Publications by authors named "Clara Maria Ausiello"

25 Publications

  • Page 1 of 1

Antibody mimicry, receptors and clinical applications.

Hum Antibodies 2017 ;25(3-4):75-85

Laboratory of Immunogenetics, Department of Medical Sciences, University of Torino, Torino 10126, Italy.

This review focuses on the concept of antibodies acting as receptor agonists and antagonists, and on the potential relevance of this notion in applied medicine. Antibodies are composed of three functional units: two antigen-binding fragments (Fabs) that confer antigen specificity and one constant fragment (Fc) linking antibodies to immune effector functions. The proof-of-concept that large amounts of highly specific and homogeneous antibodies could be produced was provided in 1975 by César Milstein and Georges Köhler. These monoclonal antibody (mAb) reagents started a revolution in medical research, diagnostics, and clinical applications. Alongside diagnostic applications, mAbs were successfully used in vivo: (i) to bind (neutralize/antagonize) antigens expressed on the surface of tumor cells; (ii) to activate immune effector mechanisms; (iii) to crosslink plasma membrane receptors and hence activate therapeutic signaling pathways; and lastly, (iv) the technique was expanded to produce bispecific mAbs, which can bind two different antigens while retaining the ability to activate immune effector functions. The abilities of mAbs to bind, transduce signals, and exert immunostimulatory agonistic capacities are the central issues of this review. The starting point is that some mAbs operate as molecular agonists, substituting for the natural ligand of the receptor. Our analysis is restricted to mAbs that act as receptor agonist/antagonists by either mimicking ligand binding, or through allosteric modulation mediated by binding sites that are topographically distinct from the orthosteric binding site. Functional considerations based on the agonistic stimulation of human CD38 by specific mAbs as surrogate ligands are described as examples of the features of such molecules.
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http://dx.doi.org/10.3233/HAB-160305DOI Listing
February 2018

Parents as source of pertussis transmission in hospitalized young infants.

Infection 2017 Apr 10;45(2):171-178. Epub 2016 Sep 10.

Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.

Purpose: This study was planned to collect evidences of familial pertussis transmission to infants younger than 6 months of age. Understanding the dynamics of transmission of pertussis in families is essential to plan effective prevention strategies that could be integrated in pertussis control.

Methods: The seroprevalence of IgG antibodies to pertussis toxin (PT-IgG) and prolonged cough symptoms were evaluated in parents of 55 infants aged <6 months hospitalized for confirmed pertussis. Parents of 33 infants with lower respiratory tract infection (LRTI) and parents of 57 healthy infants admitted as outpatients for hip ultrasound examination (HE) were enrolled as controls.

Results: Parents of pertussis cases had PT-IgG levels significantly higher as compared to LRTI and HE parents. More than 40 % were compatible as transmitters of pertussis to their babies, since they had a level of PT-IgG ≥ 100 IU/ml, which is considered diagnostic for a recent pertussis episode. Based on serology, the percentage of pertussis cases that had at least one parent as source of infection was 49.1 %. When cough symptoms were taken into account, the percentage of parents putative transmitters of the infection to their infants increased to 56.4 %.

Conclusions: Parents are scarcely aware of the household transmission of pertussis to their newborns. Our study highlights the need to advise parents about the likelihood of transmission to the newborn and to be particularly aware of coughing symptoms in the household. Since infection can be asymptomatic, a serological survey of family members should also be considered.
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http://dx.doi.org/10.1007/s15010-016-0943-6DOI Listing
April 2017

T-cell polarization: Potential serological markers in preterm and term infants.

Early Hum Dev 2016 10 11;101:69-71. Epub 2016 Jul 11.

Department of Infectious, Parasitic and Immune-Mediate Disease, Istituto Superiore di Sanità, Rome, Italy.

Background: The immaturity of immune system characterizes newborn infants. Possible serological markers of Th1 and Th2 immune response are the lymphocyte activation gene-3 (CD223) and soluble CD30, respectively (sCD30).

Aims: The aim of our study was to evaluate the relationship between Th1 and Th2 immune response and gestational age (GA), comparing data in preterm and term neonates.

Study Design: Cord blood from 20 preterm (GA: 33±2weeks, BW 1950±490g) and 20 term infants (GA: 38±1weeks, BW: 3177±330g) were tested for sCD30 and CD223 levels by ELISA. IFNγ levels produced by cord blood lymphocytes were also analyzed, both before and after stimulation with phytohaemagglutinin (PHA).

Results: sCD30 resulted significantly higher in preterm neonates when compared with term neonates (60±7.6 vs 42.6±3.9U/ml p<0.05). CD223 was undetectable in preterm neonates while resulting at a level of 176.1±112.6ng/ml in term neonates. After stimulation with PHA, a significant increase in IFNγ levels was only observed in term neonates (326.6±72.7pg/ml p<0.05).

