Anti-infective prophylaxis for primary immunodeficiencies: what is done in Italian Primary Immunodeficiency Network centers (IPINet) and review of the literature.

Primary immunodeficiencies (PIDs) are rare diseases characterized by an increased susceptibility to infections. Early diagnosis and appropriate treatment are critical for reducing morbidity and mortality. Based on available data, the efficacy of antibiotic administration for the prophylaxis of infections remains uncertain, and recommendations supporting this practice are poor. The use of antimicrobial prophylaxis is mainly based on single institution-specific experience without controlled measurements of patient safety and quality health outcomes. To address this issue an Italian Network on Primary Immunodeficiencies (IPINet) has been set up in 1999 within the Italian Association of Pediatric Hematology and Oncology (AIEOP) to increase the awareness of these disorders among physicians. Further, diagnostic and treatment guideline recommendations have been established to standardize the best clinical assistance to all patients, including antibiotic prophylaxis, and for a national epidemiologic monitoring of PIDs. The aim of this review is not only to give a scientific update on the use of antimicrobial prophylaxis in selected congenital immunological disorders but also to draw a picture of this practice in the context of the Italian Primary Immunodeficiency Network (IPINet). Controlled multicenter studies are necessary to establish if, when and how you should start an efficacious antimicrobial prophylaxis.

Hematopoietic stem cell transplantation in Omenn syndrome: a single-center experience.

We retrospectively analyzed the outcome of hematopoietic stem cell transplantations (HSCT) performed at our Center between 1991 and 2002 in 11 unselected patients with Omenn syndrome, a variant of severe combined immunodeficiency. The patients' mean age at the time of the first HSCT was 8.4 months. Two patients received two, and one patient three, HSCT procedures. The resulting 15 HSCT derived in seven cases from HLA-haploidentical parents, in four patients from matched unrelated donors, in three cases from an HLA phenotypically identical related donor, and in one case from an HLA genotypically identical family donor. Nine out of 11 patients are alive and immunoreconstituted 30-146 months after transplantation. At the time of the most recent evaluation, all of the nine survivors had normal T-cell function, and eight of them had developed normal antibody production. This study demonstrates an overall mortality of 18.2%, which is substantially lower than previously reported. Early recognition of OS, rapid initiation of adequate supportive treatment and HSCT lead to improved outcome for this otherwise fatal disease, regardless of the origin and matching of hematopoietic stem cells.

Long term follow up of children with myocarditis treated by immunosuppression and of children with dilated cardiomyopathy.

OBJECTIVE: To describe the treatment and long term outcome after immunosuppressive treatment of children with myocarditis. METHODS AND RESULTS: 114 patients with newly diagnosed dilated cardiomyopathy were divided into three groups, according to the histological pattern: group A, acute myocarditis; group B, borderline myocarditis; and group C, non-inflammatory cardiomyopathy. Groups A and B were treated with cyclosporine and prednisone in addition to conventional treatment. Survivors of the whole cohort were analysed for 13 year transplant-free survival and assessed for left ventricular function. Event-free survival at 13 years was 97 (3)% for group A, 70 (8)% for group B, and 32 (7)% for group C (p < 0.0001). It was 96 (4)% at one year and 83 (5)% at 13 years for the cumulated myocarditis group (A and B). Cardiac function recovered completely in 79% of survivors in group A, 64% in group B, and 36% in group C. The rate of complete recovery in the cumulated group (A and B) was 70%. CONCLUSIONS: The high long term survival rate of this cohort of children with myocarditis is probably due to the effect of short term immunosuppression. This result differs from previously published series of conventionally treated children, whose survival probability at one year was about 60%.

B cell function after haploidentical in utero bone marrow transplantation in a patient with severe combined immunodeficiency.

