Molecular Assessment of Staphylococcus Aureus Strains in STAT3 Hyper-IgE Syndrome Patients.

Hyper-IgE syndromes (HIES) are a group of inborn errors of immunity (IEI) caused by monogenic defects such as in the gene STAT3 (STAT3-HIES). Patients suffering from HIES show an increased susceptibility to Staphylococcus aureus (S. aureus) including skin abscesses and pulmonary infections. To assess if the underlying immune defect of STAT3-HIES patients influences the resistance patterns, pathogenicity factors or strain types of S. aureus. We characterized eleven S. aureus strains isolated from STAT3-HIES patients (n = 4) by whole genome sequencing (WGS) to determine presence of resistance and virulence genes. Additionally, we used multi-locus sequence typing (MLST) and protein A (spa) typing to classify these isolates. Bacterial isolates collected from this cohort of STAT3-HIES patients were identified as common spa types in Germany. Only one of the isolates was classified as methicillin-resistant S. aureus (MRSA). For one STAT3 patient WGS illustrated that infection and colonization occurred with different S. aureus isolates rather than one particular clone. The identified S. aureus carriage profile on a molecular level suggests that S. aureus strain type in STAT3-HIES patients is determined by local epidemiology rather than the underlying immune defect highlighting the importance of microbiological assessment prior to antibiotic treatment.

A structured patient empowerment programme for primary immunodeficiency significantly improves general and health-related quality of life.

INTRODUCTION: Primary immunodeficiencies (PIDs) are a heterogeneous group of rare diseases characterized by increased susceptibility to infections and a reduced quality of life (QoL). The influence of a patient empowerment programme for PID (PID-PEP) on general and health-related QoL was assessed in the present study. MATERIAL AND METHODS: PID-PEP is provided by a multidisciplinary team for patients with PID and immunoglobulin G (IgG) replacement therapy during a weekend course to improve patient self-management regarding chronic disease and long-term therapy. Twenty-six adult patients with PID undergoing PID-PEP were recruited. Short Form-36 (SF-36) and the Life Quality Index (LQI) were assessed as generic and disease-specific QoL instruments before as well as 6 months after the programme. RESULTS: Median visual analogue scale (VAS) values of present health status significantly increased from 68 at baseline to 76 after PID-PEP (p = 0.002). Furthermore, the SF-36 mental component summary (MCS) significantly improved from 36 to 43 following the programme (p = 0.042). Of the eight SF-36 dimensions, vitality (VT) significantly improved (p = 0.025). Median LQI index significantly increased from 77 at baseline to 86 after PID-PEP (p = 0.008). Furthermore, the LQI domains treatment interference (I) and therapy-related problems (II) significantly improved. CONCLUSIONS: Our PID-PEP significantly improved general and health-related QoL. It needs to be evaluated in future studies whether the beneficial effects of PID-PEP are sustained over longer periods of time and whether repeated PID-PEP sessions further improve QoL outcome.

Class Switch Recombination Defects: impact on B cell maturation and antibody responses.

To assess how B cell phenotype analysis correlates with antigen responses in patients with class switch recombination defects (CSRD) we quantified memory B cells by flow-cytometry and immunized CSRD patients with the neoantigen bacteriophage phiX174 (phage). CSRD patients showed uniformly absent or markedly reduced switched memory B cells (IgM-IgD-CD27+). CD40L patients had reduced CD27+ memory B cells (both non-switched and switched). In NEMO patients, results varied depending on the IKKγ gene variant. Three of four AID patients had normal percentages of CD27+ memory B cells while CD27+IgM-IgD- switched memory B cells were markedly reduced in all AID patients. Antibody response to phage was remarkably decreased with lack of memory amplification and class-switching in immunized CD40L, UNG deficient, and NEMO patients. Distinct B-cell phenotype pattern correlated with abnormal antibody responses to a T-cell dependent neoantigen, representing a powerful tool to identify CSRD patients.

High seroprevalence of antibodies against Kaposi's sarcoma-associated herpesvirus (KSHV) among HIV-1-infected children and adolescents in a non-endemic population.

