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A 4-year-old Turkish girl of consanguineous parents was hospitalized for the evaluation of headaches and recurrent febrile episodes of unknown origin. Her medical history was unremarkable except for a few episodes of uncomplicated oral thrush. Meningitis was diagnosed, and Candida albicans was the only pathogen identified by polymerase chain reaction and culture. Despite systemic antifungal multidrug therapy, a prolonged course of 16 months of therapy was necessary to clear C. albicans from the cerebrospinal fluid. Molecular genetic analysis revealed a homozygous caspase recruitment domain 9 (CARD9) mutation (Q295X), which was reported to predispose to chronic mucocutaneous candidiasis. Immunologic workup excluded predisposing B-cell and T-cell defects. In addition, T cells producing interleukin-17 were repeatedly measured within the normal range. Analyses of neutrophils demonstrated normal nicotinamide adenine dinucleotide phosphate oxidase activity in response to various stimuli including Staphylococcus aureus and C. albicans. Additional neutrophilic functional testing, however, showed a decreased cytotoxicity to nonopsonized C. albicans, indicating an impaired killing mechanism against Candida spp. independent from the production of reactive oxygen species by the nicotinamide adenine dinucleotide phosphate oxidase system. Because this defect was only demonstrated in the absence of opsonins, it might especially predispose to chronic C. albicans infections in the central nervous system where opsonin concentrations are usually low. We, therefore, suggest that due to an additional neutrophil dependent defect CARD9 deficiency predisposes not only to chronic mucocutaneous candidiasis, but also to invasive chronic Candida infections, especially of the central nervous system.

Background Increased serum IgE levels are characteristic but not specific for allergic diseases. Particularly, severe atopic dermatitis (AD) overlaps with hyper-IgE syndromes (HIES) regarding eczema, eosinophilia, and increased serum IgE levels. HIES are primary immunodeficiencies due to monogenetic defects such as in the genes DOCK8 and STAT3. As it is not known to date why allergic manifestations are not present in all HIES entities, we assessed the specificity of serum IgE of AD and HIES patients in the context of clinical and immunological findings. Methods Clinical data, skin prick tests, specific IgE to aero- and food allergens, and T helper (Th) subpopulations were compared in AD and molecularly defined HIES patients. Results Total serum IgE levels were similarly increased in STAT3-HIES, DOCK8-HIES, and AD patients. The ratio of aeroallergen-specific IgE to total IgE was highest in AD, whereas DOCK8-HIES patients showed the highest specific serum IgE against food allergens. Overall, clinical allergy and skin prick test results complied with the specific IgE results. Th2-cell numbers were significantly increased in DOCK8-HIES and AD patients compared to STAT3-HIES patients and controls. AD patients showed significantly higher nTreg-cell counts compared to STAT3-HIES and control individuals. High Th17-cell counts were associated with asthma. Specific IgE values, skin prick test, and T-cell subsets of STAT3-HIES patients were comparable with those of healthy individuals except decreased Th17-cell counts. Conclusion Hyper-IgE syndromes and atopic dermatitis patients showed different sensitization pattern of serum IgE corresponding to the allergic disease manifestations and Th-cell subset data, suggesting a key role of DOCK8 in the development of food allergy.

Autosomal dominant hyper-IgE syndrome (AD-HIES) due to heterozygous STAT3 mutation is a primary immunodeficiency characterized by eczema, elevated serum IgE, recurrent infections, and connective tissue and skeletal findings. Healing of pneumonias is often abnormal with formation of pneumatoceles and bronchiectasis. We aimed to explore whether healing after lung surgery is also aberrant. We retrospectively analyzed the medical records of 32 patients with AD-HIES who received lung surgery for the management of pulmonary infections from 1960 to 2011. We collected information including patient demographics, STAT3 mutation status, clinical history, surgical and medical procedures performed, complications, related medical treatments, and outcomes. More than 50 % of lung surgeries had associated complications, with the majority being prolonged bronchopleural fistulae. These fistulae often led to empyemas that necessitated additional interventions including prolonged antibiotics, prolonged thoracostomy tube drainage and re-operations. Lung surgery in AD-HIES patients is associated with high complication rates. STAT3 mutations likely lead to abnormalities in tissue remodelling that are further exacerbated by infection.

