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In patients with primary antibody deficiencies, subcutaneous administration of IgG (SCIG) replacement is effective, safe, well-tolerated, and can be self-administered at home. A new SCIG replacement at 20% concentration (Hizentra ® ) has been developed and has replaced Vivaglobin ® (SCIG 16%). An observational prospective multi-centric open-label study, with retrospective comparison was conducted in 15 Italian centers, in order to investigate whether and to what extent switching to Hizentra ® would affect frequency of infusions, number of infusion sites, patients' satisfaction, and tolerability in patients previously treated with Vivaglobin ® or intravenous immunoglobulins (IVIG). Any variations of dosage, frequency and duration of the infusions, and of number of infusion sites induced by Hizentra ® with respect to the former treatment were recorded. Practical advantages and disadvantages of Hizentra ® , with respect to the medicinal product formerly used, and the variations in patients' therapy-related satisfaction were monitored by means of the TSQM (Treatment Satisfaction Questionnaire for Medication); number, frequency, and duration of infectious events and adverse effects were recorded. Eighty-two patients switched to Hizentra ® : 19 (23.2%) from IVIG and 63 (76.8%) from Vivaglobin ® . The mean interval between infusions was not affected by the shift (7.0 ± 2.0 days with previous treatment versus 7.1 ± 1.2 during Hizentra ® ). A decrease in the number of infusion sites with Hizentra ® was recorded in 12 out of 56 patients for whom these data were available. At 6 months, 89.7% of patients were satisfied with Hizentra ® ; no difference in terms of effectiveness, side effects, convenience, and global satisfaction was observed. No difference in the incidence of adverse events was reported.

Background: Pollen-food syndrome (PFS) is heterogeneous with regard to triggers, severity, natural history, comorbidities, and response to treatment. Our study aimed to classify different endotypes of PFS based on IgE sensitization to panallergens., Methods: We examined 1271 Italian children (age 4-18 years) with seasonal allergic rhinoconjunctivitis (SAR). Foods triggering PFS were acquired by questionnaire. Skin prick tests were performed with commercial pollen extracts. IgE to panallergens Phl p 12 (profilin), Bet v 1 (PR-10), and Pru p 3 (nsLTP) were tested by ImmunoCAP FEIA. An unsupervised hierarchical agglomerative clustering method was applied within PFS population., Results: PFS was observed in 300/1271 children (24%). Cluster analysis identified five PFS endotypes linked to panallergen IgE sensitization: (i) cosensitization to ≥2 panallergens ('multi-panallergen PFS'); (ii-iv) sensitization to either profilin, or nsLTP, or PR-10 ('mono-panallergen PFS'); (v) no sensitization to panallergens ('no-panallergen PFS'). These endotypes showed peculiar characteristics: (i) 'multi-panallergen PFS': severe disease with frequent allergic comorbidities and multiple offending foods; (ii) 'profilin PFS': oral allergy syndrome (OAS) triggered by Cucurbitaceae; (iii) 'LTP PFS': living in Southern Italy, OAS triggered by hazelnut and peanut; (iv) 'PR-10 PFS': OAS triggered by Rosaceae; and (v) 'no-panallergen PFS': mild disease and OAS triggered by kiwifruit., Conclusions: In a Mediterranean country characterized by multiple pollen exposures, PFS is a complex and frequent complication of childhood SAR, with five distinct endotypes marked by peculiar profiles of IgE sensitization to panallergens. Prospective studies in cohorts of patients with PFS are now required to test whether this novel classification may be useful for diagnostic and therapeutic purposes in the clinical practice., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background and Objectives: Subcutaneous immunoglobulin (SCIG) therapy is becoming increasingly popular as self-administration is possible because intravenous access is unnecessary, and there is a lower frequency of systemic adverse events. The aim of this study was to evaluate the shifting from intravenous immunoglobulins (IVIGs) replacement therapy to SCIG in patients with primary immunodeficiency (PID) in a routine real-life situation., Methods: In a multicenter prospective observational study, we enrolled 50 patients suffering from PID who were monitored for 24 months; 44 patients switched from IVIG and six from different SCIG preparations. The study preparation (human IgG 16 %, Vivaglobin(®), CSL Behring GmbH, Germany) was subcutaneously infused weekly (maximum volume 15 mL/site; maximum infusion rate 22 mL/h). The study endpoints were: annual rate of severe bacterial infections (SBIs), local adverse reactions, quality of life, days off school/work, and days of hospitalization., Results: Thirty-three of 39 (84.6 %) patients who completed the study experienced an infection or signs thereof. Only five SBIs were observed, corresponding to an annual rate of 0.056 episodes per patient in 44 subjects [intention-to-treat (ITT) population]. A significant decrease in both days of hospitalization (1.93 ± 4.08 vs. 0.64 ± 2.94) and days off school/work (15.27 ± 23.17 vs. 2.26 ± 4.45) was recorded at 24 months. Local reactions were observed in 14/50 (28 %) patients, mainly consisting of skin manifestations at the injection site. Only three (6.8 %) patients discontinued due to infusion site reactions. In patients shifting from IVIG to SCIG, the total mean score of Life Quality Index (LQI) improved from 76.9 ± 16.8 to 90.7 ± 11.6 (P < 0.01) at 6 months; there was an improvement also in the overall patients' evaluation., Conclusions: A total of 93.2 % patients tolerated the new route of administration and reported a significant improvement in their LQI. Our results from a routine clinical practice in a real-life population are consistent with those of phase III clinical studies.

