Efficacy of T cell assays for the diagnosis of primary defects in cytotoxic lymphocyte exocytosis.

Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder associated with autosomal recessive variants in genes required for perforin-mediated lymphocyte cytotoxicity. A rapid diagnosis is crucial for successful treatment. Although defective cytotoxic T lymphocyte (CTL) function causes pathogenesis, quantification of natural killer (NK) cell exocytosis triggered by K562 target cells currently represents a standard diagnostic procedure for primary HLH. We have prospectively evaluated different lymphocyte exocytosis assays in 213 patients referred for evaluation for suspected HLH and related hyperinflammatory syndromes. A total of 138 patients received a molecular diagnosis consistent with primary HLH. Compared to routine K562 cell-based assays, assessment of Fc receptor-triggered NK-cell and T cell receptor-triggered CTL exocytosis displayed higher sensitivity and improved specificity for the diagnosis of primary HLH, with these assays combined providing a sensitivity of 100% and specificity of 98.3%. By comparison, NK-cell exocytosis following K562 target cell stimulation displayed a higher inter-individual variability, in part explained by differences in NK-cell differentiation or large functional reductions following shipment. We thus recommend combined analysis of T cell receptor-triggered CTL and Fc receptor-triggered NK-cell exocytosis for the diagnosis of patients with suspected familial HLH or atypical manifestations of congenital defects in lymphocyte exocytosis.

Diagnostic challenges of old diseases in the COVID-19 era: a report of two cases of carbamazepine-induced DRESS syndrome.

INTRODUCTION: A new MIS-C that develops after the acute stage of COVID-19 infection has recently been reported worldwide. Drug reaction with eosinophilia and systemic symptoms syndrome is a rare but potentially severe adverse drug-induced reaction most commonly associated with anticonvulsants. Due to variability in clinical presentation involving cutaneous and multiorgan systems, broad differential diagnosis, and lack of definitive diagnostic tests, diagnosis may be delayed. CASE REPORTS: The authors report 2 cases of pediatric patients who presented with fever, diffuse rash, and exposure to COVID-19 infection with suspected MIS-C. Both patients' medical histories revealed carbamazepine treatment for approximately 2 months. The diagnosis of DRESS syndrome was associated with the use of carbamazepine. CONCLUSIONS: Distinguishing between MIS-C and DRESS syndrome may be difficult due to similar clinical and laboratory features and the lack of definitive diagnostic tests for either condition. When encountering cases like the current report, it is important to consider DRESS syndrome for early diagnosis and medical intervention.

Effect of early atopic sensitization in children aged 0-2 years on the development of asthma symptoms at 9-11 years of age.

BACKGROUND: Personal genetic predisposition and early life environmental factors are important for the development of childhood asthma. We aimed to search whether egg, milk and mite sensitizations at 0-2 years old are risk factors for asthma symptoms at 9-11 years old. METHODS: A total of 210 wheezer children who had specific immunoglobulin (Ig) E in 2010-2012 were included in the study (followed by pediatric allergy). Patients were divided into non-atopic (group 1, n = 157) and atopic patients [groups 2-7, n = 53 (5 patients were in both group 4 and group 5)] based on sensitizations. Using the International Study of Asthma and Allergy in Childhood questionnaire, current wheeze (CW, 2nd question), exercise wheezing (EW, 7th question), and dry cough (DC, 8th question) were surveyed. Also, parental allergies, eczema at 0-2 years, current eosinophil percentage and total IgE were recorded. RESULTS: Eczema was observed as an important risk factor [CW: odds ratio (OR) = 2.83, 95% confidence interval (CI) = 1.54-5.23, P ≤ 0.001; EW: OR = 2.71, 95% CI = 1.33-5.54, P = 0.006; DC: OR = 3.03, 95% CI = 1.47-6.25, P = 0.003], whereas having no atopic sensitization at 0-2-year-old (group 1) was found as a significant protective factor for asthma at 9-11 years old (CW: OR = 0.32, 95% CI = 0.15-0.70, P = 0.004; EW: OR = 0.21, 95% CI 0.10-0.44, P ≤ 0.001; DC: OR = 0.25, 95% CI = 0.10-0.59, P = 0.002). CONCLUSION: Early personal eczema is a significant risk factor for the development of asthma symptoms at 9-11 years old, whereas not having an allergic sensitization at 0-2 years old (group 1) is an important protective factor.

