Expanding the mutation spectrum in ICF syndrome: Evidence for a gender bias in ICF2.

BACKGROUND: Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare, genetically heterogeneous, autosomal recessive disorder. Patients suffer from recurrent infections caused by reduced levels or absence of serum immunoglobulins. Genetically, 4 subtypes of ICF syndrome have been identified to date: ICF1 (DNMT3B mutations), ICF2 (ZBTB24 mutations), ICF3 (CDCA7 mutations), and ICF4 (HELLS mutations). AIM: To study the mutation spectrum in ICF syndrome. MATERIALS AND METHODS: Genetic studies were performed in peripheral blood lymphocyte DNA from suspected ICF patients and family members. RESULTS: We describe 7 ICF1 patients and 6 novel missense mutations in DNMT3B, affecting highly conserved residues in the catalytic domain. We also describe 5 new ICF2 patients, one of them carrying a homozygous deletion of the complete ZBTB24 locus. In a meta-analysis of all published ICF cases, we observed a gender bias in ICF2 with 79% male patients. DISCUSSION: The biallelic deletion of ZBTB24 provides strong support for the hypothesis that most ICF2 patients suffer from a ZBTB24 loss of function mechanism and confirms that complete absence of ZBTB24 is compatible with human life. This is in contrast to the observed early embryonic lethality in mice lacking functional Zbtb24. The observed gender bias seems to be restricted to ICF2 as it is not observed in the ICF1 cohort. CONCLUSION: Our study expands the mutation spectrum in ICF syndrome and supports that DNMT3B and ZBTB24 are the most common disease genes.

Artemis splice defects cause atypical SCID and can be restored in vitro by an antisense oligonucleotide.

Artemis deficiency is known to result in classical T-B- severe combined immunodeficiency (SCID) in case of Artemis null mutations, or Omenn's syndrome in case of hypomorphic mutations in the Artemis gene. We describe two unrelated patients with a relatively mild clinical T-B- SCID phenotype, caused by different homozygous Artemis splice-site mutations. The splice-site mutations concern either dysfunction of a 5' splice-site or an intronic point mutation creating a novel 3' splice-site, resulting in mutated Artemis protein with residual activity or low levels of wild type (WT) Artemis transcripts. During the first 10 years of life, the patients suffered from recurrent infections necessitating antibiotic prophylaxis and intravenous immunoglobulins. Both mutations resulted in increased ionizing radiation sensitivity and insufficient variable, diversity and joining (V(D)J) recombination, causing B-lymphopenia and exhaustion of the naive T-cell compartment. The patient with the novel 3' splice-site had progressive granulomatous skin lesions, which disappeared after stem cell transplantation (SCT). We showed that an alternative approach to SCT can, in principle, be used in this case; an antisense oligonucleotide (AON) covering the intronic mutation restored WT Artemis transcript levels and non-homologous end-joining pathway activity in the patient fibroblasts.

Anti-citrullinated protein antibodies have a low avidity compared with antibodies against recall antigens.

OBJECTIVES: Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA) and have been implicated in disease pathogenesis. Recent ongoing evidence indicates that the ACPA response broadens before precipitation of full-blown RA, as indicated by a more extensive isotype usage and an increased citrullinated epitope recognition profile. Nonetheless, the evolution of the ACPA response is still poorly understood and might intrinsically differ from the protective responses against pathogens. METHODS: The avidity and the avidity maturation of ACPA in relation to the avidity of antibodies against recall antigens were analysed. RESULTS: The avidity of ACPA was significantly lower than the avidity of antibodies to the recall antigens tetanus toxoid and diptheria toxoid. Moreover, ACPA did not show avidity maturation during longitudinal follow-up and ACPA avidity was also relatively low in patients who displayed extensive isotype switching. CONCLUSIONS: These observations indicate that the natural evolution of ACPA differs from the development of antibodies against recall antigens. These data also indicate that ACPA avidity maturation and isotype switching are disconnected, whereby extensive isotype switching occurs in the setting of restricted avidity maturation. Intrinsic differences between the RA-specific autoantibody system and protective antibody responses against pathogens could be of relevance for designing novel B cell-targeted therapies for RA.

