[Coagulation and complement crosstalk: molecular mechanisms of complement-mediated diseases].

The complement and coagulation systems are ancestrally related mechanisms of serine protease-induced protein activation. Recent studies have shown that the complement system enhances platelet aggregation by activating platelets and vascular endothelial cells. This system is also involved in the expression of tissue factor, which induces the coagulation reaction. Activated platelets and coagulation factors are also known to activate the complement system. In diseases involving the complement system, such as paroxysmal nocturnal hemoglobinuria, autoimmune hemolytic anemia, and atypical hemolytic uremic syndrome, excessive activation of this system contributes to complement-mediated thrombosis. The anti-C5 antibody eculizumab has shown a remarkable thromboprophylactic effect in these complement diseases. The recent surge in development of new anti-complement agents has raised expectations for the advancement of treatments and preventive measures for thrombosis associated with complement disorders. This review outlines the crosstalk between these two systems, and describes the mechanisms of several diseases featuring both thrombosis and complement activation.

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.

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.

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.

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.

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.

[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.

Safety and effectiveness of eculizumab for pediatric patients with atypical hemolytic-uremic syndrome in Japan: interim analysis of post-marketing surveillance.

BACKGROUND: In 2013, eculizumab was approved for treatment of the atypical hemolytic-uremic syndrome (aHUS) in Japan, which was defined as a thrombotic microangiopathy (TMA) excluding Shiga toxin-producing Escherichia coli-HUS and thrombotic thrombocytopenic purpura. Simultaneously, post-marketing surveillance was started to assess its safety and effectiveness. In 2016, Japanese clinical guide redefined terms to limit the use of "aHUS" to complement-mediated HUS only. Accordingly, TMA with other causes was defined as secondary TMA. Here we report the interim analysis of post-marketing surveillance of pediatric patients with aHUS and secondary TMA. METHODS: Pediatric patients treated with eculizumab from approval to 15 March 2017 were included in this observational real-world study. Clinical endpoints of effectiveness were TMA event-free status, complete TMA response, platelet count normalization, and improvement of estimated glomerular filtration rate (eGFR). Adverse reactions to eculizumab were also analyzed. RESULTS: In 27 pediatric patients with aHUS, median age at diagnosis was 4 years. Complement genes' variants were detected in 14 of 21 patients (66.7%). Median time from diagnosis to eculizumab initiation was 2.0 days. TMA event-free status, complete TMA response, platelet normalization, and improvement in eGFR were achieved in 85.2, 36.4, 78.3, and 75.0% of patients, respectively. Three patients with aHUS died. Twenty-four and 10 adverse reactions were reported in 31 aHUS patients and 17 secondary TMA patients, respectively; however, no eculizumab-related death or meningococcal infection was reported. CONCLUSIONS: This interim analysis confirmed that eculizumab is well-tolerated and effective for Japanese pediatric patients with aHUS in a real-world setting.

Safety and effectiveness of eculizumab for adult patients with atypical hemolytic-uremic syndrome in Japan: interim analysis of post-marketing surveillance.

BACKGROUND: Eculizumab has been available for the treatment of atypical hemolytic-uremic syndrome (aHUS) in Japan since 2013. To assess safety and effectiveness of eculizumab in adult aHUS patients in the real-life setting, we performed interim analysis of a post-marketing surveillance mandated by Japanese regulations. METHODS: This study enrolled any patient who was diagnosed with TMA excluding Shiga toxin-producing Escherichia coli-HUS or thrombotic thrombocytopenic purpura based on Japanese clinical guide published in 2013 as inclusion criteria and treated with eculizumab. Although the term aHUS was redefined to denote only complement-mediated HUS in the guide revised in 2016, the patients with TMA caused by other causes (secondary TMA) were included. Patient outcomes and safety were evaluated at 6 months, 12 months, and annually thereafter. RESULTS: Thirty-three patients with aHUS and 27 patients with secondary TMA were enrolled. Median treatment duration of aHUS was 24weeks. Complement genes variants were detected in 11 of 18 patients with aHUS (61.1%). Among the 29 aHUS patients with available baseline data, platelet count (PLT), lactic dehydrogenase and serum creatinine (SCr) improved within 1-month after eculizumab initiation. TMA event-free status, complete TMA response, PLT normalization, and SCr decrease were achieved in 67.9% (19/28), 27.8% (5/18), 56.5% (13/23), and 57.1% (16/28) of patients, respectively. Thirty-three and 11 adverse reactions were observed in patients with aHUS (13/33 patients) and secondary TMA (6/27 patients), respectively. CONCLUSIONS: This interim analysis confirmed the acceptable safety profile and effectiveness of eculizumab for Japanese adult aHUS patients in real-world settings.