An in utero paternal CD34(+) cell transplant was performed in a T-B+NK+ SCID fetus. We report here the results of the 3-year humoral immune reconstitution study. The methods used were ApoB VNTR typing, flow cytometry, nephelometry, hemagglutination, ELISA, ELISPOT and lymphoproliferative assays. The T cells were of donor origin whereas monocytes, B and NK cells were of host origin. Peripheral B cell counts and IgM levels were normal since birth. IVIG therapy was required at 5 months of age until 2 years old. IgA levels > or =20 mg/dl were detected from month 17 post transplantation. Isohemagglutinins were present since month 8 post transplantation, the highest titers (anti-A:1/128, anti-B:1/32) were obtained at month 33 post-transplantation. After immunization with rHBsAg, circulating anti-HBsAg IgG secreting cells and a 7.8-fold increase in serum anti-HBsAg Ab were detected. We conclude that split chimerism following in utero haploidentical BMT allows complete humoral immune reconstitution in a T-B+NK+ SCID patient.

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.

Vomiting and gastric motility in infants with cow's milk allergy.

BACKGROUND: Regurgitation and vomiting are common manifestations of cow's milk protein allergy (CMPA) in infants and are usually ascribed to gastroesophageal reflux (GER). Gastric anaphylaxis can induce antral dysmotility in the rat, and therefore the hypothesis for the current study was that cow's milk in sensitized infants may impair antral motility, thereby promoting GER and reflex vomiting. METHODS: Seven vomiting infants with CMPA and nine with primary GER underwent a challenge with cow's milk formula. Electrogastrography (EGG) was used to measure the spectral frequency (bradygastria = 1.5-2.4 cycles per minute [cpm], normogastria = 2.5-3.9 cpm, tachygastria = 4.0-9.0 cpm) and the postprandial-to-fasting power ratio of gastric electrical activity, whereas gastric half-emptying time (T1/2) was measured by electrical impedance tomography (EIT). RESULTS: In CMPA and GER, respectively, during fasting, the frequency distribution (mean +/- SD) of the EGG was as follows: normogastria 47.9%+/-12.5% versus 52.2%+/-9.8%, bradygastria 24.1%+/-5.7% versus 22.8%+/-8.3%, and tachygastria 28.0% 8.5% versus 25.0% 8.3%. In contrast, after the cow's milk challenge, the difference between the two groups was statistically significant: normogastria 33.1%+/-8.8% versus 70.6%+/-8.6% (P < 0.0001). bradygastria 38.0%+/-15.5% versus 15.7%+/-5.2% (P = 0.002), and tachygastria 28.9%+/-10.6% versus 13.4%+/-4.6% (P = 0.001. The postprandial/ fasting power ratio (mean +/- SD) was 3.2+/-1.9 in CMPA and 8.1+/-2.1 in GER (P < 0.0001). Gastric T1/2 (mean +/- SD) of the cow's milk meal was 89.0+/-26.3 minutes versus 54.0+/-12.6 minutes (P = 0.003). In infants with GER all EGG parameters and gastric T1/2 were similar to that in 10 healthy control infants. CONCLUSIONS: In sensitized infants, cow's milk induces severe gastric dysrhythmia and delayed gastric emptying, which in turn may exacerbate GER and induce reflex vomiting. Electrogastrography and EIT can be useful in the assessment of vomiting, GER, and CMPA in infants.

Combined immunodeficiencies due to defects in signal transduction: defects of the gammac-JAK3 signaling pathway as a model.

Combined immune deficiencies comprise a spectrum of genetic disorders characterized by developmental or functional defects of both T and B lymphocytes. Recent progress in cell biology and molecular genetics has unraveled the pathophysiology of most of these defects. In particular, the most common form of severe combined immune deficiency in humans, with lack of circulating T cells, a normal or increased number of B lymphocytes, and an X-linked pattern of inheritance (SCIDXI) has been shown to be due to defects of the IL2RG gene, encoding for the common gamma chain (gammac), shared by several cytokine receptors. Furthermore, defects of the JAK3 gene, encoding for an intracellular tyrosine kinase required for signal transduction through gammac-containing cytokine receptors, have been identified in patients with autosomal recessive T-B+ SCID. Characterization of the functional properties of cytokines that signal through the gammac-JAK3 signaling pathway has been favored by the detailed analysis of SCID patients. Specifically, the key role of IL-7 in promoting T cell development has been substantiated by the identification of rare patients with T-B+ SCID who have a defect in the alpha subunit of the IL-7 receptor (IL7Ralpha). The heterogeneity of genetic defects along the same signaling pathway that may lead to combined immune deficiency is paralleled by the heterogeneity of immunological phenotypes that may associate with defects in the same gene, thus creating a need for detailed immunological and molecular investigations in order to dissect the spectrum of combined immune deficiencies in humans.

