Case report: A family of atypical hemolytic uremic syndrome involving a CFH::CFHR1 fusion gene and CFHR3-1-4-2 gene duplication.

Mutations in the complement factor H (CFH) gene are associated with complement dysregulation and the development of atypical hemolytic uremic syndrome (aHUS). Several fusion genes that result from genomic structural variation in the CFH and complement factor H-related (CFHR) gene regions have been identified in aHUS. However, one allele has both CFHR gene duplication and CFH::CFHR1 fusion gene have not been reported. An 8-month-old girl (proband) presented with aHUS and was treated with ravulizumab. Her paternal grandfather developed aHUS previously and her paternal great grandmother presented with anti-neutrophil cytoplasmic antibody-associated vasculitis and thrombotic microangiopathy (TMA). However, the proband's parents have no history of TMA. A genetic analysis revealed the presence of CFH::CFHR1 fusion gene and a CFHR3-1-4-2 gene duplication in the patient, her father, and her paternal grandfather. Although several fusion genes resulting from structural variations of the CFH-CFHR genes region have been identified, this is the first report of the combination of a CFH::CFHR1 fusion gene with CFHR gene duplication. Because the CFH-CFHR region is highly homologous, we hypothesized that CFHR gene duplication occurred. These findings indicate a novel pathogenic genomic structural variation associated with the development of aHUS.

Increased infiltration of CD4+ T cell in the complement deficient lymphedema model.

BACKGROUND: Lymphedema is an intractable disease that can be caused by injury to lymphatic vessels, such as by surgical treatments for cancer. It can lead to impaired joint mobility in the extremities and reduced quality of life. Chronic inflammation due to infiltration of various immune cells in an area of lymphedema is thought to lead to local fibrosis, but the molecular pathogenesis of lymphedema remains unclear. Development of effective therapies requires elucidation of the immunological mechanisms involved in the progression of lymphedema. The complement system is part of the innate immune system which has a central role in the elimination of invading microbes and acts as a scavenger of altered host cells, such as apoptotic and necrotic cells and cellular debris. Complement-targeted therapies have recently been clinically applied to various diseases caused by complement overactivation. In this context, we aimed to determine whether complement activation is involved in the development of lymphedema. RESULTS: Our mouse tail lymphedema models showed increased expression of C3, and that the classical or lectin pathway was locally activated. Complement activation was suggested to be involved in the progression of lymphedema. In comparison of the C3 knockout (KO) mouse lymphedema model and wild-type mice, there was no difference in the degree of edema at three weeks postoperatively, but the C3 KO mice had a significant increase of TUNEL+ necrotic cells and CD4+ T cells. Infiltration of macrophages and granulocytes was not significantly elevated in C3 KO or C5 KO mice compared with in wild-type mice. Impaired opsonization and decreased migration of macrophages and granulocytes due to C3 deficiency should therefore induce the accumulation of dead cells and may lead to increased infiltration of CD4+ T cells. CONCLUSIONS: Vigilance for exacerbation of lymphedema is necessary when surgical treatments have the potential to injure lymphatic vessels in patients undergoing complement-targeted therapies or with complement deficiency. Future studies should aim to elucidate the molecular mechanism of CD4+ T cell infiltration by accumulated dead cells.

Overlapping Atypical Hemolytic Uremic Syndrome and C3 Glomerulopathy with Mutation in CFI in a Japanese Patient: A Case Report.

A 34-year-old Japanese man presented with blurred vision, headache, nausea, anemia, thrombocytopenia, and severe renal dysfunction. Thrombotic microangiopathy was initially suspected to have been caused by malignant hypertension. Antihypertensive medications did not improve his thrombocytopenia or renal dysfunction, and other diseases causing thrombotic microangiopathy were ruled out. Therefore, the patient was diagnosed with atypical hemolytic uremic syndrome. A renal biopsy revealed an overlap of thrombotic microangiopathy and C3 glomerulopathy. Genetic testing revealed c.848A>G (p.Asp283Gly), a missense heterozygous variant in the gene encoding complement factor I. Overlapping atypical hemolytic uremic syndrome and C3 glomerulopathy with complement factor I mutation is very rare, especially in Japan.