Development of a vaccine based on bacteria-mimicking tumor cells coated with novel engineered toll-like receptor 2 ligands.

For a successful tumor vaccine, it is necessary to develop effective immuno-adjuvants and identify specific tumor antigens. Tumor cells obtained from surgical or biopsy tissues are a good source of tumor antigens but, unlike bacteria, they do not induce strong immune responses. Here, we designed 2 novel lipopeptides that coat tumor cell surfaces and mimic bacterial components. Tumor cells coated with these lipopeptides (called bacteria-mimicking tumor cells [BMTC]) were prepared and their efficacy as a tumor vaccine examined. Natural bacterial lipopeptides act as ligands for toll-like receptor 2 (TLR2) and activate dendritic cells (DC). To increase the affinity of the developed lipopeptides for the negatively charged plasma membrane, a cationic polypeptide was connected to Pam2Cys (P2C), which is the basic structure of the TLR2 ligand. This increased the non-specific binding affinity of the peptides for the cell surface. Two such lipopeptides, P2CSK11 (containing 1 serine and 11 lysine residues) and P2CSR11 (containing 1 serine and 11 arginine residues) bound to irradiated tumor cells via the long cationic polypeptides more efficiently than the natural lipopeptide MALP2 (P2C-GNNDESNISFKEK) or a synthetic lipopeptide P2CSK4 (a short cationic polypeptide containing 1 serine and 4 lysines). BMTC coated with P2CSR11 or P2CSK11 were efficiently phagocytosed by DC and induced antigen cross-presentation in vitro. They also induced effective tumor-specific cytotoxic T cell responses and inhibited tumor growth in in vivo mouse models. P2CSR11 activated DC but induced less inflammation-inducing cytokines/interferons than other lipopeptides. Thus, P2CSR11 is a strong candidate antigen-specific immuno-adjuvant, with few adverse effects.

Collectin CL-P1 utilizes C-reactive protein for complement activation.

BACKGROUND: C-reactive protein (CRP) is a plasma pentraxin family protein that is massively induced as part of the innate immune response to infection and tissue injury. CRP and other pentraxin proteins can activate a complement pathway through C1q, collectins, or on microbe surfaces. It has been found that a lectin-like oxidized LDL receptor 1 (LOX-1), which is an endothelial scavenger receptor (SR) having a C-type lectin-like domain, interacts with CRP to activate the complement pathway using C1q. However it remains elusive whether other lectins or SRs are involved in CRP-mediated complement activation and the downstream effect of the complement activation is also unknown. METHODS: We prepared CHO/ldlA7 cells expressing collectin placenta-1 (CL-P1) and studied the interaction of CRP with cells. We further used ELISA for testing binding between proteins. We tested for C3 fragment deposition and terminal complement complex (TCC) formation on HEK293 cells expressing CL-P1. RESULTS: Here, we demonstrated that CL-P1 bound CRP in a charge dependent manner and the interaction of CRP with CL-P1 mediated a classical complement activation pathway through C1q and additionally drove an amplification pathway using properdin. However, CRP also recruits complement factor H (CFH) on CL-P1 expressing cell surfaces, to inhibit the formation of a terminal complement complex in normal complement serum conditions. GENERAL SIGNIFICANCE: The interaction of collectin CL-P1 with CFH might be key for preventing attack on "self" as a result of complement activation induced by the CL-P1 and CRP interaction.

M2-like macrophage polarization in high lactic acid-producing head and neck cancer.

Reprogramming of glucose metabolism in tumor cells is referred to as the Warburg effect and results in increased lactic acid secretion into the tumor microenvironment. We have previously shown that lactic acid has important roles as a pro-inflammatory and immunosuppressive mediator and promotes tumor progression. In this study, we examined the relationship between the lactic acid concentration and expression of LDHA and GLUT1, which are related to the Warburg effect, in human head and neck squamous cell carcinoma (HNSCC). Tumors expressing lower levels of LDHA and GLUT1 had a higher concentration of lactic acid than those with higher LDHA and GLUT1 expression. Lactic acid also suppressed the expression of LDHA and GLUT1 in vitro. We previously reported that lactic acid enhances expression of an M2 macrophage marker, ARG1, in murine macrophages. Therefore, we investigated the relationship between the lactic acid concentration and polarization of M2 macrophages in HNSCC by measuring the expression of M2 macrophage markers, CSF1R and CD163, normalized using a pan-macrophage marker, CD68. Tumors with lower levels of CD68 showed a higher concentration of lactic acid, whereas those with higher levels of CSF1R showed a significantly higher concentration of lactic acid. A similar tendency was observed for CD163. These results suggest that tumor-secreted lactic acid is linked to the reduction of macrophages in tumors and promotes induction of M2-like macrophage polarization in human HNSCC.

