Ultra-High Mass Resolution MALDI Imaging Mass Spectrometry of Proteins and Metabolites in a Mouse Model of Glioblastoma.

MALDI mass spectrometry imaging is able to simultaneously determine the spatial distribution of hundreds of molecules directly from tissue sections, without labeling and without prior knowledge. Ultra-high mass resolution measurements based on Fourier-transform mass spectrometry have been utilized to resolve isobaric lipids, metabolites and tryptic peptides. Here we demonstrate the potential of 15T MALDI-FTICR MSI for molecular pathology in a mouse model of high-grade glioma. The high mass accuracy and resolving power of high field FTICR MSI enabled tumor specific proteoforms, and tumor-specific proteins with overlapping and isobaric isotopic distributions to be clearly resolved. The protein ions detected by MALDI MSI were assigned to proteins identified by region-specific microproteomics (0.8 mm2 regions isolated using laser capture microdissection) on the basis of exact mass and isotopic distribution. These label free quantitative experiments also confirmed the protein expression changes observed by MALDI MSI and revealed changes in key metabolic proteins, which were supported by in-situ metabolite MALDI MSI.

Factors influencing hospital infection control policies in Italian hospitals.

A study was undertaken to determine the resources available in Italian hospitals for the control of nosocomial infections and the factors favouring a successful approach. During January-May 2000 a questionnaire about infection control was sent to the hospital health director of all Italian National Health System hospitals treating acute patients and with more than 3500 admissions in 1999. An active programme was defined as a hospital infection control committee (HICC) meeting at least four times in 1999, the presence of a doctor with infection control responsibilities, a nurse employed in infection control and at least one surveillance activity and one infection control guideline issued or updated in the past two years. There was a response rate of 87.5% (463/529). Almost fifteen percent (69/463) of hospitals had an active programme for Infection Control and 76.2% (353/463) had a HICC. Seventy-one percent (330/463) of the hospitals had a hospital infection control physician and 53% (250/463) had infection control nurses. Fifty-two percent (242/463) reported at least one surveillance activity and 70.8% (328/463) had issued or updated at least one guidance document in the last two years. The presence of regional policies [odds ratio (OR) 8.7], operative groups (OR 4.2), at least one full-time nurse (OR 4.6) and a hospital annual plan which specified infection control (OR 2.1) were statistically associated with an active programme in the multivariate analysis.

Activity of aminoglycosides against phagocytosed bacteria.

The intracellular activity of streptomycin, gentamicin, and netilmicin on Escherichia coli phagocytosed by murine peritoneal macrophages was studied using a sensitive and standardized method. Intracellular activity of streptomycin and gentamicin at therapeutic concentrations was seen after 1 h of incubation of antibiotics with macrophages containing phagocytosed bacteria, whilst for netilmicin a significant intracellular activity was observed only after 3 h exposure. The activity of these antibiotics against intraphagocytic bacteria was significantly lower than that observed against extracellular bacteria. Sub-inhibitory concentrations of streptomycin were active against intracellular E. coli. Streptomycin was also active for a phagocytosed streptomycin-resistant strain of E. coli, but this activity was eliminated when the O2-dependent killing mechanisms of macrophages were inhibited by sodium fluoride. The data demonstrate that aminoglycosides may exert a dose-dependent intraphagocytic activity against E. coli that correlates with the time of incubation of the antibiotics with infected macrophages. Streptomycin appears to be the most effective agent followed by gentamicin and the least active was netilmicin. In the case of streptomycin, the intraphagocytic activity seems to be due, at least in part, to the stimulation of O2-dependent cellular microbactericidal mechanisms.

Intracellular activity of cefamandole and aztreonam against phagocytosed Escherichia coli and Staphylococcus aureus.