Pulmonary function tests in the follow-up of children with COVID-19.

The SARS-CoV-2 virus has infected more than 235 million people since it was accepted as a pandemic in March 2020. Although a milder disease is seen in the pediatric age group, the extent of lung damage and its long-term effects are still unknown. In this study, persistent respiratory symptoms and pulmonary function tests were investigated in children with COVID-19. Fifty children with a confirmed diagnosis of COVID-19 were included in the study. Patients were evaluated for ongoing respiratory symptoms and pulmonary function tests 3 months after infection. Patients with and without persistent symptoms were compared in terms of demographic, clinical, laboratory, and radiological characteristics and also disease severity. Three months after infection, persistent respiratory symptoms were found to be present in 28% of patients; cough, chest pain and tightness, dyspnea, and exertional dyspnea were the most common symptoms. Three patients had an obstructive deficit, and one had a restrictive deficit. Four patients had impaired diffusing capacity of the lungs for carbon monoxide (DLCO). A significant decrease in FEV1/FVC and an increase in lung clearance index were found in the patients with persistent respiratory symptoms. Persistent respiratory symptoms were present in 50% of patients who had severe disease and 12.5% with non-severe disease. DLCO was also significantly lower in the severe disease group.   Conclusions: Our study suggests that the persistence of respiratory symptoms is not related to the severity of acute COVID-19 in children. The inflammatory process due to COVID-19 may continue regardless of its severity, and consequently, peripheral airways may be affected. What is Known: • As compared with adults, children with COVID-19 exhibit a milder disease course and lower mortality rates. However, due to the lack of follow-up studies on children, the long-term effects of their contracting the disease are unknown. What is New: • Although COVID-19 has been thought to have a milder course in children, respiratory system symptoms persist in approximately 30% of patients 3 months after infection. The persistent respiratory symptoms suggest that the inflammatory process due to COVID-19 may continue in some children, even if the clinical findings at admission are not severe, and that the peripheral airways may be affected accordingly.

Asthma and allergic diseases are not risk factors for hospitalization in children with coronavirus disease 2019.

BACKGROUND: Coronavirus disease 2019 (COVID-19) emerged as a pandemic toward the end of 2019, causing large numbers of people to become infected and die. OBJECTIVE: To determine whether allergic diseases are a risk factor for hospitalization in COVID-19. METHODS: We conducted a study including 107 pediatric patients after COVID-19 recovery. The International Study of Asthma and Allergies in Childhood Phase 3 questionnaires were distributed together with a detailed history of environmental factors and an allergic evaluation including skin prick tests, specific immunoglobulin E tests, and spirometry. We investigated the prevalence of allergic diseases and evaluated the factors associated with hospitalization in COVID-19. RESULTS: A total of 61 (57%) patients were hospitalized and 46 (43%) patients were followed closely in the outpatient clinic. The prevalences of allergic rhinitis, asthma, atopic dermatitis, and episodic wheezing were 10.3%, 6,5%, 4.7%, and 3.7%, respectively, within the whole study population. Although having asthma with or without allergic rhinitis, atopic dermatitis, and passive tobacco exposure were not found to be related to hospitalization because of COVID-19, having a pet at home was found to decrease the risk of hospitalization (odds ratio, 0.191; 95% confidence interval, 0.047-0.779; P = .02). Spirometry tests revealed a higher forced expiratory volume in one second to forced vital capacity ratio and a peak expiratory flow reversibility in hospitalized patients than in nonhospitalized ones (P = .02 and P = .003, respectively). CONCLUSION: Asthma and allergic diseases do not seem to be risk factors for hospitalization in children because of COVID-19, and having a pet at home can be a protective effect. Pulmonary function testing seems to be important for monitoring lung damage after COVID-19.

EuroFlow Standardized Approach to Diagnostic Immunopheneotyping of Severe PID in Newborns and Young Children.

