Defective Treg generation and increased type 3 immune response in leukocyte adhesion deficiency 1.

In 15 Turkish LAD-1 patients and controls, we assessed the impact of pathogenic ITGB2 mutations on Th17/Treg differentiation and functions, and innate lymphoid cell (ILC) subsets. The percentage of peripheral blood Treg cells, in vitro-generated induced Tregs differentiated from naive CD4+ T cells were decreased despite the elevated absolute counts of CD4+ cells in LAD-1 patients. Serum IL-23 levels were elevated in LAD-1 patients. Post-curdlan stimulation, LAD-1 patient-derived PBMCs produced more IL-17A. Additionally, the percentages of CD18-deficient Th17 cells expanded from total or naïve CD4+ T cells were higher. The blood ILC3 subset was significantly elevated in LAD-1. Finally, LAD-1 PBMCs showed defects in trans-well migration and proliferation and were more resistant to apoptosis. Defects in de novo generation of Tregs from CD18-deficient naïve T cells and elevated Th17s, and ILC3s in LAD-1 patients' peripheral blood suggest a type 3-skewed immunity and may contribute to LAD-1-associated autoimmune symptoms.

A Novel Deletion in FERMT3 Causes LAD-III in a Turkish Family.

Leukocyte adhesion deficiency-III (LAD-III) is an extremely rare autosomal recessive syndrome caused by mutations in FERMT3, the gene encoding kindlin-3. The genetic alterations in this gene lead to abnormal expression or activity of kindlin-3 in leukocytes and platelets. Kindlin-3 acts as an important regulator of integrin activation. LAD-III has features of the bleeding syndrome of Glanzmann and also of leukocyte adhesion deficiency. In this study, we report on two families, one of Turkish and one of Syrian origin, with clinical features of LAD-III, loss of kindlin-3 protein expression, and a functional leukocyte defect. A novel, homozygous deletion in FERMT3 (c.921delC, p.Ser307Argfs*21) was found in the Turkish patient. The parents were carriers of the mutation, consistent with an autosomal recessive inheritance. A common c.1525C > T (p.Arg509*) mutation was found in the Syrian patient. In conclusion, beside the variant c.1525C > T in the FERMT3 gene, which was previously found in more than 15 patients in Anatolia, our study is the first to identify the novel homozygous variant c.921delC in the FERMT3 gene.

Flow cytometric analysis of platelet surface glycoproteins in the diagnosis of thirty-two Turkish patients with Glanzmann thrombasthenia: a multicenter experience

Background/aim: Glanzmann thrombasthenia (GT) is a rare autosomal recessively inherited bleeding disorder characterized by the quantitative (type 1 and type 2) or qualitative (type 3) deficiency in platelet membrane glycoprotein (GP) IIb/IIIa (CD41a/CD61) fibrinogen receptors. In type 1, 2, and 3, CD41a/CD61 expression is 5%, 5%­20% and above 20%, respectively. In this study, diagnosis of GT was confirmed and subgroups were identified in 32 Turkish patients by flow cytometry analysis. Materials and methods: CD41a/CD61 expression levels in platelet-rich plasma (PRP) obtained from peripheral venous EDTA blood samples were analyzed with a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). GT subgroup analysis was performed by counting 50,000 events in the BD FACSDiva Software v6.1.3 program of the instrument. Results: In the present study, in blood samples of 32 patients from 23 families with GT and 22 healthy controls, co-expression levels of CD41a and CD61 in PRP was analyzed. 12 out of 23 families were consistent with type 1 GT (52.2%), 4 were consistent with type 2 GT (17.4%), and 7 were consistent with type 3 GT (30.4%). Conclusion: Especially due to consanguineous marriages, GT with various glycoprotein levels may be detected. As a result of the flow cytometry analysis of the present study with the highest GT patient population in Turkey, type 1 GT patients were the most common subgroup. In the determination of the GT subgroups; especially in the detection of type 3 GT, flow cytometry is the most sensitive glycoprotein analysis method. In addition to light transmission aggregometry, CD41a/CD61 study by flow cytometer confirms diagnosis when mutation analysis cannot be performed.

