-
Christian Leli, Elio Cenci,
Angela Cardaccia,
Amedeo Moretti,
Francesco D'Alò,
Rita Pagliochini,
Mariella Barcaccia,
Senia Farinelli,
Simona Vento,
Francesco Bistoni,
Antonella Mencacci
[show abstract]
[hide abstract]
ABSTRACT: Sepsis is a syndrome characterized by a systemic inflammatory response due to severe infection. Early detection of causal agents and appropriate antimicrobial treatment reduce mortality. Conventional microbiological methods often do not provide time critical results for an optimal early management. We used an in-house protocol based on Tween 80 to process 109 positive blood cultures for bacteria and yeast identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), and results were compared to standard reference or automated methods. MALDI-TOF MS correctly identified 91.7% of the isolates. Correct identification was obtained for 57/62 (91.9%) aerobic/facultative anaerobic Gram-positive isolates, 53 (85.5%) at species level, and 4 (6.4%) at the genus level; 32/32 (100%) aerobic/facultative anaerobic Gram-negative isolates, 31 (96.9%) at species level, and 1 (3.1%) at the genus level; 7/7 (100%) obligate anaerobes, all at the genus level; 3/7 (42.8%) fungi, all at genus level. Overall, the median identification time of MALDI-TOF MS vs reference standard methods was significantly shorter: median (interquartile range) 7.1h (4.7-10.2) vs 48.1h (32.5-50.0), p<0.0001. MALDI-TOF MS is a valuable tool for rapid identification of pathogens in septic patients. An in-house protocol based on Tween 80 can be used to process positive blood cultures.
International journal of medical microbiology: IJMM 03/2013; · 2.80 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The persistence of activated T cells in rheumatoid arthritis (RA) synovium may be attributable to increased homing, increased retention or a possible imbalance between cell proliferation and programmed cell death. Induction of apoptosis may represent a potential therapeutic approach. Galactoxylomannan (GalXM) from the opportunistic fungus Cryptococcus neoformans can interact with T cells and induce T-cell apoptosis through the inhibition of CD45 phosphatase activity. The aim of this study was to determine the effect of GalXM on circulating T cells from patients with RA and the underlying mechanisms. GalXM immunomodulating effect on apoptosis and signal transduction pathway involved in IL-17A production was evaluated on T cells. RA T-cell apoptosis, higher than that of control T cells, was further increased by GalXM through induction of caspase-3 activation. Activated T cells expressing the CD45RO molecule and producing IL-17A were the main target of GalXM-induced apoptosis. GalXM induced consistent impairment of IL-17A production and inhibition of STAT3, which was hyperactivated in RA. In conclusion, GalXM triggered apoptosis of activated memory T cells and interfered with IL-17A production in RA. These data suggest therapeutic targeting of deleterious Th17 cells in RA and other autoimmune diseases.
PLoS ONE 01/2013; 8(1):e53336. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Infective endocarditis (IE) is a life-threatening condition in which identification of the causative pathogen is pivotal for subsequent effective therapy. Blood culture (BC) is considered the gold standard for microbial diagnosis of IE, but in up to one-third of patients, it can be negative due to a number of possible factors, e.g., prior antimicrobial therapy, fastidious microorganisms, or low bacterial load. The aim of this study was to assess the diagnostic utility of the commercially available PCR based system SeptiFast (SF) for microbial diagnosis of IE from clinical blood specimens. The results indicate that, this test, in combination with BC, could represent a valuable tool for microbial diagnosis of IE. Reduction of SF detection limit set for coagulase-negative staphylococci and streptococci could improve the sensitivity of the test.
