Silvia Guglietta

Dysfunctional CFTR Alters the Bactericidal Activity of Human Macrophages against Pseudomonas aeruginosa

Chronic inflammation of the lung, as a consequence of persistent bacterial infections by several opportunistic pathogens represents the main cause of mortality and morbidity in cystic fibrosis (CF) patients. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes, additionally, mature macrophages from different tissues, although having a number of common activities, exhibit variation in some molecular and cellular functions. In order to highlight possible intrinsic macrophage defects due to CFTR dysfunction, we have focused our attention on in vitro differentiated macrophages from human peripheral blood monocytes. Here we report on the contribution of CFTR in the bactericidal activity against Pseudomonas aeruginosa of monocyte derived human macrophages. At first, by real time PCR, immunofluorescence and patch clamp recordings we demonstrated that CFTR is expressed and is mainly localized to surface plasma membranes of human monocyte derived macrophages (MDM) where it acts as a cAMP-dependent chloride channel. Next, we evaluated the bactericidal activity of P. aeruginosa infected macrophages from healthy donors and CF patients by antibiotic protection assays. Our results demonstrate that control and CF macrophages do not differ in the phagocytic activity when infected with P. aeruginosa. Rather, although a reduction of intracellular live bacteria was detected in both non-CF and CF cells, the percentage of surviving bacteria was significantly higher in CF cells. These findings further support the role of CFTR in the fundamental functions of innate immune cells including eradication of bacterial infections by macrophages.

A Combination of Antigenic Regions of Toxoplasma gondii Microneme Proteins Induces Protective Immunity against Oral Infection with Parasite Cysts

Infection with Toxoplasma gondii causes morbidity and mortality in congenitally infected and immunocompromised individuals. Both humoral and cell-mediated immunity are involved in host resistance to invasion of the parasite. Among putative vaccine candidates, the T. gondii microneme proteins appear to be promising, because they are responsible for the invasion process. The present work focused on studying the immunogenicity of microneme proteins in infected individuals and in a mouse model of chronic toxoplasmosis. We identified 5 distinct antigenic regions within MIC2, MIC4, MIC2-associated protein, and apical membrane antigen 1 gene products, which were recognized by (1) T cells from both adults with acquired infection and children with congenital infection and (2) antibodies from all patients. Finally, we demonstrated that DNA immunization with microneme fragments elicited effective protection in mice (84% reduction in brain-cyst burden), suggesting that a combination of these antigenic regions should be considered in the design of potential vaccines against toxoplasmosis.

Positive selection of cytotoxic T lymphocyte escape variants during acute hepatitis C virus infection.

Cellular immune responses are induced during hepatitis C virus (HCV) infection and acute‐phase CD8+ T cells are supposed to play an important role in controlling viral replication. In chimpanzees, failure of CD8+ T cells to control HCV replication has been associated with acquisition of mutations in MHC class I‐restricted epitopes. In humans, although selection of escape mutations in an immunodominant CTL epitope has been recently described, the overall impact of immune escape during acute HCV infection is unclear. Here, by performing an in depth analysis of the relationship between early cellular immune responses and viral evolution in a chronically evolving HCV acutely infected individual, we demonstrate: (i) the presence of a potent and focused CD8+ T cell response against a novel epitope in the NS3 protein, (ii) the elimination of the quasi‐species harboring the original amino acid sequence within this epitope, and (iii) the selection for a virus population bearing amino acid changes at a single residue within the cytotoxic T cell epitope that strongly diminished T cell recognition. These results support the view that acute‐phase CD8+ T cell responses exert a biologically relevant pressure on HCV replication and that viruses escaping this host response could have a significant survival advantage.

Long sequence duplications, repeats, and palindromes in HIV-1 gp120: Length variation in V4 as the product of misalignment mechanism

We have shown that indels in gp120 V4 are associated to the presence of duplicated and palindromic sequences, suggesting that they may be produced by strand-slippage misalignment mechanism. Indels in V4 involved region-specific duplications 9 to 15 bp long, and repeats of various lengths, associated to trinucleotides AAT. No duplications were found in V3 and C3. The frequency of palindromic sequences in individual genes was found to be significantly higher in gp120 (p < or = 3.00E-7), and significantly lower in Tat (p < or = 9.00E-7) than the average frequency calculated over the full genome. The finding of elements of misalignment in association with indels in V4 suggests that these mutations may occur in proviral DNA after integration of HIV into the host genome. It also implies that occurrence of large indels in gp120 is not random but is directed by the presence and distribution of elements of misalignment in the HIV genome.

Impact of viral selected mutations on T cell mediated immunity in chronically evolving and self limiting acute HCV infection

The ability of HCV to mutate in response to cytotoxic T lymphocyte (CTL) pressure is increasingly recognized, but the influence of such a mechanism in viral persistence and final disease outcome has not been ascertained. In this study, we performed a detailed longitudinal analysis of cell mediated immunity and HCV evolution in two self limiting and two chronically evolving HCV acutely infected patients, one of whom transiently controlled viremia. Amino acid mutations in immunodominant regions of viruses were observed in all patients, although they conferred viral escape from CTL responses only in chronically infected individuals. Resurgence of viremia coincided with the replacement of the original virus quasispecies with mutant viruses that had escaped recognition by primary CD8(+) T cell responses and infection persisted in the presence of variant viruses which were less efficiently recognized by preexisting and de novo induced T cell responses.

Length variation in HIV-1 gp120 as the product of DNA misalignment mechanism

Results Indels in V4 involved always insertion and deletion of duplicated nucleotide segments, and AAT repeats, and were associated to the presence of palindromic sequences. No duplications were detected in V3 and C3. Palindromic sequences occurred with similar frequencies in V3, C3 and V4; the frequency of palindromes in individual genes was found to be significantly higher in structural (gp120, p ≤ 3.00E-7) and significantly lower in regulatory (Tat, p ≤ 9.00E-7) genes, as compared to the average frequency calculated over the full genome.

P20-20 LB. Indels in V4 share sequence features with insertions and deletions during somatic hypermutation of immunoglobulins

Background We have shown that gp120 V4 is characterized by indels multiples of three base pairs and with no generation of stop codons. Similar insertions and deletions have also been reported during somatic hypermutation (SHM) of immunoglobulins. In this study, we have compared sequence patterns associated to indels in V4 to those reported in immunoglobulins. Our aims were i) to assess similarities between indels in V4 and SHM; ii) to assess whether sequence features of indels in V4 could also be identified in V3 and C3.