Activating PI3Kδ mutations in a cohort of 669 patients with primary immunodeficiency.

The gene PIK3CD codes for the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), and is expressed solely in leucocytes. Activating mutations of PIK3CD have been described to cause an autosomal dominant immunodeficiency that shares clinical features with common variable immunodeficiency (CVID). We screened a cohort of 669 molecularly undefined primary immunodeficiency patients for five reported mutations (four gain-of-function mutations in PIK3CD and a loss of function mutation in PIK3R1) using pyrosequencing. PIK3CD mutations were identified in three siblings diagnosed with CVID and two sporadic cases with a combined immunodeficiency (CID). The PIK3R1 mutation was not identified in the cohort. Our patients with activated PI3Kδ syndrome (APDS) showed a range of clinical and immunological findings, even within a single family, but shared a reduction in naive T cells. PIK3CD gain of function mutations are more likely to occur in patients with defective B and T cell responses and should be screened for in CVID and CID, but are less likely in patients with a pure B cell/hypogammaglobulinaemia phenotype.

Low ficolin-2 levels in common variable immunodeficiency patients with bronchiectasis.

Common variable immunodeficiency (CVID) encompasses a heterogeneous group of antibody deficiencies characterized by susceptibility to recurrent infections and sequelae, including bronchiectasis. We investigated the relevance of the lectin complement pathway in CVID patients by analysing ficolin-2 and ficolin-3 serum levels and genotyping single nucleotide polymorphisms (SNPs) in the FCN2 and FCN3 genes. Our results show that ficolin-2 levels in CVID patients are significantly lower (P < 0.0001) than in controls. The lowest ficolin-2 levels are found in CVID patients with bronchiectasis (P = 0.0004) and autoimmunity (P = 0.04). Although serum levels of ficolin-3 were similar in CVID patients and controls, CVID patients with bronchiectasis again showed lower levels when compared to controls (P = 0.0001). Analysis of single nucleotide polymorphisms in the FCN2 gene confirmed known influences on ficolin-2 serum levels, but did not support a genetic basis for the observed ficolin-2 deficiency in CVID. We found that CVID patients with bronchiectasis have very low levels of ficolin-2. The reason for the deficiency of ficolin-2 in CVID and any possible causal relationship is currently unknown. However, as bronchiectasis is a very important factor for morbidity and mortality in CVID, ficolin-2 could also serve as biomarker for monitoring disease complications such as bronchiectasis.

Intravenous sildenafil i.v. as rescue treatment for refractory pulmonary hypertension in extremely preterm infants.

BACKGROUND: Intravenous sildenafil treatment has recently shown promising results and good tolerability in the treatment of refractory pulmonary hypertension (PH) in term and near-term neonates, while comparable data in preterm infants are still lacking. However, for critically ill preterm infants suffering from PH refractory to conventional treatment, sildenafil may represent a last treatment resort. PATIENTS AND METHODS: We reviewed the records of 6 critically ill extremely preterm infants who had suffered from PH refractory to conventional treatment and had obtained intravenous sildenafil after careful consideration as ultima ratio treatment. AIM: To describe the responses to sildenafil in terms of hemodynamic and respiratory changes during treatment and outcome. RESULTS: 4/6 patients showed resolution of severe PH with full reversal of ductal shunt direction into pure left-to-right shunt within 82 ± 35 h after sildenafil start. Remarkably, 2/6 patients developed pulmonary hemorrhage at a time point when significant improvement of PH had already taken place, both of them survived. Overall 4/6 patients died, two deaths were related to treatment-refractory PH. CONCLUSION: Intravenous sildenafil treatment seems effective in improving severe PH and hemodynamic instability in extremely preterm infants with refractory PH. Pulmonary hemorrhage may represent a distinct adverse effect of sildenafil treatment in these patients, presumably due to sudden reversal of ductal shunt. Accordingly, sildenafil should be restricted to most severe and refractory cases in this population.

Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans.

The functional interaction of BAFF and APRIL with TNF receptor superfamily members BAFFR, TACI and BCMA is crucial for development and maintenance of humoral immunity in mice and humans. Using a candidate gene approach, we identified homozygous and heterozygous mutations in TNFRSF13B, encoding TACI, in 13 individuals with common variable immunodeficiency. Homozygosity with respect to mutations causing the amino acid substitutions S144X and C104R abrogated APRIL binding and resulted in loss of TACI function, as evidenced by impaired proliferative response to IgM-APRIL costimulation and defective class switch recombination induced by IL-10 and APRIL or BAFF. Family members heterozygous with respect to the C104R mutation and individuals with sporadic common variable immunodeficiency who were heterozygous with respect to the amino acid substitutions A181E, S194X and R202H had humoral immunodeficiency. Although signs of autoimmunity and lymphoproliferation are evident, the human phenotype differs from that of the Tnfrsf13b-/- mouse model.