Oral complications of pediatric hematopoietic cell transplantation: diagnosis and management.

Oral complications are a significant cause of morbidity and potential mortality for children undergoing hematopoietic cell transplant (HCT). Oral complications can occur at all stages of HCT and can interfere significantly with transplant recovery. Mucosal disease caused by conditioning regimen toxicity and infection are frequent clinical problems. Untreated dental caries and periodontal disease may result in severe infections of the mouth and/or life-threatening systemic spread of the microbial pathogens. In the course of chronic graft-versus-host disease (GVHD), which can complicate HCT, lichenoid and ulcerative lesions of the mucosa are observed. Furthermore, total-body irradiation utilized in the conditioning regimens can cause early xerostomia and consequent dental decay and also result in significant dental and skeletal developmental anomalies. The dental health care team should have a key role in the support of HCT patients. The team's primary responsibilities are those related to the prevention of severe infections originating in the mouth, which includes providing instruction on oral prophylaxis and hygiene as well as direct intervention. Prevention and/or diagnosis and management of oral complications of HCT by the dental team can improve the success of a transplant by reducing morbidity, improving the quality of life, and reducing the cost of care. The authors present specific protocols for the diagnosis and prevention and for the management of oral complications in pediatric HCT.

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.

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.

Haploidentical peripheral blood and marrow stem cell transplantation in nine cases of primary immunodeficiency.

Bone marrow transplantation (BMT) is the treatment of choice in children affected by primary immunodeficiency (PID). Because only 10-15% of affected children have a familial HLA-identical donor alternative therapeutic options are BMT from a matched unrelated donor or an haploidentical BMT. In our experience only 40% of these children find a donor within the International Registry. Therefore, the remaining 50% children affected by PID are candidates for haploidentical BMT. Unfortunately, in PID other than sever-combined immunodeficiency (SCID), low engraftment rates have been reported because of minimal residual immunity. In order to enhance engraftment rate in haploidentical BMT in PID we suggest a protocol with addition of donor peripheral stem cells after mobilization with granulocyte colony-stimulating factor (G-CSF) (16 micrograms/kg for 5 days) and bone marrow cells. This procedure increases the cell load, which allows intensification of the conditioning regimen for induction of faster engraftment. The separation of CD34+ cells from leukapheresis products was achieved in the first 6 patients by the Isolex 300 system (Baxter) with a CD34+ cell purity range of 80-95% and in another three patients by the Clinimacs System (Miltenyi). The peripheral blood stem cells were cryopreserved until BMT, 15 days after G-CSF stimulation when the bone marrow was harvested, processed and T-cell depleted with Campath 1-M in the first 6 cases while the Clinimacs System was used in the remaining cases and no T-cell depletion was required. We included 9 patients in the study protocol: SCID (4), Omenn's syndrome (3), LAD (1) and CID (1). The mean value of peripheral CD34+ cells infused was 13.42 x 10(6)/kg and the mean CD3+ cells number was 0.385 x 10(5)/kg; the mean value of BM CD34+ cells infused was 10.62 x 10(6)/kg and the mean CD3+ cell number was 2.39 x 10(5)/kg. The mean number of infused CFU was 8.1 x 10(5)/kg for PBSC and 3.59 x 10(5)/kg for BM. The 9 patients achieved more than 0.5 x 10(9) peripheral blood neutrophils/L at a mean of 14.6 days (range: 6-22 days). One patient affected by SCID showed complete chimerism, but he died after BMT of systemic CMV infection; the other 8 patients are alive and well and 4 of them show complete chimerism in all cell lines. Split chimerism was documented in 2 SCID cases (CD3+ lymphocytes were of donor origin, monocytes were autologous and granulocytes were mainly autologous); 1 patient affected by Omenn's syndrome received 3 transplants (1 from the mother and 2 from the father, T-cells alone and bone marrow) and achieved engraftment with complete chimerism after the third transplant; the patient affected by LAD also received 3 transplants (2 bone marrow infusions and 1 PBSC infusion) achieving complete chimerism after the third one. In conclusion, the engraftment achieved in all treated patients, and the acceptable conditioning-related toxicity suggest that this approach could be successfully applied to children affected by PID and candidates for haploidentical BMT.