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in RAG1 or RAG2 (n = 4, two of them presented with Omenn syndrome), IL2RG (n = 4, one of them with confirmed maternal engraftment), NHEJ1 (n = 1), CD3E (n = 1), ADA (n = 1), JAK3 (n = 3, two of them with maternal engraftment) and DCLRE1C (n = 1) and 11 other PID patients had diverse molecular defects [ZAP70 (n = 1), WAS (n = 2), PNP (n = 1), FOXP3 (n = 1), del22q11.2 (DiGeorge n = 4), CDC42 (n = 1) and FAS (n = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+HLA-DR+ and CD8+HLA-DR+ lymphocytes. Therefore, the EuroFlow SCID-RTE tube together with the previously published PIDOT tube form a sensitive and complete cytometric diagnostic test suitable for patients suspected of severe PID (SCID or CID) as well as for children identified via newborn screening programs for SCID with low or absent T-cell receptor excision circles (TRECs).

Homozygous c.130-131 ins A (pW44X) mutation in the HAX1 gene as the most common cause of congenital neutropenia in Turkey: Report from the Turkish Severe Congenital Neutropenia Registry.

BACKGROUND: Severe congenital neutropenia is a rare disease, and autosomal dominantly inherited ELANE mutation is the most frequently observed genetic defect in the registries from North America and Western Europe. However, in eastern countries where consanguineous marriages are common, autosomal recessive forms might be more frequent. METHOD: Two hundred and sixteen patients with severe congenital neutropenia from 28 different pediatric centers in Turkey were registered. RESULTS: The most frequently observed mutation was HAX1 mutation (n = 78, 36.1%). A heterozygous ELANE mutation was detected in 29 patients (13.4%) in our cohort. Biallelic mutations of G6PC3 (n = 9, 4.3%), CSF3R (n = 6, 2.9%), and JAGN1 (n = 2, 1%) were also observed. Granulocyte colony-stimulating factor treatment was given to 174 patients (80.6%). Two patients died with infectious complications, and five patients developed myelodysplastic syndrome/acute myeloblastic leukemia. The mean (± mean standard error) follow-up period was 129.7 ± 76.3 months, and overall survival was 96.8% (CI, 94.4-99.1%) at the age of 15 years. In Turkey, severe congenital neutropenia mostly resulted from the p W44X mutation in the HAX1 gene. CONCLUSION: In Turkey, mutation analysis should be started with HAX1, and if this is negative, ELANE and G6PC3 should be checked. Because of the very high percentage of consanguineous marriage, rare mutations should be tested in patients with a negative mutation screen.

The Role of Irisin, Insulin and Leptin in Maternal and Fetal Interaction

Objective: Insulin is an important hormone for intrauterine growth. Irisin is an effective myokine in the regulation of physiological insulin resistance in pregnancy. Leptin and insulin are associated with fetal growth and fetal adiposity. In this study, we aimed to investigate the relationships between irisin, insulin and leptin levels and maternal weight gain, as well as anthropometric measurements in the newborn. Methods: Eighty-four mothers and newborns were included in the study. Irisin, leptin and insulin levels were measured in the mothers and in cord blood. Anthropometric measurements in the newborn, maternal weight at the beginning of the pregnancy and at delivery were recorded. Results: Birth weight were classified as small for gestational age (SGA), appropriate for gestational age (AGA) and large for gestational age (LGA). There was no difference in irisin levels among the groups. Leptin and insulin levels were found to change significantly according to birth weight (p=0.013, and p=0.012, respectively). There was a negative correlation between the anthropometric measurements of the AGA newborns and irisin levels. This correlation was not observed in SGA and LGA babies. Leptin levels were associated with fetal adiposity. Conclusion: While irisin levels are not affected by weight gain during pregnancy nor by birth weight, they show a relationship with anthropometric measurements in AGA infants. These results may lead to the understanding of metabolic disorders that will occur in later life.

Differences in Granule Morphology yet Equally Impaired Exocytosis among Cytotoxic T Cells and NK Cells from Chediak-Higashi Syndrome Patients.

