Ida A. Casalinuovo

(1-3)-β-D-Glucan vs Galactomannan Antigen in Diagnosing Invasive Fungal Infections (IFIs)

Invasive fungal infections (IFIs) are serious and often life-threatening complications in patients with haematological malignancies. Early diagnosis and the initiation of efficacious antifungal treatments could affect the prognosis of these patients. The detection of (1-3)-β-D-Glucan (BDG) could be a promising non-culture-based, noninvasive tool for IFI analyses in haemato-oncological patients, allowing the diagnosis of the two major IFIs, invasive aspergillosis (IA) and invasive candidiasis (IC), with a single test. The aim of this work was to evaluate and compare the use of the BDG in combination with the galactomannan antigen (GAL) assay in order to exclude or confirm suspected IFIs. Sera from 46 haemato-oncological patients (24 with proven/probable IFI and 22 without IFI symptoms) were evaluated retrospectively for the detection of GAL and BDG. In 24 patients, the serum BDG levels facilitated IFI diagnosis: 18 probable IA, 3 proven IA and 3 IC. In the remaining 22 patients, the BDG level helped exclude IFIs. The BDG was positive earlier than GAL in 5/24 cases [three of probable invasive aspergillosis (IA), one of proven IA and one case of proven invasive candidiasis (IC)] and was positive at the same time as GAL in 19/24 cases; in no case was GAL positive before BDG was. The BDG detection is useful, however, the test has a great limitation because it is a completely manual procedure.

Experimental use of a new surface acoustic wave sensor for the rapid identification of bacteria and yeasts.

Aims:  Use of an electronic nose (zNoseTM) to discriminate between volatile organic molecules delivered during bacterial/fungal growth on agar and in broth media.

Combined effect of fluconazole and thymosin alpha 1 on systemic candidiasis in mice immunosuppressed by morphine treatments.

Treatment of systemic infection with Candida albicans with a combination of an antifungal agent (i.e. fluconazole) and a thymus‐derived immunostimulant (i.e. thymosin α1 (Tal)) in mice immunosuppressed by morphine treatments was investigated. In normal mice, fluconazole given after infection with 106 C. albicans cells was more effective than in mice treated with morphine. Combination treatment with fluconazole and Tal prolonged survival and reduced the fungal burden in the kidneys of immunosuppressed mice. We also investigated the influence of this combined treatment on killing properties of polymorphonuclear leucocytes (PMN) and natural killer (NK) cell activity, inhibited by morphine administrations. Treatment with TQI or fluconazole as single agents promoted a recovery of normal NK cell activity and intracellular killing of C. albicans by PMN, while the combination significantly increased both of these responses, probably through the modulation of lymphokine production. Our data suggest that the additive effect of T α 1 and fluconazole is due lo a direct antifungal action and activation of the immunocompetence.

DIFFERENTIAL EFFECTS OF ACUTE MORPHINE ADMINISTRATIONS ON POLYMORPHONUCLEAR CELL METABOLISM IN VARIOUS MOUSE STRAINS

This paper shows that an acute morphine treatment dose-dependently alters the energetic and oxidative metabolism of polymorphonuclear leukocytes obtained from BALB/c and DBA/2 mice, while phagocytic cells from C57BL/6 were not affected. In sensitive mouse strains, i.e. BALB/c and DBA/2, morphine decreased both ATP concentration and energy charge potential. At the same time, ATP catabolic products, i.e. nucleosides (inosine+adenosine) and oxypurines (hypoxanthine+xanthine+uric acid), significantly increased, indicating an imbalance between energy production and consumption. Morphine treatment also induced malondialdehyde and superoxide anions production in leukocyte cells from sensitive mice. The opiate antagonist naloxone blocked morphine-induced modifications by the lower morphine dose. The same parameters in cells from C57BL/6 mice were not affected. These findings confirm that: i) the phagocytic cells are an important target for the in vivo effects of morphine, and ii) the genotype-dependent variation influences the immunological responsiveness to opiates.

Determination of cytokine co-expression in individual splenic CD4+ and CD8+ T cells from influenza virus-immune mice.

