IKBKG is the essential modulator for nuclear factor-κB(NF-κB) signaling pathway, and mutations within this gene can lead to anhidrotic ectodermal dysplasia and immunodeficiency (EDA-ID). Here we report a male patient, who presented with mild frontal bossing, sparse hair, skin pigmentation, conical teeth, and recurrent infections involving bacteria, fungi, and viruses after one month of age, together with hypogammaglobulinemia. These symptoms were consistent with the phenotype of EDA-ID. Genetic analysis revealed a hemizygous mutation c.1249T > G (p.Cys417Gly) in exon 10 of the IKBKG gene in the patient. The mother of the patient was identified as heterozygous carriers of the same mutation. Immunological assessment revealed a significant reduction in memory B cells. The patient showed no skewed TCR diversity. PHA-induced T-cell proliferation was impaired. IFN-γ secretion by Th cells was significantly reduced after PHA stimulation. IκBα degradation rate retained the same in the patient. We summarized the clinical features of the patient and conducted immunological analysis, in order to increase pediatricians′awareness of this rare disease. For boys with early-onset recurrent infections together with ectodermal dysplasia, IKBKG mutation should be considered. In addition, genetic analysis is needed to exclude pseudogene interference and functional evaluations are required.

Objective:To investigate the clinical, cytomorphology, immunocytochemical and molecular features of metastatic melanoma in serosal cavity effusion.Methods:Cytological specimens of 14 patients with melanoma in the chest and abdomen were collected from 2017 to 2023, at the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. SOX10, S-100 protein, PRAME, BRAF V600E, HMB45, and Melan A were detected by immunocytochemical methods. Fourteen cases were tested for routine antibody combinations, including Claudin4, HEG1, Calretinin, CD68, etc. Four of the patients had biopsy or surgical samples of metastatic solid lesions of primary sites, and further next-generation sequencing (NGS) or amplification refractory mutation system (ARMS)-PCR molecular test was performed. In addition, 30 cases of serosal effusion samples were collected as control groups (10 cases of benign mesothelial cell reactive hyperplasia, 10 cases of mesothelioma, and 10 cases of metastatic lung adenocarcinoma).Results:Among the 14 cases of melanoma, there were 7 males and 7 females, with ages ranging from 35 to 86 years, and an average age of 57 years, there 10 cases aged ≥50 years. The tumor cells in the serosal effusion varied in morphology and degree of atypia. SOX10 was positive in all 14 cases (14/14), S-100 protein was positive in 10 cases (10/14), PRAME was positive in 12 cases (12/14), BRAF V600E was positive in 10 cases (10/14), HMB45 was positive in 12 cases (12/14), and Melan A was positive in 13 cases (13/14). In 4 patients with histological correlation, the cytological and histological expression of SOX10, BRAF V600E, and PRAME was positive in all 4 cases (4/4); S-100 protein was positive in 2 cases (2/4); and HMB45 and Melan A were positive in 3 cases (3/4). Using NGS or ARMS-PCR, missense mutations of BRAF V600E were detected in all 4 patients; TERT promoter mutations was detected in 1 case; and CDKN2A terminating mutations and MSI1 deletion mutations were detected in the other case. SOX10, S-100, HMB45, Melan A, PRAME and BRAF V600E were all negative in 30 control samples of serosal cavity effusion.Conclusion:By observing the morphology of tumor cells, immunocytochemical test of several combination markers, especially the expression of SOX10, BRAF V600E and PRAME, can help to improve the positive diagnosis rate of melanoma in serous cavity effusion.

