X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling.

The molecular basis of X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has remained elusive. Here we report hypomorphic mutations in the gene IKBKG in 12 males with EDA-ID from 8 kindreds, and 2 patients with a related and hitherto unrecognized syndrome of EDA-ID with osteopetrosis and lymphoedema (OL-EDA-ID). Mutations in the coding region of IKBKG are associated with EDA-ID, and stop codon mutations, with OL-EDA-ID. IKBKG encodes NEMO, the regulatory subunit of the IKK (IkappaB kinase) complex, which is essential for NF-kappaB signaling. Germline loss-of-function mutations in IKBKG are lethal in male fetuses. We show that IKBKG mutations causing OL-EDA-ID and EDA-ID impair but do not abolish NF-kappaB signaling. We also show that the ectodysplasin receptor, DL, triggers NF-kappaB through the NEMO protein, indicating that EDA results from impaired NF-kappaB signaling. Finally, we show that abnormal immunity in OL-EDA-ID patients results from impaired cell responses to lipopolysaccharide, interleukin (IL)-1beta, IL-18, TNFalpha and CD154. We thus report for the first time that impaired but not abolished NF-kappaB signaling in humans results in two related syndromes that associate specific developmental and immunological defects.

Hyper-immunoglobulin M syndrome type 3 with normal CD40 cell surface expression.

Mutations of the CD40 gene have been found in patients with autosomal recessive hyper-immunoglobulin M (HIGM) syndrome type 3. Five patients from four unrelated families with CD40 mutation have been reported so far. Clinical manifestations include recurrent sinopulmonary infections, Pneumocystis carinii pneumonia and Cryptosporidium parvum infection. Affected patients typically have very low levels of IgG and IgA and normal or high levels of IgM. Flow cytometry analysis of these five patients demonstrated that peripheral blood B lymphocytes lacked expression of surface CD40. Herein, we present two siblings from second-degree consanguineous Turkish parents with homozygous CD40 deletion of four nucleotides including the stop codon resulting presumably to a longer protein. Clinical and immunological profile of these patients is similar to the already reported HIGM3 patients except normal CD40 expression on B lymphocytes. This observation emphasizes the requirement of CD40 mutation analysis for definite diagnosis of HIGM3 despite normal flow cytometric expression of CD40, particularly if the immunological and clinical profile is suggestive for HIGM3.

Somatic hypermutation shapes the antibody repertoire of memory B cells in humans.

High-affinity antibodies produced by memory B cells differ from antibodies produced in naive B cells in two respects. First, many of these antibodies show somatic hypermutation, and second, the repertoire of antibodies expressed in memory responses is highly selected. To determine whether somatic hypermutation is responsible for the shift in the antibody repertoire during affinity maturation, we analyzed the immunoglobulin lambda light chain (Iglambda) repertoire expressed by naive and antigen-selected memory B cells in humans. We found that the Iglambda repertoire differs between naive and memory B cells and that this shift in the repertoire does not occur in the absence of somatic hypermutation in patients lacking activation-induced cytidine deaminase (AID). Our work suggests that somatic hypermutation makes a significant contribution to shaping the antigen-selected antibody repertoire in humans.

T cell clones from an X-linked hyper-immunoglobulin (IgM) patient induce IgE synthesis in vitro despite expression of nonfunctional CD40 ligand.

