Outcomes of liver transplantation for mitochondrial respiratory chain disorder in children.

AIM: Mitochondrial respiratory chain disorder (MRCD) can cause acute liver failure (ALF), which may necessitate liver transplantation (LT). However, MRCD is often difficult to diagnose before LT and the indications of LT are controversial due to the likelihood of progressive neurological disease. The present study further characterized the patient population and described the outcomes. METHODS: Thirteen patients who underwent LT for MRCD from November 2005 to May 2020 were enrolled in this study. RESULTS: Six of 13 MRCD patients were diagnosed with a mitochondrial inner membrane protein 17-related mitochondrial DNA depletion syndrome (MTDPS). Overall, nine survived with a median follow-up of 1.8 years (IQR, 1.3-5.1 years); four died within 2 years. In the long-term, seven survivors showed no progression of hypotonia after LT and attended a normal kindergarten or primary school. Neurological abnormalities were observed in two survivors, including vison loss related to Leber's hereditary optic neuropathy in one patient and psychomotor retardation related to Leigh syndrome in the other. Three non-survivors after LT were diagnosed with MTDPS and died of severe pulmonary hypertension, which had developed at 8, 9, and 18 months after LT (n=1 each). The remaining patient died of postoperative respiratory infection with respiratory syncytial virus. CONCLUSION: The long-term results support the performance of LT in patients with MRCD, although a genetic diagnosis is preferable for determining the accurate indications for LT in these patients. Furthermore, care should be taken to avoid complications due to mitochondrial dysfunction during the long-term follow-up.

High serum free IL-18 is associated with decreased omalizumab efficacy: findings from a 2-year omalizumab treatment study.

OBJECTIVE: Omalizumab is more effective in severe allergic patients with eosinophilic asthma than those with non-eosinophilic asthma. IL-18, a unique cytokine involved in allergic but non-eosinophilic inflammation, might be associated with the latter condition. We aimed to clarify the roles of IL-18 related pathways in insufficient response to omalizumab treatment. METHODS: Patients with severe allergic asthma who completed 2-year omalizumab treatments at Kyoto University Hospital were included in this study (UMIN000002389). Associations between pretreatment levels of serum free IL-18 in addition to other mediators and asthma phenotypes including responses to omalizumab treatment were analyzed. Changes in serum free IL-18, periostin and total IgE levels during the treatment were also examined. RESULTS: Twenty-seven patients (19 females, average age of 55.7 years) were examined. Fifteen incomplete responders who experienced exacerbations in the second year, were significantly and more frequently obese and showed significantly earlier asthma onset, lower blood eosinophils and more exacerbations before omalizumab treatment than complete responders. Significantly more patients showed high baseline serum free IL-18 levels (≥141 pg/mL, a threshold for the highest tertile) among the incomplete responders than complete responders. Patients with high serum free IL-18 levels shared similar characteristics with incomplete responders, showing significant reductions in serum total IgE levels during omalizumab treatment. Finally, serum free IL-18 levels negatively correlated with serum periostin levels at baseline and in change ratios. CONCLUSIONS: High baseline serum free IL-18 levels may predict reduced omalizumab efficacy in severe allergic patients with type-2 low asthma, regarding reduction of exacerbations.

Characterization of Late Acute and Chronic Graft-Versus-Host Disease according to the 2014 National Institutes of Health Consensus Criteria in Japanese Patients.