B-cell maturation and antibody responses in individuals carrying a mutated CD19 allele.

Homozygous CD19 mutations lead to an antibody deficiency due to disruption of the CD19 complex and consequent impaired signaling by the B-cell antigen receptor. We studied the effects of heterozygous CD19 mutations on peripheral B-cell development and antibody responses in a large family with multiple consanguineous marriages. Sequence analysis of 96 family members revealed 30 carriers of the CD19 mutation. Lymphocyte subset counts were not significantly different between carriers and noncarriers in three different age groups (0-10 years; 11-18 years; adults). B cells of carriers had reduced CD19 and CD21 median expression levels, and had reduced proportions of transitional (0-10 years) and CD5(+) B cells (adults). CD19 carriers did not show clinical signs of immunodeficiency; they were well capable to produce normal serum Ig levels and had normal responses to primary and booster vaccinations. The frequency of mutated Vκ alleles was not affected. Heterozygous loss of CD19 causes some changes in the naive B-cell compartment, but overall in vivo B-cell maturation or humoral immunity is not affected. Many antibody deficiencies are not monogenetic, but likely caused by a combination of multiple genetic variations. Therefore, functional analyses of immune cell function should be carried out to show whether heterozygous mutations contribute to disease.

Clinical fluctuations in MuSK myasthenia gravis are related to antigen-specific IgG4 instead of IgG1.

We studied the longitudinal relation between disease severity and titers of antigen-specific IgG subclasses in sera of patients with myasthenia gravis and antibodies to Muscle Specific Kinase (MuSK MG). Six patients were included of whom 55 samples had been collected during 2.5-13.4 years. Anti-MuSK antibodies were determined by ELISA and with a cell-based immunofluorescence assay. Disease severity was scored on a semi continuous scale. Only antigen-specific IgG4, and not IgG1, titers were significantly associated with disease severity in a linear mixed effect model (p = 0.036). Levels of IgG4 antibodies were above IgG1 in all samples except in one patient who went into clinical remission while switching from IgG4 to IgG1. The results support an important role for IgG4 in the pathogenesis of MuSK MG, in contrast to MG with anti-acetylcholine receptor antibodies.

The impact of single amino acid substitutions in CD3gamma on the CD3epsilongamma interaction and T-cell receptor-CD3 complex formation.

The human T-cell receptor-CD3 complex consists of at least eight polypeptide chains; CD3gamma- and delta-dimers associate with the disulfide linked alphabeta- and zetazeta-dimers to form a functional receptor complex. The exact structure of this complex is still unknown. We now have examined the interaction between CD3gamma and CD3 in human T-cells. For this purpose, we have generated site-directed mutants of CD3gamma that were introduced in human T-cells defective in CD3gamma expression. Cell-surface and intracellular expression of the introduced CD3gamma chains was determined, as was the association with CD3delta, CD3, and the T-cell receptor. Although the introduction of wild type CD3gamma and CD3gamma (78Y-F) fully restored T-cell receptor assembly and expression, the introduction of CD3gamma (82C-S), CD3gamma (85C-S), and CD3gamma (76Q-E) all resulted in an impaired association between CD3gamma and CD3 and a lack of cell-surface expressed CD3gamma. Finally, the introduction of CD3gamma (76Q-L) and CD3gamma (78Y-A) restored the expression of TCR-CD3deltagammazeta2 complexes, although the association between CD3gamma and CD3 was impaired. These results indicate that several amino acids in CD3gamma are essential for an optimal association between CD3gamma and CD3 and the assembly of a cell-surface expressed TCR-CD3deltagammazeta2 complex.

Graft dysfunction and delayed immune reconstitution following haploidentical peripheral blood hematopoietic stem cell transplantation.