Objective:To explore the clinical characteristics and medical nutritional therapy of 6 patients with late-onset ornithine transcarbamylase (OTC) deficiency.Methods:The clinical features, biochemical data, gene variations and treatment outcomes of 6 children with late-onset OTC deficiency admitted to the Department of Clinical Nutrition, Children′s Hospital of Nanjing Medical University from January 2020 to April 2022 were retrospectively analyzed.The 6 patients were all intervened by a long-term medical nutrition management.Results:Liver dysfunction and hyperammonemia (172.1-348.0 μmol/L) were found in all the 6 children with late-onset OTC deficiency.Serum citrulline decreased in 3 patients (3.95-5.43 μmol/L). Three patients showed increased urine orotic acid (123.48-342.60 mmol/mol Cr). Urine uracil increased in 4 patients (106.77-1 207.26 mmol/mol Cr). Variations of the OTC gene [c.364G>C p. (E122Q), c.1028C>G p. (T343R), c.664-2(IVS6)A>C, c.635G>T p. (G212V), c.929_c.931delAAG p. (E310del), c.829C>T p. (R277W)] were identified in all patients.The 6 children were all managed by individualized medical nutrition program and followed up for a long time.During the follow-up period, 3 cases developed hypoproteinemia, acute metabolic crisis and growth retardation, 3 cases had normal growth and laboratory indicators, and 1 case received liver transplantation after 3 months of nutritional management. Conclusions:The clinical manifestations of OTC deficiency are non-specific.Blood amino acids, urine organic acids and genetic tests are important for the diagnosis.Long-term regular medical nutrition management is helpful to improve the prognosis and quality of life of children.

Atopic diseases used to be considered as complex polygenic diseases with the interaction of environmental factors and genetic susceptibility.In recent years, primary atopic diseases caused by single-gene mutations have been well concerned.This study aims to review the pathogenesis and clinical characteristics of atopic diseases, thus strengthening the understanding.

Atopic dermatitis is a complex chronic, relapsing inflammatory skin disease.Atopic dermatitis in children is usually less severe than in adults, but it is with a high incidence and is susceptible to relapse.Therefore, the physical and mental health of children and their family maybe seriously affected.In the past, the treatments of atopic dermatitis have been limited to glucocorticoids and immunosuppressants.It is unsafe for children because of their toxicities.With the in-depth understanding of pathogenesis, more and more new therapies that focus on intervening in the inflammatory pathway by targeting specific cytokines or their receptors have been found and applied.This article reviews the progress of treatment of the disease to provide new insights for the optimal treatment of atopic dermatitis.

Objective:To study the pathological changes of the spleen in patients with COVID-19 and to analyze the relationship between the weakened immune system and splenic lesions.Methods:Postmortem needle autopsies from the spleen were carried out on 10 patients who died from COVID-19 in Wuhan. Routine hematoxylin and eosin (HE) staining was used to observe the pathological changes. The changes of lymphocytes were studied further with immunohistochemistry.RT-PCR was used to detect 2019-nCoV RNA in the spleen. In addition,the Epstein-Barr virus (EBV) was detected by in situ hybridization, and coronavirus particles were detected by transmission electron microscopy in 2 cases.Results:There were 7 males and 3 females, with an average age of 68.3 years.Of the 10 cases, 4 had cancer history and another 4 had other underlying diseases respectively.Cough, fever, malaise and dyspnea were the main clinical symptoms.The time from onset to death was 15-45 days.Ten cases patients had normal or slight increase in peripheral blood leukocyte count in the early stage of the disease, 6 cases had significant increase before death. Five patients′ peripheral blood lymphocyte count decreased in the early stage of the disease, and 10 patients′ peripheral blood lymphocyte count decreased significantly before the disease progressed or died. Seven cases were treated with corticosteroid (methylprednisolone ≤40?mg/d, not more than 5 days). Histopathological examination showed that the cell composition of the spleen decreased, white pulp atrophied at different levels, meanwhile lymphoid follicles decreased or absent;in addition, the ratio of red pulp to white pulp increased with varying degrees. In 7 cases, more neutrophil infiltration was found, and in 5 cases, scattered plasma cell infiltration was found. Macrophage proliferation and hemophagocytic phenomena in a few cells were found in a case. Meanwhile, necrosis and lymphocyte apoptosis were detected in 2 cases, small artery thrombosis and spleen infarction in 1 case, and fungal infection in 1 case. The results of immunohistochemistry showed that the T and B lymphocyte components of the spleen in all cases decreased in varying degrees. CD20 + B cells were found to accumulate in the lymphoid sheath around the splenic artery in 8 cases. However, CD20 and CD21 immunostaining in 2 cases showed that the number of white pulp was almost normal, and splenic nodules were atrophic. CD3 +, CD4 + and CD8 +T cells were decreased. In 9 cases,CD68 + macrophages were no significant changes in the distribution and quantity. While more CD68 + cells were found in the medullary sinuses of 1 case (related to fungal infection). Few CD56 + cells were found. EBV was negative by in situ hybridization. RT-PCR was used to detect the nucleic acid of 2019-nCoV. One of 10 cases was positive, 39 years old,who was the youngest patient in this group, and the other 9 cases were negative. Coronavirus particles were found in the cytoplasm of macrophage under electron microscope in 2 cases. Conclusions:The death of COVID-19 occurs mainly in the elderly, and some cases have no underlying diseases. Spleen may be one of the organs directly attacked by the virus in some patients who died from COVID-19. T and B lymphocyte in the spleen decrease in varying degrees, lymphoid follicles are atrophied, decreased or absent, and the number of NK cells do not change significantly. And the pathological changes of the spleen are not related to the use of low dose corticosteroid, which may be related to the direct attack of virus and the attack of immune system on its own tissues.