To characterize the incidences and outcomes of late acute (LA) and chronic graft-versus-host disease (GVHD) in East Asians according to the 2014 National Institutes of Health criteria, we retrospectively analyzed 506 consecutive Japanese patients who had a first allogeneic hematopoietic cell transplantation (HCT) at our center between 2006 and 2013. According to manifestations at onset 91 patients (60%) had LA GVHD and 60 (40%) had chronic GVHD. The cumulative incidences of LA and chronic GVHD were 20% and 17%, respectively, at 48 months after HCT. The involved sites at the onset of LA GVHD included the skin (71%), gut (13%), and liver (8%). The cumulative incidences of relapse, nonrelapse mortality (NRM), transition to chronic GVHD, and discontinued systemic treatment were 11%, 6%, 22%, and 46%, respectively, at 48 months after onset of LA GVHD. Cox models showed that prior acute GVHD was associated with NRM, and HCT from a female donor to a male patient, myeloablative conditioning, and low Karnofsky performance status were associated with a longer duration of systemic treatment after LA GVHD. The most frequently involved sites at the onset of chronic GVHD included the mouth (83%), liver (75%), skin (69%), and eyes (62%). Cox models showed that use of antithymocyte globulin in conditioning regimens was associated with a higher risk of discontinued systemic treatment after the onset of chronic GVHD. The cumulative incidences of relapse, NRM, and discontinued systemic treatment were 16%, 11%, and 41%, respectively, at 48 months after the onset of chronic GVHD. Our results suggested several potential differences between Japanese patients and those of other ethnicities. A direct comparison is needed to formally investigate ethnic differences.

Prospective Study of Live Attenuated Vaccines for Patients with Nephrotic Syndrome Receiving Immunosuppressive Agents.

OBJECTIVE: To conduct a prospective study to evaluate the immunogenicity and safety of live attenuated vaccines in patients with nephrotic syndrome receiving immunosuppressive agents. STUDY DESIGN: Patients with nephrotic syndrome receiving immunosuppressive agents with negative or borderline antibody titers (virus-specific IgG levels <4.0) against measles, rubella, varicella, and/or mumps fulfilling the criteria of cellular and humoral immunity were enrolled. Virus-specific IgG levels were measured using an enzyme immunoassay. The primary endpoint was the seroconversion rate (ie, achievement of virus-specific IgG levels ≥4.0) at 2 months after vaccination. Virus-specific IgG levels at 1 year, breakthrough infections (wild-type infections), and adverse events were also evaluated. RESULTS: A total of 116 vaccinations were administered to 60 patients. Seroconversion rates were 95.7% for measles, 100% for rubella, 61.9% for varicella, and 40.0% for mumps. More patients with a borderline antibody titer before vaccination achieved seroconversion than those with negative antibody titer, with statistical significance after varicella and mumps vaccination. The rate of patients who maintained seropositivity at 1 year after vaccination was 83.3% for measles, 94.1% for rubella, 76.7% for varicella, and 20.0% for mumps. No patient experienced breakthrough infection. No serious adverse events, including vaccine-associated infection, were observed. CONCLUSION: Immunization with live attenuated vaccines may be immunogenic and is apparently safe in our cohort of patients with nephrotic syndrome receiving immunosuppressive agents if their cellular and humoral immunologic measures are within clinically acceptable levels. TRIAL REGISTRATION: UMIN-CTR UMIN 000007710.

Combined genetic analyses can achieve efficient diagnostic yields for subjects with Alagille syndrome and incomplete Alagille syndrome.

AIM: We evaluated combined genetic analyses with targeted next-generation sequencing (NGS), multiplex ligation probe amplification (MLPA) of Jagged1 (JAG1) genes and microarray comparative genomic hybridisation (CGH) in subjects with Alagille syndrome, incomplete clinical features of Alagille syndrome and biliary atresia. METHODS: Subjects recruited from April 2013 to December 2015 underwent a targeted NGS analysis, including JAG1 and Notch homolog 2 (NOTCH2). If no mutations were detected in JAG1 or NOTCH2, or if copy number variations were suggested by the NGS analysis, we performed an MLPA analysis of JAG1. We also performed a microarray CGH analysis with whole-exon deletion detected by the MLPA analysis. RESULTS: We analysed 30 subjects with Alagille syndrome, nine with incomplete Alagille syndrome and 17 with biliary atresia and detected pathogenic mutations in JAG1 or NOTCH2 in 24/30 subjects with Alagille syndrome and in 4/9 subjects with incomplete Alagille syndrome. No pathogenic mutations were detected in subjects with biliary atresia. The frequency of JAG1 mutations was as follows: single nucleotide variants (51.9%), small insertion or deletion (29.6%) and gross deletion (18.5%). CONCLUSION: Combined genetic analyses achieved efficient diagnostic yields for subjects with Alagille syndrome and incomplete Alagille syndrome.

