Demetrio Delfino

Identification and cloning of a cryptococcal deacetylase that produces protective immune responses

ABSTRACT Cell-mediated immunity plays a crucial role in host defenses against Cryptococcus (Filobasidiella) neoformans. Therefore, the identification of cryptococcal antigens capable of producing T-cell-mediated responses, such as delayed-type hypersensitivity (DTH) reactions, may be useful in the development of immune-based strategies to control cryptococcosis. In order to characterize DTH-producing antigens, culture supernatants from the unencapsulated Cap-67 strain were separated by anion-exchange chromatography. After further fractionation by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a purified protein with an apparent molecular mass of 25 kDa was found to produce DTH, as evidenced by increased footpad swelling in mice immunized with culture supernatants, relative to unimmunized mice. The 20-amino-acid N-terminal sequence of the 25-kDa protein was used to search data of the C. neoformans Genome Project. Based on the genomic DNA sequence, a DNA probe was used to screen a λ cDNA library prepared from strain B3501. Clones were isolated containing the full-length gene (d25), which showed homology with a number of polysaccharide deacetylases from fungi and bacteria. The recombinant d25 protein expressed in Escherichia coli was similar to the natural one in DTH-producing activity. Moreover, immunization with either the natural or the recombinant protein prolonged survival and decreased fungal burden in mice challenged with the highly virulent C. neoformans strain H99. In conclusion, we have described the first cryptococcal gene whose product, a 25-kDa extracellular polysaccharide deacetylase, has been shown to induce protective immunity responses.

Molecular characterization of Italian Candida parapsilosis isolates reveals the cryptic presence of the newly described species Candida orthopsilosis in blood cultures from newborns

The authors report the molecular characterization of Candida parapsilosis isolates recovered from the blood and venous central catheter tips of patients admitted to different care units of the Polyclinic Hospital, University of Messina, Italy. Among 97 presumed C. parapsilosis isolates examined, 94 were identified as C. parapsilosis sensu stricto and the remaining 3 isolates were found to belong to the cryptic species Candida orthopsilosis which was recovered only from blood cultures of neonates (<30 days old) born prematurely. No C. metapsilosis was found in this study. This study emphasizes the role of C. parapsilosis as an important nosocomial pathogen, and it also describes, for the first time, the occurrence of C. orthopsilosis in newborns.

Microsatellite-based genotyping of Candida parapsilosis sensu stricto isolates reveals dominance and persistence of a particular epidemiological clone among neonatal intensive care unit patients.

In this study, using multilocus microsatellite analysis, we report the genetic characterization of 27 Candida parapsilosis isolates recovered in two different periods of time (2007-2009 and 2011-2012) from infants hospitalized in the neonatal intensive care unit of a hospital in Messina, Italy. The results revealed the persistence and dominance of a particular infectious genotype among NICU patients and highlight the power of the used microsatellite markers in clarifying epidemiologic associations, detect micro-evolutionary variations and facilitating the recognition of outbreaks.

Induction of tumor necrosis factor α by Leishmania infantum in murine macrophages from different inbred mice strains

The present study was undertaken to determine whether the viscerotropic species, Leishmania infantum, endemic in Italy, could induce tumor necrosis factor alpha (TNF alpha) in murine macrophages. Genetically susceptible (Lshs) and resistant (Lshr) mice were used in the attempt to correlate TNF alpha production with the ability to control parasite growth and replication. Resident peritoneal macrophages of C3H/HeN, DBA/2, CBA (Lshr), C57BL/10 and BALB/c (Lshs) mice were infected in vitro with promastigotes at a parasite to cell ratio of 8:1. No significant differences in the percentages of infected peritoneal cells of Lshs versus Lshr mice were observed until 72 h of in vitro culture. On the contrary, Kupffer cells from Lshr mice inhibited Leishmania replication. Peritoneal macrophages of resistant mice produced significantly higher amounts of TNF alpha as compared to susceptible mice. TNF alpha production of both resistant and susceptible mice peaked at about 5 h after the challenge with the parasite. No TNF alpha was found in supernatants of infected Kupffer cells from all the strains tested. The ability of macrophages from susceptible or resistant mice strains to produce TNF alpha after challenge with Leishmania infantum does not seem related to their capacity to control parasite replication in vitro.

On a Fatal Case of Candida krusei Pleural Empyema in a Pregnant Woman with Spontaneous Esophagus Perforation

IntroductionCandida empyema thoracis can be a consequence of operation, gastropleural fistula, and esophageal perforation. Case report and review of the literature. A fatal case of a 45-year-old pregnant woman with Candida krusei empyema thoracis secondary to spontaneous esophagus perforation associated with candida esophagitis is reported. The case is contextualized among similar cases found through a PubMed search.ConclusionsA suspect of esophagus rupture should arise in the presence of a fungal empyema. Candida esophagitis should be always considered as a possible cause or con-cause of esophagus rupture. An empirical systemic antimycotic therapy should always be considered in an ideal multidisciplinary approach to the management of patients with esophagus rupture.