X-linked inhibitor of apoptosis (XIAP) deficiency: the spectrum of presenting manifestations beyond hemophagocytic lymphohistiocytosis.

X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was initially described in patients with X-linked lymphoproliferative syndrome (XLP) who had no mutations in SH2D1A. In the initial reports, EBV-associated hemophagocytic lymphohistiocytosis (HLH) was the predominant clinical phenotype. Among 25 symptomatic patients diagnosed with XIAP deficiency, we identified 17 patients who initially presented with manifestations other than HLH. These included Crohn-like bowel disease (n=6), severe infectious mononucleosis (n=4), isolated splenomegaly (n=3), uveitis (n=1), periodic fever (n=1), fistulating skin abscesses (n=1) and severe Giardia enteritis (n=1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly and partial HLH. Screening by flow cytometry identified 14 of 17 patients in our cohort. However, neither genotype nor protein expression nor results from cell death studies were clearly associated with the clinical phenotype. Only mutation analysis can reliably identify affected patients. XIAP deficiency must be considered in a wide range of clinical presentations.

Clinical and immunological overlap between autoimmune lymphoproliferative syndrome and common variable immunodeficiency.

Autoimmune lymphoproliferative syndrome (ALPS) is mainly caused by defects in the CD95 pathway. Raised CD3+TCRαß+CD4-CD8- double negative T cells and impaired T cell apoptosis are hallmarks of the disease. In contrast, the B cell compartment has been less well studied. We found an altered distribution of B cell subsets with raised transitional B cells and reduced marginal zone B cells, switched memory B cells and plasma blasts in most of 22 analyzed ALPS patients. Moreover, 5 out of 66 ALPS patients presented with low IgG and susceptibility to infection revealing a significant overlap between ALPS and common variable immunodeficiency (CVID). In patients presenting with lymphoproliferation, cytopenia, hypogammaglobulinemia and impaired B cell differentiation, serum biomarkers were helpful in addition to apoptosis tests for the identification of ALPS patients. Our observations may indicate a role for apoptosis defects in some diseases currently classified as CVID.

Mannose-binding lectin gene polymorphic variants predispose to the development of bronchopulmonary complications but have no influence on other clinical and laboratory symptoms or signs of common variable immunodeficiency.

Mannose-binding lectin (MBL), activating protein of the lectin pathway of the complement system, is an important component of the non-specific immune response. MBL2 gene polymorphisms, both in the coding and promoter regions, lead to low or deficient serum MBL levels. Low serum MBL levels were shown to be associated with serious infectious complications, mainly in patients in whom other non-specific immune system barriers were disturbed (granulocytopenia, cystic fibrosis). We have analysed two promoter (-550 and -221) and three exon (codons 52, 54 and 57) MBL2 polymorphisms in a total of 94 patients with common variable immunodeficiency (CVID) from two immunodeficiency centres. Low-producing genotypes were associated with the presence of bronchiectasis (P = 0.009), lung fibrosis (P = 0.037) and also with respiratory insufficiency (P = 0.029). We could not demonstrate any association of MBL deficiency with age at onset of clinical symptoms, age at diagnosis, the number of pneumonias before diagnosis or serum immunoglobulin (Ig)G, IgA and IgM levels before initiation of Ig treatment. No association with emphysema development was observed, such as with lung function test abnormalities. No effect of MBL2 genotypes on the presence of diarrhoea, granuloma formation, lymphadenopathy, splenomegaly, frequency of respiratory tract infection or the number of antibiotic courses of the patients was observed. Our study suggests that low MBL-producing genotypes predispose to bronchiectasis formation, and also fibrosis and respiratory insufficiency development, but have no effect on other complications in CVID patients.

MicroRNA-146a reduces MHC-II expression via targeting JAK/STAT signaling in dendritic cells after stem cell transplantation.

Acute Graft-versus-host disease (GVHD) is a major immunological complication after allogeneic hematopoietic cell transplantation and a better understanding of the molecular regulation of the disease could help to develop novel targeted therapies. Here we found that a G/C polymorphism within the human microRNA-146a (miR-146a) gene of transplant recipients, which causes reduced miR-146a levels, was strongly associated with the risk of developing severe acute GVHD (n=289). In mice, deficiency of miR-146a in the hematopoietic system or transfer of recipient-type miR-146a-/- dendritic cells (DCs) enhanced GVHD, while miR-146a mimic-transfected DCs ameliorated disease. Mechanistically, lack of miR-146a enhanced JAK2-STAT1 pathway activity, which led to higher expression of class II-transactivator (CIITA) and consecutively increased MHCII-levels on DCs. Inhibition of JAK1/2 or CIITA knockdown in DCs prevented miR-146a-/- DC-induced GVHD exacerbation. Consistent with our findings in mice, patients with the miR-146a polymorphism rs2910164 in hematopoietic cells displayed higher MHCII levels on monocytes, which could be targeted by JAK1/2 inhibition. Our findings indicate that the miR-146a polymorphism rs2910164 identifies patients at high risk for GVHD before allo-HCT. Functionally we show that miR-146a acts as a central regulator of recipient-type DC activation during GVHD by dampening the pro-inflammatory JAK-STAT/CIITA/MHCII axis, which provides a scientific rationale for early JAK1/2 inhibition in selected patients.