Background: Little is known about the prevalence and clinical relevance of hypersensitivity to the plant panallergen profilin in children., Objectives: The present study aimed to investigate prevalence, risk factors and clinical relevance of profilin sensitization in a large cohort of Italian children of different ages living in different geographic areas., Methods: Children with pollen allergy enrolled by 16 pediatric outpatient clinics sited in three main geographic areas of Italy were studied. SPT were carried out with commercial pollen extracts and a commercial purified date palm pollen profilin. IgE specific for allergenic pollen molecules, Phl p 12 (grass profilin) and Pru p 3 (peach lipid transfer protein) were tested by ImmunoCAP FEIA., Results: IgE to Phl p 12 (≥0.35 kU/l) was observed in 296 of the 1,271 participants (23%), including 17 of the 108 (16%) preschool children. Profilin SPT was positive (≥3 mm) in 320/1,271 (25%) participants. The two diagnostic methods were concordant in 1,151 (91%, p < 0.0001) cases. Phl p 12 IgE prevalence declined from northern to southern Italy and was directly associated with IgE to Phl p 1 and/or Phl p 5 and Ole e 1. Among children with IgE to Phl p 12, OAS was provoked by kiwi, melon, watermelon, banana, apricot and cucumber., Conclusions: Profilin sensitization is very frequent among pollen-allergic children, occurs at a very young age and contributes to the development of childhood OAS with a typical pattern of offending foods. Pediatricians should always consider IgE sensitization to profilin while examining pollen-allergic children, even if they are at preschool age., (© 2015 S. Karger AG, Basel.)

Background: Sensitization to profilins and other cross-reacting molecules might hinder proper specific immunotherapy (SIT) prescription in polysensitized patients with pollen-related allergic rhinitis (AR). In these patients, component-resolved diagnosis (CRD) might modify SIT prescription by improving the identification of the disease-eliciting pollen sources., Objectives: We sought to measure the effect of CRD on SIT prescription in children with pollen-related AR., Methods: Children (n = 651) with moderate-to-severe pollen-related AR were recruited between May 2009 and June 2011 in 16 Italian outpatient clinics. Skin prick test (SPT) reactivity to grass, cypress, olive, mugwort, pellitory, and/or Betulaceae pollen was considered clinically relevant if symptoms occurred during the corresponding peak pollen season. IgE sensitization to Phl p 1, Phl p 5, Bet v 1, Cup a 1, Art v 1, Ole e 1, Par j 2, and Phl p 12 (profilin) was measured by using ImmunoCAP. SIT prescription was modeled on SPT responses first and then remodeled considering also CRD according to GA(2)LEN-European Academy of Allergology and Clinical Immunology guidelines and the opinions of 14 pediatric allergists., Results: No IgE to the respective major allergens was detected in significant proportions of patients with supposed clinically relevant sensitization to mugwort (45/65 [69%]), Betulaceae (146/252 [60%]), pellitory (78/257 [30%]), olive (111/390 [28%]), cypress (28/184 [15%]), and grass (56/568 [10%]). IgE to profilins, polcalcins, or both could justify 173 (37%) of 464 of these SPT reactions. After CRD, the SPT-based decision on SIT prescription or composition was changed in 277 (42%) of 651 or 315 (48%) of 651 children according to the European or American approach, respectively, and in 305 (47%) of 651 children according to the opinion of the 14 local pediatric allergists., Conclusions: In children with pollen-related AR, applying CRD leads to changes in a large proportion of SIT prescriptions as opposed to relying on clinical history and SPT alone. The hypothesis that CRD-guided prescription improves SIT efficacy deserves to be tested., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Pollen-induced allergic rhinoconjunctivitis (AR) is highly prevalent and rapidly evolving during childhood. General practitioners may not be fully aware of the nature and severity of symptoms experienced by patients and might underestimate the prevalence of moderate or severe disease. Thus, the relevance of early diagnosis and intervention may be overlooked., Objectives: To investigate the severity of pollen-induced AR and its determinants in Italian children referred to allergy specialists and who had never received specific immunotherapy (SIT)., Methods: Children (age 4-18 yr) affected by pollen-induced AR who had never undergone SIT were recruited between May 2009 and June 2011 in 16 pediatric outpatient clinics in 14 Italian cities. Recruited children's parents answered standardized questionnaires on atopic diseases (International Study of Allergy and Asthma in Childhood, Allergic Rhinitis and its Impact on Asthma, Global Initiative for Asthma). The children underwent skin-prick test (SPT) with several airborne allergens and six food allergens. Information on socio-demographic factors, parental history of allergic diseases, education, perinatal events, breastfeeding, nutrition and environmental exposure in early life was collected through an informatics platform shared by the whole network of clinical centers (AllergyCARD™)., Results: Among the 1360 recruited patients (68% males, age 10.5 ± 3.4 yr), 695 (51%) had moderate-to-severe AR, 533 (39%) asthma, and 325 (23.9%) oral allergy syndrome (OAS). Reported onset of pollen-induced AR was on average at 5.3 ± 2.8 yr, and its mean duration from onset was 5.2 ± 3.3 yr. Only 6.2% of the patients were pollen-monosensitized, and 84.9% were sensitized to ≥3 pollens. A longer AR duration was significantly associated with moderate-to-severe AR symptoms (p 0.004), asthma (p 0.030), and OAS comorbidities (p < 0.001)., Conclusions: This nationwide study may raise awareness of the severity of pollen-induced AR among Italian children who have never received pollen SIT. The strong association between pollen-induced AR duration and several markers of disease severity needs replication in longitudinal studies, while suggesting that countrywide initiatives for earlier diagnosis and intervention should be planned., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: To investigate whether an active partnership between schools, parents, and pediatricians can improve the management of asthma and quality of life of children with asthma., Methods: A comprehensive asthma program (Happy Air®), based on a strong family-physician-school relationship, was carried out over a period of 3 years in six primary schools (2765 children). This program provides educational intervention to families, school staff, and students, as well as the administration of written questionnaires to identify children with asthma, asthma diagnosis and management, and, last but not least, extracurricular activities to improve respiratory and psychological conditions. Quality of life of children and parents, at the beginning and end of the program, was assessed using PedsQL™ 4.0 (Pediatric Quality of Life Inventory) measurement model., Result: Asthma was diagnosed in 135 children, of which 37 (27%) were diagnosed de novo. In all children, both single item and total clinical asthma scores showed a significant increase (p < .001) at the end of the Happy Air® program. The average scores of both the total PedsQL™ 4.0 and the four Scales were significantly increased (p < .001)., Conclusion: Happy Air® is a model for a strategy of education- and school-based intervention for children with asthma and their families. This multi-action program for diagnosis, clinical follow-up, education, self-management, and quality-of-life control aims to minimize the socioeconomic burden of asthma disease.