The induction of immunoglobulin E (IgE) switching in B cells requires at least two signals. The first is given by either of the soluble lymphokines interleukin 4 (IL-4) or IL-13, whereas the second is contact dependent. It has been widely reported that a second signal can be provided by the CD40 ligand (CD40L) expressed on the surface of T cells, mast cells, and basophils. A defect in the CD40L has been shown recently to be responsible for the lack of IgE, IgA, and IgG, characteristic of the childhood X-linked immunodeficiency, hyper IgM syndrome (HIGM1). IgE can however be detected in the serum of some HIGM1 patients. In this study, we isolated T cell clones and lines using phytohemagglutinin (PHA) and allergen, respectively, from the peripheral blood of one such patient who expressed a truncated form of CD40L, and investigated their ability to induce IgE switching in highly purified, normal tonsillar B cells in vitro. Unexpectedly, 4 of 12 PHA clones tested induced contact-dependent IgE synthesis in the presence of exogenous IL-4. These clones were also shown to strongly upregulated IL-4-induced germline epsilon RNA and formed dense aggregates with B cells. Of the four helper clones, three were CD8+, of which two were characteristic of the T helper cell 2 (Th2) subtype. Two allergen-specific HIGM1 T cell lines, both of the Th0 subtype, could also drive IgE synthesis when prestimulated using specific allergen. All clones and lines were negative for surface expression of CD40L, and the mutated form of CD40L was confirmed for a representative clone by RNase protection assay and sequencing. The IgE helper activity could not be attributed to membrane tumor necrosis factor alpha (TNF-alpha) although it was strongly expressed on activated clones, and the addition of neutralizing anti-TNF-alpha antibody did not abrogate IgE synthesis. These results therefore suggest the involvement of T cell surface molecules other than CD40L in the induction of IgE synthesis, and that these molecules may also be implicated in other aspects of T-B cell interactions.

Immunoglobulin class switch recombination deficiencies.

Maturation of the secondary antibody repertoire is generated by means of class switch recombination and somatic hypermutation. The molecular mechanisms underlying these important processes have long remained obscure. Inherited defects in class switch recombination variably associated to defects in somatic hypermutation are a group of genetically heterogeneous diseases, the characterization of which has allowed recognition that T-B cell interaction (resulting in CD40-mediated signaling), intrinsic B cell mechanisms, and complex DNA repair machinery are involved in class switch recombination and somatic hypermutation. Elucidation of the molecular defects underlying these disorders has been essential to better understand the molecular basis of immunoglobulin diversification and has offered the opportunity to define the clinical spectrum of these diseases and to prompt more accurate diagnostic and therapeutic approaches.

Intravenous immunoglobulins--understanding properties and mechanisms.

High-dose intravenous immunoglobulin (IVIg) preparations are used currently for the treatment of autoimmune or inflammatory diseases. Despite numerous studies demonstrating efficacy, the precise mode of action of IVIg remains unclear. Paradoxically, IgG can exert both pro- and anti-inflammatory activities, depending on its concentration. The proinflammatory activity of low-dose IVIg requires complement activation or binding of the Fc fragment of IgG to IgG-specific receptors (FcgammaR) on innate immune effector cells. In contrast, when administered in high concentrations, IVIg has anti-inflammatory properties. How this anti-inflammatory effect is mediated has not yet been elucidated fully, and several mutually non-exclusive mechanisms have been proposed. This paper represents the proceedings of a session entitled 'IVIg--Understanding properties and mechanisms' at the 6th International Immunoglobulin Symposium that was held in Interlaken on 26-28 March 2009. The presentations addressed how IgG may affect the cellular compartment, evidence for IVIg-mediated scavenging of complement fragments, the role of the dimeric fraction of IVIg, the anti-inflammatory properties of the minor fraction of sialylated IgG molecules, and the genetic organization and variation in FcgammaRs. These findings demonstrate the considerable progress that has been made in understanding the mechanisms of action of IVIgs, and may influence future perspectives in the field of Ig therapy.

6th International Immunoglobulin Symposium: poster presentations.