Genetic Characteristics, Infectious, and Noninfectious Manifestations of 32 Patients with Chronic Granulomatous Disease.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare genetic disorder characterized by failure of phagocytic leukocytes to destroy certain microbes. We present a study on CGD patients enrolled at a single medical center concerning the infectious and noninfectious complications and genetic properties of the disease. METHODS: Icotinamide adenine dinucleotide phosphate oxidase activity and the expression of flavocytochrome b558 were measured by flow cytometry, and clinical outcomes of the patients were listed in relation to the genetic results. RESULTS: The clinical and genetic findings of 32 pediatric cases with CGD from 23 families were enrolled. Pneumonia and anemia were the most common infectious and noninfectious symptoms. Genetic analysis showed that 10 families (43.5%) carried CYBB variants and 13 families (56.5%) have autosomal recessive (AR) CGD, in which 6 families (26%) carried NCF1 variants, 4 (17.4%) carried CYBA variants, and 3 (13%) carried NCF2 variants. The median age of clinical onset was 3.3 and 48 months for patients with X-linked CGD (X-CGD) and AR-CGD, respectively. The onset of symptoms before age 1 year was 94% in X-CGD, 28.5% in AR-CGD, and 12.5% in patients with oxidase residual activity. Moreover, a de novo germline mutation at c.1415delG in CYBB (OMIM#300481) and a novel c.251_263del13bp in CYBA (OMIM#608508) were also investigated. CONCLUSIONS: Ihydrorhodamine-1,2,3 assay could not detect carrier mother in de novo case with CYBB variant. Most X-CGD patients have the onset of symptoms before age 1 year. Additionally, residual oxidase activity in AR-CGD causes a delay in onset, diagnosis, and prophylaxis. The protective role of residual activity is limited while the infection is ongoing and becoming serious.

Tuberculosis masked by immunodeficiency: a review of two cases diagnosed with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a genetically heterogeneous primary immunodeficiency that is characterized by recurrent and life-threatening infections resulting from defects in phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system and granuloma formation due to increased inflammatory response. The most commonly involved organs are the lungs, skin, lymph nodes, and liver due to infection. It may present with recurrent pneumonia, hilar lymphadenopathy, empyema, abscess, reticulonodular patterns, and granulomas due to lung involvement. In recent years, mycobacterial disease susceptibility has been reported in CGD cases. This article presents two male cases, one of whom is aged 18 months and the other is aged 5 years, who were diagnosed with CGD and tuberculosis during examination due to extended pneumonia. This report is presented because CGD should be considered not only in the presence of skin abscesses and Aspergillus infections, but also in the differential diagnosis for cases with BCG-itis and/or tuberculosis. It should be kept in mind that mycobacterial infections can occur during the course of the disease.

Clinical, functional, and genetic characterization of chronic granulomatous disease in 89 Turkish patients.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder of phagocytes resulting in impaired killing of bacteria and fungi. A mutation in one of the 4 genes encoding the components p22(phox), p47(phox), p67(phox), and p40(phox) of the leukocyte nicotinamide dinucleotide phosphate reduced (NADPH) oxidase leads to autosomal recessive (AR) CGD. A mutation in the CYBB gene encoding gp91(phox) leads to X-linked recessive CGD. OBJECTIVE: The aim of this study is to show the correlation between clinical, functional, and genetic data of patients with CGD from Turkey. METHODS: We report here the results of 89 patients with CGD from 73 Turkish families in a multicenter study. RESULTS: Most of the families (55%) have an AR genotype, and 38% have an X-linked genotype; patients from 5 families with a suspected AR genotype (7%) were not fully characterized. We compared patients with CGD according to the severity of NADPH oxidase deficiency of neutrophils. Patients with A22(0), A67(0) or X91(0) phenotypes with a stimulation index of 1.5 or less have early clinical presentation and younger age at diagnosis (mean, 3.2 years). However, in p47(phox)-deficient cases and in 5 other AR cases with high residual oxidase activity (stimulation index ≥ 3), later and less severe clinical presentation and older age at diagnosis (mean, 7.1 years) were found. Pulmonary involvement was the most common clinical feature, followed by lymphadenitis and abscesses. CONCLUSION: Later and less severe clinical presentation and older age at diagnosis are related to the residual NADPH oxidase activity of neutrophils and not to the mode of inheritance. CGD caused by A22(0) and A67(0) subtypes manifests as severe as the X91(0) subtype.