For many children with life-threatening hematological diseases, hematopoietic stem cell transplantation (HSCT) is the only curative option. In children lacking a matched related or unrelated donor and with the certainty that, left untreated, death will ensue alternative donors must be sought. Haplo-identical peripheral blood stem cell transplantation (PBSCT) from a healthy parent is a feasible alternative. To reduce the risk of fatal graft-versus-host disease (GvHD) as a complication of transplant across major histocompatibility antigens, intense T-cell depletion is required. Large numbers of purified, cytokine mobilized peripheral stem cells (the so-called mega-dose concept) are required to compensate for the significantly increased risk of either graft failure or early rejection. In our unit, despite this approach, graft dysfunction has, in a significant group of children, proved problematic and, despite salvage attempts at re-transplantation, usually fatal. In children with hematological malignant disease, our overall relapse-free survival is 41%. However, successful transplant outcome has been associated with considerable delays in immune reconstitution that can be implicated in subsequent viral reactivation. We are investigating new strategies to improve the outcome of haplo-identical PBSCT, which may allow us to offer this form of treatment to more children requiring urgent HSCT.

Adenovirus infection in paediatric stem cell transplant recipients: increased risk in young children with a delayed immune recovery.

Adenovirus (HAdV) infections are a frequent cause of morbidity and mortality after allogeneic human stem cell transplantation (HSCT). We report a retrospective single-centre study on 328 consecutive paediatric recipients of an allogeneic HSCT. During the first 6 months after HSCT, HAdV infection occurred in 37 children (cumulative incidence 12%). The highest incidence was found in young children up to 5 years of age, transplanted after 1994, with >2 log T-cell depletion of a graft of another than an HLA-genotypically identical related donor (actuarial frequency at 6 months 84%). Persistence of HAdV and spreading of the virus over multiple sites showed a trend towards the development of HAdV disease or death, but did not reach significance. Recovery of immunity after HSCT, that is, serum concentrations of IgM and peripheral blood counts of T cells and subsets, was delayed in children with an HAdV infection compared with noninfected children. In seven out of seven patients with HAdV DNA in serum and decreasing lymphocyte counts, the infection had a fatal course. Manipulation of cellular immunity either by tapering of immunosuppression, infusion of donor lymphocytes or immunotherapy using HAdV-specific T cells should be considered in graft recipients at risk for a severe HAdV infection.

Allogeneic stem cell transplantation in X-linked lymphoproliferative disease: two cases in one family and review of the literature.

X-linked lymphoproliferative disease (XLP) is a rare immunodeficiency caused by mutations in the signaling lymphocyte activating molecule-associated protein/SH2D1A gene and characterized by a dysregulated immune response to Epstein-Barr virus and other pathogens. The clinical presentation is heterogeneous and includes fulminant infectious mononucleosis, lymphoma, hypogammaglobulinemia and aplastic anemia. XLP is associated with a high morbidity and overall outcome is poor. At present, allogeneic stem cell transplantation (alloSCT) is the only curative treatment. XLP patients may be recognized in various stages of disease and even when symptoms are not yet evident. We here present two related XLP patients in different stages of disease that were both treated successfully with alloSCT using a matched unrelated donor. In addition, we have reviewed all reported cases of alloSCTs in XLP patients. Based on these results and in order to improve the final outcome, we conclude that alloSCT should be recommended in both symptomatic and asymptomatic XLP patients.

Adenovirus infection in children after allogeneic stem cell transplantation: diagnosis, treatment and immunity.

Human adenoviruses (HAdV) are a frequent cause of potentially fatal infections in patients after allogeneic stem cell transplantation, especially in children. Monitoring of serum/plasma by real-time quantitative PCR is a sensitive tool for the recognition of patients at risk of a potentially fatal infection and for the evaluation of the efficacy of treatment. Data from a retrospective study and from a prospective study demonstrate that recovery of immunity after transplantation is essential for the elimination of HAdV infection. The feasibility of several approaches for the manipulation of immunity in the immunocompromised host to prevent a fatal course of the infection is discussed.

Despite differential gene expression profiles pediatric MDS derived mesenchymal stromal cells display functionality in vitro.