We have studied the patterns of interleukin‐2 (IL‐2), IL‐4 and interferon‐γ (IFN‐γ) co‐expression displayed by individual splenic CD4+ and CD8+ T cells in response to influenza virus immunization. Unseparated spleen cells obtained from mice intraperitoneally (i.p.) injected with A/PR8 (H1N1) influenza virus (PR8) were cultured for 24 hr in the presence of ultraviolet‐inactivated PR8. As controls, cultures of both naive spleen cells stimulated with PR8 or of immune cells lacking the inactivated virus were used. The frequencies of CD4+ and CD8+ T cells expressing IL‐2, IL‐4 and IFN‐γ were determined by three‐colour flow cytometric analysis of fixed and saponin‐permeabilized cells fluorescent‐stained for either CD4 or CD8 surface molecules and for one of the following combinations of two intracellular cytokines: IL‐2/IL‐4, IL‐2/IFN‐γ and IL‐4/IFN‐γ. The results showed that immunization with influenza virus induces in both CD4+ and CD8+ T cells a heterogeneity of cytokine response patterns that do not follow the type 1/type 2 polarized response model, but with substantial differences between the two populations. In fact, the analysis of the phenotypes of virus‐immune CD8+ T cells revealed similar significant proportions of cells either expressing any one of the three cytokines or co‐expressing combinations of them (i.e. IL‐4/IL‐2, IL‐4/IFN‐γ and IL‐2/IFN‐γ), whereas immune CD4+ T cells were seen to express almost exclusively a single cytokine per cell. The observed patterns of cytokine production suggest that influenza virus immunization induces the expression of a type 0 cytokine pattern at both population and single cell levels in CD8+ T cells and exclusively at the population level in CD4+ T cells.

Cytokine pattern secretion by murine spleen cells after inactivated Candida albicans immunization. Effect of cocaine and morphine treatment

Abstract In the present work we have analyzed: i) the effect of heat-inactivated Candida albicans immunization on the cytokine production by murine spleen cells; ii) the effect of a subchronic cocaine and morphine treatment on this production. The treatment with a single dose of inactivated Candida blastospores induced interleukin-2 (IL-2), interferon-γ (IFNγ) and interleukin-4 (IL-4) production at 24 h after in vitro restimulation of splenocytes. In this model, the exposure to morphine (25 mg/kg, 5 days before, during and 5 days after inoculation with the yeast) significant decreased IL-2 and IL-4 levels, while secretion of IFN-γ was unaltered. The same cocaine treatment (10 mg/kg) resulted in unchanged levels of the three cytokines tested. The results showed that non-viable Candida cells of this strain induce a predominant Th0 response. This immune effect is in part impaired only by a subchronic administration of morphine.

Serum thymosin alpha 1 levels in normal and pathological conditions

ABSTRACT Introduction: Thymosin alpha 1 (Ta1) is a natural occurring peptide hormone that is crucial for the maintenance of the organism homeostasis. It has been chemically synthesized and used in diseases where the immune system is hindered or malfunctioning. Areas covered: Many clinical trials investigate the Ta1 effects in patients with cancer, infectious diseases and as a vaccine enhancer. The number of diseases that could benefit from Ta1 treatment is increasing. To date, questions remain about the physiological basal levels of Ta1 and the most effective dose and schedule of treatment. Evidence is growing that diseases characterized by deregulation of immune and/or inflammatory responses are associated with serum levels of Ta1 significantly lower than those of healthy individuals: to date, B hepatitis, psoriatic arthritis, multiple sclerosis and sepsis. The sputum of cystic fibrosis patients contains lower levels of Ta1 than healthy controls. These data are consistent with the role of Ta1 as a regulator of immunity, tolerance and inflammation. Expert opinion: Low serum Ta1 levels are predictive and/or associated with different pathological conditions. In case of Ta1 treatment, it is crucial to know the patient’s baseline serum Ta1 level to establish effective treatment protocols and monitor their effectiveness over time.

Determination of thein vitro activity of fluconazole against yeast strains using HPLC

The aim of this study was to evaluate thein vitro activity of fluconazole against two yeast strains by determining the fluconazole content via high-performance liquid chromatography (HPLC).Candida krusei ATCC 6258 andSaccharomyces cerevisiae were tested for fluconazole susceptibility by dilution tests. Fluconazole measurements by HPLC were performed in cultures of the strains at 6, 12, 24 and 48 h. InC. krusei ATCC 6258 cultures, 88.94% of the fluconazole was recovered at 48 h with respect to the level at time 0 h. The drug exhibited major activity at 12 and 24 h. An increase in cell number and elevated concentration of the drug were detected at 48 h. InS. cerevisiae cultures, the measurements revealed a fluconazole content of approximately 51.25% at 12, 24 and 48 h with respect to the level at time 0 h. However, the percentage of fluconazole detected and the minimal inhibitory concentration (MIC) value determined by agar dilution tests suggest a potential for fluconazole resistance in this susceptible strain. The decreases in fluconazole inC. krusei ATCC 6258 andS. cerevisiae cultures at each time point suggest a mechanism of resistance that is not correlated to efflux pumps.

Efficacy of the Combined Treatment with Fluconazole and Thymosin α 1 Against Candida albicans Infection in Morphine-Treated Mice

Pulmonary infections, viral hepatitis, bacterial endocarditis, bacteremia, abscesses at the site of injection, cellulitis and thrombophlebitis are reported to be common in intravenous drug abusers1 (Table 1). Parallely, there is evidence that has demonstrated that morphine, cocaine and marijuana can affect a variety of immunological functions in humans and in experimental models2,3,4, suggesting that the drug-induced inhibition of natural resistance mechanisms may represent an important risk factor in increasing susceptibility of abusers to opportunistic infections (Table 1).