Chronic granulomatous disease: A single-center experience in Central Anatolia.

BACKGROUND: Chronic granulomatous disease (CGD), one of the phagocytic cell defects, is the primary immunodeficiency caused by dysfunction of the NADPH oxidase complex in neutrophils. METHODS: The clinical, demographic and laboratory findings of 17 CGD patients who were followed-up between 2002 and 2021 were obtained retrospectively from the records of the patients. RESULTS: The number of male and female patients was 10/7. The median age at diagnosis was 5.3 months (range 4-120) for 3 patients with X-CGD, and 42.4 months (range 8-350) for 14 patients with AR-CGD. We have investigated rare CYBA exon 3-6 deletion in 7 patients and hotspot mutation with delGT at the beginning of exon 2 of NCF1 in 5 patients. The most common clinical findings were pneumonia and lymphadenitis with recurrent fever, respectively (41.2%, 35.3%). A total of 154 microbial infections requiring hospital admission (27 in 3 XL and 127 in 14 AR patients) were detected in the follow-up of the patients and median infection number for a patient was 9 in both groups. Eight of 17 patients had stem cell transplantation and the survival rate was 87.5%. CONCLUSIONS: X-CGD patients are more rapidly recognized by family history and severe infections than those with AR-CGD and early prophylaxis may decrease infectious episodes. We have investigated the large deletion suggesting a possible founder effect for CYBA exon 3-6 deletion in Central Anatolia. Additionally, HSCT transplantation leads to a high survival rate for the patients with CGD.

Adult case of chronic granulomatous disease mimicking granulomatosis with polyangiitis.

BACKGROUND: This article presents a patient who was initially diagnosed as having granulomatosis with polyangiitis (GPA), and was later diagnosed as having chronic granulomatous disease (CGD) in adulthood. We aimed to raise awareness of CGD, which can be confused with rheumatic diseases. CASE REPORT: We present a 33-year-old male patient with CGD with recurrent opportunistic bacterial and fungal infections who was diagnosed as having GPA, and had a history of recurrent lung infections and brain abscesses since childhood. The patient, who had cavitary lesions in the lung and mucosal lesions in the nose, was diagnosed as having GPA based on antineutrophil cytoplasmic antibody positivity. CGD was suspected in his last hospitalization after the patient underwent a nitro blue tetrazolium test. Accordingly, neutrophil oxidative function was tested using a dihydrorhodamine assay, which confirmed CGD. Molecular analysis of the patient revealed that the NCF1 gene had a GT deletion at the beginning of exon 2. Our patient was diagnosed as having late-onset CGD; he is currently well and taking antibiotic prophylaxis. CONCLUSION: As a result of the altered humoral immune response in CGD, there is unregulated inflammation and sustained antigen stimulation. This excessive inflammatory response can be confused with autoimmune diseases and cause delays in diagnosis. This case is important in the differential diagnosis of CGD in adult patients with recurrent opportunistic infections.

CD4+ T-Cell Depression is Linked to the Severity of COVID-19 and Predicts Mortality.

