Massimo Marconi

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.

Antibody response to pneumococcal vaccine in children receiving bone marrow transplantation

Fifty-three pediatric patients given an allogeneic or an autologous bone marrow transplantation (BMT) were immunized with a polyvalent pneumococcal capsular polysaccharide vaccine (Pneumovax II). Vaccine was administered six months or more after BMT and the pneumococcal IgM, total IgG, and IgG subclasses levels were evaluated before and three weeks after immunization. Immunization promoted a significant rise in antibody serum levels (P<0.000001), and all children vaccinated more than two years after transplantation responded to pneumococcal polysaccharides, whereas only 20–30% and 50% of patients given BMT between six months and one year and one and two years, respectively, mounted an eifective antibody production (P<0.0001). In univariate analysis, lapse of time from BMT to vaccination, chronic graft-versus-host disease occurrence, and female sex influenced the response rate. However, in multivariate analysis, only time between marrow transplant and immunization was a powerful predictor of response. Interestingly, four of 11 patients with IgG2 deficiency before immunization normalized serum levels of this IgG subclass after the pneumococcal antigenic challenge. Our study suggests that time after transplant is the major factor influencing the recovery of immune reactivity to polysaccharide antigens. This seems to confirm the hypothesis that ontogeny of the B-cell repertoire follows a predetermined sequential program in which polysaccharide antigens are some of the last to evoke an antibody response.

Phenotypical/functional characterization of in vitro-expanded mesenchymal stromal cells from patients with Crohn's disease

BACKGROUND AIMS Because of their capacity to modulate the immune response and promote tissue repair, mesenchymal stromal cells (MSC) represent a potential novel treatment for autoimmune/inflammatory diseases, including Crohns disease (CD). The aim of the study was in vitro characterization of MSC from active CD patients for future clinical application. METHODS MSC from the bone marrow (BM) of seven CD patients (median age 32 years) were expanded ex vivo in the presence of 5% platelet lysate; cells were investigated for clonogenic efficiency, proliferative capacity, morphology, immunophenotype, differentiation potential, genetic stability and ability to suppress in vitro proliferation of both autologous and allogeneic lymphocytes to polyclonal mitogens. Results were compared with those of BM MSC of four healthy donors (HD). RESULTS MSC were successfully expanded from all patients. Colony-forming unit-fibroblast (CFU-F) frequency and proliferative capacity were comparable in CD and HD MSC. CD MSC showed typical spindle-shaped morphology and differentiated into osteoblasts, adipocytes and chondrocytes. Surface immunologic markers did not differ between CD and HD MSC, with the only exception of sizeable levels of HLA-DR at early culture passages [12-84% at passage (P)1] in the former. CD MSC ceased their growth at variable passages (from P8 to P25) and entered senescence without any change in morphology/proliferation rate. Array-comparative genomic hybridization demonstrated that CD MSC do not show imbalanced chromosomal rearrangements. Both CD and HD MSC inhibited in vitro proliferation of lymphocytes to mitogens. CONCLUSIONS CD MSC show biologic characteristics similar to HD MSC and can be considered for anti-inflammatory and reparative cell therapy approaches in patients with refractory disease.

Deficiency of neutrophil phagocytosis in premature infants: effect of vitamin E supplementation

ABSTRACT. In 20 healthy premature infants, 10 of whom were administered a total dose of 120 mg/kg vitamin E intramuscularly during the first 13 days after birth, polymorphonuclear leukocyte (PMN) bactericidal activity, frequency and index of phagocytosis, NBT reduction, random movement, chemotaxis and metabolic activity were evaluated within the first 48 hours and again at 5, 14 and 30 days of age. PMN function was also assessed in 30 adult controls. In the treated and untreated infants no differences were found in PMN function before treatment with vitamin E; however phagocytosis, bactericidal activity and chemotaxis were significantly lower than in the adult controls. At 5 days of age, in the untreated infants both index and frequency of phagocytosis remained low but in the treated groups increased significantly. At 14 and 30 days phagocytosis was normal in both treated and untreated infants. No differences were found in the bactericidal activity, NBT reduction, random movement, chemotaxis or metabolic activity of the treated and untreated infants at the ages studied. This preliminary report suggests that vitamin E may be used in premature newborns for accelerating normalization of phagocytic function in the neonatal period.

Deficiency of INFgamma producing cells in adenoids of children exposed to passive smoke.