Conclusions: Our findings show that sCD30 is present and measurable in term and preterm infants, while CD223 is detectable only in term infants and that Th-cell polarization could also depend on gestational age. Our data suggest that a Th2 immune response seems predominant in preterm neonates.
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http://dx.doi.org/10.1016/j.earlhumdev.2016.03.013DOI Listing
October 2016

Evidence of increased circulation of Bordetella pertussis in the Italian adult population from seroprevalence data (2012-2013).

J Med Microbiol 2016 Jul 13;65(7):649-657. Epub 2016 Apr 13.

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

Incidence data on pertussis cases in Italy do not show pertussis resurgence as recently described in other European countries. The aim of this study was to determine the seroprevalence of IgG antibodies to pertussis toxin (PT-IgG) in selected adult age groups, who can serve as a reservoir of Bordetella pertussis and be responsible for onward transmission to vulnerable infants. The seroprevalence of PT-IgG was studied in sera collected in 2012-2013 in three age groups: 20-29 years and 30-39 years (reproductive age), and ≥60 years. These data were compared to those from sera collected in similar age groups in 1996-1997. More than 80 % of the adult population analysed in the 2012-2013 group presented detectable levels of PT-IgG (>5 IU ml). PT-IgG titres of 50-99 IU ml, considered indicative of infection in the last few years, and PT-IgG titres of  ≥100 IU ml, considered indicative of recent infection (i.e.within the last year), reached 9.1 % [95 % confidence interval (CI) 6.9-11.3‌  %; 58/639] and 5 % (95 % CI 3.3-6.7 %; 32/639) seroprevalence, respectively. Notably, the proportion of subjects with a seroprevalence indicative of recent infection increased significantly from 9.3 % (95 % CI 7.5-11.1 %; 96/1037) in 1996-1997 to 14.1 % (95 % CI 11.4-16.8 %; 90/639) in 2012-2013. Overall, our data clearly indicate a significant increase in the circulation of B. pertussis in adults in Italy; therefore, it is likely that the statutory notification system underestimates the real incidence of the disease. These findings have implications for preventive strategies.
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http://dx.doi.org/10.1099/jmm.0.000264DOI Listing
July 2016

T-cell immune responses to Bordetella pertussis infection and vaccination.

Pathog Dis 2015 Oct 4;73(7). Epub 2015 Aug 4.

Anti-Infectious Immunity Unit, Department of Infectious Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy.

The recent immunological investigations, stemming from the studies performed in the nineties within the clinical trials of the acellular pertussis vaccines, have highlighted the important role played by T-cell immunity to pertussis in humans. These studies largely confirmed earlier investigations in the murine respiratory infection models that humoral immunity alone is not sufficient to confer protection against Bordetella pertussis infection and that T-cell immunity is required. Over the last years, knowledge of T-cell immune response to B. pertussis has expanded broadly, taking advantage of the general progress in the understanding of anti-bacterial immunity and of refinements in methods to approach immunological investigations. In particular, experimental models of B. pertussis infection highlighted the cooperative role played by T-helper (Th)1 and Th17 cells for protection. Furthermore, the new baboon experimental model suggested a plausible explanation for the differences observed in the strength and persistence of protective immunity induced by the acellular or whole-cell pertussis vaccines and natural infection in humans, contributing to explain the upsurge of recent pertussis outbreaks. Despite the progress, open questions remain, the answer to them will possibly provide better tools to fight one of the hardest-to-control vaccine preventable disease.
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http://dx.doi.org/10.1093/femspd/ftv051DOI Listing
October 2015

Acellular pertussis vaccines and pertussis resurgence: revise or replace?

mBio 2014 Jun 10;5(3):e01339-14. Epub 2014 Jun 10.

Department of Experimental Medicine and Biochemical Sciences, Medical School, University of Perugia, Perugia, Italy

The resurgence of pertussis (whooping cough) in countries with high vaccination coverage is alarming and invites reconsideration of the use of current acellular pertussis (aP) vaccines, which have largely replaced the old, reactogenic, whole-cell pertussis (wP) vaccine. Some drawbacks of these vaccines in terms of limited antigenic composition and early waning of antibody levels could be anticipated by the results of in-trial or postlicensure human investigations of B- and T-cell responses in aP versus wP vaccine recipients or unvaccinated, infected children. Recent data in experimental models, including primates, suggest that generation of vaccines capable of a potent, though regulated, stimulation of innate immunity driving effective, persistent adaptive immune responses against Bordetella pertussis infection should be privileged. Adjuvants that skew Th1/Th17 responses or new wP (detoxified or attenuated) vaccines should be explored. Nonetheless, the high merits of the current aP vaccines in persuading people to resume vaccination against pertussis should not be forgotten.
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http://dx.doi.org/10.1128/mBio.01339-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4056554PMC
June 2014

Humoral and B-cell memory responses in children five years after pertussis acellular vaccine priming.