Background: the importance of dendritic cells (DCs) in the initiation of the Th2-mediated inflammatory response to allergens is well known and more recently it has been proposed that DCs have a pivotal role in maintaining tolerance to allergens. The aim of this study was to investigate whether the success of sublingual immunotherapy (SLIT) in allergic asthma is mediated by the induction of changes of DCs functions., Methods: ten children with allergic asthma sensitive to house dust mite were studied before and after 12 months of SLIT. Immature DCs were derived from peripheral blood monocytes cultured for 6 days in presence of interleukin (IL)-4 and GM-CSF and stimulated with lipopolysaccharide for the last 24 hours to induce maturation., Results: after 12 months of SLIT, mature DCs derived from SLIT-treated patients showed a statistically significant defect of CD86 up-regulation, an increase of IL-10, and a reduction of IL-12 production., Conclusion: SLIT induces changes in DCs functions that might be responsible for an impairment of T cell activation or drive T cells towards a regulatory activity, thus restoring immune tolerance to allergens.

Background: Dendritic cells (DCs) regulate the immune response to allergens in the lung; they induce either effector or regulatory T cells, which promote or suppress, respectively, the development of allergy. IL-10 is a potent immunosuppressive cytokine that induces type 1 regulatory (Tr1) T cells., Objective: To generate allergen-specific Tr1 cells in vitro from children with allergy., Methods: Monocyte-derived DCs from children with allergy to house dust mites (HDM) were generated by incubating the cells with IL-10 and pulsing them with Der p 2, a major HDM allergen, or by pulsing them with Der p 2 and incubating them with IL-10 during their last 2 days of differentiation., Results: Der p 2-specific T-cell proliferation and T(H)2 cytokine production were significantly reduced when T cells from patients with allergy to HDM were activated with autologous Der p 2-pulsed DCs that had been differentiated or incubated with IL-10. T-cell lines generated with Der p 2-pulsed DCs that were differentiated with IL-10 were hyporesponsive to reactivation with Der p 2 and able to suppress Der p 2-specific T(H)2 effector cells., Conclusion: Dendritic cells differentiated in the presence of IL-10 and pulsed with allergen gave rise to a population of tolerogenic DCs that induced allergen-specific Tr1 cells. This finding represents an important step forward to the prospective clinical application of tolerogenic DCs to modulate allergen-specific T-cell responses.

The identification of a Btk mutation in a male patient with <2% CD19(+) B cells warrants making the diagnosis of X-linked Agammaglobulinemia (XLA). Herein we report the case of a 31 year-old male with a gradual decline of peripheral B lymphocytes and low IgA and IgM but normal IgG levels. His clinical history revealed recurrent respiratory and skin infections, sclerosing cholangitis and chronic obstructive pancreatitis. Molecular studies revealed a novel aminoacidic substitution in Btk protein (T316A). His mother, maternal aunts and a maternal female cousin were heterozygotes for the same Btk mutation and were variably affected with pulmonary emphysema. This is a puzzling case where the patient's clinical history and laboratory findings divorce molecular genetics. Either this case confirms the variable expressivity of XLA disease or the T316A change in Btk SH2 domain is a novel non-pathogenic mutation and another unknown gene alteration is responsible for the disease.

Background: Common variable immunodeficiency (CVID) comprises a heterogeneous group of primary antibody deficiencies with complex clinical and immunological phenotypes. The recent discovery that some CVID patients show monogenic defects in the genes encoding ICOS, TACI or CD19 prompted us to investigate several functional candidate genes in individuals with CVID., Results: The exonic, protein coding regions of the genes encoding: APRIL, BCMA, IL10, IL10Ralpha, IL10Rbeta, IL21, IL21R, and CCL18, were analyzed primarily in familial CVID cases, who showed evidence of genetic linkage to the respective candidate gene loci and CVID families with a recessive pattern of inheritance. Two novel SNPs were identified in exon 5 and exon 8 of the IL21R gene, which segregated with the disease phenotype in one CVID family. Eleven additional SNPs in the genes encoding BCMA, APRIL, IL10, IL10Ralpha, IL21 and IL21R were observed at similar frequencies as in healthy donors., Conclusion: We were unable to identify obvious disease causing mutations in the protein coding regions of the analyzed genes in the studied cohort.

