Efficacy, safety, tolerability and pharmacokinetics of a novel human immune globulin subcutaneous, 20%: a Phase 2/3 study in Europe in patients with primary immunodeficiencies.

A highly concentrated (20%) immunoglobulin (Ig)G preparation for subcutaneous administration (IGSC 20%), would offer a new option for antibody replacement therapy in patients with primary immunodeficiency diseases (PIDD). The efficacy, safety, tolerability and pharmacokinetics of IGSC 20% were evaluated in a prospective trial in Europe in 49 patients with PIDD aged 2-67 years. Over a median of 358 days, patients received 2349 IGSC 20% infusions at monthly doses equivalent to those administered for previous intravenous or subcutaneous IgG treatment. The rate of validated acute bacterial infections (VASBIs) was significantly lower than 1 per year (0·022/patient-year, P < 0·0001); the rate of all infections was 4·38/patient-year. Median trough IgG concentrations were ≥ 8 g/l. There was no serious adverse event (AE) deemed related to IGSC 20% treatment; related non-serious AEs occurred at a rate of 0·101 event/infusion. The incidence of local related AEs was 0·069 event/infusion (0·036 event/infusion, when excluding a 13-year-old patient who reported 79 of 162 total related local AEs). The incidence of related systemic AEs was 0·032 event/infusion. Most related AEs were mild, none were severe. For 64·6% of patients and in 94·8% of IGSC 20% infusions, no local related AE occurred. The median infusion duration was 0·95 (range = 0·3-4·1) h using mainly one to two administration sites [median = 2 sites (range = 1-5)]. Almost all infusions (99·8%) were administered without interruption/stopping or rate reduction. These results demonstrate that IGSC 20% provides an effective and well-tolerated therapy for patients previously on intravenous or subcutaneous treatment, without the need for dose adjustment.

Spectrum and management of complement immunodeficiencies (excluding hereditary angioedema) across Europe.

INTRODUCTION: Complement immunodeficiencies (excluding hereditary angioedema and mannose binding lectin deficiency) are rare. Published literature consists largely of case reports and small series. We collated data from 18 cities across Europe to provide an overview of primarily homozygous, rather than partial genotypes and their impact and management. METHODS: Patients were recruited through the ESID registry. Clinical and laboratory information was collected onto standardized forms and analyzed using SPSS software. RESULTS: Seventy-seven patients aged 1 to 68 years were identified. 44 % presented in their first decade of life. 29 % had C2 deficiency, defects in 11 other complement factors were found. 50 (65 %) had serious invasive infections. 61 % of Neisseria meningitidis infections occurred in patients with terminal pathway defects, while 74 % of Streptococcus pneumoniae infections occurred in patients with classical pathway defects (p < 0.001). Physicians in the UK were more likely to prescribe antibiotic prophylaxis than colleagues on the Continent for patients with classical pathway defects. After diagnosis, 16 % of patients suffered serious bacterial infections. Age of the patient and use of prophylactic antibiotics were not associated with subsequent infection risk. Inflammatory/autoimmune diseases were not seen in patients with terminal pathway, but in one third of patients classical and alternative pathway defects. CONCLUSION: The clinical phenotypes of specific complement immunodeficiencies vary considerably both in terms of the predominant bacterial pathogen, and the risk and type of auto-inflammatory disease. Appreciation of these phenotypic differences should help both immunologists and other specialists in their diagnosis and management of these rare and complex patients.

Severe XLP Phenotype Caused by a Novel Intronic Mutation in the SH2D1A Gene.

We describe here a novel c.137 + 5G > A intronic mutation in the SH2D1A gene of the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) in association with Epstein-Barr virus (EBV)-induced fatal infectious mononucleosis (FIM) in an 8-year-old male patient and his 3-year-old step brother. The mother and the maternal grandmother of the boys are healthy and heterozygous for this sequence variant. Genetic sequencing of blood-cell-derived cDNA in the younger patient revealed a 22 bp deletion in the SH2D1A cDNA. Immunoblot and flow cytometry analysis performed in this younger patient showed the lack of SAP protein expression in peripheral blood lymphocytes. These data suggest that the novel c.137 + 5G > A mutation results in loss of function of SAP protein and leads to typical X-linked lymphoproliferative disease phenotype. We propose that intron 1 and the c.137 + 5G may be the most frequent intronic hot spot for SH2D1A splicing mutation.