The posters presented at the 6th International Immunoglobulin Symposium covered a wide range of fields and included both basic science and clinical research. From the abstracts accepted for poster presentation, 12 abstracts were selected for oral presentations in three parallel sessions on immunodeficiencies, autoimmunity and basic research. The immunodeficiency presentations dealt with novel, rare class-switch recombination (CSR) deficiencies, attenuation of adverse events following IVIg treatment, association of immunoglobulin (Ig)G trough levels and protection against acute infection in patients with X-linked agammaglobulinaemia (XLA) and common variable immunodeficiency (CVID), and the reduction of class-switched memory B cells in patients with specific antibody deficiency (SAD). The impact of intravenous immunoglobulin on fetal alloimmune thrombocytopenia, pregnancy and postpartum-related relapses in multiple sclerosis and refractory myositis, as well as experiences with subcutaneous immunoglobulin in patients with multi-focal motor neuropathy, were the topics presented in the autoimmunity session. The interaction of dendritic cell (DC)-SIGN and alpha2,6-sialylated IgG Fc and its impact on human DCs, the enrichment of sialylated IgG in plasma-derived IgG, as wells as prion surveillance and monitoring of anti-measles titres in immunoglobulin products, were covered in the basic science session. In summary, the presentations illustrated the breadth of immunoglobulin therapy usage and highlighted the progress that is being made in diverse areas of basic and clinical research, extending our understanding of the mechanisms of immunoglobulin action and contributing to improved patient care.

Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency.

In humans, interferon gamma (IFN-gamma) receptor deficiency leads to a predisposition to mycobacterial infections and impairs the formation of mature granulomas. Interleukin-12 (IL-12) receptor deficiency was found in otherwise healthy individuals with mycobacterial infections. Mature granulomas were seen, surrounded by T cells and centered with epithelioid and multinucleated giant cells, yet reduced IFN-gamma concentrations were found to be secreted by activated natural killer and T cells. Thus, IL-12-dependent IFN-gamma secretion in humans seems essential in the control of mycobacterial infections, despite the formation of mature granulomas due to IL-12-independent IFN-gamma secretion.

Isolated growth hormone deficiency in a patient with immunoglobulin class switch recombination deficiency.

Growth hormone deficiency (GHD) may be associated with a number of immunodeficiency diseases, but its association with immunoglobulin class switch recombination (Ig CSR) deficiencies is very rare. We report the case of a patient with a history of recurrent diarrhea and respiratory infections diagnosed with hyper IgM syndrome on the basis of immunological findings (low serum levels of IgG and IgA and an elevated serum level of IgM). In view of the patient's short stature, growth hormone evaluation was performed and growth hormone deficiency confirmed. The patient received growth hormone therapy in addition to Ig replacement therapy and antibiotics and responded well. As the coding regions of the genes known to be responsible for Ig CSR (CD40L, CD40, AICDA, and UNG) were intact in our patient, this might be a new form of Ig CSR deficiency.

Specific in vitro antimannan-rich antigen of Candida albicans antibody production by sensitized human blood lymphocytes.

We have developed a new antigenic system for the induction of specific in vitro antibody response in man. The antigen used was purified from the cell wall of Candida albicans strain A and contained greater than 96% polysaccharide mannan. Peripheral blood mononuclear cells from Candida-sensitized donors produced specific antimannan antibodies during a 7-d culture in the presence of mannan absorbed with methylated bovine serum albumin. Two methods were used to detect antimannan antibody responses. Antimannan antibody-producing cells were identified by radioautography with tritiated mannan. Antibody concentration in culture supernatants was measured by an enzyme-linked immunosorbent assay. In both methods, specific IgM and IgG (but not IgA) antibodies were detected. The antibody production to mannan was specific, since an antigenically unrelated polysaccharide (pneumococcal antigen S III) did not bind to methylated bovine serum albumin-mannan-induced blast cells and did not induce antimannan antibody-containing cells. Furthermore, a pulse with an excess of unlabeled mannan abolished [3H]mannan binding, whereas an excess of unlabeled S III did not. Similarly, no antimannan antibody was obtained in influenza virus-stimulated cultures and mannan-stimulated cultures were not inducing antiinfluenza antibodies. The antimannan antibody production was shown to be a T cell-dependent phenomenon. The T helper effect appeared to be radiosensitive. It was under a genetic restriction as it occurred only in autologous or semi-identical but not in allogeneic situations. This system is relatively simple, reproducible, and well suited for the study of specific secondary in vitro antibody responses to polysaccharide antigens in humans.

Dysfunctions of pokeweed mitogen-stimulated T and B lymphocyte responses induced by gammaglobulin therapy.