Evaluation of the presence of bovine proteins in human milk as a possible cause of allergic symptoms in breast-fed children.

BACKGROUND: It is generally believed that the elimination of certain foods from the diet of mothers during the lactation period produces a significant improvement in breast-fed children who develop allergic symptoms. Several studies have shown the presence of food proteins in human milk; on the other hand, no study has been able to correlate unequivocally the presence of these allergens in human milk with newborn sensitization. OBJECTIVE: The aim of this study was to evaluate the presence of bovine proteins in breast milk. METHODS: Milk samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To detect bovine proteins in human milk, immunoblotting was performed by using monoclonal antibodies (MA) specific for beta-lactoglobulin and bovine caseins. RESULTS: The results of this study do not confirm the presence of bovine proteins in breast milk suggested by other authors and shows unequivocally that the conflicting results reported in the literature about the presence of betalactoglobulin in human milk are due to cross-reactivity between bovine milk proteins and human proteins. CONCLUSIONS: Components other than bovine betalactoglobulin or caseins could be involved in the induction of allergic symptoms in exclusively breast-fed children.

Intrathymic restriction and peripheral expansion of the T-cell repertoire in Omenn syndrome.

Mutations in the human RAG genes that impair, but do not abolish, recombination activity lead to Omenn syndrome, a severe primary immune deficiency that is associated with clinical and pathological features of graft-versus-host disease and oligoclonal expansion of activated, autologous T cells. We have analyzed the mechanisms accounting for peripheral oligoclonality of the T-cell repertoire. Predominance of few T-cell receptor clonotypes (both within TCRAB- and within TCRGD-expressing lymphocytes) is already detectable in the thymus and is further selected for in the periphery, with a different distribution of clonotypes in different tissues. These data indicate that oligoclonality of the T-cell repertoire in Omenn syndrome is due both to intrathymic restriction and to peripheral expansion. Moreover, the RAG genes defect that causes Omenn syndrome directly affects early stages of V(D)J recombination, but does not alter the process of double-strand-break DNA repair, including N and P nucleotide insertion.

Cross-reactivity between milk proteins from different animal species.

BACKGROUND: Cow's milk allergy is quite frequent in the first years of human life. When breast-feeding is not possible, a cow's milk substitute must be provided for allergic subjects. Different alternatives to cow's milk have been suggested as protein sources (soy, hydrolysed proteins, goat's milk, etc.), but all these dietetic solutions are not without risks for polyallergic or more sensitive subjects. OBJECTIVE: To obtain new information on the suitability of other mammalian milks for allergic children, we evaluated the cross-reactivity between milk proteins from different animal species. METHODS: Milk samples were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). To detect antibody-antigen complexes, immunoblotting was performed by using sera from children allergic to cow's and ewe's milk (RAST class >/= 4) and monoclonal antibodies (MoAb) specific for bovine proteins (caseins and beta-lactoglobulin). RESULTS: IgEs from children allergic to cow's milk are capable of recognizing most part of milk proteins from mammals bred in European countries (ewe, goat, buffalo), while no serum used in this study contains IgEs reacting with camel's milk proteins. Camel's milk was also not recognized from circulating IgEs from a child specifically allergic to ewe's milk. Specific antibovine monoclonal antibodies cross-reacted with proteins from other mammalian species, apart from those of camel. CONCLUSIONS: Homologies in amino acidic composition could justify the cross-reactivity observed between proteins from different animal species. On the other hand, the phylogenetic difference could be responsible for the failed recognition of camel's proteins by circulating IgEs and monoclonal antibodies.