Human herpesvirus-8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS), which primarily affects human immunodeficiency virus (HIV)-infected adults with advanced immunodeficiency. Currently, only limited prevalence data for HHV-8 infection in HIV-infected children living in non-endemic areas are available. This multicenter cross-sectional study was conducted in four university hospitals in Germany specializing in pediatric HIV care. Stored serum specimens obtained from 207 vertically HIV-1-infected children and adolescents were tested for antibodies against lytic and latent HHV-8 antigens. Logistic regression was used to assess independent risk factors associated with HHV-8 seropositivity. The overall HHV-8 seroprevalence was 24.6 % (n = 51/207) without significant differences related to sex, age, or ethnicity. In univariate analysis, HHV-8 seropositivity was significantly associated with a child having being born outside Germany, maternal origin from sub-Saharan Africa, a history of breastfeeding, CDC immunologic category 3, and deferred initiation of antiretroviral therapy (>24 months of age). In multivariate analysis, a child's birth outside Germany was the only significant risk factor for HHV-8 seropositivity (odds ratio 3.98; 95 % confidence interval 1.27-12.42). HHV-8-associated malignancies were uncommon; only one patient had a history of KS. Serum specimen of vertically HIV-infected children and adolescents living in Germany showed a high HHV-8 seroprevalence. These findings suggest that primary HHV-8 infection-a risk factor for KS and other HHV-8-associated malignancies-occurs early in life. Thus, management of perinatally HIV-infected children should include testing for HHV-8 coinfection and should consider future risks of HHV-8-associated malignancies.

Successful Combination of Sequential Gene Therapy and Rescue Allo-HSCT in Two Children with X-CGD - Importance of Timing.

We report on a series of sequential events leading to long-term survival and cure of pediatric X-linked chronic granulomatous disease (X-CGD) patients after gamma-retroviral gene therapy (GT) and rescue HSCT. Due to therapyrefractory life-threatening infections requiring hematopoietic stem cell transplantation (HSCT) but absence of HLAidentical donors, we treated 2 boys with X-CGD by GT. Following GT both children completely resolved invasive Aspergillus nidulans infections. However, one child developed dual insertional activation of ecotropic viral integration site 1 (EVI1) and signal transducer and activator of transcription 3 (STAT3) genes, leading to myelodysplastic syndrome (MDS) with monosomy 7. Despite resistance to mismatched allo-HSCT with standard myeloablative conditioning, secondary intensified rescue allo-HSCT resulted in 100 % donor chimerism and disappearance of MDS. The other child did not develop MDS despite expansion of a clone with a single insertion in the myelodysplasia syndrome 1 (MDS1) gene and was cured by early standard allo-HSCT. The slowly developing dominance of clones harboring integrations in MDS1-EVI1 may guide clinical intervention strategies, i.e. early rescue allo-HSCT, prior to malignant transformation. GT was essential for both children to survive and to clear therapy-refractory infections, and future GT with safer lentiviral self-inactivated (SIN) vectors may offer a therapeutic alternative for X-CGD patients suffering from life-threatening infections and lacking HLA-identical HSC donors.

DOCK8 deficiency: clinical and immunological phenotype and treatment options - a review of 136 patients.

Mutations in DOCK8 result in autosomal recessive Hyper-IgE syndrome with combined immunodeficiency (CID). However, the natural course of disease, long-term prognosis, and optimal therapeutic management have not yet been clearly defined. In an international retrospective survey of patients with DOCK8 mutations, focused on clinical presentation and therapeutic measures, a total of 136 patients with a median follow-up of 11.3 years (1.3-47.7) spanning 1693 patient years, were enrolled. Eczema, recurrent respiratory tract infections, allergies, abscesses, viral infections and mucocutaneous candidiasis were the most frequent clinical manifestations. Overall survival probability in this cohort [censored for hematopoietic stem cell transplantation (HSCT)] was 87 % at 10, 47 % at 20, and 33 % at 30 years of age, respectively. Event free survival was 44, 18 and 4 % at the same time points if events were defined as death, life-threatening infections, malignancy or cerebral complications such as CNS vasculitis or stroke. Malignancy was diagnosed in 23/136 (17 %) patients (11 hematological and 9 epithelial cancers, 5 other malignancies) at a median age of 12 years. Eight of these patients died from cancer. Severe, life-threatening infections were observed in 79/136 (58 %); severe non-infectious cerebral events occurred in 14/136 (10 %). Therapeutic measures included antiviral and antibacterial prophylaxis, immunoglobulin replacement and HSCT. This study provides a comprehensive evaluation of the clinical phenotype of DOCK8 deficiency in the largest cohort reported so far and demonstrates the severity of the disease with relatively poor prognosis. Early HSCT should be strongly considered as a potential curative measure.

Outcome and management of pregnancies in severe chronic neutropenia patients by the European Branch of the Severe Chronic Neutropenia International Registry.

