Maurizio Caniglia

Genetic study of a new X-linked recessive immunodeficiency syndrome.

Seven forms of X-linked (XL) immunodeficiency have been described (XL agammaglobulinemia, XL severe combined immunodeficiency [SCID], Wiskott-Aldrich syndrome, XL chronic granulomatous disease, XL hyper-IgM syndrome with low IgG and IgA, and XL lymphoproliferative syndrome), and properdine deficiency. Although there are (some) phenotypic variants, diagnosis is relatively simple on the basis of clinical, immunological, and genetic characteristics. We studied a family in which several males were affected by severe infections and whose pedigree suggested recessive XL inheritance of an immunodeficiency. Immunologic and genetic studies (X inactivation patterns in females and restriction fragment length polymorphism [RFLP] segregation) were performed in order to characterize the immunodeficiency. The propositus, a 5-yr-old boy, was found to have a severe and progressive T- and B-cell functional immunodeficiency characterized by defective antigen-specific responses. No lymphocyte subsets or membrane anomalies were detected and the immunodeficiency did not correspond to usual XL forms. Studies of DNA from two of the informative females, the mother and one sister revealed nonrandom X chromosome inactivation of T cells and, partially, B cells but not PMN, a pattern similar to that observed in XL SCID carriers. RFLP studies identified a haplotype segregating with the abnormal locus that may be localized in the proximal part of the long arm of the X chromosome. We thus report the characterization of a new XL immunodeficiency that may correspond either to another XL locus or to an attenuated phenotype of XL SCID.

Revertant T lymphocytes in a patient with Wiskott-Aldrich syndrome: Analysis of function and distribution in lymphoid organs

BACKGROUND The Wiskott-Aldrich syndrome (WAS) is a rare genetic disease characterized by thrombocytopenia, immunodeficiency, autoimmunity, and hematologic malignancies. Secondary mutations leading to re-expression of WAS protein (WASP) are relatively frequent in patients with WAS. OBJECTIVE The tissue distribution and function of revertant cells were investigated in a novel case of WAS gene secondary mutation. METHODS A vast combination of approaches was used to characterize the second-site mutation, to investigate revertant cell function, and to track their distribution over a 18-year clinical follow-up. RESULTS The WAS gene secondary mutation was a 4-nucleotide insertion, 4 nucleotides downstream of the original deletion. This somatic mutation allowed the T-cell-restricted expression of a stable, full-length WASP with a 3-amino acid change compared with the wild-type protein. WASP(+) T cells appeared early in the spleen (age 10 years) and were highly enriched in a mesenteric lymph node at a later time (age 23 years). Revertant T cells had a diversified T-cell-receptor repertoire and displayed in vitro and in vivo selective advantage. They proliferated and produced cytokines normally on T-cell-receptor stimulation. Consistently, the revertant WASP correctly localized to the immunologic synapse and to the leading edge of migrating T cells. CONCLUSION Despite the high proportion of functional revertant T cells, the patient still has severe infections and autoimmune disorders, suggesting that re-expression of WASP in T cells is not sufficient to normalize immune functions fully in patients with WAS.

Role of reduced intensity conditioning in T-cell and B-cell immune reconstitution after HLA-identical bone marrow transplantation in ADA-SCID

The treatment of choice for severe combined immunodeficiency is bone marrow transplantation from an HLA-identical donor sibling without conditioning. However, this may result in low donor stem cell chimerism, leading to reduced long-term immune reconstitution. We compared engraftment, metabolic, and T-cell and B-cell immune reconstitution of HLA-identical sibling bone marrow transplantation performed in 2 severe combined immunodeficiency infants with adenosine deaminase deficiency from the same family treated with or without a reduced intensity conditioning regimen (busulfan/fludarabine). Only the patient who received conditioning showed a stable mixed chimerism in all lineages, including bone marrow myeloid and B cells. The use of conditioning resulted in higher thymus-derived naïve T cells and T-cell receptor excision circles, normalization of the T-cell repertoire, and faster and complete B-cell and metabolic reconstitution. These results suggest the utility of exploring the use of reduced intensity conditioning in bone marrow transplantation from HLA-identical donor in severe combined immunodeficiency to improve long-term immune reconstitution.

Intravenous immune globulin in lysinuric protein intolerance

In addition to systemic manifestations with skeletal, pulmonary, renal, and haematological signs, lysinuric protein intolerance (LPI), a membrane transport defect of cationic amino acids, is often complicated by severe life-threatening immunological manifestations. A 10-year-old boy with LPI who exhibited a severe systemic immunohaematological disease is described here. This patient showed cutaneous lesions similar to the subacute form of systemic lupus erythematosus, severe anaemia and dysproteinaemia, and a marked reduction of circulating T lymphocytes, mainly the CD4+ cells. In vitro bone marrow cell culture studies showed that addition of patients serum induced macrophage proliferation and inhibited erythroid progenitor cell growth. Treatment with high-dose intravenous immune globulin resolved most of the clinical and laboratory abnormalities.