Exposure to passive smoke is a very common event associated with increased susceptibility to respiratory tract infections. Many related adverse effects result from the ability of cigarette smoke extracts to interfere with the immune system, but the mechanism is not yet completly understood. The aim of the present study is to evaluate the intracellular cytokine profile in adenoids and peripheral blood cells of children exposed to passive smoke. Children undergoing adenoidectomy exposed or not exposed to passive smoke were studied. The intracellular cytokine profile of lymphocyte subsets in adenoids and in peripheral blood were evaluated by flow cytometry analysis. Children exposed to tobacco smoke showed a significantly lower percentage of INF-γ producing CD4+ and CD8+ cells in adenoids. Moreover a significant correlation was observed between the quantity of exposure and reduction in Th1 (CD4+INFγ+ and CD8+INFγ +) cells in adenoids. This reduction may be a contributing factor in the increasing susceptibility to respiratory tract infection in children exposed to tobacco smoke.

Ontogeny of CD40 expression by activated peripheral blood lymphocytes in humans

The CD40 ligand (CD40L) is a molecule expressed by activated T cells which plays a critical role in the regulation of B-cell responses, including differentiation into Ig-producing cells. Using the specific monoclonal antibody TRAP1 we have evaluated the ontogeny of CD40L expression in 97 normal individuals between birth and 50 years of age. The expression of CD40L is a function of age; it is severely reduced at birth, progressively increases during the first months of life, and reaches a plateau in the second decade. This progressive attainment of the ability to express CD40L is due to a process of maturation of the CD4 + subset, being significantly correlated with the expression of the CD45RO antigen.

Human amniotic fluid cells are able to produce IL-6 and IL-8

Problem:  In order to investigate the role of amniotic fluid cells (AFC) in the establishment of feto–maternal immune relationship, we evaluated their phenotype and capacity to produce cytokines.

Long term persistence of anti-HBs protective levels in young patients with type 1 diabetes after recombinant hepatitis B vaccine.

The aim of the present study was to evaluate the persistence of anti-hepatitis B protective levels in young patients with type 1 diabetes, successfully immunised with a recombinant hepatitis B vaccine. We re-evaluated, after a 4 year follow-up, 54 patients and 70 age and sex-matched healthy subjects. Protective antibodies levels were found in 50/54 (92%) patients and in 67/70 (96%) controls. Moreover, anti-HBs levels were similar in diabetic patients and controls (means of log-titre and (sd); 1.95 (0.88) and 2.18 (0.64) patients and controls, respectively; P=0.11). No cases of clinical hepatitis were reported and all patients and controls remained HBc negative. These data demonstrate the persistence of anti-HBs levels in children, adolescents and young patients with type 1 diabetes after recombinant hepatitis B vaccine showing evidence of longterm immunogenity and protective effect.

Neonatal outcome in patients with rheumatic disease

Rheumatic autoimmune diseases have a higher prevalence in women, particularly during their childbearing age. Due to improved management, an increasing number of patients plan and carry out one or more pregnancies. Therefore, a growing interest is being paid to the possible consequences of maternal disease and associated treatment on the fetus and newborn infant. If maternal disease is characterized by the presence of IgG isotype autoantibodies, these can cross the placenta with possible antibody-mediated damage to the fetus. This is typically the case of the so called neonatal lupus erythematosus (NLE); a similar mechanism has been shown in infants of patients with immune thrombocytopenic purpura (ITP) and, less frequently, in those from mothers with antiphospholipid syndrome (APS). Indeed, this last condition is often responsible for placental, rather than neonatal, pathology. In addition, immunosuppressive and other drugs administered to the mothers during pregnancy and lactation might affect the fetal and neonatal immune system development. Finally, mothers disease and/or treatment could be related to neuropsychological alteration reported in some of their children.

Spontaneous NBT reduction by monocytes as a marker of disease activity in children with histiocytosis

In an attempt to define a biological marker of monocyte hyperactivation in the course of infantile histiocytosis, the spontaneous nitroblue tetrazolium (NBT) reduction assay was applied to monocytes from 13 children with Langerhans cell histiocytosis (LCH), familial haemophagocytic lymphohistiocytosis (FHL), juvenile xanthogranuloma or malignant histiocytosis. Significant increase in NBT reduction was observed in the patients with both active LCH and FHL in comparison with control subjects, who were either healthy or affected by different conditions. A close relationship between spontaneous reduction rate and clinical condition of the patients was evident in patients tested at diagnosis, during remission and during the course of disease reactivation. Interleukin‐1 (IL‐1) production by monocytes was also evaluated: the patients with LCH and FHL displayed a significant increase in in vitro IL‐1 production by lipopolysaccharide‐stimulated monocytes. In our experience the spontaneous NBT reduction assay was a sensitive, quite specific, low‐cost and reproducible test for the evaluation of children with histiocytosis. Useful information may be obtained at diagnosis but also during the clinical course of disease by using this marker of monocyte spontaneous activation.