Alberto G. Ugazio

Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2).

The activation-induced cytidine deaminase (AID) gene, specifically expressed in germinal center B cells in mice, is a member of the cytidine deaminase family. We herein report mutations in the human counterpart of AID in patients with the autosomal recessive form of hyper-IgM syndrome (HIGM2). Three major abnormalities characterize AID deficiency: (1) the absence of immunoglobulin class switch recombination, (2) the lack of immunoglobulin somatic hypermutations, and (3) lymph node hyperplasia caused by the presence of giant germinal centers. The phenotype observed in HIGM2 patients (and in AID-/- mice) demonstrates the absolute requirement for AID in several crucial steps of B cell terminal differentiation necessary for efficient antibody responses.

Gene Therapy in Peripheral Blood Lymphocytes and Bone Marrow for ADA− Immunodeficient Patients

Adenosine deaminase (ADA) deficiency results in severe combined immunodeficiency, the first genetic disorder treated by gene therapy. Two different retroviral vectors were used to transfer ex vivo the human ADA minigene into bone marrow cells and peripheral blood lymphocytes from two patients undergoing exogenous enzyme replacement therapy. After 2 years of treatment, long-term survival of T and B lymphocytes, marrow cells, and granulocytes expressing the transferred ADA gene was demonstrated and resulted in normalization of the immune repertoire and restoration of cellular and humoral immunity. After discontinuation of treatment, T lymphocytes, derived from transduced peripheral blood lymphocytes, were progressively replaced by marrow-derived T cells in both patients. These results indicate successful gene transfer into long-lasting progenitor cells, producing a functional multilineage progeny.

Mutations of CD40 gene cause an autosomal recessive form of immunodeficiency with hyper IgM

CD40 is a member of the tumor necrosis factor receptor superfamily, expressed on a wide range of cell types including B cells, macrophages, and dendritic cells. CD40 is the receptor for CD40 ligand (CD40L), a molecule predominantly expressed by activated CD4+ T cells. CD40/CD40L interaction induces the formation of memory B lymphocytes and promotes Ig isotype switching, as demonstrated in mice knocked-out for either CD40L or CD40 gene, and in patients with X-linked hyper IgM syndrome, a disease caused by CD40L/TNFSF5 gene mutations. In the present study, we have identified three patients with an autosomal recessive form of hyper IgM who fail to express CD40 on the cell surface. Sequence analysis of CD40 genomic DNA showed that one patient carried a homozygous silent mutation at the fifth base pair position of exon 5, involving an exonic splicing enhancer and leading to exon skipping and premature termination; the other two patients showed a homozygous point mutation in exon 3, resulting in a cysteine to arginine substitution. These findings show that mutations of the CD40 gene cause an autosomal recessive form of hyper IgM, which is immunologically and clinically undistinguishable from the X-linked form.

In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDX1)

BACKGROUND X-linked severe combined immunodeficiency (SCIDXI) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common gamma chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDXI can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis. METHODS A male fetus was diagnosed as having SCIDXI by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E rosetting. Chimerism analysis was by HLA-DQ alpha typing and gamma-chain staining on cord blood. FINDINGS A healthy 3.6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3.5 months of age the infant is well and his T-cell counts and function are normal. INTERPRETATION In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.

Cross-reactivity between milk proteins from different animal species

Cows milk allergy is quite frequent in the first years of human life. When breast‐feeding is not possible, a cows milk substitute must be provided for allergic subjects. Different alternatives to cows milk have been suggested as protein sources (soy, hydrolysed proteins, goats milk, etc.), but all these dietetic solutions are not without risks for polyallergic or more sensitive subjects.

Ontogeny of Secretory Immunity: Levels of Secretory IgA and Natural Antibodies in Saliva

Summary: In 187 healthy subjects from 2 months to 27 years of age, secretory IgA and free secretory component were assayed in samples of whole saliva obtained before and after stimulation with lemon juice. Antibody tilers against Escherichia coli O antigens and against rabbit erythrocytes were also dosed in unstimulated saliva. Secretory IgA, undetectable in newborns, was present in all 2-month-olds tested in both unstimulated and stimulated saliva; thereafter secretory IgA levels increased progressively, reaching adult values by 6 to 8 years in unstimulated saliva and already by 2 to 4 years in stimulated saliva. The antibody titers assessed in unstimulated saliva followed a similar pattern also reaching adult values by 6 to 8 years. On the other hand, free secretory component levels showed no significant variation with age in unstimulated saliva whereas a slight increase was observed in the first year of life in stimulated saliva.Speculation: The physiologic deficiency of SIgA and of secretory antibodies in infancy and childhood might well play a role in the pathogenesis of infectious diseases of the respiratory and gastrointestinal tract frequent in this age group.

Prevalence and diagnosis of celiac disease in IgA-deficient children.