Objective: To understand the consistency of ALK Ventana-D5F3 immunohistochemistry (IHC) interpretation in Chinese lung adenocarcinoma among histopathologists from different hospitals, and to recommend solution for the problems found during the interpretation of ALK IHC in real world, with the aim of the precise selection of patients who can benefit from ALK targeted therapy. Methods: This was a multicenter and retrospective study. A total of 109 lung adenocarcinoma cases with ALK Ventana-D5F3 IHC staining were collected from 31 lung cancer centers in RATICAL research group from January to June in 2018. All cases were scanned into digital imaging with Ventana iSCANcoreo Digital Slide Scanning System and scored by 31 histopathologists from different centers according to ALK binary (positive or negative) interpretation based on its manufacturer′s protocol. The cases with high inconsistency rate were further analyzed using FISH/RT-PCR/NGS. Results: There were 49 ALK positive cases and 60 ALK negative cases, confirmed by re-evaluation by the specialist panel. Two cases (No. 2302 and No.2701) scored as positive by local hospitals were rescored as negative, and were confirmed to be negative by RT-PCR/FISH/NGS. The false interpretation rate of these two cases was 58.1% (18/31) and 48.4% (15/31), respectively. Six out of 31 (19.4%) pathologists got 100% accuracy. The minimum consistency between every two pathologists was 75.8%.At least one pathologist gave negative judgement (false negative) or positive judgement (false positive) in the 49 positive or 60 negative cases, accounted for 26.5% (13/49), 41.7% (25/60), respectively, with at least one uncertainty interpretation accounted for 31.2% (34/109). Conclusion There are certain heterogeneities and misclassifications in the real world interpretation of ALK-D5F3 IHC test, which need to be guided by the oncoming expert consensus based on the real world data.

Objective: To study the application of copy number variation (CNV) analysis based on the raw data of next-generation sequencing (NGS) in diagnosing primary immunodeficiency disease (PID). Methods: One hundred sixty-five patients with suspicious PID were tested by NGS in the Department of Rheumatology and Immunology, Shenzhen Children′s Hospital during September 2014 and Mary 2017. The raw data of the patients who got negative result were further analyzed for the CNV with CNVkit software. The pathogenic CNV were identified in the databases including Resource of Asian Primary Immunodeficiency Diseases (RAPID), Human Gene Mutation Database (HGMD) and ClinVar with the known 344 pathogenic genes of PID. The associated literature from January 2010 to May 2019 were searched in Pubmed, Weip, Wanfang and CNKI database with key words as "primary immunodeficiency disease" "copy number variation" and "next generation sequencing" . Results: Ninety-five out of 165 patients (57.6%) had negative result of the NGS test, among whom the patients with immune dysregulation had the highest negative rate (68.6%, 24/35). CNV analysis found large fragment deletion in 12 patients, within which 7 was X-linked inheritance, 3 was autosomal recessive inheritance, 2 was autosomal dominant inheritance. Partial exon deletion was found in 4 patients while whole gene deletion in 8 patients. According to the review of literature, CNV was reported in 51 pathogenic genes of PID (14.8%, 51/344) , mainly intern deletion (70.6%, 36/51), while autosomal recessive inheritance (56.9%, 29/51) was the most common pattern. Conclusions CNV is not rare in PID. When the phenotype is clear in the patients who have negative NGS test, CNV should be considered.