Journal of Medical Microbiology 03/2012; 61(Pt 6):881-3. · 2.50 Impact Factor
-
Leonella Pasqualini,
Antonella Mencacci,
Christian Leli,
Paolo Montagna,
Angela Cardaccia, Elio Cenci,
Ines Montecarlo,
Matteo Pirro,
Francesco di Filippo,
Emma Cistaro,
Giuseppe Schillaci,
Francesco Bistoni,
Elmo Mannarino
[show abstract]
[hide abstract]
ABSTRACT: Early identification of causative pathogen in sepsis patients is pivotal to improve clinical outcome. SeptiFast (SF), a commercially available system for molecular diagnosis of sepsis based on PCR, has been mostly used in patients hospitalized in hematology and intensive care units. We evaluated the diagnostic accuracy and clinical usefulness of SF, compared to blood culture (BC), in 391 patients with suspected sepsis, hospitalized in a department of internal medicine. A causative pathogen was identified in 85 patients (22%). Sixty pathogens were detected by SF and 57 by BC. No significant differences were found between the two methods in the rates of pathogen detection (P = 0.74), even after excluding 9 pathogens which were isolated by BC and were not included in the SF master list (P = 0.096). The combination of SF and BC significantly improved the diagnostic yield in comparison to BC alone (P < 0.001). Compared to BC, SF showed a significantly lower contamination rate (0 versus 19 cases; P < 0.001) with a higher specificity for pathogen identification (1.00, 95% confidence interval [CI] of 0.99 to 1.00, versus 0.94, 95% CI of 0.90 to 0.96; P = 0.005) and a higher positive predictive value (1.00, 95% CI of 1.00 to 0.92%, versus 0.75, 95% CI of 0.63 to 0.83; P = 0.005). In the subgroup of patients (n = 191) who had been receiving antibiotic treatment for ≥24 h, SF identified more pathogens (16 versus 6; P = 0.049) compared to BC. These results suggest that, in patients with suspected sepsis, hospitalized in an internal medicine ward, SF could be a highly valuable adjunct to conventional BC, particularly in patients under antibiotic treatment.
Journal of clinical microbiology 02/2012; 50(4):1285-8. · 4.16 Impact Factor
-
Elena Gabrielli,
Eva Pericolini, Elio Cenci,
Claudia Monari,
Walter Magliani,
Tecla Ciociola,
Stefania Conti,
Rita Gatti,
Francesco Bistoni,
Luciano Polonelli,
Anna Vecchiarelli
[show abstract]
[hide abstract]
ABSTRACT: We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc) of human IgG(1), is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules.
PLoS ONE 01/2012; 7(8):e43972. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Early detection of aetiological agents is pivotal for adequate therapy for bacterial infections. Although culture is still considered the mainstay for laboratory diagnosis, it often lacks sensitivity, especially in patients already treated with antibiotics. The present study investigated the potential clinical utility of the commercial real-time-PCR-based system SeptiFast (SF), originally intended for diagnosis of sepsis from blood specimens, in the aetiological diagnosis of other bacterial infections, in patients undergoing antibiotic therapy. A total of 53 non-blood specimens were analysed for microbial pathogen detection by conventional culture and with SF real-time PCR: 19 (35.8%) synovial fluids, 9 (17.0%) cardiac valve tissues and 25 (47.2%) purulent exudates from various body sites. Overall, the number of specimens positive for a pathogen by SF (26/53; 49.1%) was significantly greater (P=0.001) than that of specimens positive by culture (10/53; 18.9%). In particular, SF was superior to culture for pathogen detection in cardiac valve tissues and synovial fluids. The analysis of concordance showed a fair agreement between the two methods (kappa value=0.314; 95% confidence interval=0.531-0.097). Even with the limitation of the low number of specimens, this study confirmed the great potential of diagnosing bacterial infections by a molecular approach, and indicates that the real-time PCR SF system can be used for specimens other than blood, from patients undergoing antibiotic treatment.
Journal of Medical Microbiology 08/2011; 60(Pt 12):1774-8. · 2.50 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cryptococcus neoformans is an opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised individuals and occasionally in those with no known immune impairment. The fungus is endowed with several virulence factors, including capsular polysaccharides that play a key role in virulence. The capsule is composed of 90-95% glucuronoxylomannan (GXM), 5-8% galactoxylomannan (GalXM) and <1% mannoproteins. Capsular polysaccharides are shed into tissue where they produce many deleterious effects. Since GalXM has a smaller molecular mass, the molar concentration of GalXM in polysaccharide that is shed could exceed that of GXM in C. neoformans exopolysaccharides. Moreover, GalXM exhibits a number of unusual biologic properties both in vitro and in vivo. Here, we summarize the principal immunomodulatory effects of GalXM described during the last 20 years, particularly the mechanisms leading to induction of apoptosis in T lymphocytes, B lymphocytes and macrophages. Since the capacity of GalXM to induce widespread immune suppression is believed to contribute to the virulence of C. neoformans, this property might be exploited therapeutically to dampen the aberrant activation of immune cells during autoimmune disorders.
Immunotherapy 08/2011; 3(8):997-1005. · 1.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Breastfeeding has a major impact on CMV epidemiology. Postnatal CMV reactivation's incidence during lactation is nearby the maternal seroprevalence. Although perinatal CMV infection has practically no consequences in term newborn, it may cause, in some cases, a severe symptomatic disease in preterm newborns. The aims of the present study are to evaluate the rate and clinical expression of CMV infection breast milk transmitted in preterm infants and to check the safety of the freezing treated breast milk.
