Early hematopoietic cell transplantation for familial hemophagocytic lymphohistiocytosis in a regional treatment network in Japan.

Familial hemophagocytic lymphohistiocytosis (FHLH) is a fatal hyperinflammation syndrome arising from the genetic defect of perforin-mediated cytolysis. Curative hematopoietic cell transplantation (HCT) is needed before development of central nervous system (CNS) disease. We studied treatment outcomes of 13 patients (FHLH2 n = 11, FHLH3 n = 2) consecutively diagnosed from 2011 to 2022 by flow cytometric screening for non-myeloablative HCT in a regional treatment network in Kyushu, Japan. One patient with a novel PRF1 variant escaped screening, but all patients with FHLH2 reached diagnosis and 8 of them received HCT until 3 and 9 months of age, respectively. The earliest HCT was conducted 65 days after birth. Three pretransplant deaths occurred in newborns with liver failure at diagnosis. Ten posttransplant patients have remained disease-free, 7 of whom had no neurological involvement. Time from first etoposide infusion to HCT was shorter in patients without CNS disease or bleeding than in patients with those factors (median [range] days: 62 [50-81] vs. 122 [89-209], p = 0.016). Six of 9 unrelated patients had a PRF1 c.1090_1091delCT variant. These results suggest that the critical times to start etoposide and HCT are within 3 months after birth and during etoposide control, respectively. Newborn screening may increase the percentage of disease-free survivors without complications.

Clinical Characteristics of Cryopyrin-Associated Periodic Syndrome and Long-Term Real-World Efficacy and Tolerability of Canakinumab in Japan: Results of a Nationwide Survey.

OBJECTIVE: We assess the clinical characteristics of patients with cryopyrin-associated periodic syndrome (CAPS) in Japan and evaluate the real-world efficacy and safety of interleukin-1 (IL-1) inhibitors, primarily canakinumab. METHODS: Clinical information was collected retrospectively, and serum concentrations of canakinumab and cytokines were analyzed. RESULTS: A total of 101 patients were included, with 86 and 15 carrying heterozygous germline and somatic mosaic mutations, respectively. We identified 39 mutation types, and the common CAPS-associated symptoms corresponded with those in previous reports. Six patients (5.9% of all patients) died, with four of the deaths caused by CAPS-associated symptoms. Notably, 73.7% of patients (100%, 79.6%, and 44.4% of familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and chronic infantile neurological cutaneous articular syndrome/neonatal onset multisystem inflammatory disease, respectively) achieved complete remission with canakinumab, and early therapeutic intervention was associated with better auditory outcomes. In some patients, canakinumab treatment stabilized the progression of epiphysial overgrowth and improved height gain, visual acuity, and renal function. However, 23.7% of patients did not achieve inflammatory remission with crucial deterioration of organ damage, with two dying while receiving high-dose canakinumab treatment. Serological analysis of canakinumab and cytokine concentrations revealed that the poor response was not related to canakinumab shortage. Four inflammatory nonremitters developed inflammatory bowel disease (IBD)-unclassified during canakinumab treatment. Dual biologic therapy with canakinumab and anti-tumor necrosis factor-α agents was effective for IBD- and CAPS-associated symptoms not resolved by canakinumab monotherapy. CONCLUSION: This study provides one of the largest epidemiologic data sets for CAPS. Although early initiation of anti-IL-1 treatment with canakinumab is beneficial for improving disease prognosis, some patients do not achieve remission despite a high serum concentration of canakinumab. Moreover, IBD may develop in CAPS after canakinumab treatment.

Monogenic inflammatory bowel disease with STXBP2 mutations is not resolved by hematopoietic stem cell transplantation but can be alleviated via immunosuppressive drug therapy.

STXBP2, encoding syntaxin-binding protein 2, is involved in intracellular organelle trafficking and is associated with familial hemophagocytic lymphohistiocytosis type 5. Although STXBP2 mutations reportedly cause monogenic inflammatory bowel disease, the clinical course and underlying pathogenic mechanisms remain unclear. We identified a novel mutation in STXBP2 [c.1197delC, p.Ala400fs] in a boy with congenital intractable diarrhea and hemophagocytic lymphohistiocytosis (HLH). HLH was treated with intravenous prednisolone, cyclosporine, and dexamethasone palmitate. Hematopoietic stem cell transplantation (HSCT) along with prophylaxis for graft-versus-host-disease was performed at 5 months of age. Additionally, colonoscopies done before and after HSCT showed mild colitis with cryptitis. The patient showed elevated fecal calprotectin levels and persistent diarrhea even after HSCT and required partial parenteral nutrition. While anti-inflammatory treatment reduced diarrhea, it was not completely normalized even after HSCT, suggesting that the pathogenesis of inflammatory bowel disease associated with STXBP2 mutations involves both hyperinflammation and functional epithelial barrier defects.