Objective: Most patients with coronavirus disease (COVID-19) have abnormalities of lymphocyte subsets. This study aimed to determine the distribution of lymphocytes in patients with various severity levels of COVID-19 and to describe the relationship between the CD4+ T helper and prognosis. Materials and Methods: Adult (>18 years old) patients with COVID-19 who followed up in a tertiary hospital were included in the study prospectively. Demographic and clinical characteristics of the patients were obtained from the hospital records. Peripheral flow cytometry was studied in patients with different severity of COVID-19 and different prognoses. Next, we analyzed the characteristics and predictive values of lymphocyte subsets in COVID-19 patients. Results: Totally 86 patients were included in the study, of which 21 (24.4%) had asymptomatic, 23 (26.7%) had mild/moderate, and 42 (48.8%) had severe/critical COVID-19. Severe/critical patients had lower lymphocyte levels and older age than asymptomatic patients (p<0.001 and p<0.001, respectively). We determined that decreased CD4+ T cell ratio (p<0.001) and CD4+ /CD8+ ratio (p<0.001) were indicative of the severity of the disease. CD4+ T cell ratio on admission (odds ratio [OR]=0.858; p=0.033), day seven CD4+ T cell ratio (OR=0.840; p=0.029), and C-reactive protein (CRP) levels (OR=1.014; p=0.043) were prognostic factors for mortality. According to receiver operating characteristics (ROC) curve analysis, the area under the curve was greater than 0.9 for decreased CD4 + T cell ratio on admission and the seventh day. Conclusion: A low CD4+ T helper ratio predicts a poor prognosis. In combination with CRP, it can be used in clinical follow-up.

Endothelial Progenitor Cells and NADPH Oxidase Enzyme Activity in the Development of an Aortic Aneurysm.

INTRODUCTION: Endothelial progenitor cells (EPCs) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme activity may affect the vessel wall and have a role in development of aortic aneurysms. EPCs originate from hematopoietic stem cells and can be enumerated from peripheral blood samples by flow cytometry. In this study, we aimed to evaluate the relation of EPC number and NADPH oxidase enzyme activity in the development of thoracic aortic aneurysm (TAA). METHODS: Patients with TAA (n=30) and healthy individuals without TAA (control, n=10) were included in our study. Characterization and enumeration of EPC from peripheral blood samples were performed by flow cytometry with panels including markers of EPCs (CD34/CD133/CD309/CD146/CD144). Additionally, NADPH oxidase enzyme activity (capacity) was also measured by the dihydrorhodamine 123 (DHR 123) test. RESULTS: The enumeration of EPC with CD34+/CD146+ marker showed that the number of mean EPC/106 cells was increased in the patient group (41.5/106 cells), but not in the control group (20.50/105 cells) (P<0.01). Additionally, patients with TAA presented significantly lower NADPH oxidase activity by DHR assay than healthy controls (mean stimulation index: 60.40± 7.86 and 75.10±5.21, respectively) (P<0.01). CONCLUSION: Our results showed that the number of EPCs is significantly higher in aortic aneurysm patients and may have a role in disease progression. The crosstalk between NADPH oxidase enzyme capacity and EPC number may be useful as a parameter to explain the clinical progression of TAA.

A novel ITGB2 variant with long survival in patients with leukocyte adhesion defect type-I.

Leukocyte adhesion deficiency is an autosomal recessive primary immunodeficiency that has been divided into three types: LAD1 (beta-2 integrin (CD18) family deficiency/defect), LAD2 (absence of fucosylated carbonhydrate ligands for selectins) and LAD3 (defective activation of all beta integrins). However, recently LAD4 has been described in cystic fibrosis patients, with a defect in integrin activation reported in monocytes. LAD-I is the most common type and prevalence of 1 in 1,000,000 live births. Clinical features of LAD patients are recurrent bacterial and fungal infections, omphalitis with delayed umbilical stump separation, significant leukocytosis especially neutrophilia during infection periods, impaired pus formation, and delayed traumatic or surgical wound healing. Flow cytometry is considered a useful tool for rapid diagnosis of the disease. The study of CD18 and CD11 (a, b, c) expression patterns in peripheral blood leukocytes helps to distinguish different phenotypes of LAD-I. In general, patients with ≥ 2% CD18 expression tend to have a less severe infection and often survive until adulthood, whereas < 2% CD18 expression often results in death in infancy. In this case report, three siblings, 10, 15, and 17 years old, diagnosed with leukocyte adhesion defect type 1 in adolescence age group, are presented.

