Roberto Nisini

Cross sectional retrospective study of prevalence of atopy among Italian military students with antibodies against hepatitis a virus

Abstract Objective: To investigate the working hypothesis that common infections occurring early in life prevent atopy. Design: Cross sectional, retrospective study of young Italian men with results for hepatitis A serology and atopy. Setting: Air force school for military students in Caserta, Italy. Subjects: 1659 male students aged 17-24, most of whom (90%) were from central and southern Italy. Main outcome measures: Skin sensitisation and specific IgE antibodies to locally relevant airborne allergens; diagnosis of respiratory allergy (asthma or rhinitis, or both); hepatitis A seropositivity. Results: 443 of the 1659 subjects (26.7%) were positive for hepatitis A virus antibody. Atopy was less common among seropositive than seronegative subjects according to skin sensitization (weal reaction ≥3 mm) to one or more allergens (21.9% (97/443) v 30.2% (367/1216), P<0.001); polysensitisation (sensitive to three or more allergens) (2.7% (12/443) v 6.4% (78/1216), P<0.01); high specific IgE concentration (9.7% (43/443) v 18.4% (224/1216), P<0.00005); and lifetime prevalence of allergic rhinitis or asthma, or both (8.4% (37/443) v 16.7% (203/1216), P<0.001). Hepatitis A seropositivity remained inversely associated with atopy after adjusting for fathers education, the number of older siblings, and the area of residence (based on the number of inhabitants). The prevalence of atopy was constantly low among seropositive subjects, whatever the number of older siblings; by contrast, it increased with a decreasing number of older siblings among seronegative subjects. Conclusion: Indirect but important evidence is added to the working hypothesis as common infections acquired early in life because of the presence of many older siblings (among seronegative subjects) or because of unhygienic living conditions (among seropositive subjects) may have reduced the risk of developing atopy. Key messages Young men with antibodies to hepatitis A virus had a lower prevalence of atopy and atopic respiratory diseases, and this was independent of the number of older siblings and other relevant risk factors The prevalence of atopy was as low in seronegative as in seropositive subjects only when they had three or more older siblings Among seropositive subjects the prevalence of atopy was low, whatever the number of older siblings Common infections acquired early in life because of the presence of many older siblings (among seronegative subjects) or because of unhygienic living conditions (among seropositive subjects) may have reduced the risk of development of atopy This study adds indirect but important evidence to the hypothesis that improvements in hygiene and reduced recirculation of common infections may be a major cause of the increasing prevalence of atopy and atopic diseases in Western countries

Mycobacterium tuberculosis subverts the differentiation of human monocytes into dendritic cells

Intracellular pathogens have developed strategies for evading elimination by the defenses of the host immune system. Here we describe an escape mechanism utilized by Mycobacterium tuberculosis that involves the interference with the generation of fully competent DC from monocytes. We show that monocytes infected with live M. tuberculosis differentiated into mature, CD83+ and CCR7+ DC (Mt‐MoDC), but were characterized by a selective failure in the expression of the family of CD1 molecules. These cells also showed levels of MHC class II and CD80 (B7.1) that were reduced in comparison with LPS‐matured DC. In addition, Mt‐MoDC produced TNF‐α and IL‐10, but were unable to secrete IL‐12. The generation of Mt‐MoDC required the infection of monocytes with live M. tuberculosis, since infection with heat‐killed bacteria partially abrogated the effects on monocyte differentiation. Interestingly, Mt‐MoDC revealed an impaired antigen‐presentation function as assessed by the reduced capability to induce proliferation of cord blood T lymphocytes. Further, naive T lymphocytes expanded by Mt‐MoDC were unable to secrete cytokines, in particular IL‐4 and IFN‐γ, suggesting that they could be ineffective in helping the macrophage‐mediated killing of intracellular mycobacteria. Our results suggest that the interference with monocyte differentiation into fully competent DC is an evasion mechanism of M. tuberculosis that could contribute to its intracellular persistence by avoiding immune recognition.

Detection of interleukin-2 in addition to interferon-γ discriminates active tuberculosis patients, latently infected individuals, and controls

Effective control of tuberculosis (TB) includes discrimination of subjects with active TB from individuals with latent TB infection (LTBI). As distinct interferon (IFN)-gamma and interleukin (IL)-2 profiles of antigen-specific T-cells have been associated with different clinical stages and antigen loads in several viral and bacterial diseases, we analysed these cytokines in TB using a modified QuantiFERON-TB Gold In Tube test. Detection of IL-2 in addition to IFN-gamma distinguishes not only Mycobacterium tuberculosis-infected subjects from healthy controls, but also individuals with LTBI from active TB patients. This may help to improve diagnostic tests for TB.