BACKGROUND Reported frequencies of celiac disease in selective IgA deficiency in childhood vary widely and this is probably due to the different characteristics of the patients studied and to the different criteria used for intestinal biopsy: all patients or only those with symptoms of malabsorption. Diagnosis of celiac disease is of considerable importance in IgA deficiency because of its increased frequency and also because avoidance of dietary gluten permits elimination of the symptoms and complications of celiac disease. OBJECTIVES To obtain a more reliable estimate of the incidence of celiac disease in childhood IgA deficiency jejunal biopsies were performed in 65 consecutively diagnosed IgA-deficient children whose parents consented. Some clinical and laboratory parameters including IgA-antigliadin and IgG-antigliadin antibodies were evaluated to predict their usefulness in selecting IgA-deficient patients for intestinal biopsy. METHODS All IgA-deficient patients had serum IgA levels below 5 mg/dL and salivary IgA below 0.5 mg/dL. Jejunal biopsy was performed using a peroral Watson capsule and IgA-antigliadin and IgG-antigliadin antibodies were performed by an ELISA assay. RESULTS Biopsy findings of severe villous atrophy permitted diagnosis of celiac disease in 7.7% (5/65 children). IgG-antigliadin antibody levels, elevated in 16 patients including all five celiacs, were the best parameter for predicting celiac disease and gave no false negatives. CONCLUSIONS The 7.7% frequency of celiac disease observed in these IgA-deficient children is about 20 times higher than in the general Italian population, and the lowest among the studies biopsying all patients; this is probably attributable to the presence of a substantial proportion of healthy children (20/65) and very few (2/65) with autoimmune disorders. The elevated sensitivity and negative-predictive value of IgG-antigliadin antibodies lead us to suggest that positive IgG-antigliadin antibodies can be used to select IgA-deficient children for jejunal biopsy with a very low probability of missing celiac disease while allowing a drastic reduction in the number of biopsies performed.

Defective Expression of CD40 Ligand on T Cells Causes “X‐Linked Immunodeficiency with Hyper‐IgM (HIGM1)”

X-linked immunodeficiency with hyper-IgM (HIGM1) is a rare disorder, characterized by recurrent infections associated with very low or absent IgG and IgA, and normal to increased IgM serum levels. The disease has been earlier mapped to the q26-27 region of the X-chromosome. We have identified a novel molecule expressed on the surface of activated T cells, which was designated TRAP (Tumor necrosis factor Related Activation Protein), and could demonstrate that TRAP is a ligand for the CD40 receptor expressed on B cells. Our mapping of the TRAP gene to the Xq26.3-27.1 region suggested a causal relationship to HIGM1. Further work revealed that various mutations of the TRAP/CD40 ligand (CD40L) gene may lead to a defective expression of the TRAP/CD40L molecule on the T-cell surface in HIGM1 patients. A combination of structural and functional analyses finally demonstrated that the failure of TRAP/CD40L on T cells to interact with CD40 on B cells is responsible for the inefficient T-cell help for B cells observed in HIGM1. The observations made in HIGM1 allowed us to conclude that TRAP/CD40L is not required for IgM synthesis. In contrast, functional expression of TRAP is a prerequisite for effective immunoglobulin isotype switching and subsequent production of IgG, IgA and IgE by B cells in vivo. The interaction of TRAP/CD40L with CD40 thus provides a very critical link between the cellular and the humoral part of the immune system. The knowledge of TRAP/CD40L cDNA sequence, the availability of various reagents for the testing of expression and function of TRAP/CD40L, and our recent elucidation of the exon-intron structure of the TRAP/CD40L gene now provide all necessary tools for early diagnosis of affected patients and the detection of female carriers of HIGM1. The available information will also provide a basis for future attempts at gene therapy in this disease.

Of genes and phenotypes: the immunological and molecular spectrum of combined immune deficiency. Defects of the gamma(c)-JAK3 signaling pathway as a model.

Cytokines play a major role in lymphoid development. Defects of the common gamma chain (gamma(c)) or of the JAK3 protein in humans have been shown to result in a severe combined immune deficiency (SCID), with a profound defect in T and natural killer (NK)-cell development, whereas B-cell generation is apparently unaffected (T-B+NK-SCID). While extensive molecular and biochemical analysis of these patients has been instrumental in understanding better the biological properties of the gamma(c) and JAK3 protein, an unexpected phenotypic heterogeneity of gamma(c) and JAK3 deficiency has emerged, indicating the need for appropriate and extensive investigations even in patients with atypical presentations. At the same time, characterization of the defects has been instrumental in the development of novel therapeutic approaches, from in utero hematopoietic stem cell transplantation to gene therapy.

Serum IgG subclass concentrations in healthy subjects at different age: Age normal percentile charts

IgG subclass levels were determined in 448 normal children from 6 months to 18 years of age and in 141 healthy adults by radial immunodiffusion using monoclonal antibodies. Age-normal percentile values were calculated for each year of age up to 18 years for IgG1, IgG2, IgG3 and in adults for all four subclasses. The broad spread of IgG4 values in children did not permit calculation of reference values.