Kindlin3-Dependent CD11b/CD18-Integrin Activation Is Required for Potentiation of Neutrophil Cytotoxicity by CD47-SIRPα Checkpoint Disruption.

The CD47-signal regulatory protein-alpha (SIRPα) immune checkpoint constitutes a therapeutic target in cancer, and initial clinical studies using inhibitors of CD47-SIRPα interactions in combination with tumor-targeting antibodies show promising results. Blockade of CD47-SIRPα interaction can promote neutrophil antibody-dependent cellular cytotoxicity (ADCC) toward antibody-opsonized targets. Neutrophils induce killing of antibody-opsonized tumor cells by a process identified as trogoptosis, a necrotic/lytic type of cancer cell death that involves trogocytosis, the antibody-mediated endocytic acquisition of cancer membrane fragments by neutrophils. Both trogocytosis and killing strictly depend on CD11b/CD18-(Mac-1)-mediated neutrophil-cancer cell conjugate formation, but the mechanism by which CD47-SIRPα checkpoint disruption promotes cytotoxicity has remained elusive. Here, by using neutrophils from patients with leukocyte adhesion deficiency type III carrying FERMT3 gene mutations, hence lacking the integrin-associated protein kindlin3, we demonstrated that CD47-SIRPα signaling controlled the inside-out activation of the neutrophil CD11b/CD18-integrin and cytotoxic synapse formation in a kindlin3-dependent fashion. Our findings also revealed a role for kindlin3 in trogocytosis and an absolute requirement in the killing process, which involved direct interactions between kindlin3 and CD18 integrin. Collectively, these results identified a dual role for kindlin3 in neutrophil ADCC and provide mechanistic insights into the way neutrophil cytotoxicity is governed by CD47-SIRPα interactions.

Diagnostic Modalities Based on Flow Cytometry for Chronic Granulomatous Disease: A Multicenter Study in a Well-Defined Cohort.

BACKGROUND: Chronic granulomatous disease (CGD) is characterized by defective microbial killing due to mutations affecting subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Definitive genetic identification of disease subtypes may be delayed or not readily available. OBJECTIVE: Sought to investigate the role of intracellular staining of NADPH oxidase enzyme subunits in predicting the respective genetic defects in patients with CGD and carriers. METHODS: Thirty-four patients with genetically inherited CGD, including 12 patients with X-linked CGD (gp91phagocyte oxidase (phox) deficiency due to cytochrome b-245, beta polypeptide [CYBB] mutations) and 22 patients with autosomal-recessive CGD (p22phox, p47phox, and p67phox deficiency due to cytochrome b-245, alpha polypeptide [CYBA], neutrophil cytosolic factor 1 [NCF1] and NCF2 mutations, respectively) were recruited from different immunology centers and followed up prospectively. Dihydrorhodamine testing and NADPH oxidase subunit expression in white blood cells were determined by flow cytometry. RESULTS: gp91phox and p22phox defects, which result in simultaneous loss of both proteins due to their complex formation, were differentiated only by comparative analysis of patients' and mothers' intracellular staining. p47phox and p67phox protein expression was almost undetectable in patients compared with carrier mothers and healthy controls. The expression values of the respective subunits were found to be significantly higher in all controls as compared with carrier mothers, which in turn were higher than those of patients. CONCLUSIONS: Analysis of NADPH oxidase enzyme subunits by flow cytometry in patients and carriers is useful in the rapid prediction of the genetic defect of patients with CGD, thus guiding targeted sequencing and aiding in their early diagnosis.

Performance of Galactomannan Antigen, Beta-d-Glucan, and Aspergillus-Lateral-Flow Device for the Diagnosis of Invasive Aspergillosis.

