Giovanni Melioli

Human dendritic cells activate resting natural killer (NK) cells and are recognized via the NKp30 receptor by activated NK cells.

During the innate response to many inflammatory and infectious stimuli, dendritic cells (DCs) undergo a differentiation process termed maturation. Mature DCs activate antigen-specific naive T cells. Here we show that both immature and mature DCs activate resting human natural killer (NK) cells. Within 1 wk the NK cells increase two– to fourfold in numbers, start secreting interferon (IFN)-γ, and acquire cytolytic activity against the classical NK target LCL721.221. The DC-activated NK cells then kill immature DCs efficiently, even though the latter express substantial levels of major histocompatibility complex (MHC) class I. Similar results are seen with interleukin (IL)-2–activated NK cell lines and clones, i.e., these NK cells kill and secrete IFN-γ in response to immature DCs. Mature DCs are protected from activated NK lysis, but lysis takes place if the NK inhibitory signal is blocked by a human histocompatibility leukocyte antigen (HLA)-A,B,C–specific antibody. The NK activating signal mainly involves the NKp30 natural cytotoxicity receptor, and not the NKp46 or NKp44 receptor. However, both immature and mature DCs seem to use a NKp30 independent mechanism to act as potent stimulators for resting NK cells. We suggest that DCs are able to control directly the expansion of NK cells and that the lysis of immature DCs can regulate the afferent limb of innate and adaptive immunity.

A WAO - ARIA - GA²LEN consensus document on molecular-based allergy diagnostics

Molecular-based allergy (MA) diagnostics is an approach used to map the allergen sensitization of a patient at a molecular level, using purified natural or recombinant allergenic molecules (allergen components) instead of allergen extracts. Since its introduction, MA diagnostics has increasingly entered routine care, with currently more than 130 allergenic molecules commercially available for in vitro specific IgE (sIgE) testing.MA diagnostics allows for an increased accuracy in allergy diagnosis and prognosis and plays an important role in three key aspects of allergy diagnosis: (1) resolving genuine versus cross-reactive sensitization in poly-sensitized patients, thereby improving the understanding of triggering allergens; (2) assessing, in selected cases, the risk of severe, systemic versus mild, local reactions in food allergy, thereby reducing unnecessary anxiety for the patient and the need for food challenge testing; and (3) identifying patients and triggering allergens for specific immunotherapy (SIT).Singleplex and multiplex measurement platforms are available for MA diagnostics. The Immuno-Solid phase Allergen Chip (ISAC) is the most comprehensive platform currently available, which involves a biochip technology to measure sIgE antibodies against more than one hundred allergenic molecules in a single assay. As the field of MA diagnostics advances, future work needs to focus on large-scale, population-based studies involving practical applications, elucidation and expansion of additional allergenic molecules, and support for appropriate test interpretation. With the rapidly expanding evidence-base for MA diagnosis, there is a need for allergists to keep abreast of the latest information. The aim of this consensus document is to provide a practical guide for the indications, determination, and interpretation of MA diagnostics for clinicians trained in allergology.

The interaction between NK cells and dendritic cells in bacterial infections results in rapid induction of NK cell activation and in the lysis of uninfected dendritic cells.

NK and DC reciprocal interactions have only recently been investigated. In this study, we focused on the interplay between NK cells and DC in two models of bacterial infection. Immature monocyte‐derived DC were cultured in the presence of live Escherichia coli or bacillus Calmette–Guérin. Upon exposure to either extracellular or intracellular bacteria, DC underwent maturation as assessed by the increased levels of expression of CD80, CD86, and HLA molecules and the de novo expression of CD83 and CCR7. Significant amounts of TNF‐α and IL‐12 were released by DCupon infection, whereas IL‐2 and IL‐15 were barely detectable in culture supernatants. Both infected and uninfected DC were capable of inducing in fresh autologous NK cells the expression of CD69 and HLA‐DR and of inducing cell proliferation. Remarkably, however, infected DC were much stronger inducers of NK cell activation and proliferation than uninfected DC. Thus, after just 24 h of NK/DC coculture, only those NK cells that had been exposed to bacteria‐infected DC had acquired the ability to lyse autologous immature DC. In addition, infected DC were more resistant to NK‐mediated lysis as a consequence of the up‐regulation of HLA class I molecule expression on their surface. This study suggests a regulatory circuit involving NK cells and DC in which DC‐induced NK cell activationis effectively enhanced by the presence of pathogens. Activated NK cells, by limiting the supply of immature DC, may then exert a control on subsequent innate and adaptive immune responses.