Immunological risk and complement genetic evaluations in early onset de novo thrombotic microangiopathy after living donor kidney transplantation: A Japanese multicenter registry.

BACKGROUND: Thrombotic microangiopathy (TMA) after kidney transplantation (KTx), particularly early onset de novo (dn) TMA, requires immediate interventions to prevent irreversible organ damage. This multicenter study was performed to investigate the allogeneic clinical factors and complement genetic background of dnTMA after KTx. METHODS: Perioperative dnTMA after KTx within 1 week after KTx were diagnosed based on pathological or/and hematological criteria at each center, and their immunological backgrounds were researched. Twelve aHUS-related gene variants were examined in dnTMA cases. RESULTS: Seventeen recipients (15 donors) were enrolled, and all dnTMA cases were onset within 72-h of KTx, and 16 of 17 cases were ABO incompatible. The implementation rate of pre-transplant plasmaphereses therapies were low, including cases with high titers of anti-A/anti-B antibodies. Examination of aHUS-related gene variants revealed some deletions and variants with minor allele frequency (MAF) in Japan or East Asian genome databases in genes encoding alternative pathways and complement regulatory factors. These variants was positive in 8 cases, 6 of which were positive in both recipient and donor, but only in one graft loss case. CONCLUSIONS: Although some immunological risks were found for dnTMA after KTx, only a few cases developed into TMA. The characteristic variations revealed in the present study may be novel candidates related to dnTMA in Japanese or Asian patients, but not pathogenic variants of aHUS. Future studies on genetic and antigenic factors are needed to identify factors contributing to dnTMA after KTx.

Complement biomarkers reflect the pathological status of neuromyelitis optica spectrum disorders.

Complement is involved in the pathogenesis of neuroimmune disease, but the detailed pathological roles of the complement pathway remain incompletely understood. Recently, eculizumab, a humanized anti-C5 monoclonal antibody, has been clinically applied against neuroimmune diseases such as myasthenia gravis and neuromyelitis optica spectrum disorders (NMOSD). Clinical application of eculizumab is also being investigated for another neuroimmune disease, Guillain-Barré syndrome (GBS). However, while the effectiveness of eculizumab for NMOSD is extremely high in many cases, there are some cases of myasthenia gravis and GBS in which eculizumab has little or no efficacy. Development of effective biomarkers that reflect complement activation in these diseases is therefore important. To identify biomarkers that could predict disease status, we retrospectively analyzed serum levels of complement factors in 21 patients with NMOSD and 25 patients with GBS. Ba, an activation marker of the alternative complement pathway, was elevated in the acute phases of both NMOSD and GBS. Meanwhile, sC5b-9, an activation marker generated by the terminal complement pathway, was elevated in NMOSD but not in GBS. Complement factor H (CFH), a complement regulatory factor, was decreased in the acute phase as well as in the remission phase of NMOSD, but not in any phases of GBS. Together, these findings suggest that complement biomarkers, such as Ba, sC5b-9 and CFH in peripheral blood, have potential utility in understanding the pathological status of NMOSD.

Rac is required for the survival of cortical neurons.

Rac1, a member of small Rho GTPases, is involved in diverse cellular processes in neuronal cells. Rac1 plays especially important roles during development, and its roles have been extensively studied using Rac1-deficient mice. Rac3, a close homolog of Rac1, is ubiquitously expressed in the nervous system and may therefore compensate for Rac1 in Rac1-deficient cells. Exploration of the roles of Rac in neurons may therefore be difficult. We thus deleted both Rac1 and Rac3 in cortical neurons. Rac-deficient cerebral cortices formed slightly hypoplastic but almost normally layered structures at birth, but cortical neurons underwent apoptosis soon after birth. Rac-deficient cortical neurons had poor survivability and there was reduction in the length and the number of neurites in vitro. Activation of Pak1, a downstream effector of Rac, in Rac-deficient cortical neurons rescued the survivability in vitro. Pak1-activated Rac-deficient neurons had numerous dendrites, but no axons. Restoration of p35, a regulator of Cdk5, partly rescued the survivability of Rac-deficient neurons both in vitro and in vivo. Expression of p35 also partly rescued the length and the number of neurites in Rac-deficient neurons in vitro. Rac was shown to be indispensable for the survival of cortical neurons, and Pak1 and Cdk5/p35 work as downstream effectors of Rac to promote neuronal survival.