Severe skin inflammation and filaggrin mutation similarly alter the skin barrier in patients with atopic dermatitis.

BACKGROUND: Filaggrin (FLG) deficiency is a well-known predisposing factor for the development of atopic dermatitis (AD). Decreased FLG expression can be the result of haploinsufficiency or severe inflammation, which can cause acquired FLG alterations. FLG mutations are related to several clinical and laboratory parameters of AD; however, some recent data seem to contradict these associations. OBJECTIVES: Our aim was to determine which clinical and biochemical parameters are connected to FLG haploinsufficiency and which ones are also associated with acquired FLG alterations due to severe skin symptoms in patients with AD. METHODS: We introduced a novel classification of patients with AD, based on FLG mutations and SCORAD (SCORing Atopic Dermatitis). Based on these parameters, we created three groups of patients with AD: mild-to-moderate wild-type (A), severe wild-type (B) and severe mutant (C). In all groups, we assessed laboratory and clinical parameters and performed immunohistochemical analyses. RESULTS: Groups B and C contained patients with equally severe symptoms based on the SCORAD. The two severe groups did not differ significantly with respect to barrier-specific parameters, whereas group A had significantly better results for the barrier function measurements. However, significant differences were detected between groups B and C with respect to the allergic sensitization-specific parameters. CONCLUSIONS: These findings suggest that skin barrier function correlates with the severity of skin inflammation and can be equally impaired in patients with FLG mutant- and wild-type AD with severe symptoms. Nevertheless, our results also suggest that patients with FLG mutant-type AD may have a higher risk of allergic sensitization compared with patients with the wild-type.

Anti-cytokine autoantibodies preceding onset of autoimmune polyendocrine syndrome type I features in early childhood.

PURPOSE: Almost all patients with autoimmune polyendocrine syndrome (APS)-I have high titer neutralizing autoantibodies to type I interferons (IFN), especially IFN-ω and IFN-α2, whatever their clinical features and onset-ages. About 90 % also have antibodies to interleukin (IL)-17A, IL-17F and/or IL-22; they correlate with the chronic mucocutaneous candidiasis (CMC) that affects ~90 % of patients. Our aim was to explore how early the manifestations and endocrine and cytokine autoantibodies appear in young APS-I patients. That may hold clues to very early events in the autoimmunization process in these patients. METHODS: Clinical investigations and autoantibody measurements in 13 APS-I patients sampled before age 7 years, and 3 pre-symptomatic siblings with AIRE-mutations in both alleles. RESULTS: Antibody titers were already high against IFN-α2 and IFN-ω at age 6 months in one sibling-8 months before onset of APS-I-and also against IL-22 at 7 months in another (still unaffected at age 5 years). In 12 of the 13 APS-I patients, antibody levels were high against IFN-ω and/or IL-22 when first tested, but only modestly positive against IFN-ω in one patient who had only hypo-parathyroidism. Endocrine organ-specific antibodies were present at age 6 months in one sibling, and as early as 36 and 48 months in two of the six informative subjects. CONCLUSION: This is the first study to collate the onset of clinical features, cytokine and endocrine autoantibodies in APS-I infants and siblings. The highly restricted early autoantibody responses and clinical features they show are not easily explained by mere loss of broad-specific self-tolerance inducing mechanisms, but hint at some more sharply focused early event(s) in autoimmunization.

Molecular Diagnostic Challenges and Complex Management of Consecutive Twin Pregnancies in a Family with CD40 Ligand Deficiency.

X-linked hyper-IgM syndrome (XHIGM) is a primary immunodeficiency disorder (PID) caused by mutation in the gene encoding the CD40 ligand (CD40L) expressed on activated T cells. Prenatal genotyping in carriers with twin pregnancies is more challenging than in women with singleton pregnancies. In addition, women with twin pregnancies may decide on selective termination for which the risk of loss of the healthy foetus may exceed 7%. We report here on a family affected by XHIGM. Diagnosis of the disease was made in a male patient as late as 33 years of age. After family screening, the sister of the proband conceived male twins in two consecutive pregnancies. In the first pregnancy, one of the male foetuses was hemizygous for the c.521A>G (Q174R) mutation in the CD40L gene. In the second pregnancy, ultrasound scan showed one foetus to have exencephaly and karyotyping revealed this foetus to have trisomy 18. Several options were discussed, but the parents decided on selective termination in both pregnancies. The interventions were successful in both cases, and the mother now has two healthy sons. This report demonstrates the way in which advanced technologies in molecular medicine and obstetric interventions may assist families with decisions about possible selective termination in case of life-threatening molecular or chromosomal disorders. Diagnosis of CD40L deficiency at the age of 33 years in the proband was striking and indicated that PIDs are still neglected as disease entities in the evaluation of patients with recurrent severe infectious diseases.