The intracellular activity of cefamandole and aztreonam against phagocytosed Escherichia coli and cefamandole against phagocytosed Staphylococcus aureus was studied using a sensitive and standardized method of murine peritoneal macrophages. Cefamandole and aztreonam exerted an intracellular antibacterial activity against E. coli which was greater than their extracellular one. With concentrations of both antibiotics up to 16 x MBC a dose-dependent decrease of the initial number of intracellular E. coli which ranged from 32% to 90% was observed. However, similar antibiotic concentrations above the MBC affected the viability of extracellular E. coli by only 20% to 30%. The intracellular antibacterial activity of both antibiotics against E. coli was further enhanced by immune serum. Cefamandole at 4 x the MBC did not affect the survival of intracellular S. aureus, but killed 41% of extracellular bacteria by 1 h and 99% after 3 h. The intracellular activity of both antibiotics against E. coli was also maintained in NaF-pulsed macrophages which have an impaired oxidative metabolism. The data suggest that both cefamandole and aztreonam possess an intracellular antibacterial activity against E. coli that seems at least in part due to a positive cooperation of antibiotics with the O2-independent microbicidal system of macrophages.

Kinetics of phagocytosis and killing of E. coli by murine macrophages in presence of different serum preparations.

In this study we evaluated the kinetics of phagocytosis and killing of E. coli by thioglycollate-elicited murine peritoneal macrophages and the role of specific antibodies and complement present in different serum preparations in modulating these processes. In our system phagocytosis of E. coli by macrophage monolayer was exponential for 180 min. The killing activity was high in the first 30-60 min and then virtually ceased. The least phagocytosis and killing occurred in presence of heat-inactivated fetal calf serum (HFCS). These activities were 2-fold increased in presence of normal mouse serum (NMS) or heat-inactivated newborn calf serum (HNCS) and were highly stimulated in presence of immune mouse serum (IMS). IMS without complement was less efficient in enhancing phagocytosis and killing by macrophages. However when IMS or HNCS were deprived of specific antibodies their activity was remarkably reduced. When macrophages containing phagocytized bacteria were reincubated with different sera, multiplication of intracellular E. coli occurred with HFCS, NMS or antibody-deprived IMS or HNCS. In contrast, a significant decrease in the survival of intracellular bacteria was seen in presence of IMS, HNCS or complement-deprived IMS. The results indicated that specific bacterial antibodies play a major role in the phagocytic process and in the activation of killing mechanisms. However optimal macrophage activity resulted from the presence of both specific antibodies and complement.

Effect of beta-lactam antibiotics at subinhibitory concentrations on the phagocytosis of Staphylococcus aureus by the perfused rat liver.

We evaluated whether the exposure of Staphylococcus aureus to a subinhibitory concentration of penicillin and cefamandole could modify its susceptibility to rat serum factors and to the phagocytic activity of the isolated and perfused rat liver. Control or sub-MIC treated bacteria were added to the circulating medium which contained homologous serum, and the disappearance of bacteria from the perfusate and their recovery in the liver was determined during the 10 min experimental time. Sub-MIC treated bacteria were more susceptible to the bactericidal activity of serum present in the perfusate. However, the clearance rate of bacteria by the liver was decreased for penicillin-treated organisms and unchanged for cefamandole-treated bacteria. The data suggest that beta-lactam antibiotics at sub-MIC levels may modify S. aureus susceptibility to host defense mechanisms.

[Effect of amphotericin B on phagocytosis and intracellular killing of E. coli by rat liver perfused in vitro: preliminary results]. / Effetto dell'anfotericina B sulla fagocitosi ed uccisione intracellulare di E coli da parte del fegato di ratto perfuso in vitro: risultati preliminari.

Using isolated and perfused rat livers we found an inhibitory effect on intracellular killing of hepatic macrophages versus E. coli, in presence of therapeutic levels of amphotericin B (5 micrograms/ml). Since an interference of this antibiotic with phagocytic functions of human neutrophils has also been reported, we suggest that amphotericin B may exert a toxic effect on intraphagocytic microbicidal function, possibly by an interaction with membrane sterols.

[Bactericidal activity of the serum against Staphylococcus aureus grown in the presence of subinhibitory doses of cefamandole and gentamicin]. / Attivita' battericida del siero nei confronti di Staphylococcus aureus cresciuto in presenza di dosi subinibenti di cefamandolo e gentamicina.