Pediatric myelodysplastic syndrome (MDS) is a heterogeneous disease covering a spectrum ranging from aplasia (RCC) to myeloproliferation (RAEB(t)). In adult-type MDS there is increasing evidence for abnormal function of the bone-marrow microenvironment. Here, we extensively studied the mesenchymal stromal cells (MSCs) derived from children with MDS. MSCs were expanded from the bone-marrow of 17 MDS patients (RCC: n=10 and advanced MDS: n=7) and pediatric controls (n=10). No differences were observed with respect to phenotype, differentiation capacity, immunomodulatory capacity or hematopoietic support. mRNA expression analysis by Deep-SAGE revealed increased IL-6 expression in RCC- and RAEB(t)-MDS. RCC-MDS MSC expressed increased levels of DKK3, a protein associated with decreased apoptosis. RAEB(t)-MDS revealed increased CRLF1 and decreased DAPK1 expressions. This pattern has been associated with transformation in hematopoietic malignancies. Genes reported to be differentially expressed in adult MDS-MSC did not differ between MSC of pediatric MDS and controls. An altered mRNA expression profile, associated with cell survival and malignant transformation, of MSC derived from children with MDS strengthens the hypothesis that the micro-environment is of importance in this disease. Our data support the understanding that pediatric and adult MDS are two different diseases. Further evaluation of the pathways involved might reveal additional therapy targets.

Quantification of adenovirus DNA in plasma for management of infection in stem cell graft recipients.

We used a real-time polymerase chain reaction method for quantification of adenovirus to monitor the dynamics of viral DNA load in plasma in pediatric stem-cell graft recipients. Two cases are described to demonstrate that detection and quantification of the adenovirus DNA load at regular intervals may be important to document the stage of adenovirus infection, to make decisions on clinical intervention, and to accurately monitor the response to antiviral therapy.

Effect of ribavirin on the plasma viral DNA load in patients with disseminating adenovirus infection.

Adenovirus (AdV) infections are an increasingly frequent and potentially fatal complication in allogeneic stem cell transplant recipients. To determine the antiviral potential of ribavirin in an unbiased way, 4 patients without immune recovery were prospectively analyzed by quantitative measurement of plasma AdV DNA load. Administration of ribavirin at the first signs of AdV dissemination was not accompanied by a decrease in the plasma AdV DNA load in any of these patients, and an increase in the AdV load was even documented in 3. These observations question the potential of ribavirin to improve the outcome for patients with disseminating AdV infection and support a critical evaluation of antiviral treatments for AdV infection that involves the kinetics of virus DNA load as an objective parameter of viral replication.

IgG antibodies to ATG early after pediatric hematopoietic SCT increase the risk of acute GVHD.

Anti-thymocyte globulin (ATG), raised in rabbits, is frequently used in allogeneic hematopoietic SCT (HSCT), to prevent graft rejection and acute GVHD. In solid organ transplant patients, antibodies to rabbit IgG result in an enhanced clearance of ATG. The occurrence of such antibodies in HSCT recipients and their clinical impact is unknown. Concentrations of ATG and anti-ATG antibodies were measured in 72 pediatric HSCT recipients treated with ATG as part of the conditioning. Anti-ATG antibodies were detected in 20 children (28%), all transplanted with a non-depleted graft. IgG anti-ATG, alone or combined with IgM and/or IgA anti-ATG, appeared in 10 children. Four patients developed IgG anti-ATG antibodies early (before day 22) post-HSCT. They had steep drops in ATG levels and showed rapid T-cell recovery, which was associated with a significantly increased risk of acute GVHD. In six patients IgG anti-ATG responses occurred later (range 28-46 days) after HSCT without an increased risk of GVHD. A total of 10 children only mounted an IgM (and IgA) anti-ATG response, which was without major impact on ATG levels. These results indicate that early development of IgG anti-ATG antibodies has a major impact on acute GVHD. Routine analysis ATG/anti-ATG Ab measurement should be considered.

Mannose-binding lectin levels and infections in children after allogeneic hematopoietic SCT.

Mannose-binding lectin (MBL) is an activator of the lectin pathway of the C' system and hence an important component of the innate immune system. Although reports are conflicting, MBL deficiency has been reported to influence the infection susceptibility in hematology/oncology patients or recipients of allogeneic hematopoietic SCT (HSCT). MBL levels and the occurrence of infections were retrospectively analyzed in 98 pediatric HSCT patients. MBL deficiency in recipients was not corrected by HSCT using a donor with normal MBL production. In addition, low serum MBL concentrations were not associated with increased susceptibility to any type of infections post-HSCT in this cohort of pediatric HSCT recipients.