Molecular Genetic Dissection and Neonatal/Infantile Intrahepatic Cholestasis Using Targeted Next-Generation Sequencing.

OBJECTIVES: To ascertain a molecular genetic diagnosis for subjects with neonatal/infantile intrahepatic cholestasis (NIIC) by the use of next-generation sequencing (NGS) and to perform a genotype-phenotype correlation. STUDY DESIGN: We recruited Japanese subjects with NIIC who had no definitive molecular genetic diagnosis. We developed a diagnostic custom panel of 18 genes, and the amplicon library was sequenced via NGS. We then compared clinical data between the molecular genetically confirmed subjects with NIIC. RESULTS: We analyzed 109 patients with NIIC ("genetic cholestasis," 31 subjects; "unknown with complications" such as prematurity, 46 subjects; "unknown without complications," 32 subjects), and a molecular genetic diagnosis was made for 28 subjects (26%). The rate of positive molecular genetic diagnosis in each category was 22 of 31 (71%) for the "genetic cholestasis" group, 2 of 46 (4.3%) for the "unknown with complications" group, and 4 of 32 (12.5%) for the "unknown without complications" group. The grouping of the molecular diagnoses in the group with genetic cholestasis was as follows: 12 with Alagille syndrome, 5 with neonatal Dubin-Johnson syndrome, 5 with neonatal intrahepatic cholestasis caused by citrin deficiency, and 6 with progressive familial intrahepatic cholestasis or benign recurrent intrahepatic cholestasis with low gamma-glutamyl transpeptidase levels. Several clinical datasets, including age of onset, direct bilirubin, and aminotransferases, were significantly different between the disorders confirmed using molecular genetic diagnosis. CONCLUSION: Targeted NGS can be used for molecular genetic diagnosis in subjects with NIIC. Clinical diagnosis should be accordingly redefined in the view of molecular genetic findings.

Serum eosinophil-derived neurotoxin: correlation with persistent airflow limitation in adults with house-dust mite allergic asthma.

BACKGROUND: The serum level of eosinophil-derived neurotoxin (EDN), a protein present in eosinophil granules, correlates with the severity of childhood asthma. However, the relationship between the serum EDN level and the severity of adult asthma has not been sufficiently investigated. OBJECTIVE: This study aimed to elucidate the correlation between the serum EDN level and markers of severity in adult asthma. METHODS: The subjects comprised 83 adult patients who had asthma and who were undergoing treatment. Of these patients, 40 were positive for house-dust-specific immunoglobulin E (IgE) antibodies; 9 patients with severe adult asthma who were treated with omalizumab were included in the study. We measured the blood eosinophil count, serum EDN, and eosinophil cationic protein levels before investigating the correlations of these parameters with lung function and symptom score. RESULTS: There were no significant correlations between the blood eosinophil count or serum EDN or eosinophil cationic protein level with lung function and the symptom score in patients with asthma. However, serum EDN level was inversely correlated with the decrease percentage forced expiratory volume in 1 second (%FEV1) in patients positive for house-dust-specific IgE antibody (R = -0.54; p < 0.05), whereas no such correlation was observed in patients with negative results for house-dust-specific IgE antibody (R = 0.11; p = 0.468). A significant correlation was observed between a decrease in serum EDN level from baseline and lung function improvement after 8 weeks of omalizumab therapy (R = -0.77; p = 0.015). CONCLUSION: Serum EDN level may be a useful marker for monitoring persistent airflow limitation in adult patients with asthma who had positive results for house-dust-specific IgE antibodies.

[Effects of Instruction on Inhalation Techniques Using iPads - Web Application "Inhalation Lessons"].