Assessment of type I interferon signatures in undifferentiated inflammatory diseases: A Japanese multicenter experience.

Purpose: Upregulation of type I interferon (IFN) signaling has been increasingly detected in inflammatory diseases. Recently, upregulation of the IFN signature has been suggested as a potential biomarker of IFN-driven inflammatory diseases. Yet, it remains unclear to what extent type I IFN is involved in the pathogenesis of undifferentiated inflammatory diseases. This study aimed to quantify the type I IFN signature in clinically undiagnosed patients and assess clinical characteristics in those with a high IFN signature. Methods: The type I IFN signature was measured in patients' whole blood cells. Clinical and biological data were collected retrospectively, and an intensive genetic analysis was performed in undiagnosed patients with a high IFN signature. Results: A total of 117 samples from 94 patients with inflammatory diseases, including 37 undiagnosed cases, were analyzed. Increased IFN signaling was observed in 19 undiagnosed patients, with 10 exhibiting clinical features commonly found in type I interferonopathies. Skin manifestations, observed in eight patients, were macroscopically and histologically similar to those found in proteasome-associated autoinflammatory syndrome. Genetic analysis identified novel mutations in the PSMB8 gene of one patient, and rare variants of unknown significance in genes linked to type I IFN signaling in four patients. A JAK inhibitor effectively treated the patient with the PSMB8 mutations. Patients with clinically quiescent idiopathic pulmonary hemosiderosis and A20 haploinsufficiency showed enhanced IFN signaling. Conclusions: Half of the patients examined in this study, with undifferentiated inflammatory diseases, clinically quiescent A20 haploinsufficiency, or idiopathic pulmonary hemosiderosis, had an elevated type I IFN signature.

Recurrent tandem duplication of UNC13D in familial hemophagocytic lymphohistiocytosis type 3.

Familial hemophagocytic lymphohistiocytosis type 3 is a fatal inborn error of immunity due to abnormal cytotoxic activity of T and NK cells and is caused by variants in UNC13D, which encodes Munc13-4. One published case was reported to carry a tandem duplication of UNC13D exons 7-12, and we here present another case with the exact same duplication breakpoints. The patient carried the tandem duplication from maternal origin, and a c.2346_2349 variant on the paternal allele. Single nucleotide polymorphism analysis around UNC13D revealed that the allele with tandem duplication was most likely a founder allele. Transposable element analysis showed that the breakpoints occurred within Alu elements in introns 12 and 6. Multiple sequence alignment revealed that Alu elements containing the truncated points are highly homologous. Sequence homology was thought to be a factor predisposing to the tandem duplication variant.

Case Report: A Pediatric Case of Familial Mediterranean Fever Concurrent With Autoimmune Hepatitis.

Familial Mediterranean fever (FMF) is a hereditary, autoinflammatory disease that causes recurrent fever, arthritis, and serositis. The diagnosis of FMF is based on the presentation of typical clinical symptoms and the Mediterranean fever gene (MEFV) test. However, the challenge lies in diagnosing atypical cases. In this report, we have described a pediatric patient with complex FMF whose diagnosis required trio-whole exome sequencing (WES) and functional validation of a rare MEFV variant. A 3-year-old boy presented with recurrent episodes of elevated liver enzymes and arthralgia. He was diagnosed with autoimmune hepatitis (AIH), and his liver enzymes improved rapidly with steroid treatment. However, he exhibited recurrent arthralgia and severe abdominal attacks. Trio-WES identified compound heterozygous mutations in MEFV (V726A and I692del). Ex vivo functional assays of the patient's monocytes and macrophages, which had been pre-treated with Clostridium difficile toxin A (TcdA) and colchicine, were comparable to those of typical FMF patients, thereby confirming the diagnosis of FMF. Although he was intolerant to colchicine because of liver toxicity, subsequent administration of canakinumab successfully ameliorated his abdominal attacks. However, it was ineffective against liver injury, which recurred after steroid tapering. Therefore, in this case, the pathogenesis of AIH was probably interleukin-1ß (IL-1ß)-independent. In fact, AIH might have been a concurrent disease with FMF, rather than being one of its complications. Nevertheless, further studies are necessary to determine whether FMF-induced inflammasome activation contributes to AIH development. Moreover, we must consider the possibility of mixed phenotypes in such atypical patients who present distinct pathologies simultaneously.