Lymphocytes obtained from nonimmuno deficient children treated with commercially available preparations of gammaglobulin failed to proliferate and to mature into plasma cells in vitro after stimulation with pokeweed mitogen. The influence of the treatment on lymphocyte functions varied according to the cell population considered. A T helper cell activity was detected in these patients but only in the cell subset bearing receptors for IgG after irradiation. T lymphocytes exerted a suppressive effect that disappeared after irradiation or incubation at 37 degrees C. The suppressive cells were found among E rosette-forming cells depleted of leukocytes bearing receptors for IgG. Their suppressive effect was expressed only in the presence of normal radioresistant T lymphocytes that did not bear Fc receptors for IgG. Similar dysfunctions could be induced in vitro by incubation of normal T and B lymphocytes with gammaglobulin preparations. Because F(ab)'2 fragments or deaggregated preparations of gammaglobulin failed to activate T suppressor lymphocytes, this activation was likely triggered by attachment of Fc portion of denatured IgG to the corresponding membrane receptor. This activation step was prostaglandin E(2)-dependent, suggesting that activated monocytes were involved in the activation process. B lymphocyte responses appeared directly inhibited by attachment of denatured gammaglobulin on membrane Fc receptor. Our observations suggest that immunological effects of gammaglobulin therapy are not limited to antibody transfer, since it also induces subtle modifications of in vitro pokeweed mitogen-stimulated T and B cell responses. These modifications must be considered in interpreting results obtained in immunodeficient patients investigated under gamma-globulin therapy.

Specific binding of antigen onto human T lymphocytes.

Human T lymphocytes sensitized to Candida albicans (CA) were shown to proliferate in cultures induced with mannan, a ramified polysaccharide extracted from the cell well of CA. We presently describe that, when we used strongly labeled [3H]mannan, antigen-specific T blast cells were able to bind the labeled mannan on their membrane. The observations that irrelevant blast cells did not bind [3H]mannan, and that mannan-specific blast cells did not bind tritiated pneumococcal polysaccharide SIII, indicate the specificity of mannan binding. Mannan binding was reversible and saturable. Mannan binding on T blast cells was inhibited by preincubation with monoclonal antibodies to T3 but not to other T cell-related molecules. The characteristics of this receptor suggest its identity with the T cell receptor for antigen. The direct binding of mannan could be either due to a cross-linking of the receptor by multivalent mannan or to a recognition of mannan in association with HLA-DQ molecules, as suggested by partial blocking of mannan binding using anti-HLA-DQ monoclonal antibodies.

Control of human B cell tumor growth in severe combined immunodeficiency mice by monoclonal anti-B cell antibodies.

Severe combined immunodeficiency (scid) mice develop EBV (+)B cell tumors after infusion of EBV(+)B cells or of B cells and EBV. In this study, scid mice were infused with B cell lines derived from three patients who developed a B lymphocyte proliferative disorder after bone marrow or organ transplantation. Intraperitoneal injection of 5 x 10(6) B cells induced tumor growth in all mice, leading to death within 60 d. Human B cells were identified in spleen and bone marrow by means of immunofluorescence or EBV genome amplification, and human IgM was detected in serum. Infusion of murine monoclonal antibodies specific for human B cell membrane antigens CD21, CD24, and CD23 was effective in 80% of animals, against two of the three cell lines preventing tumor development or inducing remission according to the time of treatment. The effect was antibody dose dependent and was optimal with four intravenous infusions of at least 0.1 mg 4 d apart. Human IgM in serum and human B cells in spleen and bone marrow became undetectable when peritoneal tumors regressed completely. Infusions of IgG1 isotype-matched anti-CD4 antibody or anti-CD3 antibody had no effect. Tumors developed or recurred in 50% of these animals injected with one of the B cell line 3 mo after treatment was stopped. The same anti-CD21 and anti-CD24 antibodies had been used to treat the three patients, and shown similar degrees of effectiveness as in the scid mouse model. These results indicate that scid mice may be suitable for assessing therapeutic approaches to human B cell proliferation.

A defect in the regulation of major histocompatibility complex class II gene expression in human HLA-DR negative lymphocytes from patients with combined immunodeficiency syndrome.

