 96.2 W*kg-1; dog diets: PW 37.7 kg*h-1, PS 65.6 kg*h-1, EW 0.03 kg*h-1, ES 0.8 kg*h-1, SME 110.1 W*kg-1; averages of all treatments). Kibble characteristics (length, diameter, SEI, weight, volume, and density) were similar among all the cat diets. However, dog diets with B had larger (P < 0.05) diameter and SEI than M and C. Diets with B tended (P > 0.10) to have slightly greater volume than M and greater density than C. These results indicate that M behaved similar to C during extrusion of cat and dog diets and was consistent with the fiber profile between these two ingredients. The small differences that were observed can be adjusted with modest process modifications. In conclusion, the extrusion parameters were not affected by fiber source, and small differences in kibble characteristics can be corrected with modest process adjustments. [ABSTRACT FROM AUTHOR]

Two experiments were conducted to evaluate the effects of dietary amino acid density and exogenous protease inclusion on growth performance and amino acid digestibility in poultry. In experiment 1, Cobb 500 broiler chicks (n = 480) were fed to 21 d of age. Treatments were arranged as a 2 3 4 factorial design with main effects of commercial protease (with or without) and amino acid density which corresponded to approximately 95, 97.5, 100, or 102.5% of breeder recommendations for digestible Lys. In experiment 2, hybrid turkey poults (n = 780) were fed diets formulated to provide low or adequate amino acid density (approximately 91 and 100% of the NRC-recommended requirement for digestible Lys, respectively) with each diet being fed with or without an exogenous protease. Poults received experimental diets from day 1 to 42 of age. Growth performance metrics were calculated from pen weights and feed consumption recorded throughout each experiment, and digestibility data were obtained from analysis of ileal contents. Data were analyzed using SAS 9.4 with pen as the experimental unit and pen location as the blocking factor. Results of these experiments demonstrated broilers fed 1.12 and 1.21% digestible Lys diets with added protease had a 2-point improvement in feed conversion ratio (FCR) compared with chicks fed these diets without protease. Increasing amino acid density improved FCR in broilers and poults and improved BW, ADG, and ADFI in poults. There was no evidence that added protease had an effect on BW, ADG, or ADFI in broilers or poults. Finally, ileal amino acid digestibility was not affected by amino acid density or protease inclusion for either 20-day-old broiler chicks or 42-day-old turkey poults. [ABSTRACT FROM AUTHOR]

Background: Between 5 and 50% of atypical hemolytic uremic syndrome (aHUS) cases in children are caused by autoantibodies against complement factor H (CFH). Given the acquired autoimmune nature of the disease, plasma exchange (PE) and various immunosuppressive treatments have been used. More recently, eculizumab has been proposed., Methods: In this multicenter, retrospective study, we report outcomes of 12 children with anti-FH antibody-associated HUS treated with eculizumab associated with various immunosuppressive regimens., Results: Patients were treated with eculizumab for 15.5 [9.5;23.0] months and 3 received PE or IgG adsorption. Three patients received mycophenolate mofetil (MMF) alone, 1 patient received MMF and steroids, 1 patient received MMF and rituximab, 3 patients received MMF/steroids and rituximab, and 4 patients did not receive any immunosuppression. Anti-FH antibody levels significantly decreased but no difference was observed based on the immunosuppressive regimen. Eculizumab was discontinued in 7/10 patients after 11 [7.5;15.5] months and MMF in 6/8 patients after 36 [35;40] months. Anti-FH titers at MMF discontinuation ranged from 257 to 3425 UI/L. None of these patients relapsed and eGFR at last follow-up was above 70 mL/min/1.73 m 2 in all patients., Conclusions: Eculizumab is effective and safe in inducing and maintaining remission in aHUS secondary to anti-FH antibodies and renders reduction of anti-FH titers less urgent. Anti-FH antibody titers decreased in most patients irrespective of the immunosuppressive treatment chosen, so that a strategy consisting of combining eculizumab with MMF monotherapy seems sufficient at least in non-Indian or less severe forms of anti-FH antibody-associated HUS., (© 2024. The Author(s), under exclusive licence to International Pediatric Nephrology Association.)