Vaccine 2014 Apr 18;32(18):2093-9. Epub 2014 Feb 18.

Anti-Infectious Immunity Unit, Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità (ISS), Viale Regina Elena 299, Rome, Italy. Electronic address:

The resurgence of pertussis suggests the need for greater efforts in understanding the long-lasting protective responses induced by vaccination. In this paper we dissect the persistence of humoral and B-cell memory responses induced by primary vaccination with two different acellular pertussis (aP) vaccines, hexavalent Hexavac(®) vaccine (Hexavac) (Sanofi Pasteur MSD) and Infanrix hexa(®) (Infanrix) (GlaxoSmithKline Biologicals). We evaluated the specific immune responses in the two groups of children, 5 years after primary vaccination by measuring the persistence of IgG and antibody secreting cells (ASC) specific for vaccine antigens. Part of the enrolled children received only primary vaccination, while others had the pre-school boost dose. A similar level of antigen-specific IgG and ASC was found in Infanrix and Hexavac vaccinated children. The mean IgG levels were significantly higher in children that received the pre-school boost as compared with children that did not receive the boost dose. A longer persistence after the pre-school boost of IgG-Pertussis Toxin (PT) and IgG-pertactin levels was observed in Infanrix primed children, but it was not statistically significant. More than 80% of children presented a positive ASC B memory response. Around 50% of children still presented protective IgG-PT levels which are reduced to 36% in no-boosted children. The pre-school booster dose restores the percentage of protected children above 50%. In conclusion our data underline the importance of giving a booster dose 5 years after primary vaccination and suggest the need for a new vaccine able to induce a long lasting protective response.
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http://dx.doi.org/10.1016/j.vaccine.2014.02.005DOI Listing
April 2014

CD38 ligation in peripheral blood mononuclear cells of myeloma patients induces release of protumorigenic IL-6 and impaired secretion of IFNγ cytokines and proliferation.

Mediators Inflamm 2013 30;2013:564687. Epub 2013 Dec 30.

Department of Infectious, Parasitic and Immune-mediated Diseases, Anti-infectious Immunity Unit, Istituto Superiore di Sanità, 00161 Rome, Italy.

CD38, a surface receptor that controls signals in immunocompetent cells, is densely expressed by cells of multiple myeloma (MM). The immune system of MM patients appears as functionally impaired, with qualitative and quantitative defects in T cell immune responses. This work answers the issue whether CD38 plays a role in the impairment of T lymphocyte response. To this aim, we analyzed the signals implemented by monoclonal antibodies (mAb) ligation in peripheral blood mononuclear cells (PBMC) obtained from MM patients and compared to benign monoclonal gammopathy of undetermined significance (MGUS). PBMC from MM both failed to proliferate and secrete IFNγ induced by CD38 ligation while it retained the ability to respond to TCR/CD3. The impaired CD38-dependent proliferative response likely reflects an arrest in the progression of cell cycle, as indicated by the reduced expression of PCNA. CD38 signaling showed an enhanced ability to induce IL-6 secretion. PBMC from MM patients displays a deregulated response possibly due to defects of CD38 activation pathways and CD38 may be functionally involved in the progression of this pathology via the secretion of high levels of IL-6 that protects neoplastic cells from apoptosis.
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http://dx.doi.org/10.1155/2013/564687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3892939PMC
June 2014

Different T cell memory in preadolescents after whole-cell or acellular pertussis vaccination.

Vaccine 2013 Dec 29;32(1):111-8. Epub 2013 Oct 29.

Laboratory of Vaccinology and Mucosal Immunity, Université Libre de Bruxelles (ULB), Brussels, Belgium; Immunobiology Clinic, Hôpital Erasme, Université Libre de Bruxelles (ULB), Brussels, Belgium. Electronic address:

To better understand vaccine-induced protection and its potential failure in light of recent whooping cough resurgence, we evaluated quantity as well as quality of memory T cell responses in B. pertussis-vaccinated preadolescent children. Using a technique based on flow cytometry to detect proliferation, cytokine production and phenotype of antigen-specific cells, we evaluated residual T cell memory in a cohort of preadolescents who received a whole-cell pertussis (wP; n=11) or an acellular pertussis vaccine (aP; n=13) during infancy, and with a median of 4 years elapsed from the last pertussis booster vaccine, which was aP for all children. We demonstrated that B. pertussis-specific memory T cells are detectable in the majority of preadolescent children several years after vaccination. CD4(+) and CD8(+) T cell proliferation in response to pertussis toxin and/or filamentous hemagglutinin was detected in 79% and 60% of the children respectively, and interferon-γ or tumor necrosis factor-α producing CD4(+) T cells were detected in 65% and 53% of the children respectively. Phenotyping of the responding cells showed that the majority of antigen-specific cells, whether defined by proliferation or cytokine production, were CD45RA(-)CCR7(-) effector memory T cells. Although the time since the last booster vaccine was significantly longer for wP-compared to aP-vaccinated children, their proliferation capacity in response to antigenic stimulation was comparable, and more children had a detectable cytokine response after wP- compared to aP-vaccination. This study supports at the immunological level recent epidemiological studies indicating that infant vaccination with wP induces longer lasting immunity than vaccination with aP-vaccines.
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http://dx.doi.org/10.1016/j.vaccine.2013.10.056DOI Listing
December 2013

The virulence factors of Bordetella pertussis: talented modulators of host immune response.

Arch Immunol Ther Exp (Warsz) 2013 Dec 18;61(6):445-57. Epub 2013 Aug 18.

Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy,

Approximately 40 million whooping cough cases and between 200,000 and 400,000 pertussis-linked deaths are recorded each year. Although several types of vaccines are licensed and widely used, Bordetella pertussis continues to circulate in populations with high vaccine coverage of infants and children due to the waning of protection induced by the vaccination. B. pertussis typically expresses a wide array of virulence factors which promote bacterial adhesion and invasion by altering the local environment, including pertussis toxin, tracheal cytotoxin, adenylate cyclase toxin, filamentous hemagglutinin, and the lipooligosaccharide. The virulence factors of B. pertussis also possess immunomodulatory properties, exerted through their enzymatic and receptor-binding activities. Both pro- and anti-inflammatory effects are mediated, that can subvert host innate and adaptive immunity and favor the onset of a long-term infection. This review describes the capacities of B. pertussis virulence factors to modulate host immune responses and the mechanisms employed, which have been the subject of extensive research in the recent years, both in murine and human experimental systems. Knowledge of these mechanisms is gaining increasing importance, since it could provide in the near future the basis for the identification of therapeutic agents for modulating the immune system as well as novel molecular targets to treat pertussis.
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http://dx.doi.org/10.1007/s00005-013-0242-1DOI Listing
December 2013

Hepatitis B specific T cell immunity induced by primary vaccination persists independently of the protective serum antibody level.

Vaccine 2013 Jan 19;31(3):506-13. Epub 2012 Nov 19.

Anti-Infectious Immunity Unit, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy.

In 2005, in accordance with recommendations made by the European Medicines Agency, the Italian Drug Agency ordered withdrawal of the hexavalent Hexavac(®) vaccine (Sanofi Pasteur MSD) from the market. Concerns had been raised about the low immunogenicity of the hepatitis B virus component of the vaccine, assessed by measurement of serum antibody levels, and its potential consequences on long-term protection against hepatitis B infection. We evaluated memory T cell response to establish whether there are differences in the protective mechanisms among children who had received either Hexavac(®) or Infanrix-hexa(®) (GlaxoSmithKline) as their primary vaccination. Immunological memory was determined by measuring the ability of T cells to proliferate and secrete IFNγ by ELISA and intracellular cytokines (IFNγ and IL-2) when cultured with hepatitis B surface antigen (HBsAg). The different memory subsets of T cells were also measured. The results indicate that, although they generate different serum antibody levels, both vaccines are efficient in generating T recall responses in vitro five years after the primary vaccination. The less immunogenic Hexavac(®) vaccine induces a strong T antigen response, as indicated by increased blast proliferation and the enhanced presence of memory subsets after HBsAg recall stimulation. These findings suggest that cellular immune response should be considered alongside serological markers as a surrogate of protection.
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http://dx.doi.org/10.1016/j.vaccine.2012.11.029DOI Listing
January 2013

Antigen-specific responses assessment for the evaluation of Bordetella pertussis T cell immunity in humans.

Vaccine 2012 Feb 9;30(9):1667-74. Epub 2012 Jan 9.

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy.

Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.
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http://dx.doi.org/10.1016/j.vaccine.2011.12.104DOI Listing
February 2012

Identity and ranking of colonic mesenchymal stromal cells.

J Cell Physiol 2012 Sep;227(9):3291-300

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.
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http://dx.doi.org/10.1002/jcp.24027DOI Listing
September 2012

Attenuated Bordetella pertussis vaccine candidate BPZE1 promotes human dendritic cell CCL21-induced migration and drives a Th1/Th17 response.