Long-term granulocyte-colony stimulating factor treatment has been shown to be safe and effective in severe chronic neutropenia patients. However, data on its use during pregnancy are limited. To address this issue, we analyzed all pregnancies reported to the European branch of the Severe Chronic Neutropenia International Registry since 1994. A total of 38 pregnancies in 21 women with chronic neutropenia (16 pregnancies in 10 women with congenital, 10 in 6 women with cyclic, 12 in 5 women with idiopathic neutropenia) were reported. Granulocyte-colony stimulating factor was administered throughout pregnancy in 16 women and for at least one trimester in a further 5 women. No major differences were seen between treated and untreated women with respect to pregnancy outcome, newborn complications and infections. In addition, we evaluated the genetic transmission of known or suspected genetic defects in 16 mothers having 22 newborns as well as in 8 men fathering 15 children. As a proof of inheritance, neutropenia was passed on to the newborn in 58% from female and in 62% from male patients with ELANE mutations, but also to some newborns from parents with unknown gene mutation. Based on our results, granulocyte-colony stimulating factor therapy has been shown to be safe for mothers throughout pregnancies and for newborns without any signs of teratogenicity. With an increasing number of adult patients, genetic counseling prior to conception and supportive care of mothers during pregnancy are crucial. The acceptance of having affected children may reflect the high quality of life obtained due to this treatment.

HIV-1 seroreversion in HIV-1-infected children: do genetic determinants play a role?

BACKGROUND: HIV-1 seroreversion in infants with vertically transmitted HIV-1 infection who started ART in the first months of life has been reported in only a subset of patients. However, the reason why most infants remain seropositive despite similar treatment response is not understood. Here, we assessed whether HIV-1 seroreversion in maternally infected infants is associated with genetic determinants. METHODS: HIV-1-infected infants with a history of documented HIV-1 seroreversion were identified throughout Germany using a standardized questionnaire. At study entry immune reconstitution and anti-HIV-1 antibody expression were monitored as clinical parameters. To search for genetic determinants high-resolution HLA genotyping was performed. In addition, the coding sequence of the chemokine receptor CCR5 was analyzed by Sanger sequencing regarding potential mutations. RESULTS: Patients showed normal numbers and frequencies of lymphocyte subpopulations. Five out of eight patients still had seronegative HIV-1 antibody status at study entry. HLA genotyping revealed the enrichment of HLA-DQB1*03 and DQB1*06 alleles within the patient cohort. Only one patient was found to carry a 32 bp-deletion within the CCR5 gene. CONCLUSION: Our results indicate that the phenotype of HIV-1 seroreversion in infants might correlate with the presence of HLA class II alleles DQB1*03 and DQB1*06. This finding supports the idea of genetic predisposition determining HIV-1 seroreversion in vertically infected infants effectively treated with ART.

A novel gain-of-function IKBA mutation underlies ectodermal dysplasia with immunodeficiency and polyendocrinopathy.

PURPOSE: This study reports the identification of a novel heterozygous IKBA missense mutation (p.M37K) in a boy presenting with ectodermal dysplasia with immunodeficiency (EDA-ID) who had wild type IKBKG gene encoding NEMO. Our aim was to characterize the clinical course of this IκB-α gain-of-function mutant and to investigate if the p.M37K substitution affects NF-κB activation by interfering with IκB-α degradation, thus impairing NF-κB signaling and causing the EDA-ID phenotype. METHODS: NF-κB signaling was evaluated by measuring IκB-α degradation in patient fibroblasts. In addition, transiently transfected HeLa cells expressing either the M37K-mutant IκB-α allele, the previously characterized S36A-mutant IκB-α allele, or wild type IκB-α were evaluated for IκB-α degradation and NF-κB nuclear translocation following stimulation with TNF-α. RESULTS: Clinical findings revealed a classical ectodermal dysplasia phenotype complicated by recurrent mucocutaneous candidiasis, hypothyroidism, hypopituitarism, and profound combined immunodeficiency with decreased numbers of IL-17 T cells. IκB-α degradation after TNF-α and TLR agonist stimulation was abolished in patient fibroblasts as well as in HeLa cells expressing M37K-IκB-α similar to cells expressing S36A-IκB-α resulting in impaired nuclear translocation of NF-κB and reduced NF-κB dependent luciferase activity compared to cells expressing wild type IκB-α. Patient whole blood cells failed to secrete IL-6 in response to IL-1ß, Pam2CSK4, showed reduced responses to LPS and PMA/Ionomycin, and lacked IL-10 production in response to TNF-α. CONCLUSION: The novel heterozygous mutation p.M37K in IκB-α impairs NF-κB activation causing autosomal dominant EDA-ID with an expanded clinical phenotype.

Phylodynamic and phylogeographic patterns of the HIV type 1 subtype F1 parenteral epidemic in Romania.