The study included fifty-seven preterm infants and their CMV seropositive mothers. Fresh breast milk samples have been collected from 1(st) to 9(th) postpartum week. Both fresh breast milk and 72, 96, 120 hours frozen samples have been examined, checking the presence of CMV; urine samples have been tested too.
70.2% of tested mothers showed reactivation of the infection, and CMV-positive breast milk during the six weeks postpartum has been found. However, only one infant was infected by CMV, developing hepatic affection concomitantly with a multi-system involvement, as shown CMV DNA detection in urine, saliva, blood, gastric aspirate, and stools.
Freezing breast milk at -20°C and pasteurization may respectively reduce or eliminate the viral load.
Italian Journal of Pediatrics 01/2011; 37:6.
-
[show abstract]
[hide abstract]
ABSTRACT: Abstract Background Breastfeeding has a major impact on CMV epidemiology. Postnatal CMV reactivation's incidence during lactation is nearby the maternal seroprevalence. Although perinatal CMV infection has practically no consequences in term newborn, it may cause, in some cases, a severe symptomatic disease in preterm newborns. The aims of the present study are to evaluate the rate and clinical expression of CMV infection breast milk transmitted in preterm infants and to check the safety of the freezing treated breast milk. Methods The study included fifty-seven preterm infants and their CMV seropositive mothers. Fresh breast milk samples have been collected from 1st to 9th postpartum week. Both fresh breast milk and 72, 96, 120 hours frozen samples have been examined, checking the presence of CMV; urine samples have been tested too. Results 70.2% of tested mothers showed reactivation of the infection, and CMV-positive breast milk during the six weeks postpartum has been found. However, only one infant was infected by CMV, developing hepatic affection concomitantly with a multi-system involvement, as shown CMV DNA detection in urine, saliva, blood, gastric aspirate, and stools. Conclusion Freezing breast milk at -20°C and pasteurization may respectively reduce or eliminate the viral load.
The Italian Journal of Pediatrics. 01/2011;
-
[show abstract]
[hide abstract]
ABSTRACT: Previously, we reported that Galactoxylomannan (GalXM) activates the extrinsic and intrinsic apoptotic pathways through an interaction with the glycoreceptors on T cells. In this study we establish the role of the glycoreceptor CD45 in GalXM-induced T cell apoptosis, using CD45(+/+) and CD45(-/-) cell lines, derived from BW5147 murine T cell lymphoma. Our results show that whereas CD45 expression is not required for GalXM association by the cells, it is essential for apoptosis induction. In CD45(+/+) cells, CD45 triggering by GalXM reduces the activation of Lck, ZAP70 and Erk1/2. Conversely, in CD45(-/-) cells, Lck was hyperphosphorylated and did not show any modulation after GalXM stimulation. On the whole, our findings provide evidence that the negative regulation of Lck activation occurs via CD45 engagement. This appears to be related to the capacity of GalXM to antagonize T cell activation and induce T cell death. Overall this mechanism may be responsible for the immune paralysis that follows GalXM administration and could explain the powerful immunosuppression that accompanies cryptococcosis.
PLoS ONE 01/2010; 5(9):e12720. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: For many years the development of new azole antifungals has been quite empirically based. More recently, the publication of the crystal structure of CYP51 of Mycobacterium tuberculosis (MT-CYP51) provided new opportunities to rationalize the knowledge about antifungal action of this class of compounds. Recent studies reported that a 'channel 2 opened' conformation of the enzyme could better explain the interaction with ketoconazole (KTZ)-like drugs. Conformational changes were made on our model of Candida albicans CYP51 (CA-CYP51) previously reported and docking experiments were performed. The results allowed new KTZ analogues to be designed, by predicting that the 1,4-benzoxazine moiety could replace the KTZ aryl-piperazinyl chain. The synthesis of derivatives 12 and 13 was planned. The in vitro antifungal activity was evaluated against different Candida species and low and high capsulated strains of Cryptococcus neoformans. Since the in vitro activity do not necessarily correlate with the in vivo antifungal activity the newly synthesized compounds were also tested in a murine model of systemic C. albicans infection. The therapeutic effect was evaluated in terms of animal survival and of fungal growth in the kidneys, the target organ in systemic candidiasis.