Patients with an autosomal recessive combined immunodeficiency are characterized by an HLA negative phenotype of activated T and B lymphocytes. To determine the molecular basis of this syndrome we have studied the biosynthesis of class I and II antigens and the expression of relevant genes in these patients. The synthesis of the HLA A, B, and C heavy chain is markedly decreased, while beta 2 microglobulin is made in normal amounts. Biosynthesis of HLA-DR alpha-chain and beta-chain is abolished in the lymphocytes of these patients and there is a total absence of mRNA for either alpha-chains or beta-chains of HLA-DR. This indicates that the lack of class II antigen on these lymphocytes results from a block in the expression of HLA-DR genes. The Ii-chain, the invariant polypeptide associated intracellularly with HLA-DR, and its mRNA are made in normal amounts. Since the structural genes coding for class II polypeptides do not seem to be affected, the reported genetic defect in the patients concerns the regulation of the expression of HLA-DR genes.

Deficiency of the adhesive protein complex lymphocyte function antigen 1, complement receptor type 3, glycoprotein p150,95 in a girl with recurrent bacterial infections. Effects on phagocytic cells and lymphocyte functions.

A patient presenting delayed umbilical cord detachment, severe recurrent bacterial infections, and inability to form pus exhibited a profound defect in the expression of alpha- and beta-chains of the receptor for the C3bi fragment of C3 (CR3), lymphocyte function antigen 1 (LFA-1) molecule, and the p150,95 molecule found on neutrophils, monocytes, and lymphocyte membranes. This was shown by immunofluorescence studies using specific monoclonal antibodies, rosette formation with C3bi-coated erythrocytes, and immunoprecipitation for the LFA-1 complex. These membrane defects were responsible for abnormal phagocytic cell functions including adherence to nylon wool, cell movement, phagocytosis, and opsonized particle-induced oxidative response and for defective natural killer cell activity. In addition, lymphocyte function deficiencies previously unobserved in this disease were found. Cytolytic T lymphocyte activity was profoundly reduced; alpha- and gamma-interferon production were impaired. Finally, there was no antibody production to vaccinal antigens whereas the antibody responses to polysaccharides and to cytomegalovirus were found to be normal. The cytotoxic T cell deficiency could be expected from previous blocking experiments of this function with monoclonal antibodies to LFA-1 and is probably related to an extremely severe deficiency in LFA-1 expression in this patient. Anomalies in interferon and in antibody production suggest additional role(s) of the LFA-1 complex in monocyte/T lymphocyte/B lymphocyte cell interactions that have not yet been envisaged.

Inherited interleukin 12 deficiency in a child with bacille Calmette-Guérin and Salmonella enteritidis disseminated infection.

Interferon-gamma receptor ligand-binding chain (IFN-gammaR1) or signaling chain (IFN-gammaR2) deficiency, like interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency, predispose to severe infections due to poorly virulent mycobacteria and salmonella. A child with bacille Calmette-Guérin and Salmonella enteritidis infection was investigated. Mutations in the genes for IFN-gammaR1, IFN-gammaR2, IL-12Rbeta1, and other molecules implicated in IL-12- or IFN-gamma-mediated immunity were sought. A large homozygous deletion within the IL-12 p40 subunit gene was found, precluding expression of functional IL-12 p70 cytokine by activated dendritic cells and phagocytes. As a result, IFN-gamma production by lymphocytes was markedly impaired. This is the first discovered human disease resulting from a cytokine gene defect. It suggests that IL-12 is essential to and appears specific for protective immunity to intracellular bacteria such as mycobacteria and salmonella.

Human genetic defects in class-switch recombination (hyper-IgM syndromes).

Several genetic defects in class-switch recombination, leading to hyper-IgM syndromes, have been recently described in humans. Besides the well-known role of interaction between CD40-ligand and CD40, these pathological conditions definitively demonstrate the requirement of CD40-mediated NF-kappaB activation and the essential role of a newly described molecule, activation-induced cytidine deaminase (AID), in B cell terminal differentiation.