Instruction on inhalation techniques for chronic obstructive pulmonary disease(COPD)and asthma patients being treated with inhalants have sufficient therapeutic effects and are important to maintain adherence. However, problems continue to exist, including time constraints of medical staff that have a large number of patients and a lack of knowledge on inhalation instruction methods. A web application,"Inhalation Lessons,'for the iPad has been developed. It explains inhalation methods, and consists of videos and review tests. Instruction on inhalation techniques was performed using this application for patients that use Diskus, and the effects were examined. As a result, there are significant improvements in the inhalation techniques of patients after viewing the"Inhalation Lessons'application. Uniform instruction on inhalation techniques can be performed even in the field of homecare.

Romiplostim treatment allows for platelet transfusion-free liver transplantation in pediatric thrombocytopenic patient with primary sclerosing cholangitis.

Thrombocytopenia is a major risk factor for cirrhotic liver disease. Patients with thrombocytopenia may have esophageal or gastric varices secondary to portal hypertension, leading to variceal bleeding which exposes the liver to further damage. Here, we present a female pediatric patient with PSC and CD, whose progressive thrombocytopenia was successfully controlled by romiplostim, a TPO receptor agonist. The patient developed bloody diarrhea at four yr of age, and was subsequently diagnosed with PSC and CD when seven yr old. While CD was well-controlled by immunomodulators, the patient's thrombocytopenia gradually progressed resulting in petechiae (platelet count of 11 × 10(9) /L) when she was 10 yr and four months old. She responded poorly to immunoglobulin and corticosteroids. Weekly subcutaneous injection of romiplostim was therefore initiated, and platelet counts were maintained over at 50 × 10(9) /L. She was able to undergo successful LDLT without platelet transfusion seven months after the initiation of romiplostim. Romiplostim was not required after LDLT with improved platelet counts. This case report suggests that romiplostim may be effective in the treatment of thrombocytopenic children with liver cirrhosis and portal hypertension, and in eliminating the need for platelet transfusion during the peri-transplant period.

Development of assay for determining free IgE levels in serum from patients treated with omalizumab.

BACKGROUND: Omalizumab, a monoclonal anti-IgE antibody, is currently indicated for the treatment of moderate-to-severe allergic asthma. To measure active IgE levels in sera from patients treated with omalizumab, the IgE subfraction in complex with omalizumab should be eliminated from total IgE, and free IgE levels can then be determined. With the aim of therapeutic monitoring for anti-IgE therapy, we developed a new ELISA for free IgE. METHODS: We used recombinant human soluble FcεRIα as a capture antigen and a biotinylated polyclonal anti-IgE antibody for detection. Using the newly developed ELISA, we measured the serum free IgE levels weekly in four asthmatic patients after their first omalizumab injection. We also measured the serum free IgE levels in 54 patients treated with omalizumab for over 4 weeks. RESULTS: This assay was technically robust, the mean recovery rate in serum was 93.16% ± 5.34%. For all patients, omalizumab treatment significantly reduced serum free IgE levels prior to the second omalizumab injection. To maintain the benefit of omalizumab, serum free IgE concentrations should be <50 ng/ml. However, in 14 of 54 patients treated with omalizumab for over 4 weeks, serum free IgE concentrations measured by our ELISA were >50 ng/ml. CONCLUSIONS: Our data suggest that the measurement of free IgE levels using our newly developed ELISA would be useful for monitoring serum free IgE levels during omalizumab therapy.

Validity study of the Japanese version of the Nijmegen Questionnaire for verifying dysfunctional breathing in Japanese asthma patients.