Objective: To evaluate the role of mitochondrion-dependent apoptosis in reduction of bupivacaine-induced cardiotoxicity by lipid emulsion in rats. Methods: Forty-five healthy adult male Sprague-Dawley rats, weighing 300-350 g, were divided into 3 groups by a random number table method: sham operation group (Sham group, n=5), bupivacaine group (B group, n=20), and lipid emulsion group (L group, n=20). Cardiac arrest was induced by intravenously injecting 0.4% bupivacaine 30 mg/kg over 20 s to establish the cardiotoxicity model.Twenty percent lipid emulsion was intravenously injected in a dose of 5 ml/kg during resuscitation in group L, and normal saline was intravenously injected in a loading dose of 5 ml/kg during resuscitation in group B, followed by a 3-min infusion of 1 ml·kg^-1·min^-1in two groups.The successful resuscitation and survival rate at 120 min of return of spontaneous circulation (ROSC) were recorded.Systolic blood pressure, heart rate, mean arterial pressure, rate-pressure product (RPP) and ratio of RPP at each time point after recovery of spontaneous heart beat to baseline value (RPPh) were recorded every 10 min after ROSC.The time from administration to cardiac arrest (T₀), time from beginning of cardiopulmonary resuscitation to appearance of the first spontaneous heart beat (T_s) and time from beginning of cardiopulmonary resuscitation to appearance of ROSC (T_r) were recorded.Rats were sacrificed at 120 min of ROSC, and left ventricular tissues were obtained for determination of the expression of Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3, cytochrome C (Cyt c) in cytoplasm and mitochondria (by Western blot) and expression of Bax and Bcl-2 mRNA (by real-time polymerase chain reaction) and for examination of myocardial ultrastructure. Results: Compared with Sham group, the expression of Bcl-2 protein and mRNA and mitochondrial Cyt c was significantly down-regulated, and the expression of Bax protein and mRNA, cleaved caspase-9, cleaved caspase-3 and cytoplasmic Cyt c was up-regulated in B group (P<0.05). Compared with B group, the rate of successful resuscitation and survival rate were significantly increased, T_r was shortened, systolic blood pressure, heart rate, RPP and RPPh were increased after ROSC, the expression of Bcl-2 protein and mRNA and mitochondrial Cyt c was up-regulated, the expression of Bax protein and mRNA, cleaved caspase-9, cleaved caspase-3 and cytoplasmic Cyt c was down-regulated (P<0.05), no significant change was found in T_o or T_s (P>0.05), and the pathological changes of myocardium were significantly attenuated in L group. Conclusion The mechanism by which lipid emulsion reduces bupivacaine-induced cardiotoxicity may be related to inhibiting mitochondrion-dependent apoptosis in rats.

Objective To analyze the clinical,radiological features and risk factors of diplopia in patients with acute lacunar infarction (ALI).Methods Retrospectively retrieved patients of ALI (lesion diameter was less than 1.5 cm in DWI sequence)diagnosed by MR and clinical.We further summarized 13 ALI patients with diplopia and randomly selected 13 ALI patients without diplopia as the control group. SPSS22.0 statistical software was used for statistical analysis.The general clinical data such as sex and age was compared by Ch-i square test and t-test.The risk factors were primarily analyzed by one-way ANOVA and then the risk factors with statistical significance were brought into the logistic regression model for multivariate analysis.Results The incidence of diplopia in ALI patients was about 2.7%(13/489). The infarct sites were all located in the brain stem of the oculomotor-related brain nucleus and the dorsolateral medulla oblongata.Hypertension and hematocrit were negatively correlated with diplopia after infarction (P＜0.05 ).Conclusion The incidence of diplopia is low in ALI patients.The medial longitudinal tract of the dorsolateral medulla is an important area causing diplopia.Hypertension and hematocrit are non-risk factors for diplopia after ALI.