Influenza vaccine administration in rheumatoid arthritis patients under treatment with TNFα blockers: Safety and immunogenicity

Twenty-eight patients with low-moderate, stable rheumatoid arthritis (RA), under treatment with tumor necrosis factor (TNF) alpha blockers, were immunized at least once with non-adjuvanted trivalent influenza vaccine during three consecutive influenza seasons. Antibodies toward A influenza antigens significantly increased and reached protective levels, still detectable 6 months after vaccination, both in RA patients and healthy controls. Response to B antigen instead was only observed from the second year for healthy controls and in the third year for patients. No significant difference in disease activity and anti-nuclear antibodies was observed as a consequence of vaccine administration, whereas T regulatory cells showed a significant increase 30 days after immunization in RA patients. This study confirms safety of influenza vaccine administration in RA patients treated with TNFalpha blockers. The cohort follow-up revealed the overcoming of poor B vaccine antigen immunogenicity via repeated vaccinations. Finally, protective antibody response was still observed 6 months after vaccination.

Monocyte-derived dendritic cells generated after a short-term culture with IFN-α and granulocyte-macrophage colony-stimulating factor stimulate a potent epstein-barr virus-specific CD8+ T cell response

Cellular immune responses are crucial for the control of EBV-associated lymphoproliferative diseases. To induce an anti-EBV cell-mediated immunity, we have used dendritic cells (DCs) generated by a 3-day culture of human CD14+ monocytes in the presence of GM-CSF and type I IFN (IFN-DCs) and pulsed with peptides corresponding to CTL EBV epitopes. The functional activity of IFN-DCs was compared with that of APCs differentiated by culturing monocytes for 3 days with GM-CSF and IL-4 and indicated as IL-4-DCs. Stimulation of PBLs from EBV-seropositive donors with EBV peptide-pulsed autologous IFN-DCs resulted in a stronger expansion of specific T lymphocytes producing IFN-γ with respect to stimulation with peptide-loaded IL-4-DCs, as assessed by ELISPOT assays. When purified CD8+ T cells were cocultured with EBV peptide-pulsed IFN-DCs or IL-4-DCs, significantly higher levels of specific cytotoxic activity were observed in CD8+ T cell cultures stimulated with IFN-DCs. Injection of peptide-pulsed IFN-DCs into SCID mice transplanted with autologous PBLs led to the recovery of a significantly greater number of EBV-specific human CD8+ T cells from the spleen and the peritoneal cavity with respect to that recovered from mice injected with peptide-pulsed IL-4-DCs. Moreover, a significant delay in lymphoma development was observed when peptide-pulsed IFN-DCs were injected into SCID mice reconstituted with PBMCs endowed with a high capability of lymphoma induction, whereas injection of unpulsed IFN-DCs was ineffective. Our results indicate that IFN-DCs efficiently promote in vitro and in vivo the expansion of CD8+ T lymphocytes acting as cytotoxic effectors against EBV-transformed cells.

Human Dendritic Cells following Aspergillus fumigatus Infection Express the CCR7 Receptor and a Differential Pattern of Interleukin-12 (IL-12), IL-23, and IL-27 Cytokines, Which Lead to a Th1 Response

ABSTRACT Aspergillus fumigatus is the most prevalent airborne fungal pathogen and causes fatal invasive aspergillosis in immunocompromised patients. Given the essential role of dendritic cells (DC) in initiating and regulating immune responses, we investigated the impact of A. fumigatus conidial infection on human DC. A. fumigatus conidia were rapidly internalized and induced the release of tumor necrosis factor alpha within the first 8 h. After A. fumigatus infection, the majority of DC underwent full maturation, although CCR7 expression was observed only in DC that had internalized the conidia. Additionally, the analysis of regulatory cytokines showed that infected DC simultaneously produced interleukin-12p70 (IL-12p70) and significant amounts of IL-10. IL-10 neutralization was not able to further increase IL-12p70 production from infected DC. Whereas the central role of IL-12 in the generation of Th1 cells has long been appreciated, recently two other members of the IL-12 family, IL-23 and IL-27, were reported to play important roles in the regulation of gamma interferon (IFN-γ) production from naïve and memory T cells. A. fumigatus-infected DC were also able to express high levels of IL-23p19 and low levels of IL-27p28 at later stages of infection. According to this expression pattern, A. fumigatus-infected DC were able to prime IFN-γ production of naïve T cells. Thus, this study on the expression of the new IL-12 family members controlling the Th1 response sheds light on a novel aspect of the contribution of DC to anti-Aspergillus immunity.