Metabolic profiling analysis of head and neck squamous cell carcinoma.

OBJECTIVE: Tumor cells can acquire a large amount of energy and structural components by reprogramming energy metabolism; moreover, metabolic profiles slightly differ according to cancer type. This study compared and assessed the metabolic profile of head and neck squamous cell carcinoma (HNSCC) and normal tissues, which were collected from patients without cancer. SUBJECTS AND METHODS: Overall, 23 patients with HNSCC and 6 patients without cancer were included in the analysis. Metabolomic profiles were analyzed using capillary electrophoresis-mass spectrometry. Gene expression was evaluated using real-time reverse transcription-polymerase chain reaction. RESULTS: Glycolysis, the pentose phosphate pathway, tricarboxylic acid cycle, and glutamine metabolism were upregulated in HNSCC tissues based on gene expression analysis. HNSCC could then have enhanced energy production and structural component. The levels of lactate, succinate, glutathione, 2-hydroxyglutarate, and S-adenosylmethionine, considered as oncometabolites, increased and these had accumulated in HNSCC tissues. CONCLUSIONS: The level of metabolites and the expression of enzymes differ between HNSCC and normal tissues. Reprogramming metabolism in HNSCC provides an energy source as well as structural components, creating a system that offers rapid proliferation, progression, and is less likely to be eliminated.

Sequential hydrolysis of FAD by ecto-5' nucleotidase CD73 and alkaline phosphatase is required for uptake of vitamin B2 into cells.

Extracellular hydrolysis of flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to riboflavin is thought to be important for cellular uptake of vitamin B2 because FAD and FMN are hydrophilic and do not pass the plasma membrane. However, it is not clear whether FAD and FMN are hydrolyzed by cell surface enzymes for vitamin B2 uptake. Here, we show that in human cells, FAD, a major form of vitamin B2 in plasma, is hydrolyzed by CD73 (also called ecto-5' nucleotidase) to FMN. Then, FMN is hydrolyzed by alkaline phosphatase to riboflavin, which is efficiently imported into cells. We determined that this two-step hydrolysis process is impaired on the surface of glycosylphosphatidylinositol (GPI)-deficient cells due to the lack of these GPI-anchored enzymes. During culture of GPI-deficient cells with FAD or FMN, we found that hydrolysis of these forms of vitamin B2 was impaired, and intracellular levels of vitamin B2 were significantly decreased compared with those in GPI-restored cells, leading to decreased formation of vitamin B2-dependent pyridoxal 5'-phosphate and mitochondrial dysfunction. Collectively, these results suggest that inefficient uptake of vitamin B2 might account for mitochondrial dysfunction seen in some cases of inherited GPI deficiency.

A visualization system for erectile vascular dynamics.

Erection is an essential process which requires the male penis for copulation. This copulatory process depends on the vascular dynamic regulation of the penis. The corpus cavernosum (CC) in the upper (dorsal) part of the penis plays a major role in regulating blood flow inside the penis. When the CC is filled with blood, the sinusoids, including micro-vessels, dilate during erection. The CC is an androgen-dependent organ, and various genital abnormalities including erectile dysfunction (ED) are widely known. Previous studies have shown that androgen deprivation by castration results in significantly decreased smooth muscles of the CC. Experimental works in erectile biology have previously measured intracavernosal penile pressure and mechanical tension. Such reports analyze limited features without assessing the dynamic aspects of the erectile process. In the current study, we established a novel explant system enabling direct visual imaging of the sinusoidal lumen to evaluate the dynamic movement of the cavernous space. To analyze the alternation of sinusoidal spaces, micro-dissected CC explants by patent blue dye injection were incubated and examined for their structural alternations during relaxation/contraction. The dynamic process of relaxation/contraction was analyzed with various external factors administered to the CC. The system enabled the imaging of relaxation/contraction of the lumens of the sinusoids and the collagen-containing tissues. Histological analysis on the explant system also showed the relaxation/contraction. Thus, the system mimics the regulatory process of dynamic relaxation/contraction in the erectile response. The current system also enabled evaluating the erectile pathophysiology. In the current study, the lumen of sinusoids relaxed/contracted in castrated mice similarly with normal mice. These results suggested that the dynamic erectile relaxation/contraction process was similarly retained in castrated mice. However, the system also revealed decreased duration time of erection in castrated mice. The current study is expected to promote further understanding of the pathophysiology of ED, which will be useful for new treatments in the future. Hence, the current system provides unique information to investigate the novel regulations of erectile function, which can provide tools for analyzing the pathology of ED.