Rapid T-cell receptor CD4+ repertoire reconstitution and immune recovery in unrelated umbilical cord blood transplanted pediatric leukemia patients

Umbilical cord blood transplantation has been successfully employed for treatment of many immune and hematologic disorders. The aim of this study was to evaluate the quality of immune reconstitution after umbilical cord blood transplantation in 6 leukemia children. T-cell receptor Vβ third complementary region spectratyping was used for monitoring the contribution of the thymic pathway in patients’ immune reconstitution. Absolute numbers of lymphocyte subsets (T, B, and natural killer), and lymphoproliferative in vitro response to mitogens, recovered within 12 months after transplantation. Furthermore, an overall diversification of T-cell receptor complexity in the repopulating T cells, with a polyclonal Gaussian profiles in most (74%) of total families was observed. Noteworthy, we showed a wider and more rapid reconstitution of T-cell receptor CD4+ T cell families compared with T-cell receptor CD8+ T ones still exhibiting some perturbations at 24 months. These data show that umbilical cord blood transplantation allows immune reconstitution already within 12 months with generation of newly diversified CD4+ T lymphocyte subsets.

NK cell effector functions in a Chédiak-Higashi patient undergoing cord blood transplantation: Effects of in vitro treatment with IL-2

NK cell cytotoxicity in Chédiak-Higashi syndrome (CHS) is strongly impaired as lytic granules are not released upon NK-target cell contact, contributing to several defects typical of this severe immunodeficiency. Correction of NK cell defects in CHS should improve the outcome of hematopoietic stem-cell transplantation, proposed as therapy. We investigated NK cell functions in a CHS patient before and after cord-blood transplantation, and the ability of in vitro IL-2 treatment to restore them. Before the transplant, the strong defect in NK cell-mediated natural and antibody-dependent cytotoxicity, as well as in IFN-γ production, could be restored up to normal levels by in vitro IL-2 treatment. This cytokine also caused the appearance of smaller lysosomal granules and their orientation towards the NK-target cell contact area, thus suggesting that IL-2 had a more general capacity to restore NK cell effector functions. Moreover after the transplant, although the successful engraftment, NK cell cytotoxicity resulted still partially impaired at one year, almost normal at ten years and, anyhow, fully recovered by in vitro IL-2 treatment. Taken together, our results indicate that IL-2 had a wide capacity to restore NK cell effector functions, being able to reverse the altered cytotoxic activity, lytic granule pattern, and cytokine production observed in the CHS patient.

Early-onset monocyte–B–natural killer–dendritic cells’ deficiency successfully treated with hematopoietic stem cell transplantation

it extremely difficult to prove efficacy of a drug for long-term treatment in seasonal trials with pre-established time points. In this study no significant difference in the total number of flare-ups between the first and second years were noted; however, we observed a marked reduction in the number of flare-ups in the cyclosporine-treated group during the second year of the study. Because no significant differences were reported in airborne allergen concentrations in Italy during the study, we can exclude that environmental triggers played a major role in the results of the crossover year. The difference in the number of flare-ups during the second year of the study might have been due to the crossover design. In fact, we observed a marked difference in the dropout rate between the 2 experimental treatments during the first year. In line with this hypothesis, 4 patients dropped out for inefficacy in the ketotifen group during the second year. In this study 0.05% cyclosporine significantly ameliorated the signs and symptoms of allergy (ie, itching, photophobia, and hyperemia) but was equivalent to ketotifen in controlling other secondary signs and symptoms. The composite sum scores were not significantly different. The only other comparable randomized clinical trial that investigated the efficacy of 0.5% cyclosporine eye drops versus an antiallergic agent, 0.5% ketorolac tromethamine, did not report significant differences in controlling the signs and symptoms of VKC. The most clinically relevant finding of this study involved the results obtained with long-term, daily preventative treatment with a low concentration of cyclosporine and how this significantly affected the course of VKC, demonstrating it to be a valid steroidsparing therapy. The results of the nested study demonstrated an inferior efficacy of the higher 0.1% cyclosporine dose compared with 0.15% dexamethasone. Steroid use led to a greater reduction in signs and symptoms, as well as a significantly lower number of patients (30%) requiring a second week of open-label rescue therapy than cyclosporine (64%). Cyclosporine was safe and well tolerated, confirming data from a recent study reporting no side effects in 594 patients with VKC or atopic keratoconjunctivitis treated with 0.1% cyclosporine for 1 year. In conclusion, in this randomized controlled study we report the safety and efficacy of topical 0.05% cyclosporine for the longterm prevention of VKC relapses. This finding is relevant to the long-term management of pediatric patients at risk of visual impairment, whether because of steroid abuse or continued recurrences of acute inflammation. Alessandro Lambiase, MD, PhD* Andrea Leonardi, MD* Marta Sacchetti, MD, PhD* Velika Deligianni, MD Sabrina Sposato, MD, PhD Stefano Bonini, MD*