A multi-centre study of efficacy and safety of Intratect®, a novel intravenous immunoglobulin preparation.

We studied the efficacy, safety and pharmacokinetic profiles of Intratect®, a recently developed polyvalent intravenous immunoglobulin (IVIG) preparation. Fifty-one patients (aged 6-48 years) with primary immunodeficiencies (PID) and established replacement therapy using a licensed IVIG were enrolled and treated for 12 months with Intratect®. Retrospective patient data served as prestudy controls. The primary efficacy variable was the annual rate of acute serious bacterial infection (ASBI) per patient. Secondary parameters were annual rate of acute relevant infection (ARI), days with antibiotic use, fever, absence from school/work and hospitalization. The average IVIG dose was 0·49 g/kg, with an average infusion rate of 2·4 ml/kg/h. The annual ASBI rate/patient was 0·02 and ARIs were detected 128 times during the 630 adverse events in 40 patients, specified mainly as bronchitis, sinusitis, respiratory tract infection, rhinitis and pharyngitis. The annual rate of respiratory ARIs/patient was 2·0 and the rates/patient for days with fever >38°C, school/work absence and hospitalization were 1·81, 3·99 and 0·36, respectively. A total of 630 adverse events (AEs) were observed in 50 of 51 (98·0%) of patients. In 46 of 51 patients the AEs were not related to infusion. Pharmacokinetic studies after the first infusion revealed a mean elimination half-life of 50·8 ± 30·3 days. During this study, 19 of 649 (2·9%) IgG trough levels were below 6 g/l, better than that of reference IVIGs during the 6 months before study start (10 of 201). These data suggest that Intratect® is a well tolerated, safe and effective IgG concentrate for the treatment of patients with PID.

Novel primary immunodeficiencies relevant to internal medicine: novel phenotypes.

Primary immunodeficiencies (PIDs) are often recognized in adults, either because of delayed diagnosis of a paediatric illness, or increasingly because of the recognition of adult onset forms of these diseases. Moreover, a growing fraction of children diagnosed with PIDs reach adulthood. It has become clear that many of these conditions affect various organs and therefore will be referred to professionals from various fields of internal medicine. It is well known that infectious diseases, allergy, auto-immunity and cancer may result from PIDs. Surprisingly, other clinical manifestations were recently found to reflect inborn errors of immunity. Ground-breaking discoveries suggest that atypical haemolytic uraemic syndrome, Crohn's disease, and alveolar proteinosis may actually be manifestations of novel PIDs.

Multiple independent second-site mutations in two siblings with somatic mosaicism for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disorder associated with microthrombocytopenia, eczema, autoimmunity and predisposition to malignant lymphoma. Although rare, few cases of somatic mosaicism have been published in WAS patients to date. We here report on two Ukrainian siblings who were referred to us at the age of 3 and 4 years, respectively. Both patients suffered from severe WAS caused by a nonsense mutation in exon 1 of the WAS gene. In both siblings, flow cytometric analysis revealed the presence of Wiskott-Aldrich syndrome protein (WASp)-positive and WASp-negative cell populations among T and B lymphocytes as well as natural killer (NK) cells. In contrast to previously described cases of revertant mosaicism in WAS, molecular analyses in both children showed that the WASp-positive T cells, B cells, and NK cells carried multiple different second-site mutations, resulting in different missense mutations. To our knowledge, this is the first report describing somatic mosaicism in WAS patients caused by several independent second-site mutations in the WAS gene.

Deficient interferon-gamma receptor-mediated signaling in neonatal macrophages.