Introduction: Complement factor H (FH) is a major regulator of the complement alternative pathway, its mutations predispose to an uncontrolled activation in the kidney and on blood cells and to secondary C3 deficiency. Plasma exchange has been used to correct for FH deficiency and although the therapeutic potential of purified FH has been suggested by in vivo experiments in animal models, a clinical approved FH concentrate is not yet available. We aimed to develop a purification process of FH from a waste fraction rather than whole plasma allowing a more efficient and ethical use of blood and plasma donations., Methods: Waste fractions from industrial plasma fractionation (pooled human plasma) were analyzed for FH content by ELISA. FH was purified from unused fraction III and its decay acceleration, cofactor, and C3 binding capacity were characterized in vitro. Biodistribution was assessed by high-resolution dynamic PET imaging. Finally, the efficacy of the purified FH preparation was tested in the mouse model of C3 glomerulopathy (Cfh-/- mice)., Results: Our purification method resulted in a high yield of highly purified (92,07%), pathogen-safe FH. FH concentrate is intact and fully functional as demonstrated by in vitro functional assays. The biodistribution revealed lower renal and liver clearance of human FH in Cfh-/- mice than in wt mice. Treatment of Cfh-/- mice documented its efficacy in limiting C3 activation and promoting the clearance of C3 glomerular deposits., Conclusion: We developed an efficient and economical system for purifying intact and functional FH, starting from waste material of industrial plasma fractionation. The FH concentrate could therefore constitute possible treatments options of patients with C3 glomerulopathy, particularly for those with FH deficiency, but also for patients with other diseases associated with alternative pathway activation., Competing Interests: Authors FM, CF, and AL were employed by Kedrion. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mori, Pascali, Berra, Lazzarotti, Panetta, Rocchiccioli, Ceccherini, Norelli, Morlando, Donadelli, Clivio, Farina, Noris, Salvadori and Remuzzi.)

For over six decades, nutritional science has provided well-developed, peer-reviewed nutrient recommendations to support the health of dogs and cats. These guidelines are updated based on new scientifically valid research and appropriate peer-review. Recent regulatory and scientific positions around health issues have resulted in strong opinions and desires for rapid regulatory action surrounding mineral nutrition, but with limited and conflicting scientific evidence. Pet Food Institute nutrition experts have come together to jointly author an article on the complexities of establishing mineral tolerances of dogs and cats to illustrate the limitations in defining mineral tolerances. This discussion covers how mineral requirements were determined, including the opportunities and pitfalls encountered. Scientific councils must review and clarify any proposed changes in conducting mineral nutrition research that might impact complete and balanced foods and surrounding regulations. It is important to clarify the multiple issues in mineral nutrition research and the necessity for thorough evaluation of data while avoiding arbitrary and potentially harmful guidelines., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited disease of the kidney. It occurs in adulthood but is also rarely diagnosed in early childhood. The majority of the disease-causing variants observed in ADPKD patients are in two genes: PKD1 and PKD2., Methods: 237 patients from 198 families with a clinical diagnosis of ADPKD were screened for PKD1 and PKD2 genetic variants using Sanger sequencing and multiple ligation-dependent probe amplification analysis., Results: Disease-causing (diagnostic) variants were identified in 173 families (211 patients), 156 on PKD1 and 17 on PKD2. Variants of unknown significance were detected in 6 additional families, while no mutations were found in the remaining 19 families. Among the diagnostic variants detected, 51 were novel. In ten families, seven large rearrangements were found and the molecular breakpoints of 3 rearrangements were identified. Renal survival was significantly worse for PKD1-mutated patients, particularly those carrying truncating mutations. In patients with PKD1 truncating (PKD1-T) mutations, disease onset was significantly earlier than in patients with PKD1 non-truncating variants or PKD2-mutated patients., Conclusions: Comprehensive genetic testing confirms its utility in diagnosing patients with ADPKD and contributes to explaining the clinical heterogeneity observed in this disease. Moreover, the genotype-phenotype correlation can allow for a more accurate disease prognosis., (© 2023 S. Karger AG, Basel.)