Chediak-Higashi syndrome (CHS) is caused by autosomal recessive mutations in LYST, resulting in enlarged lysosomal compartments in multiple cell types. CHS patients display oculocutaneous albinism and may develop life-threatening hemophagocytic lymphohistiocytosis (HLH). While NK cell-mediated cytotoxicity has been reported to be uniformly defective, variable defects in T cell-mediated cytotoxicity has been observed. The latter has been linked to the degree of HLH susceptibility. Since the discrepancies in NK cell- and T cell-mediated cellular cytotoxicity might result from differences in regulation of cytotoxic granule release, we here evaluated perforin-containing secretory lysosome size and number in freshly isolated lymphocytes from CHS patients and furthermore compared their exocytic capacities. Whereas NK cells from CHS patients generally contained a single, gigantic perforin-containing granule, cytotoxic T cells predominantly contained several smaller granules. Nonetheless, in a cohort of 21 CHS patients, cytotoxic T cell and NK cell granule exocytosis were similarly impaired upon activating receptor stimulation. Mechanistically, polarization of cytotoxic granules was defective in cytotoxic lymphocytes from CHS patients, with EEA1, a marker of early endosomes, mislocalizing to lysosomal structures. The results leads to the conclusion that lysosome enlargement corresponds to loss of distinct organelle identity in the endocytic pathway, which on a subcellular level more adversely affects NK cells than T cells. Hence, vesicular size or numbers do not per se dictate the impairment of lysosomal exocytosis in the two cell types studied.

Increased plasma soluble human leukocyte antigen-G in persistent wheezy infants.

BACKGROUND: Human leukocyte antigen (HLA)-G is a non-classical major histocompatibility complex class I antigen characterized by limited polymorphism in its coding region, unique tissue expression pattern in physiologic conditions and immunomodulatory properties. Recently, the level of soluble (s)HLA-G was found to be higher in atopic asthma and allergic rhinitis, but this remains to be clarified in wheezy infants. The aim of the present study was therefore to investigate sHLA-G in wheezy infants. METHODS: The subjects consisted of infants with persistent wheezing and positive modified asthma predictive index (mAPI; n = 30; persistent group) and those with transient wheezing and negative mAPI (n = 17; transient group). sHLA-G was measured in plasma using enzyme-linked immunosorbent assay. Total immunoglobulin E (IgE) and eosinophil count were measured, and skin testing was performed with a battery of 13 antigens with appropriate positive and negative controls. RESULTS: sHLA-G was significantly higher in the persistent wheezing (positive mAPI) group compared with the transient wheezing (negative mAPI) group (P = 0.008). There was no significant difference in peripheral blood eosinophil count and total IgE between the groups. CONCLUSIONS: The increased sHLA-G in infants with persistent wheeze suggests that sHLA-G may be able to be used to distinguish persistent from transient wheeze. Further comprehensive studies are needed on this topic.

DOCK8 deficiency in a boy who presented with a giant aortic aneurysm between aortic root and iliac bifurcation.

Dedicator of cytokinesis 8 protein (DOCK8) deficiency is an autosomal recessive, inherited form of hyper-immunoglobulin E (hyper-IgE) syndrome, characterized by persistent cutaneous viral infections, elevated IgE, eosinophilia, and allergic manifestations. The case of a 10-year-old boy who presented with giant aortic aneurysm between the aortic root and iliac bifurcation is described in the present report. Aortic aneurysm of this size has not yet been reported.

Plasma glutamine and cystine are decreased and negatively correlated with endomysial antibody in children with celiac disease.

BACKGROUND AND OBJECTIVES: Glutamine is a nonessential amino acid which improves intestinal mucosal regeneration and absorption. Glutathione is a vital molecule for antioxidant reactions and is synthesized from cystine. The first aim of the study is to measure the plasma glutamine and cystine in children with celiac disease (CD) and compare them with controls. The second aim of this study is to investigate whether these amino acids are correlated with endomysial antibody (EMA) or not. METHODS AND STUDY DESIGN: Fifty children with CD were compared to 50 healthy, age, and sex matched normal children as control. Plasma glutamine and cystine levels of the children were measured by using tandem mass spectrometry. RESULTS: Plasma glutamine (808 vs 870 µmol/L) and cystine (19 vs 48.5 µmol/L) were significantly lower in the celiac group than the controls (p<0.05). The levels of plasma glutamine (797 vs 928 µmol/L, n=42) and cystine (18 vs 31.5 µmol/L, n=8) were lower (p<0.05) in the EMA-positive than the EMA-negative celiac patients. We could not find any statistically significance between EMA-negative celiac patients and controls for the plasma glutamine (928 vs 870 µmol/L) and cystine (31.5 vs 48.5 µmol/L) (p>0.05). Serum EMA was negatively correlated with plasma cystine (r=-0,321, p=0.023), glutamine (r=-0.413, p=0.003). CONCLUSIONS: Our study indicated that plasma glutamine and cystine were significantly lower in the celiac children than the controls. Also, these amino acids were negatively correlated with EMA.