AIM: To describe functional and molecular characteristics of interferon-gamma (IFN-gamma) activation in neonatal mononuclear phagocytes (monocytes and macrophages). METHODS AND RESULTS: Exposure of cord and adult macrophages to IFN-gamma gave quantitatively different results in Candida killing, as well as in release of superoxide anion (O2-). At concentrations of 100 U ml(-1) IFN-gamma, maximal increase in these functions with adult macrophages was achieved, whereas no enhancement of killing and O2- release by cord macrophages could be detected. Expression of IFN-gamma receptors was comparable on cord and adult cells and specific binding of [125I]IFN-gamma to cord monocytes and macrophages was even higher compared with adult cells. By flow cytometry, elements of IFN-gamma receptor-mediated signaling in cord and adult monocytes and macrophages were studied. Monoclonal antibodies against the native form of the signal transducer and activator of transcription-1 (STAT-1) revealed comparable expression of this protein in cord and adult macrophages. However, STAT-1 phosphorylation in response to IFN-gamma was significantly decreased in neonatal monocytes (p < 0.05) and macrophages (p < 0.01) compared with adult cells. CONCLUSION: These data suggest deficient cytokine receptor signaling in neonatal mononuclear phagocytes exposed to IFN-gamma.

No short-term immunological effects of Pneumococcus vaccination in patients with systemic lupus erythematosus.

OBJECTIVE: to investigate the early immunological effects of Pneumococcus vaccination in SLE patients and healthy controls. METHODS: First-four-week follow-up of 18 patients and 9 healthy controls by repeated measurements of anti-nuclear antibodies, anti-dsDNA, C-reactive protein, complement factor 3 (C3) and 4 (C4), total IgG, IgA and IgM. Specific antibody response, percentage of blood lymphocyte populations and whole blood chemiluminescence measurements were carried out in six patients and six controls. RESULTS: No disease flare was detected in the vaccinated patients. all side effects were mild. The concentrations of serum IgG, IgA, C3 and C4 decreased significantly, but still remained within the normal range. The other changes were statistically non-significant. The specific antibody responses to 6B and 23F Pneumococcus serotypes showed striking individual differences. CONCLUSION: There was no short-term immunological effect of Pneumococcus vaccination in the patients with SLE. The non-responders. without any sign of disease activation should possibly be given more immunogenic, new vaccines to avoid life-threatening Pneumococcus infections.

Cytokine receptor signalling in neonatal macrophages: defective STAT-1 phosphorylation in response to stimulation with IFN-gamma.

We reported earlier that neonatal monocyte-derived macrophages (MDM) could not be fully activated with IFN-gamma, a finding that could not be attributed to lower expression of IFN-gamma receptors on the neonatal cells. In this study we explored elements of IFN-gamma R-mediated signalling in cord monocytes and MDM. Intracellular expression of STAT-1 was analysed by flow cytometry. We have assessed phosphorylation of STAT-1 by using MoAbs that distinguish native and phosphorylated forms of STAT-1 on a discrete cell basis. Using MoAbs against the native form of STAT-1 revealed comparable expression of this protein in cord and adult cells (both monocytes and MDM). However, STAT-1 phosphorylation in response to IFN-gamma was significantly decreased in neonatal monocytes (P < 0.05) and MDM (P < 0.01) compared to adult cells (n > 5 for each). These data suggest deficient cytokine-receptor signalling in neonatal mononuclear phagocytes exposed to IFN-gamma. We propose that decreased STAT-1 phosphorylation and activation may represent developmental immaturity and may contribute to the unique susceptibility of neonates to infections by intracellular pathogens.

A mannose-binding receptor is expressed on human keratinocytes and mediates killing of Candida albicans.

Human keratinocytes are known to kill Candida albicans in vitro, but the mechanism of killing is not yet understood. Here, we demonstrate that spontaneous, ultraviolet-B-light-induced, alpha-melanocyte-stimulating-hormone-induced, and interleukin-8-induced Candida killing by keratinocytes can be inhibited with mannan and mannosylated bovine serum albumin (Man-BSA). A polyclonal goat serum raised against the human macrophage mannose receptor stained suprabasal keratinocytes, but no staining was observed on keratinocytes with a monoclonal antibody (mAb15) specific for the human macrophage mannose receptor. Mannose-affinity chromatography of keratinocyte extract isolated a 200 kDa protein, and on the Western blot the goat antiserum reacted with a 200 kDa protein. In radioligand binding studies, the binding of 125I-Man-BSA to human keratinocytes was inhibited by mannan in a concentration-dependent manner. Analysis of the binding revealed a single class keratinocyte mannose receptor with a KD of 1.4 x 10(-8) M and a Bmax of 1 x 10(4) binding sites per cell. The binding of 125I-Man- BSA to keratinocytes proved to be time-dependent, acid-precipitable, and Ca2+- and trypsin-sensitive. After trypsinization the receptors underwent a rapid recovery at 37 degrees C. These results demonstrate the presence of mannose receptor on human keratinocytes, and its active involvement in the killing of Candida albicans.