Background Dedicator of cytokinesis 8 (DOCK8) mutations are responsible for a rare primary combined immunodeficiency syndrome associated with severe cutaneous viral infections, increased IgE levels, autoimmunity, and malignancy. Natural killer (NK) cells are essential for tumor surveillance and defense against virally infected cells. NK cell function relies on Wiskott-Aldrich syndrome protein for filamentous actin (F-actin) accumulation at the lytic NK cell immunologic synapse. DOCK8 activates cell division cycle 42, which, together with Wiskott-Aldrich syndrome protein, coordinates F-actin reorganization. Although abnormalities in T- and B-cell function have been described in DOCK8-deficient patients, the role of NK cells in this disease is unclear. Objectives We sought to understand the role of DOCK8 in NK cell function to determine whether NK cell abnormalities explain the pathogenesis of the clinical syndrome of DOCK8 deficiency. Methods A cohort of DOCK8-deficient patients was assembled, and patients' NK cells, as well as NK cell lines with stably reduced DOCK8 expression, were studied. NK cell cytotoxicity, F-actin content, and lytic immunologic synapse formation were measured. Results DOCK8-deficient patients' NK cells and DOCK8 knockdown cell lines all had decreased NK cell cytotoxicity, which could not be restored after IL-2 stimulation. Importantly, DOCK8 deficiency impaired F-actin accumulation at the lytic immunologic synapse without affecting overall NK cell F-actin content. Conclusions DOCK8 deficiency results in severely impaired NK cell function because of an inability to form a mature lytic immunologic synapse through targeted synaptic F-actin accumulation. This defect might underlie and explain important attributes of the DOCK8 deficiency clinical syndrome, including the unusual susceptibility to viral infection and malignancy.

Dedicator of cytokinesis 8 protein (DOCK8) deficiency is a combined immunodeficiency disorder characterized by an expanding clinical picture with typical features of recurrent respiratory or gastrointestinal tract infections, atopic eczema, food allergies, chronic viral infections of the skin, and blood eosinophilia often accompanied by elevated serum IgE levels. The only definitive treatment option is allogeneic hematopoietic stem cell transplantation (HSCT). We report a patient with early severe manifestation of DOCK8 deficiency, who underwent unrelated allogeneic HSCT at the age of 3 years following a reduced toxicity conditioning regimen. The transplant course was complicated by pulmonary aspergilloma pretransplantation, adenovirus (ADV) reactivation, and cytomegalovirus (CMV) pneumonitis 4 weeks after transplantation. With antifungal and antiviral treatment the patient recovered. Seven months after transplantation the patient is in excellent clinical condition. Eczematous rash, chronic viral skin infections, and food allergies have subsided, associated with normalization of IgE levels and absolute numbers of eosinophils. Chimerism analysis shows stable full donor chimerism. DOCK8 deficiency can be successfully cured by allogeneic HSCT. This treatment option should be considered early after diagnosis, as opportunistic infections and malignancies that occur more frequently during the natural course of the disease are associated with higher morbidity and mortality.

Whole-exome sequencing reveals activating STAT1 mutations in some patients with autosomal dominant chronic mucocutaneous candidiasis disease., Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) IL-17F deficiency or autosomal recessive (AR) IL-17RA deficiency. Here, using whole-exome sequencing, we identified heterozygous germline mutations in STAT1 in 47 patients from 20 kindreds with AD CMCD. Previously described heterozygous STAT1 mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-dependent cellular responses to IFN-γ. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-α/β, IFN-γ, IFN-λ, and IL-27. In contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as IL-6 and IL-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent IL-17 inhibitors IFN-α/β, IFN-γ, and IL-27, and stronger STAT1 activation in response to the STAT3-dependent IL-17 inducers IL-6 and IL-21, hinder the development of T cells producing IL-17A, IL-17F, and IL-22. Gain-of-function STAT1 alleles therefore cause AD CMCD by impairing IL-17 immunity.