Identification of the phosphorylation site on human erythrocyte band 7 integral membrane protein: implications for a monotopic protein structure.

Band 7 membrane protein was metabolically labelled with [32P]phosphate in the presence of cAMP, isolated and digested, the labelled peptides were purified and sequenced. Ser-9 was identified as the only phosphorylation site. This proves that the N-terminal region is located at the cytoplasmic side of the membrane and implies a monotopic rather than the predicted bitopic structure.

Isolation, molecular characterization, and tissue-specific expression of ECP-51 and ECP-54 (TIP49), two homologous, interacting erythroid cytosolic proteins.

We isolated two proteins, ECP-51 and ECP-54, from human erythrocyte cytosol by affinity chromatography using a peptide of the integral membrane protein stomatin as bait. Partial amino acid sequence information obtained by microsequencing allowed us to clone the respective cDNAs. Analysis of the nucleotide sequences revealed that ECP-51 and ECP-54 are homologous (44.2% amino acid identity) and contain ATP-binding sites. ECP-54 was identified as TIP49/RUVBL1/NMP238, which is a component of a large nuclear protein complex, possibly the RNA polymerase II holoenzyme; ECP-51 is a novel protein. Using the two-hybrid system, we showed that these proteins interact with each other. The interaction of ECP-51 and ECP-54 with the stomatin peptide and the localization to the nucleus and cytoplasm suggest an additional function for these proteins as chaperone components.

Isolation, molecular characterization, and tissue-specific expression of a novel putative G protein-coupled receptor.

We isolated a 40 kDa integral membrane protein (p40) from human erythrocyte ghosts by affinity chromatography, using a C-terminal peptide of stomatin, and obtained partial sequences which enabled us to isolate two full-length cDNAs from human bone marrow and fetal brain cDNA libraries. The cDNA sequences were identical and encoded a novel putative G protein-coupled receptor (399 amino acids). Northern and RNA dot blot analyses demonstrated that the major 4.8 kb-transcript is predominantly expressed in brain. In situ hybridization studies of tissue sections revealed high expression in neurons of the brain and spinal cord, in thymocytes, megakaryocytes, and macrophages.

A novel compound heterozygous TACI mutation in an autosomal recessive common variable immunodeficiency (CVID) family.

Common variable immunodeficiency (CVID) is a primary immune disorder characterized by low immunoglobulin serum levels and increased susceptibility to infections. Underlying genetic causes are only known in less than 15% of patients and encompass mutations in the genes encoding for ICOS, TACI, BAFF-R, CD19, CD20, CD81 and MSH5. TACI is the most frequently mutated gene among CVID patients. We report on two pediatric Italian male siblings with hypogammaglobulinemia and recurrent respiratory and gastrointestinal infections in association with a novel compound heterozygous TACI mutation. Both patients carry the I87N/C104R mutation that has not been reported yet. This results in aberrant TACI expression and abrogates APRIL binding on EBV B cells. This study identifies a novel combined mutation in TNFRSF13B increasing the spectrum of TACI mutations associated with CVID.

Sequence variants of the TNFRSF13B gene in Czech CVID and IgAD patients in the context of other populations.

Mutations in the TNFRSF13B gene, encoding TACI, have been found in common variable immunodeficiency (CVID) and selective IgA deficient (IgAD) patients, but only the association with CVID seems to be significant. In this study, Czech CVID, IgAD and primary hypo/dysgammaglobulinemic (HG/DG) patients were screened for all TNFRSF13B sequence variants. The TNFRSF13B gene was mutated in 4/70 CVID patients (5.7%), 9/161 IgAD patients (5.6%), 1/17 HG/DG patient (5.9%) and none of 195 controls. Eight different mutations were detected, including the most frequent p.C104R and p.A181E mutations as well as 1 novel missense mutation, p.R189K. A significant association of TNFRSF13B gene mutations was observed in both CVID (p=0.01) and IgAD (p=0.002) Czech patients. However, when combined with all published data, only the association with CVID remained significant compared with the controls (9.9% vs. 3.2%, p<10(-6)), while statistical significance disappeared for IgAD (5.7% vs. 3.2%, p=0.145). The silent mutation p.P97P was shown to be associated significantly with CVID compared with the controls in both Czech patients (allele frequency 4.3% vs. 0.2%, p=0.01) and in connection with the published data (5.1% vs. 1.8%, p=0.003). The relevance of some TNFRSF13B gene variants remains unclear and needs to be elucidated in future studies.