[Extracorporeal membrane oxygenation in critically ill neonates and children]. / Place de l'assistance respiratoire et circulatoire extracorporelle de courte durée (ECMO), post-cardiotomie exclue, dans la prise en charge des défaillances graves du nouveau-né et de l'enfant.

Extracorporeal membrane oxygenation is used as a last resort during neonatal and pediatric resuscitation in case of refractory circulatory or respiratory failure under maximum conventional therapies. Different types of ECMO can be used depending on the initial failure. The main indications for ECMO are refractory respiratory failure (acute respiratory distress syndrome, status asthmaticus, severe pneumonia, meconium aspiration syndrome, pulmonary hypertension) and refractory circulatory failure (cardiogenic shock, septic shock, refractory cardiac arrest). The main contraindications are a gestational age under 34 weeks or birth weight under 2kg, severe underlying pulmonary disease, severe immune deficiency, a neurodegenerative disease and hereditary disease of hemostasis. Neurological impairment can occur during ECMO (cranial hemorrhage, seizure or stroke). Nosocomial infections and acute kidney injury are also frequent complications of ECMO. The overall survival rate of ECMO is about 60 %. This survival rate can change depending on the initial disease: from 80 % for meconium aspiration syndrome to less than 10 % for out-of-hospital refractory cardiac arrest. Recently, mobile ECMO units have been created. These units are able to perform ECMO out of a referral center for untransportable critically ill patients.

A syndrome associating partial albinism and immunodeficiency.

Two unrelated patients with partial albinism, frequent pyogenic infections and acute episodes of fever, neutropenia and thrombocytopenia are described. Their pigmentary dilution was characterized by large clumps of pigments in the hair shafts and an accumulation of melanosomes in melanocytes. Melanocytes had few short dendritic expansions, and keratinocytes were hypopigmented. No or few Langerhans' cells were detected in skin by electron microscopy and ATP-ase reactions. This pigmentary dilution, different from all other human albinisms, resembles the unique defect of the mutant dilute (d-d) mouse. Despite the presence of an adequate number of T and B lymphocytes, the patients were hypogammaglobulinemic, deficient in antibody production and incapable of manifesting delayed skin hypersensitivity or of rejecting skin grafts. Their leukocytes did not stimulate normal lymphocytes and could not generate cytotoxic cells during mixed leukocyte reaction. T lymphocytes of one patient were unable to exert a helper effect on the maturation of B lymphocytes into immunoglobulin-containing cells following in vitro stimulation with pokeweed mitogen. This suggests that the humoral deficiency might be secondary to a defect of helper T lymphocytes. Granulocytes did not show any morphologic abnormality, and their bactericidal activity was only moderately reduced. An increased number of polymorphonuclear leukocytes with polar distribution of Concanavaline A (Con A) receptors (capping) was found in one patient and her parents. The family histories suggest that this syndrome is transmitted as an autosomal recessive character.

Restriction of the in vitro anti-mannan antibody response by HLA-DQ molecules.

In humans, the in vitro antibody response directed towards mannan, a polysaccharide extracted from the cell wall of Candida albicans, has been previously shown to be dependent on the presence of T lymphocytes and monocytes. Evidence is now given for the existence of a genetic restriction governing this response since antibody production is achieved provided that monocytes and T lymphocytes on one side and monocytes and B lymphocytes on the other side are of the same origin. In order to delineate the restriction element governing these interactions, blocking experiments have been designed using well-defined monoclonal antibody, anti-HLA class II molecules. The results clearly indicate that the restriction element belongs to the HLA-DQ molecular series, as shown in T-cell proliferation and antibody production assays in the presence of either T cells or T-cell supernatants. Incubation of isolated cell populations (T, B lymphocytes and monocytes) with the monoclonal antibody have indicated that DQ determinants are involved in the mannan presentation by monocytes to T and B cells. The HLA-DQ mediated restriction of the in vitro immune response to mannan has been observed in all the subjects tested, suggesting that mannan epitopes are preferentially, or even only, recognized in association with an unique group of HLA-class II molecules, namely HLA-DQ.