Cytometric detection of antigen-specific IFN-γ/IL-2 secreting cells in the diagnosis of tuberculosis

BackgroundThe purpose of this study was to further characterize the immune response to Mycobacterium tuberculosis (Mtb) antigens, in order to provide new insight into host-pathogen interactions in tuberculosis (TB), and to offer tools for a more accurate diagnosis of the different stages of TB.MethodsT-cell responses to Bacillus Calmette-Guérin (BCG), purified protein derivative (PPD), early secretory antigenic target-6 (ESAT-6) protein and culture filtrate protein-10 kDa (CFP-10) were measured in terms of interferon (IFN)-γ and interleukin (IL)-2 release, using a novel flow cytometric cell-secreting cytokine detection technique. The study was conducted on peripheral blood mononuclear cells (PBMC) obtained from active TB patients, latently TB infected individuals, and healthy donors. IL-10 and IL-17 were also measured to test their possible role as indicators of disease activity.ResultsWe confirmed that the enumeration of IFN-γ releasing cells upon Mtb-specific stimulation is sufficient to identify TB patients and that CD8+ T cells concur to IFN-γ secretion. IL-2 secreting cells were more frequently observed in latent TB infected individuals compared to active TB patients, suggesting that measurement of cells secreting this cytokine could be a marker of disease stage. No discriminating role was associated to IL-10 and IL-17 release in TB patients.ConclusionOur data indicate that the flow cytometric cytokine-secreting cell detection technique may be envisaged as an additional tool for TB diagnosis allowing the analysis of the immune response to M. tuberculosis-related antigens in the different stages of TB.

Cell wall-associated alpha-glucan is instrumental for Mycobacterium tuberculosis to block CD1 molecule expression and disable the function of dendritic cell derived from infected monocyte

We previously described an escape mechanism exploited by Mycobacterium tuberculosis (Mtb) to prevent the generation of fully competent dendritic cells (DC). We have now tested the effect of isolated mycobacterial components on human monocyte differentiation into DC and demonstrated that cell wall (CW)‐associated alpha‐glucan induces monocytes to differentiate into DC (Glu‐MoDC) with the same altered phenotype and functional behaviour of DC derived from Mtb‐infected monocytes (Mt‐MoDC). In fact, Glu‐MoDC lack CD1 molecule expression, fail to upregulate CD80 and produce IL‐10 but not IL‐12. We also showed that Glu‐MoDC are not able to prime effector T cells or present lipid antigens to CD1‐restricted T‐cell clones. Thus, we propose a mechanism of Mtb–monocyte interaction mediated by CW‐associated alpha‐glucan, which allows the bacterium to evade both innate and acquired immune responses.

The interaction of human dendritic cells with yeast and germ-tube forms of Candida albicans leads to efficient fungal processing, dendritic cell maturation, and acquisition of a Th1 response-promoting function

T helper cell type 1 (Th1) cell‐mediated immunity plays a rical role in protection against the opportunistic pathogen Candida albicans. Virulence of the fungus is closely associated with its ability to form germ‐tubes (GT), the early phase of the dimorphic transition from the commensal yeast (Y) to the more invasive hyphal (H) form. In this study, we examined the functional outcome of the interaction of Y or GT forms with human dendritic cells (DCs), professional antigen‐presenting cells, which are pivotal for initiation and modulation of T cell responses. DCs phagocytosed and killed Y and GT cells with a comparable efficiency, becoming able to trigger strong proliferative responses by Candida‐specific, autologous T cell clones. Both fungal forms induced DC maturation, as indicated by up‐regulation of CD83, CD80, CD86, CD40, and major histocompatibility complex classes I and II surface antigens. Chemokine receptors were also modulated in Candida–DCs, which showed increased CCR7/CXCR4 and decreased CCR5 expression. Y‐ and GT‐activated DCs differed in the pattern of cytokine expression. In particular, GT cells, in common with fully differentiated H cells, induced significantly more elevated levels of interleukin (IL)‐10 than Y cells. Nevertheless, Y‐, GT‐, or H‐pulsed DCs secreted comparable amounts of IL‐12p70. In addition, irrespective of the fungal form triggering DC activation, Candida–DCs acquired the ability to prime naive T lymphocytes with a defined Th1 phenotype. Overall, our findings highlight the induction of substantially similar functional patterns in human DCs encountering the different forms of growth of C. albicans, both seemingly activating the Th1‐type immunity which is characteristic of the healthy human subjects, naturally immunized and protected against the fungus.

Generation of a Recombinant 65-Kilodalton Mannoprotein, a Major Antigen Target of Cell-Mediated Immune Response to Candida albicans

ABSTRACT A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogenCandida albicans. An expression library of C. albicans was screened with serum from mice immunized with ScMp65 (ScW10), a Saccharomyces cerevisiae recombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract of C. albicans. After cloning and sequencing of the relevant C. albicans cDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designatedCaMP65) was localized to chromosome R of C. albicans as determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His6-tagged protein (rCaMp65) was expressed inEscherichia coli under an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4+ T-cell clones were generated using a C. albicans mannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinant C. albicans Mp65 but not to ScMp65. Thus, the recombinant Mp65 of C. albicans retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.