Bioorganic & medicinal chemistry 07/2009; 17(11):3838-46. · 2.82 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The major virulence factor of Cryptococcus neoformans is its capsular polysaccharide, which is also released into tissues. The shed polysaccharide is composed of glucuronoxylomannan, galactoxylomannan (GalXM), and mannoproteins. In a previous study, we demonstrated a direct interaction of purified soluble GalXM with T cells that induced their apoptosis. In this study, we focus on the mechanisms involved in the apoptotic effect of GalXM. In our experimental system, we analyzed the effect of GalXM on purified human T cells and Jurkat cells, a T cell line routinely used for apoptotic studies. Our results reveal that GalXM activates the extrinsic and intrinsic apoptotic pathways through the cleavage and recruitment of caspase-8. Caspase-8 elicits the downstream executioner caspase-3, caspase-6, and caspase-7 both directly and indirectly, via Bid cleavage and caspase-9 activation. These effects appeared to be primarily mediated by the interaction of GalXM with the glycoreceptors, which differed in human T and Jurkat cells. CD45 was primarily involved in Jurkat cells apoptosis while CD7 and CD43 mediated human T cell apoptosis. Our results highlight a new mechanism by which a microbial product can contribute to virulence through direct interaction with T cell glycoreceptors, thereby triggering lymphocyte apoptosis.
The Journal of Immunology 06/2009; 182(10):6003-10. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Dectin-1 is a pattern recognition receptor found on monocytes and dendritic cells (DC) able to recognize beta-1,3 and beta-1,6 glucans. It is thought to act via the spleen tyrosine kinase (Syk) to initiate immune response against infectious agents such as Candida albicans, one of the leading causes of invasive fungal disease in immunocompromised individuals. This study addresses the importance of this receptor in the context of human DC response to C. albicans. Upon blockage of Dectin-1, immature DC are less able than untreated cells to bind, phagocytose, and kill C. albicans via oxidative burst. In fact, a consistent decrease in superoxide anion, but not nitric oxide production, was manifested when the Syk pathway was inhibited. C. albicans-induced cytokine production via Dectin-1 recognition is mediated by the Syk activation pathway. Indeed, specific Syk inhibition significantly suppressed the production of IL-12, IL-6, and TNF-alpha. Finally, we observed that Dectin-1 engagement was also involved in DC maturation and subsequent lymphocyte activation. Collectively, these findings identify Dectin-1 as a key receptor influencing critical biological functions of DC in response to C. albicans leading to T cells response alteration. These effects are largely, though not completely, mediated by Syk activation.
Microbes and Infection 05/2009; 11(6-7):661-70. · 3.10 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The glucocorticoid-induced TNFR-related (GITR) protein is a member of the tumor necrosis factor receptor superfamily influencing natural and acquired immune response. GITR is activated by its ligand, GITRL, mainly expressed on antigen presenting cells. Previously, we demonstrated that GITR plays a role in regulating immune response to Candida albicans. Here we analyzed whether GITRL-GITR interaction influences the recognition of C. albicans by regulating the expression of pattern recognition receptors on splenic dendritic cells. Our report demonstrates that under physiological conditions and during candidiasis the GITRL-GITR system affects TLR-2 and TLR-4 expression on DC. These changes correlate with decrease in: MyD88 activation; CD80 and CD40 expression on DC; T cell activation response, including CD28 expression, IL-2 and IFN-gamma production. Our results point out that, during fungal infection, GITRL-GITR interaction modulates TLR-4 and TLR-2 expression, thereby altering the antigen presentation process, and suggesting a role of GITRL-GITR interaction in resistance against infectious diseases.
Cellular Immunology 04/2009; 257(1-2):13-22. · 1.97 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It has been documented that, independently from the specificity of the native antibody (Ab) for a given antigen (Ag), complementarity determining regions (CDR)-related peptides may display differential antimicrobial, antiviral and antitumor activities.
In this study we demonstrate that a synthetic peptide with sequence identical to V(H)CDR3 of a mouse monoclonal Ab (mAb) specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. Significantly, V(H)CDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties.
These results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscent of regulatory peptides of innate immunity.
PLoS ONE 01/2009; 4(12):e8187. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In this study, we analyzed the possibility that Indinavir (IDV), a well-known protease inhibitor (PI) used in highly active antiretroviral therapy, could affect immune response against the opportunistic fungus Cryptococcus neoformans. In particular, the quality of dendritic cell (DC) response was analyzed. The results reported here show that IDV treatment induces an expansion of DC with CD8alpha phenotype in spleens of infected hosts. Splenic CD11c+ DC expressed elevated costimulatory molecules such as CD40 and CD80, showed an increased expression of mRNA for proinflammatory cytokines, and secreted abundant IL-12. Integration of all aforementioned regulatory effects results in development of an efficient, T cell-protective response that reflects a consistent reduction in fungus colonization at a cerebral level. These results could help to elucidate the immunoregulatory activity of PI and point out the beneficial effects of IDV in regulating DC functions and antifungal activity. Therefore, although new PI are being introduced in the clinical setting, nevertheless, given its low cost and proven efficacy, IDV could still be considered a potential key compound in the treatment of HIV in resource-limited settings.