Growth of S. aureus in the presence of subinhibitory concentrations of cefamandole alone (at 1/5 the MIC) or with gentamicin (both at 1/5 the MIC) produced large, globular cells with reduced viability (50%) as compared to untreated bacteria. Gentamicin (1/5 the MIC) did not modify bacteria and also reduced viability of inoculum (57% vs. controls). Cefamandole treated bacteria were more susceptible to the rat serum bactericidal activity then control bacteria. The growth of S. aureus in the presence of cefamandole plus gentamicin did not modify further bacterial susceptibility to serum opsonins. The data suggest that even at subinhibitory concentrations cefamandole may exert an antibacterial effect by acting in cooperation with serum factors.

Increased phagocytosis and killing of Escherichia coli treated with subinhibitory concentrations of cefamandole and gentamicin in isolated rat livers.

Our purpose was to study whether treatment of Escherichia coli with subinhibitory concentrations of either cefamandole or gentamicin could change bacterial susceptibility to the serum bactericidal effect and to the phagocytic and killing activity of the rat liver reticuloendothelial system. Bacteria were grown overnight with 1/5 or 1/10 of the MIC of each antibiotic. At one-fifth of the MIC, cefamandole induced filamentous elongated bacteria whose viability was decreased by 75%. The susceptibility of control and antibiotic-treated bacteria to serum was tested by measuring the survival of organisms exposed to different concentrations of rat serum in vitro. Susceptibility of bacteria to hepatic macrophage activity was tested by following the hepatic clearance of bacteria after they were added to the perfusate of the isolated rat liver. E. coli treated with subinhibitory concentrations of cefamandole or gentamicin appeared somewhat more resistant to the lytic activity of serum at a concentration of 4%, but not at 20%. Bacteria treated with 1/5 or 1/10 of the MIC of cefamandole or with 1/5 of the MIC of gentamicin were significantly more susceptible to phagocytosis and to the bactericidal activity of liver macrophages. Cefamandole appeared more potent than gentamicin in inducing these effects. The results suggest that subinhibitory levels of antibiotics may alter bacterial cell surface (cefamandole) or may impair the expression of antiphagocytic material (gentamicin), thus favoring phagocytosis and killing by macrophages. Our study provides evidence that antibiotics at subinhibitory concentrations may cooperate with host defence mechanisms against bacterial infections.

[Calcium dependent hepatotoxicity of the ionophore A23187 studied by the experimental model of rat liver isolated and perfused in vitro]. / Epatotossicita' calcio-dipendente dello ionoforo A23187 studiata col modello sperimentale del fegato di ratto isolato e perfuso in vitro.

Ionophore A23187, an agent that promoves Ca2+ influx into cells, significantly increases enzymatic leakage of AST and LDH into the medium by perfused rat livers. This work provides evidence that Ca2+ dependent hepatotoxicity can be studied conveniently in vitro whit isolated and perfused rat livers, since this model respects integrity of organ and duplicates many its in vivo functions.

[Sensitivity of bacteria exposed to subinhibitory concentrations of antibiotics to the action of serum and liver phagocytes]. / Sensibilita' di germi esposti a concentrazioni subinibenti di antibiotici alla azione del siero e dei fagociti epatici.

Incubation of E.coli and S. aureus with subinhibitory concentration (1/5 MIC) of cefamandole modified bacterial morphology and resistance to host defence mechanisms. In fact, cefamandole induced filamentous forms of E. coli, when added to the perfusing medium of the isolated rat liver system, were phagocytized at a much fortes rate then control bacteria, but appeared less sensitive to serum bactericidal activity. In contrast, S.aureus, after exposure to the antibiotic, was more sensitive to the bactericidal activity of serum then controls, while treated and untreated cells were phagocytized at the some rate. The data suggest that even at low doses some antibiotics may alter bacterial structure and increase their susceptibility to host factors.