Uncontrolled activation of the alternative pathway (AP) of complement, due to genetic and/or acquired defects, plays a primary pathogenetic role in C3 glomerulopathy (C3G), a rare and heterogeneous disease characterised by predominant C3 fragment deposition within the glomerulus, as well as glomerular damage. There are currently no approved disease-specific treatments for C3G, but new drugs that directly counteract AP dysregulation, targeting components of the pathway, have opened promising new perspectives for managing the disease. Complement factor B (FB), which is primarily synthesised by hepatocytes, is a key component of the AP, as it drives the central amplification loop of the complement system. In this study we used a GalNAc (N-Acetylgalactosamine)-conjugated siRNA to selectively target and suppress liver FB expression in two mouse models characterised by the complete (Cfh -/- mice) or partial (Cfh +/- ) loss of function of complement factor H (FH). Homozygous deletion of FH induced a severe C3G phenotype, with strong dysregulation of the AP of complement, glomerular C3 deposition and almost complete C3 consumption. Mice with a heterozygous deletion of FH had intermediate C3 levels and exhibited slower disease progression, resembling human C3G more closely. Here we showed that FB siRNA treatment did not improve serum C3 levels, nor limit glomerular C3 deposition in Cfh -/- mice, while it did normalise circulating C3 levels, reduce glomerular C3 deposits, and limit mesangial electron-dense deposits in Cfh +/- mice. The present data provide important insights into the potential benefits and limitations of FB-targeted inhibition strategies and suggest RNA interference-mediated FB silencing in the liver as a possible therapeutic approach for treating C3G patients with FH haploinsufficiency., Competing Interests: Declaration of Competing Interest Marina Noris has received honoraria from Alexion Pharmaceuticals for giving lectures, and for participating in advisory boards, and she has received research grants from Omeros, Gemini, Novartis and BioCryst Pharmaceuticals. Ariela Benigni and Giuseppe Remuzzi have consultancy agreements with Alexion Pharmaceuticals and BioCryst Pharmaceuticals., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenia purpura (TTP) are rare diseases sharing a common pathological feature, thrombotic microangiopathy (TMA). TMA is characterized by microvascular thrombosis with consequent thrombocytopenia, microangiopathic hemolytic anemia and/or multiorgan dysfunction. In the past, the distinction between HUS and TTP was predominantly based on clinical grounds. However, clinical presentation of the two syndromes often overlaps and, the differential diagnosis is broad. Identification of underlying pathogenic mechanisms has enabled the classification of these syndromes on a molecular basis: typical HUS caused by Shiga toxin-producing Escherichia coli (STEC-HUS); atypical HUS or complement-mediated TMA (aHUS/CM-TMA) associated with genetic or acquired defects leading to dysregulation of the alternative pathway (AP) of complement; and TTP that results from a severe deficiency of the von Willebrand Factor (VWF)-cleaving protease, ADAMTS13. The etiology of TMA differs between pediatric and adult patients. Childhood TMA is chiefly caused by STEC-HUS, followed by CM-TMA and pneumococcal HUS (Sp-HUS). Rare conditions such as congenital TTP (cTTP), vitamin B12 metabolism defects, and coagulation disorders (diacylglycerol epsilon mutation) present as TMA chiefly in children under 2 years of age. In contrast secondary causes and acquired ADAMT13 deficiency are more common in adults. In adults, compared to children, diagnostic delays are more frequent due to the wide range of differential diagnoses. In this review we focus on the three major forms of TMA, STEC-HUS, aHUS and TTP, outlining the clinical presentation, diagnosis and management of the affected patients, to help highlight the salient features and the differences between adult and pediatric patients which are relevant for management., Competing Interests: Declaration of Competing Interest RD has received honoraria from Alexion Pharmaceuticals for contributing to a publishing project. MN has received honoraria from Alexion Pharmaceuticals for giving lectures and for participating in advisory boards, and she has received research grants from