Rac-deficient cerebellar granule neurons die before they migrate to the internal granule layer.

Granule neurons are the most common cell type in the cerebellum. They are generated in the external granule layer and migrate inwardly, forming the internal granule layer. Small Rho GTPases play various roles during development of the nervous system and may be involved in generation, differentiation and migration of granule neurons. We deleted Rac1, a member of small Rho GTPases, by GFAP-Cre driver in cerebellar granule neurons and Bergmann glial cells. Rac1flox/flox; Cre mice showed impaired migration and slight reduction in the number of granule neurons in the internal granule layer. Deletion of both Rac1 and Rac3 resulted in almost complete absence of granule neurons. Rac-deficient granule neurons differentiated into p27 and NeuN-expressing post mitotic neurons, but died before migration to the internal granule layer. Loss of Rac3 has little effect on granule neuron development. Rac1flox/flox; Rac3+/-; Cre mice showed intermediate phenotype between Rac1flox/flox; Cre and Rac1flox/flox; Rac3-/-; Cre mice in both survival and migration of granule neurons. Rac3 itself seems to be unimportant in the development of the cerebellum, but has some roles in Rac1-deleted granule neurons. Conversely, overall morphology of Rac1+/flox; Rac3-/-; Cre cerebella was normal. One allele of Rac1 is therefore thought to be sufficient to promote development of cerebellar granule neurons.

Moyamoya Disease Associated with a Deficiency of Complement Component 6.

OBJECTIVES: Complement component 6 (C6) deficiency is a very rare genetic defect that leads to significantly diminished synthesis, secretion, or function of C6. In the current report, we demonstrate a previously undescribed, homozygous missense mutation in exon 17 of the C6 gene (c.2545A>G p.Arg849Gly) in a 35-year-old Japanese woman with moyamoya disease and extremely low levels of CH50 (<7.0 U/mL). MATERIALS AND METHODS: The complement gene analysis using hybridization capture-based next generation sequencing was performed. CH50 was determined in patient's plasma mixed with plasma from a healthy donor or purified human C6 protein. Western blot was performed on patient's plasma using polyclonal antibodies against C6, with healthy donor's plasma and purified human C6 protein as positive controls while C6-depleted human serum as a negative control. The carriage of ring finger protein 213 variant (c.14576G>A p.Arg4859Lys), a susceptibility gene for moyamoya disease, was examined by direct sequencing. RESULTS: CH50 mixing test clearly showed a deficiency pattern, being rescued by addition of only 1% healthy donor's plasma or 1 µg/mL purified human C6 protein (1/50-1/100 of physiological concentration). Western blot revealed the absence of C6 protein in the patient's plasma, confirming a quantitative deficiency of C6. The ring finger protein 213 variant was not detected. CONCLUSIONS: Our data implies that unrecognized complement deficiencies would be harbored in cerebrovascular diseases with unknown etiologies.

Potent adjuvant effect elicited for tumor immunotherapy by a liposome conjugated pH-sensitive polymer and dendritic cell-targeting Toll-like-receptor ligand.