In the late 1980s an HIV-1 epidemic emerged in Romania that was dominated by subtype F1. The main route of infection is believed to be parenteral transmission in children. We sequenced partial pol coding regions of 70 subtype F1 samples from children and adolescents from the PENTA-EPPICC network of which 67 were from Romania. Phylogenetic reconstruction using the sequences and other publically available global subtype F sequences showed that 79% of Romanian F1 sequences formed a statistically robust monophyletic cluster. The monophyletic cluster was epidemiologically linked to parenteral transmission in children. Coalescent-based analysis dated the origins of the parenteral epidemic to 1983 [1981-1987; 95% HPD]. The analysis also shows that the epidemic's effective population size has remained fairly constant since the early 1990s suggesting limited onward spread of the virus within the population. Furthermore, phylogeographic analysis suggests that the root location of the parenteral epidemic was Bucharest.

X-linked lymphoproliferative disease due to SAP/SH2D1A deficiency: a multicenter study on the manifestations, management and outcome of the disease.

X-linked lymphoproliferative disease (XLP1) is a rare immunodeficiency characterized by severe immune dysregulation and caused by mutations in the SH2D1A/SAP gene. Clinical manifestations are varied and include hemophagocytic lymphohistiocytosis (HLH), lymphoma and dysgammaglobulinemia, often triggered by Epstein-Barr virus infection. Historical data published before improved treatment regimens shows very poor outcome. We describe a large cohort of 91 genetically defined XLP1 patients collected from centers worldwide and report characteristics and outcome data for 43 patients receiving hematopoietic stem cell transplant (HSCT) and 48 untransplanted patients. The advent of better treatment strategies for HLH and malignancy has greatly reduced mortality for these patients, but HLH still remains the most severe feature of XLP1. Survival after allogeneic HSCT is 81.4% with good immune reconstitution in the large majority of patients and little evidence of posttransplant lymphoproliferative disease. However, survival falls to 50% in patients with HLH as a feature of disease. Untransplanted patients have an overall survival of 62.5% with the majority on immunoglobulin replacement therapy, but the outcome for those untransplanted after HLH is extremely poor (18.8%). HSCT should be undertaken in all patients with HLH, because outcome without transplant is extremely poor. The outcome of HSCT for other manifestations of XLP1 is very good, and if HSCT is not undertaken immediately, patients must be monitored closely for evidence of disease progression.

ORAI1 deficiency and lack of store-operated Ca2+ entry cause immunodeficiency, myopathy, and ectodermal dysplasia.

BACKGROUND: Defects in the development or activation of T cells result in immunodeficiency associated with severe infections early in life. T-cell activation requires Ca2+ influx through Ca2+-release activated Ca2+ (CRAC) channels encoded by the gene ORAI1. OBJECTIVE: Investigation of the genetic causes and the clinical phenotype of immunodeficiency in patients with impaired Ca2+ influx and CRAC channel function. METHODS: DNA sequence analysis for mutations in the genes ORAI1, ORAI2, ORAI3, and stromal interaction molecule (STIM) 1 and 2, as well as mRNA and protein expression analysis of ORAI1 in immunodeficient patients. Immunohistochemical analysis of ORAI1 tissue distribution in healthy human donors. RESULTS: We identified mutations in ORAI1 in patients from 2 unrelated families. One patient is homozygous for a frameshift nonsense mutation in ORAI1 (ORAI1-A88SfsX25), and a second patient is compound heterozygous for 2 missense mutations in ORAI1 (ORAI1-A103E/L194P). All 3 mutations abolish ORAI1 expression and impair Ca2+ influx and CRAC channel function. The clinical syndrome associated with ORAI1 deficiency is characterized by immunodeficiency with a defect in the function but not in the development of lymphocytes, congenital myopathy, and anhydrotic ectodermal dysplasia with a defect in dental enamel calcification. In contrast with the limited clinical phenotype, we found ORAI1 protein expression in a wide variety of cell types and organs. CONCLUSION: Ca2+ influx through ORAI1 is crucial for lymphocyte function in vivo. Despite almost ubiquitous ORAI1 expression, the channel has a nonredundant role in only a few cell types judging from the limited clinical phenotype in ORAI1-deficient patients.

Common variable immunodeficiency disorders in children: delayed diagnosis despite typical clinical presentation.