J Immunol 2011 May 23;186(9):5388-96. Epub 2011 Mar 23.

Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanità, 00161 Rome, Italy.

New vaccines against pertussis are needed to evoke full protection and long-lasting immunological memory starting from the first administration in neonates--the major target of the life-threatening pertussis infection. A novel live attenuated Bordetella pertussis vaccine strain, BPZE1, has been developed by eliminating or detoxifying three important B. pertussis virulence factors: pertussis toxin, dermonecrotic toxin, and tracheal cytotoxin. We used a human preclinical ex vivo model based on monocyte-derived dendritic cells (MDDCs) to evaluate BPZE1 immunogenicity. We studied the effects of BPZE1 on MDDC functions, focusing on the impact of Bordetella-primed dendritic cells in the regulation of Th and suppressor T cells (Ts). BPZE1 is able to activate human MDDCs and to promote the production of a broad spectrum of proinflammatory and regulatory cytokines. Moreover, conversely to its parental wild-type counterpart BPSM, BPZE1-primed MDDCs very efficiently migrate in vitro in response to the lymphatic chemokine CCL21, due to the inactivation of pertussis toxin enzymatic activity. BPZE1-primed MDDCs drove a mixed Th1/Th17 polarization and also induced functional Ts. Experiments performed in a Transwell system showed that cell contact rather than the production of soluble factors was required for suppression activity. Overall, our findings support the potential of BPZE1 as a novel live attenuated pertussis vaccine, as BPZE1-challenged dendritic cells might migrate from the site of infection to the lymph nodes, prime Th cells, mount an adaptive immune response, and orchestrate Th1/Th17 and Ts responses.
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http://dx.doi.org/10.4049/jimmunol.1003765DOI Listing
May 2011

Bordetella pertussis commits human dendritic cells to promote a Th1/Th17 response through the activity of adenylate cyclase toxin and MAPK-pathways.

PLoS One 2010 Jan 15;5(1):e8734. Epub 2010 Jan 15.

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA), an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC), an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT) or a mutant lacking CyaA (BpCyaA-), or the BpCyaA- strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR) 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008734PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2806909PMC
January 2010

Genetically detoxified pertussis toxin induces Th1/Th17 immune response through MAPKs and IL-10-dependent mechanisms.

J Immunol 2009 Aug 13;183(3):1892-9. Epub 2009 Jul 13.

Department of Infectious, Istituto Superiore di Sanità, Rome, Italy.

Genetically detoxified pertussis toxin (dPT) maintains the protein structure and the immunological properties, but not the enzymatic activity. In search of an adjuvant able to direct polarization of T cells to induce/potentiate protective immune response to a variety of infectious disease, we investigated the role played by dPT on human dendritic cell-driven Th polarization and analyzed the intracellular signaling events. To reach these aims, we used a highly purified dPT preparation devoid of contamination and monocyte-derived dendritic cells, a well-characterized model to study ex vivo the polarization of the immune responses. First, we analyzed dPT-induced monocyte-derived dendritic cell maturation, longevity, and cytokine production and, in a second step, we analyzed TLR4/2 engagement by dPT, the connected signaling events, and their relevance to the skewing of Th cell polarization. These approaches allowed us to clarify some of the mechanisms that are responsible for dPT-driven regulation of T cell polarization. We demonstrated that dPT acts utilizing TLR4/TLR2 engagement, being the signaling induced by the former stronger. dPT, through a crucial role played by MAPK and IL-10, favors the expansion of the Th1/Th17 immunity. Indirect evidences indicated that dPT-induced Th17 expansion is counterregulated by the PI3K pathway. For its properties and being already used in humans as vaccine Ag in pertussis, dPT may represents a valid candidate adjuvant to foster immune protective response in vaccines against infectious diseases where Th1/Th17 are mediating host immunity.
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http://dx.doi.org/10.4049/jimmunol.0901071DOI Listing
August 2009

A natural pertactin deficient strain of Bordetella pertussis shows improved entry in human monocyte-derived dendritic cells.