Omeros, Gemini, Novartis and BioCryst Pharmaceuticals. GR has consultancy agreements with AbbVie, Alexion Pharmaceuticals, Novartis Pharma and BioCryst Pharmaceuticals., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: Atypical hemolytic uremic syndrome (aHUS) is a rare disease that manifests with microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure, and is associated with dysregulation of the alternative complement pathway. The chromosomal region including CFH and CFHR1-5 is rich in repeated sequences, favoring genomic rearrangements that have been reported in several patients with aHUS. However, there are limited data on the prevalence of uncommon CFH-CFHR genomic rearrangements in aHUS and their impact on disease onset and outcomes., Methods: In this study, we report the results of CFH-CFHR Copy Number Variation (CNV) analysis and the characterization of resulting structural variants (SVs) in a large cohort of patients, including 258 patients with primary aHUS and 92 with secondary forms., Results: We found uncommon SVs in 8% of patients with primary aHUS: 70% carried rearrangements involving CFH alone or CFH and CFHR (group A; n=14), while 30% exhibited rearrangements including only CFHRs (group B; n=6). In group A, 6 patients presented CFH::CFHR1 hybrid genes, 7 patients carried duplications in the CFH-CFHR region that resulted either in the substitution of the last CFHR1 exon(s) with those of CFH ( CFHR1::CFH reverse hybrid gene) or in an internal CFH duplication. In group A, the large majority of aHUS acute episodes not treated with eculizumab (12/13) resulted in chronic ESRD; in contrast, anti-complement therapy induced remission in 4/4 acute episodes. aHUS relapse occurred in 6/7 grafts without eculizumab prophylaxis and in 0/3 grafts with eculizumab prophylaxis. In group B, 5 subjects had the CFHR3 1-5 ::CFHR4 10 hybrid gene and one had 4 copies of CFHR1 and CFHR4 . Compared with group A, patients in group B exhibited a higher prevalence of additional complement abnormalities and earlier disease onset. However, 4/6 patients in this group underwent complete remission without eculizumab treatment. In secondary forms we identified uncommon SVs in 2 out of 92 patients: the CFHR3 1-5 ::CFHR4 10 hybrid and a new internal duplication of CFH ., Discussion: In conclusion, these data highlight that uncommon CFH-CFHR SVs are frequent in primary aHUS and quite rare in secondary forms. Notably, genomic rearrangements involving the CFH are associated with a poor prognosis but carriers respond to anti-complement therapy., Competing Interests: MN has received honoraria from Alexion Pharmaceuticals for giving lectures, and for participating in advisory boards, and she has received research grants from Omeros, Gemini, Novartis and BioCryst Pharmaceuticals. AB has received honoraria from Alexion Phamaceuticals and BioCryst Pharmaceuticals. GR has consultancy agreements with AbbVie, Alexion Pharmaceuticals, Novartis Pharma and BioCryst Pharmaceuticals. Since 1st May 2022, the co-author EV has been employed by Frontiers Media SA. EV declared her affiliation with Frontiers, and the handling Editor states that the process nevertheless met the standards of a fair and objective review. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2023 Piras, Valoti, Alberti, Bresin, Mele, Breno, Liguori, Donadelli, Rigoldi, Benigni, Remuzzi and Noris.)

The skin is the first barrier the body has to protect itself from the environment. There are several bacteria that populate the skin, and their composition may change throughout the dog's life due to several factors, such as environmental changes and diseases. The objective of this research was to determine the skin microbiome changes due to a change in diet on healthy pet dogs. Healthy client-owned dogs (8) were fed a fresh diet for 30 days then dry foods for another 30 days after a 4-day transition period. Skin bacterial population samples were collected after each 30-day feeding period and compared to determine microbiome diversity. Alpha diversity was higher when dogs were fed the fresh diet compared to the dry foods. Additionally, feeding fresh food to dogs increased the proportion of Staphylococcus and decreased Porphyromonas and Corynebacterium . In conclusion, changing from fresh diet to dry foods promoted a relative decrease in skin microbiome in healthy pet dogs.