Background: Dysfunctional breathing (DB) is a clinical condition characterized by irregular breathing patterns presenting a sensation of dyspnea and a feeling of chest tightness. DB is a known comorbidity of asthma that is difficult to control, leading to poor quality of life, so early diagnosis and therapeutic intervention are essential to improve the clinical condition of asthma. The Nijmegen Questionnaire (NQ), developed to screen for DB and translated into various languages, is used worldwide. However, a Japanese NQ (JNQ) is unavailable, so DB has not been clinically verified in people with asthma in Japan. Objective: This study aimed to prepare a JNQ, verify its reliability and validity, and demonstrate its clinical benefits in asthma treatment. Methods: The JNQ was prepared by back-translating the NQ with the author's consent. The answers to self-administered questionnaires, including the JNQ, Asthma Control Test (ACT), Asthma Control Questionnaire (ACQ), Mini Asthma Quality of Life Questionnaire (Mini-AQLQ), and Patient Health Questionnaire 9 (PHQ-9), were obtained with the consent of 68 people with asthma (average age ± SD, 52.04 ± 12.43 years) who visited Nihon University Itabashi Hospital. The reliability of the JNQ was analyzed by the Cronbach alpha coefficient. A comparative test was conducted for each questionnaire (ACT, ACQ, Mini-AQLQ, PHQ-9), considering a JNQ score of 23 as the cutoff value. Patients with a score of 23 or more were assigned to the DB group, whereas patients with a score of less than 23 were assigned to the non-DB group. We analyzed the correlation between the JNQ and each questionnaire. Results: The JNQ showed sufficient reliability (Cronbach alpha = 0.875). Correlation analysis between the JNQ score and each questionnaire revealed negative correlations with the ACT score (r = 0.262) and Mini-AQLQ score (r = -0.453) and positive correlations with the ACQ score (r = 0.337) and PHQ-9 score (r = 0.539). All of these correlations were statistically significant. As a result of the comparative test, the DB and non-DB groups showed a significant difference in Mini-AQLQ (P = .023) and PHQ-9 (P = .003) scores. No significant difference was observed between ACT (P = .294) and ACQ (P = .177) scores. Conclusions: The JNQ validates DB in Japanese people with asthma and reflects the deterioration of asthma control, decreased quality of life, and depression. Using the JNQ, early diagnosis and therapeutic intervention (eg, breathing exercises and a psychosomatic approach) for DB in people with asthma may help suppress the severity of asthma in Japan.

Proteomic profiling of FFPE specimens: Discovery of HNRNPA2/B1 and STT3B as biomarkers for determining formalin fixation durations.

Recent advancements in proteomics technologies using formalin-fixed paraffin-embedded (FFPE) samples have significantly advanced biomarker discovery. Yet, the effects of varying sample preparation protocols on proteomic analyses remain poorly understood. We analyzed mouse liver FFPE samples that varied in fixatives, fixation duration, and storage temperature using LC/MS. We found that variations in fixation duration significantly affected the abundance of specific proteins, showing that HNRNPA2/B1 demonstrated a significant decrease in abundance in samples fixed for long periods, whereas STT3B exhibited a significant increase in abundance in samples fixed for long durations. These findings were supported by immunohistochemical analysis across liver, spleen, and lung tissues, demonstrating a significant decrease in the nuclear staining of HNRNPA2/B1 in long-duration acid formalin(AF)-fixed FFPE samples, and an increase in cytoplasmic staining of STT3B in long-duration neutral buffered formalin-fixed liver and lung tissues and granular staining in all long-duration AF-fixed FFPE tissue types. Similar trends were observed in the long-duration fixed HeLa cells. These results demonstrate that fixation duration critically affects the proteomic integrity of FFPE samples, emphasizing the urgent need for standardized fixation protocols to ensure consistent and reliable proteomic data. SIGNIFICANCE: The quality of FFPE samples is primarily influenced by the fixation and storage conditions. However, previous studies have mainly focused on their impact on nucleic acids and the extent to which different fixation conditions affect changes in proteins has not been evaluated. In addition, to our knowledge, proteomic research focusing on differences in formalin fixation conditions has not yet been conducted. Here, we analyzed FFPE samples with different formalin fixation and storage conditions using LC/MS and evaluated the impact of different fixation conditions on protein variations. Our study unequivocally established formalin fixation duration as a critical determinant of protein variation in FFPE specimens and successfully identified HNRNPA2/B1 and STT3B as potential biomarkers for predicting formalin fixation duration for the first time. The study findings open new avenues for quality assessment in biomedical research and diagnostics.