Three pentraxins C-reactive protein, serum amyloid p component and pentraxin 3 mediate complement activation using Collectin CL-P1.

BACKGROUND: Pentraxins (PTXs) are a superfamily of multifunctional conserved proteins involved in acute-phase responses. Recently, we have shown that collectin placenta 1 (CL-P1) and C-reactive protein (CRP) mediated complement activation and failed to form terminal complement complex (TCC) in normal serum conditions because of complement factor H inhibition. METHODS: We used CL-P1 expressing CHO/ldlA7 cells to study the interaction with PTXs. Soluble type CL-P1 was used in an ELISA assay for the binding, C3 and TCC deposition experiments. Furthermore, we used our previously established CL-P1 expressing HEK293 cells for the C3 fragment and TCC deposition assay. RESULTS: We demonstrated that CL-P1 also bound serum amyloid p component (SAP) and pentraxin 3 (PTX3) to activate the classical pathway and the alternative pathway using factor B. CRP and PTX3 further amplified complement deposition by properdin. We found that CRP and PTX3 recruit CFH, whereas SAP recruits C4 binding protein on CL-P1 expressing cell surfaces to prevent the formation of TCC in normal serum conditions. In addition, depletion of CFH, C4BP and complement factor I (CFI) failed to prevent TCC formation both in ELISA and cell experiments. Furthermore, soluble complement receptor 1, an inhibitor of all complement pathways prevents PTX induced TCC formation. CONCLUSION: Our current study hypothesizes that the interaction of pentraxins with CL-P1 is involved in complement activation. GENERAL SIGNIFICANCE: CL-P1 might generally inhibit PTX induced complement activation and host damage to protect self-tissues.

Glycosylphosphatidylinositol (GPI) anchor deficiency caused by mutations in PIGW is associated with West syndrome and hyperphosphatasia with mental retardation syndrome.

BACKGROUND: Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors 150 or more kinds of proteins to the human cell surface. There are at least 26 genes involved in the biosynthesis and remodelling of GPI anchored proteins (GPI-APs). Recently, inherited GPI deficiencies (IGDs) were reported which cause intellectual disability often accompanied by epilepsy, coarse facial features and multiple anomalies that vary in severity depending upon the degree of defect and/or step in the pathway of affected gene. METHODS AND RESULTS: A patient born to non-consanguineous parents developed intractable seizures with typical hypsarrhythmic pattern in electroencephalography, and was diagnosed as having West syndrome. Because the patient showed severe developmental delay with dysmorphic facial features and hyperphosphatasia, characteristics often seen in IGDs, the patient was tested for GPI deficiency. The patient had decreased surface expression of GPI-APs on blood granulocytes and was identified to be compound heterozygous for NM_178517:c.211A>C and c.499A>G mutations in PIGW by targeted sequencing. CONCLUSION: Here we describe the first patient with deficiency of PIGW, which is involved in the addition of the acyl-chain to inositol in an early step of GPI biosynthesis. Therefore, IGD should be considered in West syndrome and flow cytometric analysis of blood cells is effective in screening IGD.

Toll-like receptor 3 signaling converts tumor-supporting myeloid cells to tumoricidal effectors.