Unlabelled: Temporary henna tattoos have become increasingly popular as a safe alternative to permanent tattoos among American and European children and teenagers during the summer holidays. Currently, temporary henna tattoos contain not only henna, but also other additives such as para-phemylenediamine (PPD), which is considered to be the chemical agent that most frequently causes skin reactions associated with the use of commercial black henna. In this report, we describe an 11-year-old boy who applied a temporary black henna tattoo on his right arm during the summer holidays in Greece and developed a severe contact dermatitis at the tattoo site with residual hypopigmentation. He had no previous history of contact dermatitis, however he did suffer from seasonal allergic rhinitis and atopic dermatitis. Patch testing revealed a strong reaction to PPD, a substance commonly contained in temporary henna tattoo preparations., Conclusion: Henna tattoos are an increasing problem worldwide since they carry an increased risk of severe skin reactions; therefore we suggest that the use of temporary henna tattoos in children be discouraged.

HyperIgM syndrome is a heterogenous immunodeficiency characterized by impaired class-switch recombination due to different molecular abnormalities. We report on two female patients affected by a novel syndrome associating HIGM, growth and pubertal disturbances, and severe lymphoid hyperplasia with eventual development into lymphomas, suggesting a DNA repair defect.

Psoriasis (PS) is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and altered differentiation. Atopic dermatitis (ATOD) is a chronic inflammatory, pruritic and eczematous disease frequently associated with respiratory atopy. These diseases are associated with distinct immunologic abnormalities and represent typical examples of complex diseases triggered by both genetic and environmental factors, as demonstrated by independent twin studies. Genome wide linkage studies have mapped susceptibility loci on several chromosomes (PSORS1-9; ATOD1-5). Four of them overlap on chromosomes 1q21, 3q21, 17q25 and 20p although ATOD is quite distinct from PS and these two diseases rarely occur together in the same patient. An association fine-mapping study has been performed to refine PSORS4 and ATOD2 susceptibility loci on chromosome 1q21 analyzing two independently collected cohorts of 128 PS and 120 ATOD trios. Genotype and haplotype analysis of PSORS4 and ATOD2 led us to detect significant p value for haplotypes defined by MIDDLE and ENDAL16 markers in both PS (p = 0.0000036) and ATOD (p = 0.0276), suggesting a strict co-localization within an interval of 42 kb. This genomic interval contains a single gene, LOR, encoding for loricrin. Polymorphic markers mapping in regulatory and coding regions did not show evidence of association in neither of the two diseases. However, expression profiles of LOR in skin biopsies have shown reduced levels in PS and increased levels in ATOD, suggesting the existence of a specific misregulation in LOR mRNA production., (Copyright (c) 2006 S. Karger AG, Basel)

The formation of trans FA residues in membrane phospholipids may be due to a free radical-catalyzed isomerization process occurring to the cis unsaturated FA moieties. Radical stress is well documented in inflammatory processes of atopic diseases, but no data are yet available about a possible association with trans FA detected in these patients. We investigated the presence of trans lipid isomers in the erythrocyte and T-lymphocyte membranes of 26 children affected by atopic eczema/dermatitis syndrome (AEDS). Trans lipid isomers were found in both cell membranes, up to a total content of 2.7 and 4.9% of the FA composition, respectively. By using the geometrical trans lipid library derived from in vitro models of thiyl radical-catalyzed isomerization, oleic and arachidonic acid isomers were detected. The statistical significance was evaluated by comparison with an age-matched control group. These results suggest the role of an endogenous free radical isomerization path occurring to membrane unsaturated lipids, complementary to the dietary contribution, which can be involved in the lipid impairment in AEDS. This study contributes to lipidomic research regarding the double bond structure and the influence of a geometrical change of membrane lipids in physiology and diseases.

Primary immunodeficiencies are rare diseases, characterized by an increased susceptibility to infections. Early diagnosis and appropriate treatment are critical for reducing morbidity and mortality. Given the rarity of these diseases, the awareness of these disorders by physicians is often insufficient, leading to delayed diagnosis and inappropriate treatment which are the major causes of severe long term complications. In an attempt to address and resolve these problems an Italian Network on primary immunodeficiencies has been established with the aim to increase the awareness of these disorders among physicians and to provide the best clinical assistance to all patients on the national territory.

Recurrent infections are a common cause of morbidity in childhood. Several reports have associated this condition to low levels of IgA and IgG subclasses and/or lack of specific antipolysaccharide antibody response, although the relevance of these defects in terms of prognosis and therapeutic approach is still unclear. The aim of our study was to determine the frequency and the clinical relevance of humoral immunodeficiency (HID) other than hypogammaglobulinemia in children affected by recurrent infections. We recruited 67 pediatric patients affected by recurrent infections. Serum IgG, IgA, IgM, IgG2, IgG3, and specific anti-Haemophilus influenzae (anti-Hib) antibodies were determined. Thirty-seven out of 67 patients showed antibody defects (55%). IgA deficiency was observed in 21 out of 67 patients (31%), followed by IgG2 (18%), IgG3 (15%) and IgM (6%) defects. Anti-Hib deficiency was present in three out of 44 patients (7%). A tendency for a higher occurrence of pneumonia and otitis, although not statistically significant (p > 0.05), was observed in HID patients compared to children with normal humoral function. No statistical difference as to the frequency of mild infections (URI) was found between HID and non-HID patients. We therefore suggest that the therapeutic program is based on the clinical status of the patients. Long-term follow-up with repeated determinations of antibody levels is crucial, however, to detect those defects that might evolve into more complex immunodeficiencies.