XLF deficiency results in reduced N-nucleotide addition during V(D)J recombination.

Repair of DNA double-strand breaks (DSBs) by the nonhomologous end-joining pathway (NHEJ) is important not only for repair of spontaneous breaks but also for breaks induced in developing lymphocytes during V(D)J (variable [V], diversity [D], and joining [J] genes) recombination of their antigen receptor loci to create a diverse repertoire. Mutations in the NHEJ factor XLF result in extreme sensitivity for ionizing radiation, microcephaly, and growth retardation comparable to mutations in LIG4 and XRCC4, which together form the NHEJ ligation complex. However, the effect on the immune system is variable (mild to severe immunodeficiency) and less prominent than that seen in deficiencies of NHEJ factors ARTEMIS and DNA-dependent protein kinase catalytic subunit, with defects in the hairpin opening step, which is crucial and unique for V(D)J recombination. Therefore, we aimed to study the role of XLF during V(D)J recombination. We obtained clinical data from 9 XLF-deficient patients and performed immune phenotyping and antigen receptor repertoire analysis of immunoglobulin (Ig) and T-cell receptor (TR) rearrangements, using next-generation sequencing in 6 patients. The results were compared with XRCC4 and LIG4 deficiency. Both Ig and TR rearrangements showed a significant decrease in the number of nontemplated (N) nucleotides inserted by terminal deoxynucleotidyl transferase, which resulted in a decrease of 2 to 3 amino acids in the CDR3. Such a reduction in the number of N-nucleotides has a great effect on the junctional diversity, and thereby on the total diversity of the Ig and TR repertoire. This shows that XLF has an important role during V(D)J recombination in creating diversity of the repertoire by stimulating N-nucleotide insertion.

Targeted high-throughput sequencing for genetic diagnostics of hemophagocytic lymphohistiocytosis.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a rapid-onset, potentially fatal hyperinflammatory syndrome. A prompt molecular diagnosis is crucial for appropriate clinical management. Here, we validated and prospectively evaluated a targeted high-throughput sequencing approach for HLH diagnostics. METHODS: A high-throughput sequencing strategy of 12 genes linked to HLH was validated in 13 patients with previously identified HLH-associated mutations and prospectively evaluated in 58 HLH patients. Moreover, 2504 healthy individuals from the 1000 Genomes project were analyzed in silico for variants in the same genes. RESULTS: Analyses revealed a mutation detection sensitivity of 97.3%, an average coverage per gene of 98.0%, and adequate coverage over 98.6% of sites previously reported as mutated in these genes. In the prospective cohort, we achieved a diagnosis in 22 out of 58 patients (38%). Genetically undiagnosed HLH patients had a later age at onset and manifested higher frequencies of known secondary HLH triggers. Rare, putatively pathogenic monoallelic variants were identified in nine patients. However, such monoallelic variants were not enriched compared with healthy individuals. CONCLUSIONS: We have established a comprehensive high-throughput platform for genetic screening of patients with HLH. Almost all cases with reduced natural killer cell function received a diagnosis, but the majority of the prospective cases remain genetically unexplained, highlighting genetic heterogeneity and environmental impact within HLH. Moreover, in silico analyses of the genetic variation affecting HLH-related genes in the general population suggest caution with respect to interpreting causality between monoallelic mutations and HLH. A complete understanding of the genetic susceptibility to HLH thus requires further in-depth investigations, including genome sequencing and detailed immunological characterization.

Three faces of recombination activating gene 1 (RAG1) mutations.

Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation.

A patient developing anaphylaxis and sensitivity to two different GnRH analogues and a review of literature.

Gonadotropin-releasing hormone analogues are used in the treatment of prostate cancer, breast cancer, endometriosis, and uterine leiomyomas in adults and often in the treatment of precocious puberty in children. Many adverse effects have been reported for gonadotropin-releasing hormone analogues, but anaphylaxis is rarely reported as an adverse effect. Frequent cross-reactions, particularly during childhood, and diversity of the time of onset of anaphylactic manifestations complicate the diagnosis. A patient who exhibited anaphylactic allergic reactions to two different agents used in the treatment of central precocious puberty presented here because the case has an atypical course and is the first in the literature.