Aspergillus lateral-flow device (LFD) was recently introduced as a practical tool for the diagnosis of invasive aspergillosis (IA). We investigated the performance of Aspergillus-LFD as a point-of-care test for the diagnosis of IA. Serum samples were collected twice weekly from patients who received intensive chemotherapy for acute leukemia, or recepients of allogeneic stem cell transplantation. Aspergillus galactomannan (GM) antigen, 1,3-beta-d-glucan and Aspergillus-LFD tests were carried out according to manufacturers' recommendations. GM testing was repeated with a modified procedure which was proven to increase the sensitivity. Aspergillus-LFD was performed without applying any pretreatment procedure to allow the kit to fit as a point-of-care test. Fungal infections were categorized according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria. A total of 75 neutropenia episodes in 64 patients were prospectively followed between February 2012 and January 2013. Probable IA was diagnosed in 11 patients, probable pulmonary fungal disease was diagnosed in one patient, and rhinocerebral aspergillosis was diagnosed in one patient. Fungemia was detected in two patients. Aspergillus-LFD was positive in serum of a patient with probable IA and in the bronchoalveolar lavage fluid of an other patient with probable IA. Aspergillus-LFD was false positive in serum of two patients. Although there was no radiological finding of IA or documented fungemia, fever resolved after empirical caspofungin therapy in one of these patients. The sensitivity of Aspergillus-LFD as a point-of-care test without any pretreatment of serum sample is low.

Endothelial Progenitor Cells and NADPH Oxidase Enzyme Activity in the Development of an Aortic Aneurysm

Abstract Introduction: Endothelial progenitor cells (EPCs) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme activity may affect the vessel wall and have a role in development of aortic aneurysms. EPCs originate from hematopoietic stem cells and can be enumerated from peripheral blood samples by flow cytometry. In this study, we aimed to evaluate the relation of EPC number and NADPH oxidase enzyme activity in the development of thoracic aortic aneurysm (TAA). Methods: Patients with TAA (n=30) and healthy individuals without TAA (control, n=10) were included in our study. Characterization and enumeration of EPC from peripheral blood samples were performed by flow cytometry with panels including markers of EPCs (CD34/CD133/CD309/CD146/CD144). Additionally, NADPH oxidase enzyme activity (capacity) was also measured by the dihydrorhodamine 123 (DHR 123) test. Results: The enumeration of EPC with CD34+/CD146+ marker showed that the number of mean EPC/106 cells was increased in the patient group (41.5/106 cells), but not in the control group (20.50/105 cells) (P<0.01). Additionally, patients with TAA presented significantly lower NADPH oxidase activity by DHR assay than healthy controls (mean stimulation index: 60.40± 7.86 and 75.10±5.21, respectively) (P<0.01). Conclusion: Our results showed that the number of EPCs is significantly higher in aortic aneurysm patients and may have a role in disease progression. The crosstalk between NADPH oxidase enzyme capacity and EPC number may be useful as a parameter to explain the clinical progression of TAA.

The association of endothelial progenitor cell markers with arteriovenous fistula maturation in hemodialysis patients.

AIMS: Arteriovenous fistula (AVF) failure is one of the most important clinical problems in end-stage renal disease. Endothelial progenitor cells (EPCs) have a role on vascular angiogenesis and endothelialization. We aimed to investigate the association markers of EPCs on AVF maturation by measuring the surface expressions of CD34, CD309 and CD133 on the monocytes. METHODS: This prospective observational study was conducted in 54 voluntary patients with end-stage renal disease who were admitted for their first renal replacement therapy and were available for AVF creation. Venography was performed in all patients before AVF creation. Six patients were excluded due to inadequate veins after venographic imaging, and also seven patients were excluded due to postoperative thrombosis. The blood samples were analyzed a day before the fistula operation, and the expressions of CD34, CD133 and CD309 on the surface of monocytes were measured. RESULTS: Patients were divided into two groups after the evaluation of AVF maturation, as the mature group and the failure group. The CD309 expression level on the monocytes was 338.00 (35.00-479.00) in the mature group; however, it was 36.00 (5.50-237.00) (p 0.031) in the failure group. Multiple logistic regression analyses showed that both BMI and the mean fluorescence intensity level of CD309 expression on monocytes independently predicted AVF maturation. CONCLUSIONS: The presence of DM and increased BMI negatively correlated with AVF maturation. High intensity of CD309 expression on monocytes was observed in patients with successful AVF maturation.