A questionnaire-based retrospective clinical and immunological survey was conducted in 73 males with a definite diagnosis of X-linked agammaglobulinemia based on BTK sequence analysis. Forty-four were sporadic and 29 familial cases. At December 2000, the patients' ages ranged from 2 to 33 years; mean age at diagnosis and mean duration of follow-up were 3.5 and 10 years respectively. After the mid-1980s all but 2 were on intravenous immunoglobulin (IVIG) substitution therapy, with residual IgG >500 mg/dl in 94% of the patients at the time of enrollment. Respiratory infections were the most frequent manifestation both prior to diagnosis and over follow-up. Chronic lung disease (CLD) was present in 24 patients, in 15 already at diagnosis and in 9 more by 2000. The cumulative risk to present at diagnosis with CLD increased from 0.17 to 0.40 and 0.78 when the diagnosis was made at the ages of 5, 10, and 15 years respectively. For the 9 patients who developed CLD during follow-up, the duration of follow-up, rather than age at diagnosis; previous administration of intramuscular immunoglobulin; and residual IgG levels had a significant effect on the development of CLD. Chronic sinusitis was present in 35 patients (48%), in 15 already at diagnosis and in 20 by 2000. Sistemic infections such as sepsis and meningitis/meningoencephalitis decreased over follow-up, probably due to optimal protection provided by high circulating IgG levels reached with IVIG.

This study is aimed at detecting perinatal infections of Parvovirus B19 (PV B19). The authors have analyzed specific antibodies (IgM and IgG) in the cord serum of 647 babies, born consecutively at the St. Eugenio Hospital in Rome. 156 of them (24%) were positive to IgG. The analysis of viral genome by PCR methods showed three positive subjects (2%). The three newborn babies did not develop any pathologies during a two-year follow-up and PCR became negative within the first 6 months. Data show that a mass pregnancy test is not useful for PV B19 detection, and that specific antibody analysis should be limited to pregnancies at risk. However healthy the infected patients may appear at birth, though, it is advisable to have them monitored for at least one year so as to avoid the risk of chronic infections.

Background: The diagnosis of X-linked agammaglobulinemia (XLA) is not always clearcut. Not all XLA conform to the classic phenotype and less than 50% of affected boys have a family history of immunodeficiency. Mutations in the gene for Bruton's tyrosine kinase (BTK) are responsible for the majority of agammaglobulinemia cases. However, a certain proportion of patients may have mutations involving other genes, although they show with an XLA phenotype. We performed BTK gene mutation analysis in 37 males with presumed XLA and analyzed the pattern of X-chromosome inactivation (XCI) in 31 mothers to evaluate the relevance of these approaches to diagnosis and genetic counseling., Materials and Methods: Twenty affected males with a sporadic occurrence and 17 familial cases belonging to nine families were enrolled within the framework of the Italian Multicenter Clinical Study on XLA. We used non-isotopic RNase cleavage assay (NIRCA), followed by cDNA sequence determination to screen for BTK mutations and X-chromosome inactivation analysis for carrier detection., Results: Using the cDNA-based approach, the identification of BTK gene abnormalities confirmed the clinical diagnosis of XLA in 31 of 37 affected infants. Missense was the most frequent mutational event and the kinase domain was mostly involved. In addition, nine novel mutations were identified. In sporadic cases, BTK gene abnormalities were identified in 9 out of 10 patients whose mothers had a nonrandom pattern of XCI and in 5 out of 6 patients whose mother had a random pattern of XCI. With the exception of one family, all patients with a familial occurrence and born to mothers with a nonrandom pattern of XCI had mutations of the BTK gene., Conclusions: Our findings indicate that in sporadic cases BTK gene sequencing is the only reliable tool for a definitive diagnosis of XLA and support XCI as the first diagnostic tool in the mothers of affected males in multiple generations. Furthermore, our molecular analysis confirms that 10-20% of BTK-unaltered patients have disorders caused by defects in other genes.

Mutations in the Bruton's tyrosine kinase (BTK ) gene are responsible for X-linked Agammaglobulinemia (XLA), an immunodeficiency caused by a block in B cell differentiation. Non Isotopic RNAse Cleavage Assay (NIRCA), followed by sequencing was used to screen for BTK mutations in 11 Italian XLA patients. Nine novel mutations were identified: 6 missense (Y39S, L512P, L512Q, R544G, S578Y, E589K), one non-sense (Q260X), one frameshift (1599-1602del GCGC) and one in-frame insertion (2037-2038insTTTTAG), that represents the first case of premature stop codon introduction in the BTK coding frame. These data support the high molecular heterogeneity of BTK gene in XLA disease and provide new insight to the diagnosis and to the role of BTK domain in XLA and in B cell signal transduction and development. Hum Mutat 15:117, 2000., (Copyright 2000 Wiley-Liss, Inc.)

Bruton's tyrosine kinase (Btk) is a non-receptor protein tyrosine kinase (PTK) that is expressed in all haemopoietic lineages except mature T cells and plasma cells. Despite the broad range of expression. mutations that inactivate this molecule affect primarily the development of the B-cell lineage. As a PTK, Btk could potentially be involved directly or indirectly in the processes that relate to the malignant transformation of all the cell lineages where this molecule is expressed. Previous studies have failed to demonstrate mutations in patients with B-cell origin acute lymphoblastic leukaemia (ALL). We have utilized a recently developed method that enables the rapid and convenient detection of mutations at the cDNA level, namely, the non-isotopic RNase cleavage assay (NIRCA) to analyse Btk sequences from 27 patients with different types of acute myeloid leukaemia (AML). The only alteration that we observed was a polymorphism at position 2031. This polymorphism has already been seen in previous studies. Furthermore, using the same methodology, we identified the Btk mutations in six XLA (X-linked agammaglobulinaemia) patients. Our results, although they do not exclude the involvement of Btk mutations in the development or progression of some type of AML, nevertheless suggest that such mutations do not constitute a major co-factor in the development of myeloid malignancies.