Journal of Leukocyte Biology 06/2008; 83(5):1286-94. · 4.99 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Tipranavir (TPV) is a non-peptidic protease inhibitor (PI) that represents one of the latest options approved in the salvage setting for HIV-infected multi-drug resistant patients. In this study, we explored whether TPV affects virulence of opportunistic fungi such as Cryptococcus neoformans and Candida albicans.
C. neoformans and C. albicans were cocultured in the presence or absence of TPV for various time periods. Subsequently, growth inhibition, phospholipases, proteases and capsule size were examined. In selected in vivo experiments, TPV was administered in immunocompetent and immunosuppressed mice. Survival rate and colony forming units from organs were evaluated in mice systemically challenged with C. neoformans or C. albicans.
Indeed, when cultured in the presence of TPV, both fungi showed significant reduction in protease and phospholipase production, but TPV showed an opposite effect on the major virulence factors of C. neoformans and C. albicans by inhibiting capsule while promoting mycelial transition, respectively. TPV impaired in vitro growth of C. neoformans, but not of C. albicans. Moreover, TPV-treated C. neoformans, but not C. albicans, resulted more susceptible to killing by human neutrophils. Finally, TPV showed a therapeutic effect in experimental systemic cryptococcosis, as evaluated by reduced fungal burden in brain and liver of immunocompetent and immunodepressed mice.
These new data indicate that TPV could act in multiple ways by diversifying its effects on various opportunistic pathogenic fungi.
The Journal of infection 02/2008; 56(1):58-64. · 4.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The PMT gene family in Candida albicans encodes five isoforms of the protein mannosyltransferases that initiate O-mannosylation of secretory proteins. Mutations at the Pmt level have been associated with differences in pathogenicity, e.g. in contrast to pmt5/pmt5, pmt2/PMT2 mutants showed poor virulence. Our objective was to determine whether these differences were related to the capacity of pmt2/PMT2 and pmt5/pmt5 to (i) express differences in selected virulence factors, and (ii) stimulate the natural immune system. The results show that pmt mutants (i) form hyphae in serum, (ii) show defective production of proteases but not of phospholipases with respect to the parental strain, (iii) undergo mycelial transition in the kidneys of hematogenously infected animals, (iv) are phagocytosed and killed by macrophages similar to the parental strain, although neutrophils are unable to destroy pmt5/pmt5, (v) engage TLR4 and stimulate MyD88 leading to NF-kappaB activation, and (vi) stimulate cytokine production by macrophages. Collectively our findings suggest that the defect in protein O-mannosylation in C. albicans cause attenuation of the virulence although the antigenic factors that retain the capacity to stimulate an efficient immune response are preserved.
Medical Mycology 01/2008; 45(8):709-19. · 2.46 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We report a case of pulmonary cryptococcosis in a 21-year-old Italian female smoker with no apparent immune disorder. In this study we demonstrated that: (i) patient's neutrophils and monocytes manifested a significant reduction of killing activity against Cryptococcus neoformans as well as Candida albicans; (ii) the suppression was more pronounced in monocytes than in neutrophils; (iii) neutrophils and monocytes showed a significant impairment of TNF-alpha, IL-1beta, and nitric oxide production. These results suggest that the apparent immunocompetent host with pulmonary cryptococcosis could have specific defects in natural immune system mechanisms.
The Journal of infection 02/2007; 54(1):e5-8. · 4.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A multidrug-resistant Pseudomonas aeruginosa (r-Pa) was isolated from a lethal case of sepsis in a bone marrow transplant recipient. Genotypic analysis of P. aeruginosa isolates demonstrated that sepsis was secondary to gut colonization. The interactions between r-Pa and patient's neutrophils were studied. The results indicate that: (1) the patient's neutrophil killing activity and nitric oxide production against r-Pa or drug sensitive P. aeruginosa (s-Pa) were profoundly impaired; (2) r-Pa cells, but not s-Pa cells or their filtered culture supernatants, induced necrosis of healthy donor neutrophils. Neutrophil necrosis emerges as a remarkable event in the pathogenesis of P. aeruginosa sepsis.
The Journal of infection 01/2007; 53(6):e259-64. · 4.13 Impact Factor