Induction of interleukin 1α in murine macrophages infected in vitro with different species and strains of Leishmania

It is now generally agreed that several cytokines released by immunocompetent cells such as macrophages play a crucial role in the outcome of infections caused by protozoa belonging to the genus Leishmania. In particular, tumor necrosis factor (TNF) induction during the course of cutaneous leishmaniasis has been related to resistance to L. major infection in mice. However, the role played by interleukin 1 (IL-1) in the host response to leishmaniasis has yet to be completely elucidated. The aim of this work was to study whether different species and strains of Leishmania could induce IL-1 alpha in murine macrophages in vitro. Resident peritoneal macrophages of BALB/c and C3H/HeN mice were infected with L. donovani, L. major, or different strains of L. infantum. It was found that L. donovani did not induce IL-1 alpha in macrophages from either mice strain. Infection with L. major or with three out of six strains of L. infantum induced consistent amounts of IL-1 alpha, but only in macrophages from genetically resistant C3H/HeN mice. No relationship was found between the rate of infection of macrophages and the amount of IL-1 alpha detected in the supernatants of infected macrophages. Data obtained confirm that the release of IL-1 alpha by murine macrophages infected in vitro with Leishmania is influenced by the genetic background of the cells as well as by the parasite species.

Inhibition of normal rat macrophage functions by soluble tumor products

SummaryThe phagocytic and chemotactic activities of normal rat peritoneal macrophages were inhibited by sera from tumor-bearing rats (TBR) and 3 M KCl extracts of tumor mass. However, sera from Corynebacterium parvum- or Listeria monocytogenes-treated TBR did not inhibit phagocytosis. On the other hand, sera from C. parvum-treated, but not from L. monocytogenes-treated TBR still inhibited the chemotactic response of the normal macrophages. Furthermore, 3 M KCl extracts of tumors from C. parvum-treated TBR did not inhibit phagocytosis and chemotactic response of the same cells. Similar results were obtained with extracts of tumor masses from L. monocytogenes-treated rats. It is suggested that treatment with bacterial immunomodulators can influence the release from neoplastic cells of soluble products influencing normal macrophage functions.

Selective Depression of Phagocytes Intracellular Killing Activity

Valid resistance to infections is the result of a number of complex interactions between infectious agents and host’s immune response. Virulence of microorganisms, genetic factors, host’s immune mechanisms may in different ways influence the infectious agent host relationship. Phagocytes, including polymorphonucleates and macrophages, are the final effector cells in the elimination of a variety of bacteria and fungi.l

Neonatal liver abscesses associated with candidemia: three cases and review of literature.

Abstract Background: Our aim was to identify risk factors for the development of neonatal Candida liver abscess and to find useful information to better manage this potentially fatal complication. Methods: A computerized search was conducted using PubMed. Overall, three articles describing the history of seven infants were finally considered. The characteristics of these seven cases were analyzed together with those of three new cases that we treated in the recent past. Results: All the neonates were premature. Previous antibiotic use was reported in all the cases, umbilical venous catheterization in 9/10 and total parenteral nutrition in 8/10. Candida albicans was isolated in 9/10. All the patients presented with aspecific signs of sepsis. Liver abscesses were described as “microabscesses” or “miliary abscesses” in three cases, as solitary lesion in two cases. In one case two lesions and in one four lesions were reported. Three infants died. Conclusions: Liver ultrasonography should be performed in all the neonates with signs of sepsis, especially in the presence of candidemia and/or hepatomegaly and/or significant change in liver enzymes. Umbilical venous catheter should be removed, and peripheral IV access should be used until there is documented clearance from the blood with three or more negative blood cultures.

Multilocus microsatellite analysis of European and African Candida glabrata isolates

This study aimed to elucidate the genetic relatedness and epidemiology of 127 clinical and environmental Candida glabrata isolates from Europe and Africa using multilocus microsatellite analysis. Each isolate was first identified using phenotypic and molecular methods and subsequently, six unlinked microsatellite loci were analyzed using automated fluorescent genotyping. Genetic relationships were estimated using the minimum-spanning tree (MStree) method. Microsatellite analyses revealed the existence of 47 different genotypes. The fungal population showed an irregular distribution owing to the over-representation of genetically different infectious haplotypes. The most common genotype was MG-9, which was frequently found in both European and African isolates. In conclusion, the data reported here emphasize the role of specific C. glabrata genotypes in human infections for at least some decades and highlight the widespread distribution of some isolates, which seem to be more able to cause disease than others.