New Microbiol 2009 Apr;32(2):159-66

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

The invasion and the immunomodulatory effect of a Bordetella pertussis natural deficient strain 00141(PRN-) on human dendritic cells (MDDC) and its in vivo infection ability in a mouse model were evaluated in comparison with the reference B. pertussis strain ATCC 97-97 (18323). The mutant was isolated from a case of pertussis which occurred in a 22-month-old infant with typical symptoms of the disease. The results showed that this natural B. pertussis PRN deficient strain presented higher invasion ability of human MDDC compared to the reference strain. This natural mutant similar to the B. pertussis reference strain had immunomodulatory properties, inducing maturation in the DC phenotype which resulted in the acquisition of potent T cell-activating properties and down-regulated IL-12 production, and secretion of IL-10. The ability of PRN- strain to infect the lungs of CD1 mice was comparable to the reference strain and no difference was observed in the kinetics of clearance. Overall, these results show that the enhanced ability of the PRN- strain to invade/infect MDCC suggest that the PRN antigen may play a role in survival of the microorganism in the host.
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April 2009

IFN-gamma arms human dendritic cells to perform multiple effector functions.

J Immunol 2008 Feb;180(3):1471-81

Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

Dendritic cells (DCs) are central players in immunity and are used in immune-adoptive vaccine protocols in humans. IFN-gamma, mandatory in Th-1 polarization and endowed with regulatory properties, is currently used to condition monocyte-derived DCs (MDDC) in cancer therapy and in clinical trials to treat chronic infectious diseases. We therefore performed a wide analysis of IFN-gamma signaling consequences on MDDC multiple effector functions. IFN-gamma itself induced IL-27p28 expression and survival but did not promote relevant CCR7-driven migration or activated Th-1 cell recruitment capacity in MDDC. Administered in association with classical maturation stimuli such as CD40 or TLR-4 stimulation, IFN-gamma up-regulated IL-27 and IL-12 production, CCR7-driven migration, and activated Th-1 cell recruitment, whereas it decreased IL-10 production and STAT3 phosphorylation. CD38 signaling, which orchestrates migration, survival, and Th-1 polarizing ability of mature MDDC, was involved in IFN-gamma-mediated effects. Thus, IFN-gamma is a modulator of multiple DC effector functions that can be helpful in MDDC-based vaccination protocols. These data also help understand the dual role exerted by this cytokine as both an inducer and a regulator of inflammation and immune response.
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http://dx.doi.org/10.4049/jimmunol.180.3.1471DOI Listing
February 2008

Lipooligosaccharide from Bordetella pertussis induces mature human monocyte-derived dendritic cells and drives a Th2 biased response.

Microbes Infect 2007 Jun 12;9(7):855-63. Epub 2007 Mar 12.

Department of Infectious, Parasitic and Immune-mediated Diseases, Anti-infectious Immunity Unit, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

Bordetella pertussis has a distinctive cell wall lipooligosaccharide (LOS) that is released from the bacterium during bacterial division and killing. LOS directly participates in host-bacterial interactions, in particular influencing the dendritic cells' (DC) immune regulatory ability. We analyze LOS mediated toll-like receptor (TLR) activation and dissect the role played by LOS on human monocyte-derived (MD)DC functions and polarization of the host T cell response. LOS activates TLR4-dependent signaling and induces mature MDDC able to secrete IL-10. LOS-matured MDDC enhance allogeneic presentation and skew T helper (Th) cell polarization towards a Th2 phenotype. LOS protects MDDC from undergoing apoptosis, prolonging their longevity and their functions. Compared to Escherichia coli lipopolysaccharide (LPS), the classical DC maturation stimulus, LOS was a less efficient inducer of TLR4 signaling, MDDC maturation, IL-10 secretion and allogeneic T cell proliferation and it was not able to induce IL-12p70 production in MDDC. However, the MDDC apoptosis protection exerted by LOS and LPS were comparable. In conclusion, LOS treated MDDC are able to perform antigen presentation in a context that promotes licensing of Th2 effectors. Considering these properties, the use of LOS in the formulation of acellular pertussis vaccines to potentiate protective and adjuvant capacity should be taken into consideration.
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http://dx.doi.org/10.1016/j.micinf.2007.03.002DOI Listing
June 2007

Expression and role of phosphatidylcholine-specific phospholipase C in human NK and T lymphocyte subsets.

Eur J Immunol 2006 Dec;36(12):3277-87

Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.

We recently reported evidence of phosphatidylcholine-specific phospholipase C (PC-PLC) involvement in NK cell-mediated cytotoxicity and in lytic granule exocytosis. In the present study, different subpopulations of human PBL were investigated in relation to PC-PLC enzyme expression. While a substantial intracellular amount of PC-PLC was detected in all lymphoid subsets, expression of this enzyme on the outer membrane surface reached high levels only in NK cells, was present at low levels in B lymphocytes and in some TCR gamma/delta T cells and was practically absent in CD4(+) and CD8(+ )T lymphocytes. Moreover, in NK cells two different subpopulations were identified, CD56(dim) PC-PLC(bright) and CD56(bright) PC-PLC(low/-) cells, corresponding to distinct subsets with cytolytic and immunoregulatory functions, respectively. Interestingly, the PC-PLC expression level on the NK membrane surface correlated closely with that of the CD16 receptor, suggesting a possible relationship between enzyme externalization and NK cell maturation. In summary, our results suggest that a high PC-PLC expression on the cell membrane surface of PBL is a peculiarity of NK cytolytic cells, in which the enzyme is apparently involved in the ability of this subset to lyse sensitive target cells.
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http://dx.doi.org/10.1002/eji.200635927DOI Listing
December 2006

Surface layer proteins from Clostridium difficile induce inflammatory and regulatory cytokines in human monocytes and dendritic cells.