[Perforin gene mutation in middle-age onset hemophagocytic lymphohistiocytosis].

A 54-year-old woman was referred to our hospital for pancytopenia and liver dysfunction, and with no personal or family history of hemophagocytic lymphohistiocytosis (HLH). Although the etiology was unknown, she was diagnosed with HLH. She experienced exacerbation of HLH even after initiating systemic chemotherapy with etoposide, dexamethasone, and cyclosporine. Furthermore, flow cytometric analysis of the natural killer cells revealed a reduction in perforin expression, and DNA sequencing of the perforin gene (PRF1) revealed two known mutations, confirming the diagnosis of late-onset familial HLH type 2. She received an allogeneic stem cell transplant from an unrelated human leukocyte antigens identical donor, but developed thrombotic microangiopathy, and succumbed to septic shock shortly after the transplant. Previously, HLH in adults was believed to develop from underlying diseases. However, as in our case, several reports demonstrated that HLH gene mutations could be found even after adolescence. Adult with HLH with no underlying disorders should undergo early HLH-associated gene testing for confirmatory diagnosis.

Rapid Flow Cytometry-Based Assay for the Functional Classification of MEFV Variants.

PURPOSE: Pathogenic MEFV variants cause pyrin-associated autoinflammatory diseases (PAADs), which include familial Mediterranean fever (FMF), FMF-like disease, and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). The diagnosis of PAADs is established by clinical phenotypic and genetic analyses. However, the pathogenicity of most MEFV variants remains controversial, as they have not been functionally evaluated. This study aimed to establish and validate a new functional assay to evaluate the pathogenicity of MEFV variants. METHODS: We transfected THP-1 monocytes with 32 MEFV variants and analyzed their effects on cell death with or without stimulation with Clostridium difficile toxin A (TcdA) or UCN-01. These variants were classified using hierarchical cluster analysis. Macrophages were obtained from three healthy controls and two patients with a novel homozygous MEFVP257L variant, for comparison of IL-1ß secretion using a cell-based assay and a novel THP-1-based assay. RESULTS: Disease-associated MEFV variants induced variable degrees of spontaneous or TcdA/UCN-01-induced cell death in THP-1. Cell death was caspase-1 dependent and was accompanied by ASC speck formation and IL-1ß secretion, indicating that pathogenic MEFV variants induced abnormal pyrin inflammasome activation and subsequent pyroptotic cell deaths in this assay. The MEFV variants (n = 32) exhibiting distinct response signatures were classified into 6 clusters, which showed a good correlation with the clinical phenotypes. Regarding the pathogenicity of MEFVP257L variants, the results were consistent between the cell-based assay and the THP-1-based assay. CONCLUSION: Our assay facilitates a rapid and comprehensive assessment of the pathogenicity of MEFV variants and contributes to a refined definition of PAAD subtypes.

Analysis of Trichophyton antigen-induced contact hypersensitivity in mouse.

BACKGROUND: Trichophyton-induced superficial skin mycosis is a common infectious human disease, but the immunological mechanism against Trichophyton infection is unclear with regard to many points. Since Trichophyton cannot colonize mice, guinea pigs were used in previous experiments on Trichophyton infection. However, it is difficult to perform immunological and genetic analyses in guinea pigs. OBJECTIVE: The objective of this study was to establish a mouse Trichophytin-associated inflammation model of superficial skin mycosis in which immunological and genetic analyses can be performed. METHODS: We established a mouse Trichophyton-induced contact hypersensitivity model by applying Trichophytin, the Trichophyton antigen, extracted from Trichophyton mentagrophytes, to mice. Using a Th1-dominant strain, C57BL/6, and a Th2-dominant strain, BALB/c, we investigated the expression of inflammatory cytokines and receptors of the innate immune system for fungi, TLR4, TLR2, and dectin-1, and their influences on responses of the acquired immune system. RESULTS: In C57BL/6 mice, expressions of IFN-γ and IL-17 A in regional lymph nodes and IL-1ß, IFN-γ, IL-6, and IL-23 in the inflammatory auricular skin were enhanced by Trichophytin challenge, suggesting that not only Th1 cells but also Th17 cells were induced. In BALB/c mice, expressions of IL-4 in regional lymph nodes, and TSLP and IL-4 in the auricular skin were enhanced by Trichophytin challenge. Interestingly, dectin-1-neutralizing antibody inhibited the promotion of IFN-γ production in C57BL/6 mice, and dectin-1-expressing immune cells had crucial actions in Trichophyton-induced IFN-γ production. CONCLUSION: These results suggest that inflammatory mediators differently regulate Trichophytin-induced contact hypersensitivity on the basis of the status of host immunity.