Preemptive alloimmune intervention in high-risk pediatric acute lymphoblastic leukemia patients guided by minimal residual disease level before stem cell transplantation.

Relapse of pediatric acute lymphoblastic leukemia (ALL) remains the main cause of treatment failure after allogeneic stem cell transplantation (alloSCT). A high level of minimal residual disease (MRD) before alloSCT has been shown to predict these relapses. Patients at risk might benefit from a preemptive alloimmune intervention. In this first prospective, MRD-guided intervention study, 48 patients were stratified according to pre-SCT MRD level. Eighteen children with MRD level >or=1 x 10(-4) were eligible for intervention, consisting of early cyclosporine A tapering followed by consecutive, incremental donor lymphocyte infusions (n=1-4). The intervention was associated with graft versus host disease >or=grade II in only 23% of patients. Event-free survival in the intervention group was 19%. However, in contrast with the usual early recurrence of leukemia, relapses were delayed up to 3 years after SCT. In addition, several relapses presented at unusual extramedullary sites suggesting that the immune intervention may have altered the pattern of leukemia recurrence. In 8 out of 11 evaluable patients, relapse was preceded by MRD recurrence (median 9 weeks, range 0-30). We conclude that in children with high-risk ALL, immunotherapy-based regimens after SCT are feasible and may need to be further intensified to achieve total eradication of residual leukemic cells.

Cellular and humoral responses to tetanus vaccination in Gabonese children.

Protection to tetanus is often not optimal in developing countries due to incomplete vaccination schemes, or decreased efficacy of vaccination. In this study we investigated the immunological response to tetanus booster vaccination in school children living in a semi-urban or in a rural area of Gabon. Tetanus-specific total IgG as well as antibody subclasses of the IgG1, IgG2, IgG3 and IgG4 isotype and the avidity of the dominating IgG1 subclass were determined both before and 1 month after the booster vaccination. In addition, tetanus-specific cytokine responses were determined. We found a polarization towards a T helper 1 (Th1) profile in the semi-urban children, whereas the cytokine responses of the rural children showed a T helper 2 (Th2) skewed response. Furthermore, tetanus-specific antibodies of the different IgG subclasses were all increased upon a tetanus booster vaccination and levels of IgG1 and IgG3 were higher in the rural children. In conclusion, a tetanus booster vaccination induced a stronger Th2 over Th1 cytokine profile to tetanus toxoid (TT) in rural children who showed the highest levels of IgG1 and IgG3 anti-TT antibody responses.

ABO incompatible stem cell transplantation in children does not influence outcome.

BACKGROUND: Although delayed red cell engraftment and/or hemolysis have been thoroughly documented in association with ABO incompatibility between donor and recipient in patients undergoing hematopoietic stem cell transplantation (HSCT), there are no studies defining the general, long term clinical outcome in a large group of pediatric patients. METHODS: We undertook a retrospective single center analysis of children undergoing pediatric allogeneic stem cell transplantation to determine the influence of ABO donor/recipient incompatibility. Outcome was analyzed according to donor type and included survival, graft versus host disease (GvHD), relapse, days of infection, antibiotic use, transfusion requirement and duration of hospital stay. RESULTS: Two hundred and sixteen children (136 males; 80 females, aged 0-19) transplanted between January 1992 and December 2003 were included in the study. Indications for transplantation were hematological malignancies (n=179) and aplastic conditions (n=37). ABO compatibility was documented in 121 donor/recipient pairs. ABO incompatibility was documented in 95 donor/recipient pairs with 40 major, 40 minor and 15 bi-directional incompatible pairs. ABO incompatibility did not influence survival rate (P=0.3762), the incidence of GvHD (P=0.253) or rate of relapse (P=0.930). Recovery of leucocytes was influenced by ABO incompatibility (P=0.0493), but the rate of infection, transfusion requirements and duration of hospital stay are not. CONCLUSION: In the pediatric setting, ABO major and/or minor mismatch between donor and recipient did not significantly influence the outcome of HSCT. The choice of donor should be determined by the degree of HLA match and CMV status in preference to ABO blood group compatibility.