Phosphatidic acid and diacylglycerol directly activate NADPH oxidase by interacting with enzyme components.

The enzyme NADPH oxidase is regulated by phospholipase D in intact neutrophils and is activated by phosphatidic acid (PA) plus diacylglycerol (DG) in cell-free systems. We showed previously that cell-free NADPH oxidase activation by these lipids involves both protein kinase-dependent and -independent pathways. Here we demonstrate that only the protein kinase-independent pathway is operative in a cell-free system of purified and recombinant NADPH oxidase components. Activation by PA + DG was ATP-independent and unaffected by the protein kinase inhibitor staurosporine, indicating the lack of protein kinase involvement. Both PA and DG were required for optimal activation to occur. The drug reduced activation of NADPH oxidase by either arachidonic acid or PA + DG, with IC(50) values of 46 and 25 microm, respectively. The optimal concentration of arachidonic acid or PA + DG for oxidase activation was shifted to the right with, indicating interference of the drug with the interaction of lipid activators and enzyme components. inhibited the lipid-induced aggregation/sedimentation of oxidase components p47(phox) and p67(phox), suggesting a disruption of the lipid-mediated assembly process. The direct effects of on NADPH oxidase activation complicate its use as a "specific" inhibitor of DG kinase. We conclude that the protein kinase-independent pathway of NADPH oxidase activation by PA and DG involves direct interaction with NADPH oxidase components. Thus, NADPH oxidase proteins are functional targets for these lipid messengers in the neutrophil.

[Disorders of interferon-gamma activation pathways: new group of primary immune deficiency diseases]. / Az interferon-gamma aktivációs utak defektusai: a primer immunhiányos betegségek új csoportja.

Protective immunity to intracellular bacteria such as mycobacteria and salmonella depends on intact cell-mediated immunity. Major effector mechanisms of cell-mediated immunity involve activation of macrophages by T helper-1 cytokines, particularly interferon-gamma. Patients with genetic deficiency of T helper-1 cytokines (IFN-gamma, IL-12) or T helper-1 cytokine receptors (IFN-gamma receptor, IL-12 receptor) are susceptible to infections with poorly pathogenic mycobacteria, and salmonella, suggesting that T helper-1 cytokines are essential in host defense against these pathogens. This review reports on the genetic and clinical characteristics of primary deficiencies of interferon-gamma activation pathways.

Activation of human granulocytes by intravenous immunoglobulin preparations is mediated by FcgammaRII and FcgammaRIII receptors.

Previous studies from our laboratory have shown that i.v. Ig (IVIG) exposure triggers superoxide anion (O2) generation by and increases bactericidal capacity of human blood granulocytes. However, the molecular interactions between IVIG and granulocytes have not been evaluated before. The objective of this study was to investigate the role of FcgammaRII and FcgammaRIII receptors in the immunomodulatory effects of IVIG concentrates on granulocytes. We found that four different IVIG preparations (concentration range, 1-10 mg/mL) shared the ability to stimulate O2- release in vitro by granulocytes in a dose-dependent manner. Dimers fractionated from IVIG were significantly more potent in inducing activity of the respiratory burst than were monomers. MAb (concentration range, 0.1-10 microg/mL) specific for FcgammaRII and FcgammaRIII receptors inhibited the IVIG-induced O2- release, with a more profound inhibitory effect observed with anti-FcgammaRIII. These findings suggest the involvement of Fcgamma receptors in triggering O2- release by granulocytes exposed to IVIG. We also report that IVIG added to granulocyte suspensions elicited a rapid and vigorous increase in the concentration of cytosolic free calcium, a finding suggesting direct activation and not priming of granulocytes by IVIG through FcgammaRII and FcgammaRIII receptors. The in vitro effects described here might occur in patients treated with IVIG and may, in part, be responsible for inflammatory reactions evoked by infused Ig concentrates as well as the immunomodulatory effect of Ig in patients with autoimmune and inflammatory diseases.