Factors influencing human immunodeficiency virus type 1 (HIV-1) mother-to-child transmission include both immunological and virological parameters: higher viral loads have been associated with clinical stage of HIV-1-infected individuals as well as higher risk of mother-to-child transmission. Furthermore, we have shown that transmitting mothers more frequently harbour HIV-1 isolates with rapid/high syncytium-inducing (SI) biological phenotype than non-transmitting mothers do. Genetically homogeneous virus populations have been found in HIV-1-infected children at birth, in contrast to the heterogeneous virus populations often found in their infected mothers. This observation suggests that a few virus variants are transmitted or initially are replicating in the child. By comparing the HIV-1 gp120 V3 region of sequentially obtained samples from infected children with samples obtained from their mothers at delivery we found, however, that multiple variants of HIV-1 with different outgrowth kinetics can be transmitted. In addition, we have obtained results indicating an impaired ability of the immune response to adapt to the sequence evolution of HIV-1 in transmitting mothers, as assessed by measuring serum reactivities to peptides representing selected yet closely related V3 sequences. By analysing the presence of antibodies in maternal serum at delivery, which neutralize autologous isolates as well as other primary virus isolates, we have indications that a protective immunity in HIV-1 mother-to-child transmission might exist. Immunotherapy has been assessed in infected adult individuals by passive immunization with a variety of HIV-1-specific antibody products. Data from these studies indicated a differential response to therapy according to the stage of the disease. Active vaccine strategies, including envelope glycoproteins, pursued so far in seronegative adult subjects have shown limitations because broadly neutralizing antibodies, such as can be found in infected individuals, have not been evoked. Further investigations are therefore needed to give support for the potential use of either passive and/or active immunization for the prevention of HIV-1 mother-to-child transmission.

The elective treatment for allergy to cow's milk protein is the elimination of these proteins from the diet. The present study with a follow-up of over two years took the form of a comparison between different replacement formulas based on soya (group A), hydrolysate of soya and bovine collagen (group B), and hydrolysate of casein (group C), randomly administered to 55 children (30 males and 25 females, aged between 2-48 months) with documented allergy to cow's milk proteins, but with different clinical symptoms. Tests to evaluate the acquisition of clinical tolerance to cow's milk proteins were performed using a day-hospital regime every 6 months. Sensitivity reactions were observed in 22% of cases in group A, 8% in group B and 37.5% in group C. It is worth underlining that 5 of the 6 children with reactions to soya protein then showed an excellent tolerance to hydrolysate of soya when it was administered subsequently until tolerance was achieved. Weight and statutory growth was uniformly good in all three groups. A high percentage of children achieved tolerance after 24 months (72%); the mean time taken to acquire clinical tolerance was 11.6 +/- 4.8 in group A, 11.6 +/- 6.02 in group B, and 14 +/- 5.6 in group C. No correlation was found between the type of initial symptoms, age at onset, method of response to first challenge and the time taken to acquire tolerance.

It has been demonstrated that patients with atopic disease have anomalies of fatty acid composition, as a result of altered metabolism or abnormal incorporation of fatty acids into the tissues. In the present study, in 57 newborns 'at risk' for atopic disease, the polyunsaturated fatty acid (PUFA) levels were found to be lower in cord blood in infants who subsequently developed atopic disease than in non-atopics. In all babies, levels of arachidonic acid and dihomo-gamma-linolenic acid in sera at 1 and 3 months of age were lower than those in cord blood. These changes were more marked in children who subsequently developed atopic disease, and in those who, independently of signs and/or symptoms of atopic disease, were formula-fed. A comparison between IgE and PUFA levels revealed no significant differences at any tested time interval. In conclusion, our data suggest that in children 'at risk' for atopy, PUFA levels may be predictive of atopic disease.

Seven children suffering from severe atopic dermatitis, unresponsive to standard therapy, received an iv bolus dose of methylprednisolone (20 mg/kg/day) for three days. Immunological parameters were evaluated before and after treatment. At the end of bolus therapy both skin lesions and itching improved for several months in five of seven patients. No side effects were observed, but a significant and transient lymphopenic response occurred, with lower CD4+ than CD8+ lymphocyte counts. Our data suggest that this therapy may be a novel and safe therapeutic approach in severe atopic dermatitis.

The aim of the present study was to evaluate the efficacy of an oral specific hyposensitization therapy in children with atopic dermatitis and positive prick skin tests and/or RAST to Dermatophagoides pteronyssinus (D.pt.). A total of 60 patients, in three different clinical groups, were selected for a three years clinical trial. Group A: children with atopic dermatitis and allergic asthma and/or rhinitis; groups B and C: children with exclusively atopic dermatitis. Groups A and B received specific hyposensitization therapy. Group C was the control group. The clinical evaluation of the dermatological lesions, at the end of our study, didn't show any significant difference among the three groups. Moreover, the onset of respiratory symptoms between the two groups with exclusively atopic dermatitis was similar and not related to the positivity of prick skin tests and/or RAST to seasonal allergens. Our study suggests that specific hyposensitisation therapy with extracts of D.pt., although with no side effects, does not affect the natural history of atopic dermatitis.