OBJECTIVE: To characterize common variable immunodeficiency disorder (CVID) in childhood. STUDY DESIGN: We retrospectively investigated clinical findings in 32 children with primary CVID by questionnaire and file review. RESULTS: Clinical presentation included recurrent or chronic respiratory tract infections (88%), sinusitis (78%), otitis media (78%), and intestinal tract infections (34%), mainly with encapsulated bacteria. Meningitis was found in 25%, sepsis in 16%, and pyelonephritis in 16% of patients. Poliomyelitis after vaccination occurred in 2 patients and opportunistic infections occasionally. Allergic disorders were present in 38%, and autoimmune disease in 31% of patients. Eighty percent of the patients underwent surgical procedures because of recurrent infections. Growth retardation was seen in 28% of patients, and 16% showed retarded mental development. Bronchiectasis developed in 34%, and lymphoid proliferative disease in 13%. Incidence of allergic and autoimmune diseases was increased in first-degree relatives with normal immunologic findings. Mean time between symptoms and induction of immunoglobulin substitution therapy was 5.8 years (0.2-14.3). CONCLUSIONS: CVID in children presents with comparable symptoms and disorders as in adults. We found a significant influence on growth and development. The marked delay of diagnosis may be due to overlap with common pediatric disorders, while also reflecting insufficient awareness of these disorders.

Long-term posaconazole treatment and follow-up of rhino-orbital-cerebral mucormycosis in a diabetic girl.

To demonstrate that the 2-yr clinical follow-up of our patient strongly suggests that long-term therapy with posaconazole (POS) is safe and beneficial in treatment and prevention of relapses of, otherwise fatal, central nervous system mucormycosis. Mucormycosis is a very rare opportunistic mycotic infection of diabetic children. We present the 30-month follow-up of a 12-yr-old girl affected by diabetic ketoacidotic coma, complicated by rhinocerebral mucormycosis and successfully treated with POS at the initial daily dose of 5 mg/kg t.i.d. with fatty food for 3 wk, followed by a daily dose of 10 mg/kg in four doses for 2 months and then 20 mg/kg/d in four doses for 16 months and in two doses for further 5 months. The previous amphotericin B, granulocyte colony-stimulating factor, hyperbaric oxygen and nasal and left maxillary sinus surgical debridement therapy was ineffective in stopping the progression of the infection to the brain. The patient improved within 10 d with reduced ocular swelling and pain, and 6 months after therapy stop, she is in good health and cultures are sterile. This article demonstrates that POS may be a useful drug in mucormycosis in children. We also strongly draw the attention to the main preventive procedure against invasive fungal infection that is the correct management of antidiabetic therapy that prevents the predisposing temporary neutrophils activity deficit, contributing to a better survival rate of diabetic children.

Dysregulation of innate immune receptors on neutrophils in chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is the most common inherited disorder of neutrophil function, is caused by mutations in the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and results in recurrent bacterial infections. OBJECTIVE: We sought to investigate the expression and function of innate immune receptors on neutrophils in patients with CGD. METHODS: We quantified mRNA and protein expression of Toll-like receptors (TLRs), complement receptors, and chemokine receptors on neutrophils from 15 patients with CGD compared with that seen in healthy control subjects (n = 15) and control patients with bacterial pneumonia (n = 15). Phagocytosis, chemotaxis, and TLR function of isolated neutrophils were analyzed. The effect of NADPH oxidase inhibition on receptor expression and function was analyzed in control neutrophils. RESULTS: Neutrophils from patients with CGD had lower expression levels of TLR5, TLR9, CD11b, CD18, CD35, and CXCR1 compared with those from healthy control subjects, whereas similar or increased receptor expressions were found in patients without CGD but with bacterial pneumonia. Reduced TLR5 expression resulted in impaired neutrophil activation by bacterial flagella, reduced CD11b/CD18 expression was associated with impaired phagocytosis of Staphylococcus aureus, and reduced CXCR1 expression was associated with decreased chemotaxis. TLR5 and CD18 expression levels correlated with disease severity in patients with CGD. TLR5 and TLR9 expression were greater in patients with residual NADPH oxidase activity. Inhibition of the NADPH oxidase in control neutrophils in vitro decreased TLR5 and TLR9 expression and impaired TLR5 function. CONCLUSION: These results provide the first evidence that innate immune receptors are dysregulated in patients with CGD.

"Hair-on-end" skull induced by long-term G-CSF treatment in severe congenital neutropenia.

"Hair-on-end" skull changes are typically seen in individuals suffering from thalassaemia. They are induced by widening of the diploic space due to marrow expansion that is a consequence of ineffective and excessive erythropoiesis. We present a child with severe congenital neutropenia who exhibited the typical hair-on-end sign on plain skull radiographs and MRI. In this patient the skull changes were very likely induced by the expansion of white blood cell precursors induced by long-term daily injections of recombinant human granulocyte colony stimulating factor (G-CSF) to treat his confounding disease. This case report is the first description of hair-on-end changes associated with the use of G-CSF.