The generation of DCs with augmented functions is a strategy for obtaining satisfactory clinical outcomes in tumor immunotherapy. We developed a novel synthetic adjuvant comprising a liposome conjugated with a DC-targeting Toll-like-receptor ligand and a pH-sensitive polymer for augmenting cross-presentation. In an in vitro study using mouse DCs, these liposomes were selectively incorporated into DCs, significantly enhanced DC function and activated immune responses to present an epitope of the incorporated antigen on the major histocompatibility complex class I molecules. Immunization of mice with liposomes encapsulating a tumor antigen significantly enhanced antigen-specific cytotoxicity. In tumor-bearing mice, vaccination with liposomes encapsulating a tumor antigen elicited complete tumor remission. Furthermore, vaccination significantly enhanced cytotoxicity, targeting not only the vaccinated antigen but also the other antigens of the tumor cell. These results indicate that liposomes are an ideal adjuvant to develop DCs with considerably high potential to elicit antigen-specific immune responses; they are a promising tool for cancer therapy with neoantigen vaccination.

Early Elevation of Complement Factor Ba Is a Predictive Biomarker for Transplant-Associated Thrombotic Microangiopathy.

Transplant-associated thrombotic microangiopathy (TA-TMA) is a fatal complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Previous reports suggest that TA-TMA is caused by complement activation by complement-related genetic variants; however, this needs to be verified, especially in adults. Here, we performed a nested case-control study of allo-HSCT-treated adults at a single center. Fifteen TA-TMA patients and 15 non-TA-TMA patients, matched according to the propensity score, were enrolled. Based on a previous report showing an association between complement-related genes and development of TA-TMA, we first sequenced these 17 genes. Both cohorts harbored several genetic variants with rare allele frequencies; however, there was no difference in the percentage of patients in the TA-TMA and non-TA-TMA groups with the rare variants, or in the average number of rare variants per patient. Second, we measured plasma concentrations of complement proteins. Notably, levels of Ba protein on Day 7 following allo-HSCT were abnormally and significantly higher in TA-TMA than in non-TA-TMA cases, suggesting that complement activation via the alternative pathway contributes to TA-TMA. All other parameters, including soluble C5b-9, on Day 7 were similar between the groups. The levels of C3, C4, CH50, and complement factors H and I in the TA-TMA group after Day 28 were significantly lower than those in the non-TA-TMA group. Complement-related genetic variants did not predict TA-TMA development. By contrast, abnormally high levels of Ba on Day 7 did predict development of TA-TMA and non-relapse mortality. Thus, Ba levels on Day 7 after allo-HSCT are a sensitive and prognostic biomarker of TA-TMA.

Anti-complement factor H (CFH) antibodies and a novel CFH gene mutation in an atypical hemolytic uremic syndrome patient with complement activation of the classical pathway.

Atypical hemolytic uremic syndrome (aHUS) is a rare disease caused by overactivation of the complement alternative pathway. aHUS involves the presence of antibodies against complement factor H and its mutations in the complement genes. A 2-month-old boy presented with discoid rash, hemolytic anemia, thrombocytopenia, multiple antibodies, and hypocomplementemia with a very low level of C4 (< 3 mg/dL), indicating activation of the complement pathway, together fulfilling the systemic lupus erythematosus (SLE) criteria of the American College of Rheumatology at 5 months of age. However, most of these findings normalized spontaneously without any intervention. Further investigations revealed a high level of anti-complement factor H antibodies and a novel heterozygous missense mutation (p.Glu1172Ala, located in exon 22) in a complement gene, CFH. At 2 years of age, his SLE-like symptoms have not recurred, but hematuria and schistocytes were persistent. Eventually, aHUS was diagnosed rather than SLE. Our findings suggest that multiple antibody complex, including anti-complement factor H antibody, may temporarily activate the classical pathway, resulting in SLE-like findings.

Disseminated gonococcal infection in a Japanese man with complement 7 deficiency with compound heterozygous variants: A case report.