Treatment of EBV-Related Post-Renal Transplant Lymphoproliferative Disease with a Tailored Regimen Including EBV-Specific T Cells

The treatment of EBV‐associated post‐transplant lymphoproliferative disease (PTLD) poses a considerable challenge. Efforts have been made to define regimens based on combination of the available therapeutic agents, chosen and tailored on a patient‐by‐patient basis, with the aim of augmenting event‐free patient and graft survival. Recently, autologous EBV‐specific cytotoxic T‐lymphocytes (CTL) have proved effective in enhancing EBV‐specific immune responses and reducing viral load in organ transplant recipients with active infection. We investigated the use of a tailored combined approach including autologous EBV‐specific CTL for the treatment of EBV‐related PTLD developing after pediatric kidney transplantation.

Heterogeneity of Tonsillar Subepithelial B Lymphocytes, the Splenic Marginal Zone Equivalents

The VH4 genes expressed by both resting and in vivo-activated subepithelial (SE) B cells from human tonsils were studied. Resting SE B cells were subdivided according to the presence (IgDlow) or absence (IgM-only) of surface IgD. CD27 was abundant on activated SE B cells and low on resting IgM-only B cells. Resting IgDlow SE B cells could be subdivided into CD27low and CD27high cell fractions. Resting IgDlow SE B cells displayed VH4 genes with a substantial number of mutations (13/29 of the molecular clones were mutated), whereas 25/26 of the clones from resting IgM-only SE B cells were unmutated. Moreover, mutated VH4 genes were detected mainly within the CD27high cell fraction of the IgDlow SE B cells. Several identical unmutated VH4DJH sequences (11/32) were found in different molecular clones from resting IgM-only SE B cells, suggesting local cellular expansion. Both unmutated (14/25) and mutated (11/25) sequences were found in μ transcripts of activated SE B cells. Extensive mutation was observed in the γ transcripts of activated SE B cells. Therefore, SE B cells are heterogeneous, being comprised of B cells with mutated Ig VH4 genes, that are Ag-experienced B cells, and a subset of B cells with unmutated VH4 genes that are either virgin cells or cells driven by Ags that did not induce or select for V gene mutations.

Epidemiology and clinical features of Mycoplasma pneumoniae infection in children

Mycoplasma pneumoniae (MP) is, considered to affect rarely children less than 5 yrs of age. This study was performed to describe the epidemiology and the clinical features of MP lower respiratory tract infection (LRTI) in children, presenting to a tertiary children hospital. Eleven month-longitudinal study of LRTI due to MP, diagnosed by polymerase chain reaction (PCR) on throat swab specimen, was performed. Out of 866 children with LRTI admitted to the Gaslini Pediatric Institute in Genoa, 102 had a positive PCR for MP. We found 39 preschool-aged children, 42 school-aged children and 21 young adolescent [6.20 (3.81) yrs old]. Interestingly, eight MP+ infants had <8 months of age. The commonest presentations were cough and/or fever (76.5%). Tachypnoea, upper respiratory tract involvement, diarrhoea and vomiting were more common in the <5 yr Gr as compared to the other groups. Chest X-ray was found abnormal in 76 children: consolidations were the commonest finding. Laboratory test showed that the preschool-aged children had a higher number of lymphocytes (p<0.0001) and monocytes (p=0.009). Thrombocytosis was found in 35.7% of children and was more frequent in the preschool-aged children (p=0.013). MP infection is common in preschool-aged children, including young infants, and may have different clinical presentation, as compared to older children.