Alternative pathway complement dysregulation with abnormal glomerular C3 deposits and glomerular damage is a key mechanism of pathology in C3 glomerulopathy (C3G). No disease-specific treatments are currently available for C3G. Therapeutics inhibiting complement are emerging as a potential strategy for the treatment of C3G. In this study, we investigated the effects of N -acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C3 component of complement that inhibits liver C3 expression in the C3G model of mice with heterozygous deficiency of factor H ( Cfh +/- mice). We showed a duration of action for GalNAc-conjugated C3 siRNA in reducing the liver C3 gene expression in Cfh +/- mice that were dosed s.c. once a month for up to 7 mo. C3 siRNA limited fluid-phase alternative pathway activation, reducing circulating C3 fragmentation and activation of factor B. Treatment with GalNAc-conjugated C3 siRNA reduced glomerular C3d deposits in Cfh +/- mice to levels similar to those of wild-type mice. Ultrastructural analysis further revealed the efficacy of the C3 siRNA in slowing the formation of mesangial and subendothelial electron-dense deposits. The present data indicate that RNA interference-mediated C3 silencing in the liver may be a relevant therapeutic strategy for treating patients with C3G associated with the haploinsufficiency of complement factor H., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Microvascular thrombosis is associated with multiorgan failure and mortality in coronavirus disease 2019 (COVID-19). Although thrombotic complications may be ascribed to the ability of SARS-CoV-2 to infect and replicate in endothelial cells, it has been poorly investigated whether, in the complexity of viral infection in the human host, specific viral elements alone can induce endothelial damage. Detection of circulating spike protein in the sera of severe COVID-19 patients was evaluated by ELISA. In vitro experiments were performed on human microvascular endothelial cells from the derma and lung exposed to SARS-CoV-2-derived spike protein 1 (S1). The expression of adhesive molecules was studied by immunofluorescence and leukocyte adhesion and platelet aggregation were assessed under flow conditions. Angiotensin converting enzyme 2 (ACE2) and AMPK expression were investigated by Western Blot analysis. In addition, S1-treated endothelial cells were incubated with anti-ACE2 blocking antibody, AMPK agonist, or complement inhibitors. Our results show that significant levels of spike protein were found in the 30.4% of severe COVID-19 patients. In vitro , the activation of endothelial cells with S1 protein, via ACE2, impaired AMPK signalling, leading to robust leukocyte recruitment due to increased adhesive molecule expression and thrombomodulin loss. This S1-induced pro-inflammatory phenotype led to exuberant C3 and C5b-9 deposition on endothelial cells, along with C3a and C5a generation that further amplified S1-induced complement activation. Functional blockade of ACE2 or complement inhibition halted S1-induced platelet aggregates by limiting von Willebrand factor and P-selectin exocytosis and expression on endothelial cells. Overall, we demonstrate that SARS-CoV-2-derived S1 is sufficient in itself to propagate inflammatory and thrombogenic processes in the microvasculature, amplified by the complement system, recapitulating the thromboembolic complications of COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Perico, Morigi, Galbusera, Pezzotta, Gastoldi, Imberti, Perna, Ruggenenti, Donadelli, Benigni and Remuzzi.)

Hemolytic uremic syndrome (HUS) is a rare disease characterized by hemolytic anemia, thrombocytopenia, and renal impairment mostly triggered by strains of Shiga-like toxin-producing Escherichia coli (STEC-HUS). A rarer form of HUS, defined as atypical HUS (aHUS), is associated with genetic or acquired dysregulation of the alternative pathway of the complement system and presents a poorer prognosis than STEC-HUS. Factor H autoantibodies (anti-FHs) have been reported in aHUS in 5-11% of cases and are strongly associated with the homozygous deletion of CFHR3-CFHR1 genes. In the large majority of patients, anti-FH-associated aHUS is commonly preceded by gastrointestinal or respiratory tract infections. Here, we described the clinical case of a 3-year-old boy who was hospitalized for aHUS preceded by Mycoplasma pneumoniae (MP) infection. He resulted positive for anti-FHs and carried the homozygous deletion of CFHR3-CFHR1. Of relevance, he also showed a variant of unknown significance in the C5 gene. The patient was successfully treated with eculizumab and achieved hematological and renal remission. The anti-FH titer decreased, became negative after 6 months of mycophenolate mofetil (MMF) treatment, and remained negative for 21-month follow-up indicating that immunosuppression was effective and could prevent the reappearance of anti-FHs. We hypothesized that MP, likely through an evasion strategy of immunosurveillance based on binding of pathogen to FH, triggers anti-FH antibody generation and aHUS in a subject genetically predisposed. In conclusion, to the best of our knowledge, here, we reported the first case of anti-FH-mediated aHUS after an MP infection who benefited from eculizumab and immunosuppressive therapy based on MMF. Hence, monitoring of anti-FHs in patients with post-MP infection glomerulonephritis could be recommended, especially in those with low C3 plasma levels., (© 2022 S. Karger AG, Basel.)