RATIONALE: Complement deficiency are known to be predisposed to disseminated gonococcal infection (DGI). We herein present a case of DGI involving a Japanese man who latently had a complement 7 deficiency with compound heterozygous variants. PATIENT CONCERNS: A previously healthy 51-year-old Japanese man complained of sudden-onset high fever. Physical examination revealed various skin lesions including red papules on his trunk and extremities, an impetigo-like pustule on left forearm, and tendinitis of his right forefinger. DIAGNOSIS: Blood culture testing detected gram-negative cocci, which was confirmed to be Neisseria gonorrhoeae based on mass spectrometry and a pathogen-specific PCR test. INTERVENTIONS: Screening tests for underlying immunocompromised factors uncovered that complement activities (CH50) was undetectable. With a suspicion of a congenital complement deficiency, genetic analysis revealed rare single nucleotide variants in complement 7 (C7), including c.281-1G>T and a novel variant c.1454C>T (p.A485V). CH50 was normally recovered by adding purified human C7 to the patient's serum, supporting that the patient has C7 deficiency with compound heterozygous variants. OUTCOMES: Under a diagnosis of DGI, the patient underwent an antibiotic treatment with cefotaxime for a week and was discharged without any sequela. LESSONS: DGI is a rare sexually-transmitted infection that potentially induces systemic complications. Complement immunity usually defeats N. gonorrhoeae and prevents the organism from causing DGI. This case highlighted the importance of suspecting a complement deficiency when a person develops DGI.

Paroxysmal nocturnal hemoglobinuria caused by CN-LOH of constitutional PIGB mutation and 70-kbp microdeletion on 15q.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell (HSC) disorder characterized by defective synthesis of the glycosylphosphatidylinositol (GPI) anchors as a result of somatic mutations in the X-linked PIGA gene. The disease is acquired. No constitutional PNH has been described. Here, we report familial PNH associated with unusual inflammatory symptoms. Genetic analysis revealed a germline heterozygous PIGB mutation on chromosome 15 without mutations in PIGA or any of the other genes involved in GPI biosynthesis. In vitro data confirmed that transfection of the mutant PIGB could not restore the surface expression of GPI-anchored proteins (APs) in PIGB-deficient Chinese hamster ovary cells. Homozygosity was caused by copy number-neutral loss of heterozygosity (CN-LOH) of the germline PIGB mutation, leading to deficient expression of GPI-APs in the affected blood cells of the index patient and her mother. The somatic event leading to homozygosity of the germline mutant PIGB gene involved a 70-kbp microdeletion of chromosome 15q containing the TM2D3 and TARSL2 genes, which was implicated in chromosome 15q mosaicism. Interestingly, we detected the deletion in both the patient and her mother. A sister of the mother, who carried the same germline PIGB mutation but without this microdeletion involving TM2D3 and TARSL2, did not have a PNH clone or CN-LOH. In conclusion, we describe PNH caused by CN-LOH of a germline heterozygous PIGB mutation in a patient and her mother and hypothesize that the 70-kbp microdeletion may have contributed to the PNH clone in both.

[The Warburg Effect and M2 Macrophage Polarization in Head and Neck Cancer].

Reprogramming of glucose metabolism in tumor cells is referred to as the Warburg effect. The Warburg effect is caused by tumor cells not only to adapt their metabolism to the demand for and limited supply of oxygen but also to obtain large amounts of nucleotides, amino acids and lipids for excessive proliferation of tumor cells. The Warburg effect results in increased production of lactic acid, as the final product of glycolysis, in the tumor microenvironment. Lactic acid secreted by tumor cells functions as an immunosuppressive mediator and converts macrophages into M2 macrophages. M2 macrophages reduce inflammatory responses and adaptive Th1 immunity, and promote angiogenesis and tissue remodeling. Tumor-associated macrophages(TAMs)polarize into the M2 phenotype and suppress the host anti-cancer immune response, leading to tumor progression. We have demonstrated that tumor-secreted lactic acid is linked to the induction of M2-macrophage polarization in human head and neck squamous cell carcinoma(HNSCC). FDG, which is a glucose analog, uptake measured by positron emission tomography/computed tomography(PET/CT)indicates the Warburg effect in tumor tissue. M2-macrophage polarization is promoted in HNSCC with increased glucose uptake, maximum standardized uptake value(SUVmax), mean SUV(SUVmean). Tumor cells mediate an immunosuppressive microenvironment via inducing M2-macrophage polarization by reprogramming of glucose metabolism, called Warburg effect.