HLA Class I molecule expression is up-regulated during maturation of dendritic cells, protecting them from natural killer cell-mediated lysis

It has been widely demonstrated that natural killer (NK) cells are able to discriminate between normal and abnormal cells on the basis of the interaction of their NKRs with the MHC molecules expressed on the target cells. Recent studies showed that also normal dendritic cells are susceptible to NK cell-mediated lysis. In this work, the potential relationships between the expression of different surface molecules on both immature and mature DC and susceptibility to lysis have been investigated. The reduced density of HLA class I on the surface of immature DC resulted in the only permissive signal to NK cell mediated killing. On the contrary, the remarkable increase in HLA class I molecules detected during DC maturation was strictly related to the resistance to NK cell. Contemporary, no clear evidences of a role for other surface molecules modulated during DC maturation (CD80, CD83, CD86 and CD40), were obtained.

The ImmunoCAP ISAC molecular allergology approach in adult multi-sensitized Italian patients with respiratory symptoms

OBJECTIVES To evaluate the performances of an allergen microarray in multi-sensitized allergic patients with respiratory symptoms. DESIGN AND METHODS 321 patients and 92 controls were included in this study. Specific serum IgE were assayed using ImmunoCAP ISAC, a microarray containing 103 components derived from 47 allergens and results were compared with extract-based ImmunoCAP Allergens sIgE to 15 common airborne allergens. RESULTS The reproducibility of ISAC was good. The Positive Percent Agreement (PPA) varied between 75% and 100% for sIgE levels above 1 kUA/l. For samples with sIgE levels below 0.1 kUA/l, the Negative Percent Agreement (NPA) ranged between 90% and 100%. Notably, 58% of respiratory allergy patients had IgE to food-specific proteins and 52% resulted sensitized to cross-reactive pan-allergens. CONCLUSION ImmunoCAP ISAC detects allergen sensitization at component level and adds important information by defining both cross and co-sensitization to a large variety of allergen molecules.

Proteome Profiling of Neuroblastoma-Derived Exosomes Reveal the Expression of Proteins Potentially Involved in Tumor Progression

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, with grim prognosis in a half of patients. Exosomes are nanometer-sized membrane vesicles derived from the multivesicular bodies (MVBs) of the endocytic pathway and released by normal and neoplastic cells. Tumor-derived exosomes have been shown in different model systems to carry molecules that promote cancer growth and dissemination. In this respect, we have here performed the first characterization and proteomic analysis of exosomes isolated from human NB cell lines by filtration and ultracentrifugation. Electron microscopy demonstrated that NB-derived exosomes exhibited the characteristic cup-shaped morphology. Dynamic light scattering studies showed a bell-shaped curve and a polydispersity factor consistent with those of exosomes. Zeta potential values suggested a good nanoparticle stability. We performed proteomic analysis of NB-derived exosomes by two dimension liquid chromatography separation and mass spectrometry analyses using the multidimensional protein identification technology strategy. We found that the large majority of the proteins identified in NB derived exosomes are present in Exocarta database including tetraspanins, fibronectin, heat shock proteins, MVB proteins, cytoskeleton-related proteins, prominin-1 (CD133), basigin (CD147) and B7-H3 (CD276). Expression of the CD9, CD63 and CD81 tetraspanins, fibronectin, CD133, CD147 and CD276 was validated by flow cytometry. Noteworthy, flow cytometric analysis showed that NB-derived exosomes expressed the GD2 disialoganglioside, the most specific marker of NB. In conclusion, this study shows that NB-derived exosomes express a discrete set of molecules involved in defense response, cell differentiation, cell proliferation and regulation of other important biological process. Thus, NB-derived exosomes may play an important role in the modulation of tumor microenvironment and represent potential tumor biomarkers.

The IgE repertoire in children and adolescents resolved at component level: a cross-sectional study.

To cite this article: Melioli G, Marcomini L, Agazzi A, Bazurro G, Tosca M, Rossi GA, Minale P, Rossi R, Reggiardo G, Canonica GW, Passalacqua G. The IgE repertoire in children and adolescents resolved at component level: A cross‐sectional study. 
Pediatr Allergy Immunol 2012: 23: 433–440.