Microbes Infect 2006 Sep 8;8(11):2640-6. Epub 2006 Aug 8.

Department of Infectious, Parasitic and Immune-mediated Diseases, Anti-Infectious Immunity Unit, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

Clostridium difficile, an etiological agent of most cases of antibiotic-associated diarrhea, exerts its pathological action mainly by the activity of toxin A and toxin B. Less known is the role that S-layer proteins (SLPs), predominant surface components of the bacterium, may play in pathogenesis. Here, we evaluate the ability of SLPs to modulate the function of human monocytes and dendritic cells (DC) and to induce inflammatory and regulatory cytokines, influencing the natural and adaptive immune response. To this aim, SLPs were extracted from the clinical isolate C253 and characterized for their effects on immune cells. SLPs induced the release of elevated amounts of interleukin (IL)-1beta and IL-6 pro-inflammatory cytokines by resting monocytes, induced maturation of human monocyte-derived DC (MDDC), and enhanced proliferation of allogeneic T cells. C253-SLP-treated MDDC also secreted large amounts of IL-10 and IL-12p70 and induced a mixed Th1/Th2 orientation of immune response in naïve CD4 T cells. In conclusion, C. difficile SLPs may contribute to the pathogenicity of the bacterium by perturbing the fine balance of inflammatory and regulatory cytokines. These data are of interest also in the light of the possible use of SLPs in a multicomponent vaccine against C. difficile infections for high-risk patients.
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http://dx.doi.org/10.1016/j.micinf.2006.07.009DOI Listing
September 2006

Bordetella pertussis inhibition of interleukin-12 (IL-12) p70 in human monocyte-derived dendritic cells blocks IL-12 p35 through adenylate cyclase toxin-dependent cyclic AMP induction.

Infect Immun 2006 May;74(5):2831-8

Istituto Superiore di Sanità, Department of Infectious, Parasitic, and Immune-Mediated Diseases, Rome, Italy.

Bordetella pertussis, the causative agent of whooping cough, possesses an array of virulence factors, including adenylate cyclase toxin (ACT), relevant in the establishment of infection. Here we better define the impact of cyclic AMP (cAMP) intoxication due to the action of ACT on dendritic cell (DC)-driven immune response, by infecting monocyte-derived DC (MDDC) with an ACT-deficient B. pertussis mutant (ACT- 18HS19) or its parental strain (WT18323). Both strains induced MDDC maturation and antigen-presenting cell functions; however, only ACT- 18HS19 infected MDDC-induced production of interleukin-12 (IL-12) p70. Gene expression analysis of the IL-12 cytokine family subunits revealed that both strains induced high levels of p40 (protein chain communal to IL-12 p70 and IL-23) as well as p19, a subunit of IL-23. Conversely only ACT- 18HS19 infection induced consistent transcription of IL-12 p35, a subunit of IL-12 p70. Addition of the cAMP analogous D-butyril-cAMP (D-cAMP) abolished IL-12 p70 production and IL-12 p35 expression in ACT- 18HS19-infected MDDC. ACT- 18HS19 infection induced the expression of the transcription factors interferon regulatory factor 1 (IRF-1) and IRF-8 and of beta interferon, involved in IL-12 p35 regulation, and the expression of these genes was inhibited by D-cAMP addition and in WT18323-infected MDDC. The concomitant expression of IL-12 p70 and IL-23 allowed ACT- 18HS19 to trigger a more pronounced T helper 1 polarization compared to WT18323. The present study suggests that ACT-dependent cAMP induction leads to the inhibition of pathways ultimately leading to IL-12 p35 production, thus representing a mechanism for B. pertussis to escape the host immune response.
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http://dx.doi.org/10.1128/IAI.74.5.2831-2838.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1459734PMC
May 2006

60-kDa heat shock protein of Chlamydia pneumoniae promotes a T helper type 1 immune response through IL-12/IL-23 production in monocyte-derived dendritic cells.

Microbes Infect 2006 Mar 11;8(3):714-20. Epub 2006 Jan 11.

Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena, 299-00161 Rome, Italy.

Infection, in particular by Chlamydia pneumoniae (Cp), has been associated with atherosclerosis and coronary heart disease. Immune reactions to heat shock proteins (HSPs) have been advocated to link infection to atherosclerosis and its acute sequelae based on molecular mimicry with host HSPs. We have here evaluated the role played by recombinant Cp-HSP60 and Cp-HSP10 for their ability to induce maturation of human monocyte-derived dendritic cells (MDDC) and T cell polarization. Cp-HSP60, but not Cp-HSP10, induced a strong MDCC maturation, as assessed by the expression of co-stimulatory molecules and other markers. Secretion of regulatory cytokines and enhancement of antigen presenting ability of mature (m)MDDC toward a clear T helper (Th) 1 pattern were also induced by Cp-HSP60. An analysis of the IL-12 cytokine family demonstrated that Cp-HSP60-matured MDDC were able to express p35 and p40 mRNA subunits to form IL-12, and p19 and p40 subunits to form IL-23. Thus, preferential Th1 polarization of immune response induced by Cp-HSP60-matured MDDC appears to be due to the concomitant expression of IL-12 and IL-23. Our data suggest that Cp-HSP60-matured DC may contribute to T-cell mediated immunopathology of atherosclerosis via a chronic stimulation of Th1 immune responses.
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http://dx.doi.org/10.1016/j.micinf.2005.09.007DOI Listing
March 2006

CD38 orchestrates migration, survival, and Th1 immune response of human mature dendritic cells.

Blood 2006 Mar 17;107(6):2392-9. Epub 2005 Nov 17.

Department of Infectious, Parasitic, and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

CD38, an ectoenzyme and a signaling receptor, is a novel marker of human mature monocyte-derived dendritic cells (MDDCs). The working hypothesis is that CD38 is not only a marker but also contributes to functions specifically gained by MDDCs with maturation. This was tested by assessing the role(s) of CD38 after signaling with agonistic anti-CD38 monoclonal antibodies or by blocking the interactions taking place between CD38 and CD31, its counterreceptor. The results indicate the following: (1) CD38 engagement in MDDCs ensures efficient chemotaxis and transendothelial migration driven by CC chemokine ligand 21 (CCL21); (2) CD38 is laterally associated with the CCL21-specific CC chemokine receptor 7 and with CD83 and CD11b; (3) CD38 localizes in membrane lipid domains; (4) CD38 signaling contributes to support longevity of lipopolysaccharide (LPS)-matured MDDCs after growth factor withdrawal; and (5) IFN-gamma is produced by cocultured T lymphocytes, thus affecting T-helper 1 (Th1) polarization. These data suggest that the localization of CD38 in lipid rafts and its multiple interactions with signaling receptors rule innate and adaptive immune responses by tuning DC migration, survival, and Th1-polarization ability. These findings may lay out the basis to assess the functional role(s) of human CD38 in infections, autoimmune diseases, and neoplastic disorders.
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http://dx.doi.org/10.1182/blood-2005-07-2913DOI Listing
March 2006

CD38 is expressed on human mature monocyte-derived dendritic cells and is functionally involved in CD83 expression and IL-12 induction.

Eur J Immunol 2004 May;34(5):1342-50

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.

Dendritic cell (DC) maturation is characterized by the gain or loss of immunological functions and by expression of distinctive surface receptors. CD38 is an ectoenzyme that catalyzes the synthesis of cyclic ADP ribose (a potent second messenger for Ca(2+) release), as well as a receptor that initiates transmembrane signaling upon engagement with its counter-receptor CD31 or with agonistic monoclonal antibodies. Since CD38 is expressed by resting monocytes, we aimed to monitor CD38 expression during the differentiation of human monocyte-derived DC (MDDC) and to investigate the possibility that CD38 plays a functional role during DC maturation. CD38 is down-modulated during differentiation into immature MDDC and expressed again upon maturation. The extent of CD38 expression is dependent on the stimulus adopted (LPS > IFN-gamma > CD40 cross-linking). Although weak, IFN-gamma consistently induces DC maturation. De novo-synthesized CD38 is enzymatically active, and its expression in mature (m) MDDC is dependent on NF-kappa B activity. However, CD38 is not merely a maturation marker but also mediates signaling in mMDDC, where it maintains its functions as a receptor. Activation via agonistic anti-CD38 mAb induces up-regulation of CD83 expression and IL-12 secretion, whereas disruption of CD38/CD31 interaction inhibits CD83 expression, IL-12 secretion and MDDC-induced allogeneic T cell proliferation.
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http://dx.doi.org/10.1002/eji.200324728DOI Listing
May 2004