Novel ribbon-type nuclear factor of activated T cells decoy oligodeoxynucleotides preclude airways hyperreactivity and Th2 cytokine expression in experimental asthma.

BACKGROUND: Nuclear factor of activated T cells (NFAT) is required for the differentiation of Th2 responses, so we examined its role in mouse experimental asthma and tested the hypothesis that an NFAT blockade with a decoy against NFAT can prevent asthma progression. OBJECTIVE: To determine the effects of the NFAT decoy oligodeoxynucleotides (ODNs) on the development of airway inflammation, we designed a novel ribbon-type ODN containing two binding sites for NFAT in a single decoy molecule without an open end, which is more stable than a conventional decoy, and largely preserved its structural integrity in the presence of nucleases. METHODS: Ribbon-type NFAT decoy ODNs were transfected into ovalbumin (OVA)-sensitized CD3+ T cells in vitro. OVA-immunized mice received these cells by intraperitoneal injection. Airway hyperreactivity (AHR) was measured and the transfected CD3+ T cells' responses to the airways were characterized. RESULTS: Development of AHR after OVA challenge was effectively abolished after adoptive transfer of ribbon-type NFAT decoy ODN transfected CD3+ T cells. Transfer of ribbon-type decoy significantly reduced the number of inflammatory cells and the concentrations of IL-4, IL-5 and IL-13, but not IFN-γ, in the bronchoalveolar lavage of the recipient mice. CONCLUSION: These results suggest the inhibitory effect of ribbon-type decoy ODNs against NFAT on the induction of bronchial asthma. Adoptively transferred CD3+ T cells, which are transfected with NFAT decoy, may be an effective strategy for the treatment of asthma.

[Impact of the "Doctor-Heli" system for emergency and critical care medicine in otolaryngology].

We reported the "Doctor-Heli" (medical service helicopter) system at the center of emergency and critical care medicine at Tokai University Hospital. From October 1999 to March 2001, the service had transported 485 patients, shortening the time to critical care and improving patient-prognosis. We report a case of cervical and laryngeal trauma occurring during a suicide attempt successfully treated thanks to the rapid start of critical care enabled by use of the helicopter. The service has proven its utility in otolaryngology and head and neck surgery.

Itch-associated response induced by experimental dry skin in mice.

The present study was conducted to establish a new mouse model of dry skin pruritus. The rostral back was treated daily with cutaneous application of acetone/ether (1:1) mixture (AE), water following AE (AEW), 1% sodium lauryl sulfate (SLS) or tape stripping (TS). On the day after 5-day treatment, although all four treatments significantly decreased stratum corneum (SC) hydration and increased transepidermal water loss (TEWL), only AEW treatment significantly increased spontaneous scratching. An increase in the frequency of TS produced the marked increase of TEWL, without significant effects on SC hydration and spontaneous scratching. In AEW-treated mice, changes in SC hydration and TEWL were marked in the initial 2-day period, while spontaneous scratching increased gradually from 3 days after starting the treatment. The degranulation of cutaneous mast cells was increased by SLS treatment but not by other treatments. There was no apparent difference in AEW-induced spontaneous scratching between mast cell-deficient mice (WBB6F1-W/Wv) and normal littermates (WBB6F1-+/+). Opioid antagonists, naloxone and naltrexone, (1 mg/kg, subcutaneously) significantly suppressed spontaneous scratching in AEW-treated mice. It is suggested that spontaneous scratching of AEW-treated mice is an itch-related response and a useful model for studying the mechanisms of dry skin pruritus.

Intradermal cholinergic agonists induce itch-associated response via M3 muscarinic acetylcholine receptors in mice.

In the present study, we examined whether cholinergic agonists would elicit an itch-associated response in mice. When mice were given an intradermal injection of carbachol (1-10 nmol) or bethanechol (0.3-100 nmol) into the rostral back, they showed the dose-dependent increase of scratching. Nicotine (1-10 nmol) showed no effect. Pretreatment with naloxone, but not with terfenadine, significantly suppressed the carbachol-induced scratching. When intradermally co-injected with carbachol, atropine and 4-DAMP but neither methoctramine nor pancuronium significantly inhibited the carbachol-induced scratching. Muscarinic agonists are suggested to produce itch through activation of M3 muscarinic receptors in the skin.