Smoldering inflammation often increases the risk of progression for malignant tumors and simultaneously matures myeloid dendritic cells (mDCs) for cell-mediated immunity. PolyI:C, a dsRNA analog, is reported to induce inflammation and potent antitumor immune responses via the Toll-like receptor 3/Toll-IL-1 receptor domain-containing adaptor molecule 1 (TICAM-1) and melanoma differentiation-associated protein 5/IFN-ß promoter stimulator 1 (IPS-1) pathways in mDCs to drive activation of natural killer cells and cytotoxic T lymphocytes. Here, we found that i.p. or s.c. injection of polyI:C to Lewis lung carcinoma tumor-implant mice resulted in tumor regression by converting tumor-supporting macrophages (Mfs) to tumor suppressors. F4/80(+)/Gr1(-) Mfs infiltrating the tumor respond to polyI:C to rapidly produce inflammatory cytokines and thereafter accelerate M1 polarization. TNF-α was increased within 1 h in both tumor and serum upon polyI:C injection into tumor-bearing mice, followed by tumor hemorrhagic necrosis and growth suppression. These tumor responses were abolished in TNF-α(-/-) mice. Furthermore, F4/80(+) Mfs in tumors extracted from polyI:C-injected mice sustained Lewis lung carcinoma cytotoxic activity, and this activity was partly abrogated by anti-TNF-α Ab. Genes for supporting M1 polarization were subsequently up-regulated in the tumor-infiltrating Mfs. These responses were completely abrogated in TICAM-1(-/-) mice, and unaffected in myeloid differentiation factor 88(-/-) and IPS-1(-/-) mice. Thus, the TICAM-1 pathway is not only important to mature mDCs for cross-priming and natural killer cell activation in the induction of tumor immunity, but also critically engaged in tumor suppression by converting tumor-supporting Mfs to those with tumoricidal properties.

Development of a dendritic cell-targeting lipopeptide as an immunoadjuvant that inhibits tumor growth without inducing local inflammation.

Materials used for the past 30 years as immunoadjuvants induce suboptimal antitumor immune responses and often cause undesirable local inflammation. Some bacterial lipopeptides that act as Toll-like receptor (TLR) 2 ligands activate immune cells as immunoadjuvants and induce antitumor effects. Here, we developed a new dendritic cell (DC)-targeting lipopeptide, h11c (P2C-ATPEDNGRSFS), which uses the CD11c-binding sequence of intracellular adhesion molecule-1 to selectively and efficiently activate DCs but not other immune cells. Although the h11c lipopeptide activated DCs similarly to an artificial lipopeptide, P2C-SKKKK (P2CSK4), via TLR2 in vitro, h11c induced more effective tumor inhibition than P2CSK4 at low doses in vivo with tumor antigens. Even without tumor antigens, h11c lipopeptide significantly inhibited tumor growth and induced tumor-specific cytotoxic T cells. P2CSK4 was retained subcutaneously at the vaccination site and induced severe local inflammation in in vivo experiments. In contrast, h11c was not retained at the vaccination site and was transported into the tumor within 24 hr. The recruitment of DCs into the tumor was induced by h11c more effectively, while P2CSK4 induced the accumulation of neutrophils leading to severe inflammation at the vaccination site. Because CD11b+ cells, but not CD11c+ cells, produced neutrophil chemotactic factors such as macrophage inflammatory protein (MIP)-2 in response to stimulation with TLR2 ligands, the DC-targeting lipopeptide h11c induced less MIP-2 production by splenocytes than P2CSK4. In this study, we succeeded in developing a novel immunoadjuvant, h11c, which effectively induces antitumor activity without adverse effects such as local inflammation via the selective activation of DCs.

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.

Dichloroacetate improves immune dysfunction caused by tumor-secreted lactic acid and increases antitumor immunoreactivity.

The activation of oncogenic signaling pathways induces the reprogramming of glucose metabolism in tumor cells and increases lactic acid secretion into the tumor microenvironment. This is a well-known characteristic of tumor cells, termed the Warburg effect, and is a candidate target for antitumor therapy. Previous reports show that lactic acid secreted by tumor cells is a proinflammatory mediator that activates the IL-23/IL-17 pathway, thereby inducing inflammation, angiogenesis and tissue remodeling. Here, we show that lactic acid, or more specifically the acidification it causes, increases arginase I (ARG1) expression in macrophages to inhibit T-cell proliferation and activation. Accordingly, we hypothesized that counteraction of the immune effects by lactic acid might suppress tumor development. We show that dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinases, targets macrophages to suppress activation of the IL-23/IL-17 pathway and the expression of ARG1 by lactic acid. Furthermore, lactic acid-pretreated macrophages inhibited CD8+ T-cell proliferation, but CD8+ T-cell proliferation was restored when macrophages were pretreated with lactic acid and DCA. DCA treatment decreased ARG1 expression in tumor-infiltrating immune cells and increased the number of IFN-γ-producing CD8+ T cells and NK cells in tumor-bearing mouse spleen. Although DCA treatment alone did not suppress tumor growth, it increased antitumor immunotherapeutic activity of Poly(IC) in both CD8+ T cell- and NK cell-sensitive tumor models. Therefore, DCA acts not only on tumor cells to suppress glycolysis but also on immune cells to improve the immune status modulated by lactic acid and to increase the effectiveness of antitumor immunotherapy.