The prognostic and protective role of antibodies mediating cellular cytotoxicity (ADCC) and neutralization was evaluated in sera of HIV-1-infected mothers and their consecutively followed children. The presence and titres of ADCC mediating and/or neutralizing antibodies in maternal sera did not predict HIV-1 infection in their respective children. No significant difference in the sera from the children was seen when comparing the presence of neutralizing antibodies between the uninfected and infected children. Stratification of the infected group according to clinical status revealed differences. Only one of 24 AIDS patients had a high neutralizing titre against IIIB. Four patients had a very low titre and the remaining had no detectable neutralizing antibodies at all. In contrast, 10/17 infected non-AIDS children had neutralizing antibodies. Similarly, no significant difference was seen when comparing the presence of ADCC-mediating antibodies between the uninfected and the infected group of children. However, a significantly higher frequency of ADCC was seen in the seropositive non-AIDS children compared with the AIDS children. This study clearly shows that the presence of antibodies mediating ADCC and neutralization in infected children, 0-2 years old, is associated with a better clinical status and delayed disease progression.

A simple, rapid, reproducible and sensitive peptide-Time-Resolved-Fluoroimmunoassay (TR-FIA) method is described which allows the detection of antibodies to the Human Immunodeficiency Virus type 1 (HIV-1). By using a panel of synthetic peptide antigens that covered env, gag and pol amino acid sequences, a 20 amino acid peptide (GIWGCSGKLICTTAVPWNAS) describing an immunodominant and conserved domain on the gp41 region of the BH10 clone was found to be the most reactive in this study. Optimal conditions for antigen concentration, serum dilution and incubation time were established. The peptide-TR-FIA is specific, as assessed by testing HIV-1 positive sera which included samples from AIDS, ARC patients and HIV-positive drug users. The test was used to detect HIV antibodies in 250 well characterized HIV-1 positive sera and 50 normal sera. Peptide-TR-FIA results indicate that the env peptide was highly reactive with HIV-positive sera showing a sensitivity of 100%. None of the 50 control sera showed positive reactivity against the synthetic peptide. Furthermore the peptide-TR-FIA allowed a fine titration of antibodies to defined epitopes of immunodominant HIV structural proteins that usually cannot be achieved by peptide-ELISA assays.

Transmission of HIV virus from an infected mother to her offspring is becoming the leading cause of spreading in the pediatric population. This is particularly burdensome in the pattern II countries where HIV infection has reached epidemic proportions and infection rates affect more women of childbearing age than men. During the last few years considerable effort has been directed at defining the optimal methods for identifying infants of HIV-infected mothers who will themselves prove to be infected because conventional serological assessment is impaired due to placental transfer of maternal IgG. At present, detection of specific IgA to HIV proteins, evaluation of autochthonous antibody production in vitro from infants' lymphocytes, and gene amplification of HIV DNA/RNA by polymerase chain reaction are the most promising procedures for early diagnosis within the first 6 months of life. Nevertheless all these techniques are cumbersome when evaluating specimens from the newborn child. Many studies have been performed to identify those maternal factors affecting the risk of vertical transmission. So far, none of them has shown real prognostic value. However, evaluation of maternal antibody response to functional epitopes of HIV envelope proteins such as the principal neutralizing domain, in some epidemiological settings, seems to correlate to a reduced rate of mother-to-child transmission.

In children born to human immunodeficiency virus (HIV)-infected mothers, factors that determine disease outcome and progression are unclear. Also, early diagnosis is hampered by maternally transferred antibodies. Children aged 0-24 months were retrospectively divided into two groups based on HIV seroreactivity or nonreactivity at age 15 months and analyzed for the presence of antibodies that mediate cellular cytotoxicity (ADCC) and virus neutralization. No difference was seen in the presence of these functional antibodies between groups. The persistently seropositive group was further divided into non-AIDS and AIDS groups according to clinical status at serum collection. The ADCC antibody frequencies were much higher (70%) in the non-AIDS group than in the AIDS group (30%). Of the non-AIDS children, 63% had neutralizing antibodies; no children with AIDS had these antibodies. HIV-specific ADCC and neutralizing antibodies do not seem to protect against transmission of HIV from mother to child but are significantly correlated with a better clinical stage of childhood HIV infection.

The present study demonstrates that maternal antibodies to certain epitopes of human immunodeficiency virus 1 (HIV-1) proteins are associated with a defined outcome for at-risk pregnancies of HIV-infected women. An initial retrospective analysis of antibodies to synthetic peptides and recombinant proteins representing env, pol, and gag regions of HIV-1 was carried out. Sera studied were from 33 children who were born to HIV-infected mothers and whose clinical outcome was known at the time of analysis. Sera, collected within the first 6 months of life, of uninfected at-risk children were found to selectively contain maternal antibodies to certain peptides containing epitopes of the HIV envelope glycoprotein gp120. To confirm the predictive role of maternal antibodies to defined HIV-1 epitopes, a prospective analysis was then performed on sera from 21 HIV-seropositive mothers and their infants, whose clinical and immunological status was then followed up for a period of at least 15 months. As expected, antibodies to the same envelope protein peptides were detected almost exclusively in sera from mothers of uninfected children. Our data suggest that antibodies against select epitopes of HIV envelope protein gp120 might play an important role in preventing mother-to-child transmission of HIV-1 infection. Accordingly, site-directed serology might be used to predict the outcome of an at-risk pregnancy of an HIV-infected woman.