Background: Complement activation contributes to lung dysfunction in coronavirus disease 2019 (COVID-19). We assessed whether C5 blockade with eculizumab could improve disease outcome., Methods: In this single-centre, academic, unblinded study two 900 mg eculizumab doses were added-on standard therapy in ten COVID-19 patients admitted from February 2020 to April 2020 and receiving Continuous-Positive-Airway-Pressure (CPAP) ventilator support from ≤24 hours. We compared their outcomes with those of 65 contemporary similar controls. Primary outcome was respiratory rate at one week of ventilator support. Secondary outcomes included the combined endpoint of mortality and discharge with chronic complications., Results: Baseline characteristics of eculizumab-treated patients and controls were similar. At baseline, sC5b-9 levels, ex vivo C5b-9 and thrombi deposition were increased. Ex vivo tests normalised in eculizumab-treated patients, but not in controls. In eculizumab-treated patients respiratory rate decreased from 26.8±7.3 breaths/min at baseline to 20.3±3.8 and 18.0±4.8 breaths/min at one and two weeks, respectively (p<0.05 for both), but did not change in controls. Between-group changes differed significantly at both time-points (p<0.01). Changes in respiratory rate correlated with concomitant changes in ex vivo C5b-9 deposits at one (rs = 0.706, p = 0.010) and two (rs = 0.751, p = 0.032) weeks. Over a median (IQR) period of 47.0 (14.0-121.0) days, four eculizumab-treated patients died or had chronic complications versus 52 controls [HRCrude (95% CI): 0.26 (0.09-0.72), p = 0.010]. Between-group difference was significant even after adjustment for age, sex and baseline serum creatinine [HRAdjusted (95% CI): 0.30 (0.10-0.84), p = 0.023]. Six patients and 13 controls were discharged without complications [HRCrude (95% CI): 2.88 (1.08-7.70), p = 0.035]. Eculizumab was tolerated well. The main study limitations were the relatively small sample size and the non-randomised design., Conclusions: In patients with severe COVID-19, eculizumab safely improved respiratory dysfunction and decreased the combined endpoint of mortality and discharge with chronic complications. Findings need confirmation in randomised controlled trials., Competing Interests: M.G. reported grants from Omeros Corporation, Alexion Pharmaceuticals, F. Hoffman-La Roche Ltd and Novartis Pharma AG (payments were made to her institution), all outside the submitted work; M.N. reported grants from Omeros Corporation, Novartis Pharma AG, F. Hoffman-La Roche Ltd and BioCryst Pharmaceuticals (payments were made to her institution) as well as personal fees from Inception Sciences, BioCryst Pharmaceuticals and Alexion Pharmaceuticals, all outside the submitted work; A.B. reported personal fees from Akebia Pharmaceuticals, Alexion Pharmaceuticals, BioCryst Pharmaceuticals, Janssen Research & Development LLC, as well as speaker honorarium/travel reimbursements from Alnylam, Boehringer Ingelheim and Inception Sciences Canada, all outside the submitted work; G.R. reported personal fees from Akebia Pharmaceuticals, Alexion Pharmaceuticals, BioCryst Pharmaceuticals and Janssen Research & Development LLC, as well as speaker honorarium/travel reimbursements from Alnylam, Boehringer Ingelheim and Inception Sciences Canada, all outside the submitted work. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All the other authors have nothing to disclose.