Immune reconstitution after bone marrow transplantation for combined immunodeficiencies: down-modulation of Bcl-2 and high expression of CD95/Fas account for increased susceptibility to spontaneous and activation-induced lymphocyte cell death.

We have studied the regeneration of T cell subsets and function after BMT in 21 children affected by combined immunodeficiency after BMT. In the first months, the striking predominance of CD4+ cells displayed the primed CD45R0+ phenotype and a high number of activated (HLA-DR+) T cells were observed. Regeneration of naive CD4+CD45RA+ cells correlated with the recovery of proliferative responses to mitogens (r = 0.64, P<0.001). Peripheral blood lymphocytes circulating after BMT undergo an increased process of in vitro cell death, resulting from two mechanisms: spontaneous apoptosis (SA), a consequence of defective production of IL-2 and down-regulation of Bcl-2 (P = 0.02 vs. healthy controls), and high susceptibility to activation-induced cell death (AICD) after restimulation with mitogens. In accordance with the role of CD95/Fas in this latter process, we have observed a high level of CD95 expression (P<0.001 vs. healthy controls), correlated with AICD (P<0.001) but not with SA, and decreasing with time after BMT (P<0.001). Both SA and AICD levels correlated with the presence of activated T cells and decreased with the progressive recovery of T cell proliferative response. Therefore, the lymphocyte hyperactivated status might explain their susceptibility to apoptosis and contribute to the genesis of immunodeficiency that follows BMT.

Prenatal molecular diagnosis of Wiskott-Aldrich syndrome by direct mutation analysis.

We have performed prenatal diagnosis for Wiskott Aldrich syndrome (WAS) in two unrelated families by direct gene analysis. Using a combined non-radioactive analysis of single-strand conformational polymorphism (SSCP) and heteroduplex formation (HD), followed by automated sequencing, we studied DNA from chorionic villus sampling (CVS), allowing the diagnosis of one affected and one healthy male at the 12th week of gestation.

[Prenatal and postnatal transplantation of hematopoietic stem cells in children with primary immunodeficiency]. / Il trapianto prenatale e postnatale di cellule staminali emopoietiche in bambini affetti da immunodeficienza primitiva.

Primary immunodeficiencies are inherited diseases characterized by impaired immune responses. In case of severe impairment of immunity bone marrow transplantation is the only therapeutic option. The molecular defect is known for several primary immunodeficiencies allowing prenatal diagnosis. This paper summarizes the clinical experience treating these pathologies by bone marrow transplantation.

Monocytes from Wiskott-Aldrich patients display reduced chemotaxis and lack of cell polarization in response to monocyte chemoattractant protein-1 and formyl-methionyl-leucyl-phenylalanine.

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by trombocytopenia, eczema, and progressive decline of the immune function. In addition, lymphocytes and platelets from WAS patients have morphologic abnormalities. Since chemokines may induce morphologic changes and migration of leukocytes, we investigated the monocyte response to chemoattractants in cells from WAS patients with an identified mutation in the WAS protein gene. Here, we report that monocytes derived from four patients with molecularly defined typical WAS have a severely impaired migration in response to FMLP and to the chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha compared with normal donors. Conversely, neither MCP-1 binding to monocytes nor induction of the respiratory burst by MCP-1 and FMLP is significantly different between WAS patients and normal donors. Within a few minutes of stimulation, monocytes respond to chemokines with increased expression of adhesion molecules and with morphologic changes such as cell polarization. Although up-regulation of CD11b/CD18 expression following stimulation with FMLP or MCP-1 is preserved in WAS patients, cell polarization is dramatically decreased. Staining of F-actin by FITC-phalloidin in monocytes stimulated with chemoattractants shows F-actin to have a rounded shape in WAS patients, as opposed to the polymorphic distribution of F-actin in the polarized monocytes from healthy donors. These results suggest that WAS protein is involved in the monocyte response to the chemokines MCP-1 and macrophage inflammatory protein-1alpha.