We analysed HIV-specific immunoglobulin G (IgG) responses to gag and env peptides in infants born to HIV-positive mothers. Questions of interest were whether there are early specific markers for prognosis, and whether any specific IgG is related to the prevention of vertical transmission of infection. Fifty-three children, 0-24 months old and born to HIV-1-infected mothers, were retrospectively divided into two groups based on HIV seroreactivity or non-reactivity at 15 months of age. Their sera were used to find reactivities important in diagnosis and/or prediction of the putative HIV disease. Three important findings emerged. First, a low IgG titer against the very immunodominant penv9 in newborns was found to be associated with rapid progression to AIDS. This difference was clearly reflected in the reactivity to a small peptide representing amino acid (aa) 598-606. The second interesting finding was the putative hypervariable loop on gp120 (especially aa 324-338), reactivity to which was found only in the uninfected group, and was seen in six out of 19 uninfected children under 6 months of age. This specific response was not caused by a generally high total anti-HIV reactivity, and may indicate a role of protective antibodies against vertical transmission. The response to this region in the infected group, on the other hand, was directed to the amino terminal half of the putative loop, in particular peptide 53, aa 304-318. Finally, response to a part of the amino terminal end of P17 was seen in seven out of eight infected children over 6 months of age.(ABSTRACT TRUNCATED AT 250 WORDS)

A study was conducted to assess the occurrence of latent infection with the human immunodeficiency virus (HIV) among seronegative people at high risk of infection. The presence of HIV genomes was analysed by molecular techniques in two seronegative children born to mothers infected with HIV and in three regular sexual partners of seropositive drug addicts. The adults were selected from a seronegative cohort at high risk of infection because of their sexual contacts and the children selected because of impaired growth. HIV retroviral sequences were detected in four of the five subjects directly at the cellular level by in situ hybridisation in peripheral blood mononuclear cells. HIV genomic sequences were confirmed by in vitro amplification of viral DNA with the polymerase chain reaction technique. The existence of a latent viral infection state in these seronegative subjects indicates the unreliability of standard serological analysis in people who have been in regular contact with infected patients.

Monocytic features can be induced in the myeloid cell line HL60 in order to provide a suitable in vitro model for the investigation of in vivo activity in mononuclear phagocytes. 1,25 dihydroxyvitamin D3 (Calcitriol) induced the HL60 cell line to express the monocytic differentiation antigen Leu M3 in about 30-50% of the cells along with an increase (up to 20%) in the expression of the HLA class II antigen recognized by the monoclonal antibody (MoAb) HLADR, but not HLADQ. Functional investigation showed that Calcitriol-treated cells formed rosettes with sheep erythrocytes coated with a specific IgG2a mouse MoAb and readily ingested them. In addition, these same sensitized erythrocytes were lysed in an 18 hrs antibody-dependent cellular cytotoxicity (ADCC) assay. All together these data indicate the presence of functionally active Fc gamma Receptors (FcR). Sorting experiment demonstrated that only Leu M3+ HLADR+ cells contained the effector cell population. The phenotypic profile was not per se predictive of FcR presence and function, as TPA induced HL60 cells neither formed rosettes nor phagocytosed nor exhibited ADCC activity, although they express Leu M3+ and HLADR+ (as well as HLADQ) antigens. These results suggest that Calcitriol and TPA cause the differentiation of HL60 cells along distinct pathways. On the other hand, different subpopulations with given predetermined differentiation capabilities may coexist in HL60 cell line.

Impairment of cell-mediated immunity in patients with primary central nervous system (CNS) tumors has repeatedly been reported but data to demonstrate the underlying immunological defect are not univocal. This report concerns a series of 31 patients harboring a glioma in which we studied: peripheral blood T-lymphocyte subpopulations by monoclonal antibody analysis; cellular responsiveness to mitogens; serum immunoglobulin values. The same parameters were also evaluated in 7 cases of intracranial meningioma and in 8 patients affected by non-proliferative, non-inflammatory CNS diseases. Thirty age-matched healthy volunteers formed the control group. Neither impairment of T-cells as regard to number, responsiveness and subsets, nor abnormal Ig values were found in these groups. However two patients, harboring respectively a third ventricle low grade astrocytoma and an anterior callosal glioblastoma, presented a striking T-cells impairment. These findings might suggest a correlation between hypothalamus and immune system, as already postulated by several previous experimental and clinical studies.

Peripheral blood monocytes are comprised of a heterogeneous population of cells with respect to phenotype and functional activity. In this investigation we confirm the presence of two distinct subsets of peripheral blood adherent monocytes with respect to MHC class II antigens HLA DR and HLA DQ using two color FACS analysis. In the attempt to mimic the in vivo situation promyelocytic cells lines have been established which can be induced to undergo characteristic monocytic differentiation in response to a variety of agents. The HL 60 cell line can be induced in vitro by phorbol esters (TPA) to display membrane HLA DR and HLA DQ antigens along with the mature monocytic marker Leu M3. Physiological concentrations of 1,25 dihydroxy Vitamin D3 leads to the expression of HLA DR and Leu M3 antigens but not HLA DQ. These data suggest that these inducers may cause the differentiation of phenotypically distinct monocytic subpopulations.

The random motility of polymorphonuclear leucocytes (PMN), cellular chemotaxis and chemokinesis in kinetic fashion in 4 patients with immotile cilia syndrome (ICS) have been evaluated. No impairment of granulocyte ability of orientation and migration was found. Ultrastructural alterations of cilia which are the primary factor in the pathogenesis of respiratory tract disease in patients with ICS do not impair the PMN function.