Predictive Model for Occurrence of Febrile Neutropenia after Chemotherapy in Patients with Diffuse Large B-Cell Lymphoma: A Multicenter, Retrospective, Observational Study.

Febrile neutropenia (FN) is a major concern in patients undergoing chemotherapy for diffuse large B-cell lymphoma (DLBCL); however, the overall risk of FN is difficult to assess. This study aimed to develop a model for predicting the occurrence of FN in patients with DLBCL. In this multicenter, retrospective, observational analysis, a multivariate logistic regression model was used to analyze the association between FN incidence and pretreatment clinical factors. We included adult inpatients and outpatients (aged ≥ 18 years) diagnosed with DLBCL who were treated with chemotherapy. The study examined 246 patients. Considering FN occurring during the first cycle of chemotherapy as the primary outcome, a predictive model with a total score of 5 points was constructed as follows: 1 point each for a positive hepatitis panel, extranodal involvement, and a high level of soluble interleukin-2 receptor and 2 points for lymphopenia. The area under the receiver operating characteristic curve of this model was 0.844 (95% confidence interval: 0.777-0.911). Our predictive model can assess the risk of FN before patients with DLBCL start chemotherapy, leading to better outcomes.

Case of an infant with hepatic cirrhosis caused by mitochondrial respiratory chain disorder.

The patient had hepatomegaly with liver dysfunction at the age of 1 month. Magnetic resonance imaging performed at the age of 1 year showed multiple nodules of varying size in his liver. We were able to examine the mitochondrial respiratory chain function in the liver biopsy samples because all other differential diagnoses for hepatic cirrhosis had been ruled out. Complex I and IV activities were below the normal level (<30%) of the citrate synthase (CS) ratio. Liver blue native polyacrylamide gel electrophoresis showed an extremely weak complex I and IV band. Liver respiratory chain complexes I and IV were found to be deficient in this patient. The histologic findings were highly suggestive of mitochondrial respiratory chain disorder. Findings of progressive liver cirrhosis changes were observed in magnetic resonance imaging at the age of 5 years. An examination of the mitochondrial respiratory chain function should be performed along with a liver biopsy if mitochondrial respiratory chain disorder is suspected as a possible differential diagnosis of idiopathic hepatitis.

Etiology of Severe Acute Hepatitis in a Pediatric Transplant Center in Japan.

The proportion of pediatric cases with severe acute hepatitis of unknown etiology in the coronavirus disease 2019 era was higher than that in the pre-coronavirus disease 2019 era in Japan's largest pediatric transplant center. Further research and monitoring are essential.

Comprehensive genetic analysis of histological components of combined small cell carcinoma.

BACKGROUND: Combined small-cell lung cancer (cSCLC) is a rare type of small-cell lung cancer (SCLC) that includes both SCLC and non-small-cell lung cancer (NSCLC). The molecular biological mechanisms underlying the heterogeneity of histological types in combined or metachronously transformed SCLC (mtSCLC) remain unclear. This study aimed to investigate the relationship between genetic alterations and each histological component heterogeneously detected in cSCLC and mtSCLC. METHODS: This study included four cSCLC cases and one mtSCLC case. Formalin-fixed and paraffin-embedded sections of each histological component of these tumors were subjected to next-generation sequencing (NGS) and quantitative reverse transcription-polymerase chain reaction to investigate the genetic mutations and expression levels of neuroendocrine cell-specific transcription factors (achaete-scute homolog-1 [ASCL1], brain-2 [BRN2] also known as POU domain class 3 transcription factor 2, nuclear factor 1 B [NF1B], insulinoma-associated protein 1 [INSM1], and thyroid transcription factor-1 [TTF-1]). RESULTS: NGS analysis revealed that SCLC and NSCLC components share the same somatic mutations detected most frequently in TP53, and also in RB1 and EGFR. Gene expression analysis showed ASCL1 expression was significantly lower in the NSCLC component than in the SCLC component. CONCLUSION: We conclude that the morphological evolution of heterogeneous histological components in cSCLC may be associated with differences in ASCL1 expression levels, but not in acquired somatic gene mutations.