BackgroundHyper-IgE syndromes (HIES) are primary immunodeficiency disorders characterized by elevated serum IgE, eczema, and recurrent infections. Despite the availability of confirmatory molecular diagnosis of several distinct HIES entities, the differentiation of HIES particularly from severe forms of atopic dermatitis remains a challenge. The two most common forms of HIES are caused by mutations in the genes STAT3 and DOCK8. MethodsHere, we assess the clinical and immunologic phenotype of DOCK8- and STAT3-HIES patients including the cell activation, proliferation, and cytokine release after stimulation. ResultsExisting HIES scoring systems are helpful to identify HIES patients. However, those scores may fail in infants and young children due to the age-related lack of clinical symptoms. Furthermore, our long-term observations showed a striking variation of laboratory results over time in the individual patient. Reduced memory B-cell counts in concert with low specific antibody production are the most consistent findings likely contributing to the high susceptibility to bacterial and fungal infection. In DOCK8-HIES, T-cell lymphopenia and low IFN-gamma secretion after stimulation were common, likely promoting viral infections. In contrast to STAT3-HIES, DOCK8-HIES patients showed more severe inflammation with regard to allergic manifestations, elevated activation markers (HLA-DR, CD69, CD86, and CD154), and significantly increased inflammatory cytokines (IL1-beta, IL4, IL6, and IFN-gamma). ConclusionDifferentiating HIES from other diseases such as atopic dermatitis early in life is essential for patients because treatment modalities differ. To expedite the diagnosis process, we propose here a diagnostic workflow.

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.

The authors describe two young brothers and a 12-year-old male with long-standing thrombocytopenia with normal sized platelets, in whom novel mutations of the WAS gene were identified. Their clinical picture and the in vitro assessment of the T-cell function were consistent with X-linked thrombocytopenia (XLT). A high index of suspicion for XLT is required, even in the setting of normal sized platelets for males with affected maternally-related male family members, and males with moderately severe chronic thrombocytopenia that have failed to respond to treatments that are usually effective for immune thrombocytopenia.

The authors describe two young brothers and a 12-year-old male with long-standing thrombocytopenia with normal sized platelets, in whom novel mutations of the WAS gene were identified. Their clinical picture and the in vitro assessment of the T-cell function were consistent with X-linked thrombocytopenia (XLT). A high index of suspicion for XLT is required, even in the setting of normal sized platelets for males with affected maternally-related male family members, and males with moderately severe chronic thrombocytopenia that have failed to respond to treatments that are usually effective for immune thrombocytopenia.

Background Hyper-IgE syndromes (HIES) are primary immunodeficiency disorders characterized by Staphylococcus aureus abscesses, recurrent pneumonia, increased serum IgE levels, and eczema. The association of heterozygous signal transducer and activator of transcription 3 (STAT3) mutations with autosomal dominant (AD)–HIES allows the differentiation of AD-HIES from disorders associated with eczema and increased serum IgE levels, such as other primary immunodeficiencies and atopic dermatitis. Objective To facilitate early diagnosis of AD-HIES to initiate appropriate therapy. Methods The clinical phenotype (suggested by a National Institutes of Health [NIH] score of ≥40 points), STAT3 genotype, and T H 17 cell counts were compared in a cohort of 78 patients suspected of having HIES. Results Heterozygous STAT3 missense mutations and in-frame deletions were identified in 48 patients, all but 2 with an NIH score ≥40 points. Patients with STAT3 mutations with HIES showed significantly lower T H 17 cell counts compared with patients with wild-type STAT3 and control subjects. Only 1 patient with wild-type STAT3 had both an NIH score ≥40 points and abnormal T H 17 cell counts (≤0.2% of CD4 + cells), with this exception being identified with a homozygous dedicator of cytogenesis 8 protein (DOCK8) mutation. Pathologic shedding of primary teeth was present in 3 patients with wild-type STAT3 and 33 patients with STAT3 mutations. Internal abscesses and severe infections were exclusively seen in patients with STAT3 mutations, who also had increased pneumatocele formation and skeletal or connective tissue manifestations compared with patients with wild-type STAT3 . Conclusion We expanded the number of STAT3 mutations and validated that the NIH score sensitively identifies patients with HIES. Based on our patient cohort, we propose key findings that, when combined with T H 17 cell numbers, predict patients with AD-HIES with STAT3 mutations, supporting early diagnosis of AD-HIES.