Active vaccination in patients with common variable immunodeficiency (CVID).

Active vaccination of CVID patients with standard vaccines has rarely been studied in depth although some patients have been shown to develop transient vaccine-specific immunity. We addressed the question whether these patients can be identified by functional classification of their B cell subsets in vitro. Twenty-one CVID patients receiving regular IgG substitution were immunized with anti-peptide and anti-polysaccharide vaccines. Humoral vaccination responses were compared to the numbers of circulating memory B cells, CD21(low) B cells and the capacity to produce antibodies in vitro. Our findings allow four conclusions: (1) positive vaccination responses are not contradictory to the diagnosis of CVID; they occurred against polypeptide vaccines in 23% and against polysaccharide antigens in 18% of all vaccinations. (2) Class-switched antibody responses occur preferentially in patients of CVID group II. (3) A normal percentage of IgM memory B cells is necessary but not sufficient for a vaccination response to polysaccharide antigens. (4) Active vaccination in addition to IgG replacement therapy should be performed in patients of CVID type II - especially in case of vaccines for which passive protection cannot be guaranteed.

The analysis of essential oils and extracts (oleoresins) from seasonings--a critical review.

A critical review of the analytical methods employed for the determination of the relevant components of seasonings is presented. Where the available methods were inadequate, new ones have been devised. Particular emphasis has been placed on those methods of analysis that provide a rapid and sufficiently accurate appraisal of seasoning extracts and essential oils from seasonings under routine control laboratory conditions. At the same time, the margin of error of these methods has been determined. The individual seasoning extracts were assessed according to the following criteria: (1) essential oil--cardamom, laurel leaves, cloves, origanum (marjoram), sage, and thyme; (2) essential oil and nonvolatile lipids--dillseed, coriander, caraway, mace, nutmeg, pimento (allspice), and celery seed; (3) essential oil and/or pungent ingredients--capsicum, ginger, and pepper; (4) essential oil and/or coloring matter--turmeric (curcuma) and paprika; (5) essential oil and other components--garlic, onion, and cinnamon.

Stomatin, flotillin-1, and flotillin-2 are major integral proteins of erythrocyte lipid rafts.

Lipid rafts are sphingolipid- and cholesterol-rich membrane microdomains that are insoluble in nonionic detergents, have a low buoyant density, and preferentially contain lipid-modified proteins, like glycosyl phosphatidylinositol (GPI)-anchored proteins. The lipid rafts were isolated from human erythrocytes and major protein components were identified. Apart from the GPI-anchored proteins, the most abundant integral proteins were found to be the distantly related membrane proteins stomatin (band 7.2b), flotillin-1, and flotillin-2. Flotillins, already described as lipid raft components in neurons and caveolae-associated proteins in A498 kidney cells, have not been recognized as red cell components yet. In addition, it was shown that the major cytoskeletal proteins, spectrin, actin, band 4.1, and band 4.2, are partly associated with the lipid rafts. Stomatin and the flotillins are present as independently organized high-order oligomers, suggesting that these complexes act as separate scaffolding components at the cytoplasmic face of erythrocyte lipid rafts.

[Adult-onset primary immunodeficiencies]. / Adult-manifestierende primäre Immundefizienzen.

Different individuals with the same kind of primary immunodeficiency may start having symptoms from early childhood on, or alternatively much later in adult life, or never. The differences in phenotype can only partly be deduced from genotype-analysis or--in case of female patients with X-linked diseases--from age-related skewing of lyonisation. The role of compensatory immune mechanisms is less clear. The microbial spectrum of infections is usually the same for both adult and infantile forms of a special primary immunodeficiency syndrome. Yet, many of the adult forms are associated with non-infectious complications, such as granuloma formation, autoimmunity or tumors. Besides standard antibiotic treatment and IgG replacement therapy, there are now different cytokine- or enzyme-replacement regimens available for some of the primary immunodeficiencies. However, exact diagnostic classification of the immunodeficiency should be obtained before such treatment modalities are used. Adult primary immunodeficiency syndromes therefore represent a challenge to both clinicians and molecular biologists.

Magnetic resonance imaging of absent pulmonary valve syndrome.

A neonate with absent pulmonary valve syndrome was examined by means of echocardiography and magnetic resonance imaging (MRI). MRI provided an excellent evaluation of the intracardial morphology, vessel anatomy, mediastinal structures, and shunt flow; and it was superior to echocardiography for detection of aortopulmonary collaterals and vessel abnormalities. This case report of an uncommon cardiac abnormality confirms that MRI is a useful complementary modality to echocardiography in neonates and is recommended for problems with central pulmonary vessels and their relation to tracheobronchial structures.