Myeloperoxidase (MPO) deficiency is one of the most common inherited phagocyte defects, but it is rarely associated with clinical symptoms [1]. MPO, which is abundant in azurophilic granules of neutrophils and in the lysosomes of monocytes, plays a key role in amplifying the toxicity of hydrogen peroxide generated by the respiratory burst [1,2]. The diagnosis of MPO deficiency was rare before 1979; currently, the diagnosis is easily made due to the widespread use of automated flow cytochemical analysis in clinical hematology laboratories for enumerating peripheral blood neutrophils with peroxidase activity [2]. The MPO gene is encoded on chromosome 17q23 [3]. Primary deficiency of MPO is inherited as an autosomal recessive disorder. A secondary form of MPO deficiency has been described in various disorders, including lead poisoning, severe infections, neuronal lipofuscinosis, obstructive jaundice, diabetes mellitus, and such disseminated cancers as acute and chronic myeloid leukemia, myelodysplastic syndrome, and Hodgkin’s lymphoma [1,2,4]. MPO-deficient neutrophils are markedly less efficient at killing Candida albicans and Aspergillus hyphae [1]. Although MPO is involved in killing certain microorganisms, to date, no particular susceptibility to persistent or severe infections has been noted in the vast majority of MPO-deficient patients [1,2]. Recurrent Candida infection has been observed predominantly in MPO-deficient patients that also have diabetes mellitus. In addition to infectious manifestations, the relationship between congenital MPO deficiency and the occurrence of malignancy remains controversial [1,2]. There is no specific treatment for MPO deficiency; however, in symptomatic patients antifungal treatment is crucial. Herein we present a 17-year-old male that presented to our hematology department with neutropenia. Anamnesis showed that the patient did not have any recurrent or severe infection, but had been receiving benzathine penicillin prophylaxis due to acute rheumatic fever for years. Physical examination showed the following: weight: 58 kg (3-10p); height: 169 cm (10-25p); 1-2-degree/6 systolic murmur in the mesocardial region; the remainder of the examination was unremarkable. Complete blood count results were as follows: hemoglobin: 13.1 g/dL; leucocyte count: 6140/mm3; platelet count: 503,000/mm3. The formulation of the leucocytes was interesting, with an absolute neutrophil count of 20/mm3 and a high absolute unstained cell count of 3020/mm3 (nearly 50%). Although the absolute neutrophil count was 20/mm3, peripheral blood smear showed normal formulation of leucocytes (49% neutrophils, 47% lymphocytes, and 4% monocytes). The inconsistency between the absolute neutrophil count and the peripheral blood smear suggested the possibility of MPO deficiency, and we therefore performed cytochemical staining of peroxidase and flow cytometry analysis. Cytochemical staining of peroxidase was negative, as compared to the control (Figure 1a, 1b), and flow cytometry analysis showed a dramatic decrease in MPO(+) neutrophils (Figure 2). In addition, we identified a compound heterozygous MPO gene mutation [c.1705C> T];[c.2031-2A>C]. The patient was followed-up for 12 months after his initial examination, during which time he was asymptomatic and healthy. In conclusion, we think that MPO deficiency must be considered in patients with incompatibility of neutropenia between smear and counter, especially when the flag of unstained cell is remarked. Figure 1 The cytochemical staining of the neutrophil from patient (a) shows negativity when compared to the control (b). Figure 2 The flow cytometry analysis showed reduced positivity of myeloperoxidase at the gate of neutrophils.

Introduction DOCK8 mutations are responsible for a rare autosomal recessive immunodeficiency syndrome associated with severe cutaneous viral infections, elevated IgE levels, environmental allergies, autoimmunity, and malignancy. DOCK8 activates CDC42, which is important for cell signalling and actin reorganisation. Natural killer cells play a vital role in tumour surveillance and defence against virally infected cells. NK cell function relies on the accumulation of actin at the NK cell immunologic synapse formed with target cells. Although abnormalities in T and B cell function have been described in DOCK8-deficient patients, the role of NK cells in this disease is poorly understood. Objectives/Aims Given the susceptibility to severe cutaneous viral infection and malignancy, we hypothesised there was a substantive defect in NK cell function in patients with DOCK8 deficiency. Methods 10 patients with genetically confirmed DOCK8 deficiency as well as NK cell lines with stably reduced DOCK8 expression were evaluated experimentally using in vitro NK cell cytotoxicity, F-actin content, and confocal immunofluorescence microscopy assays. Results DOCK8-deficient patients and cell lines all had decreased NK cell cytotoxicity and function could not be restored after IL-2 stimulation. Importantly, DOCK8 deficiency did not affect NK cell F-actin content, but impaired F-actin accumulation at the lytic immunological synapse. Conclusions DOCK8 deficiency results in severely deficient NK cell function owing to an inability to form a mature lytic immunological synapse via focal F-actin accumulation. This defect may underlie important and previously perplexing attributes of the DOCK8 deficiency clinical syndrome including the unusual susceptibility to viral infection.