Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria.

BACKGROUND: Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state. A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment. METHODS: We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis. After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex. A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome. Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively. RESULTS AND CONCLUSIONS: With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology.

Monitoring of urinary excretion of modified nucleosides in cancer patients using a set of six monoclonal antibodies.

Monoclonal antibodies were produced and characterized in order to allow the monitoring of the urinary excretion of six modified nucleosides. The specificity of each antibody was determined and competitive solid-phase enzyme-linked immunoassays were designed, the sensitivity of which lay in the pmol range. Detection and quantitation of 5-methylcytidine (5-MeCyd), 4-acetylcytidine (4-AcCyd), 1-methylinosine (1-MeIno), 1-methyladenosine (1-MeAdo), 7-methylguanosine (7-MeGuo) and pseudouridine (psi-Urd) can be performed in small volumes (70 microliters) of untreated urine. Results can be obtained from as many as 20 different samples, for one molecule, within 3 h. With this technique, values observed for three commonly measured nucleosides in urine from healthy subjects (psi-Urd, 1-MeAdo and 1-MeIno) are in good agreement with those reported by other authors after analysis by high performance liquid chromatography. Results obtained in urine from cancer patients show significantly increased levels of the six haptens quantitated by this immunoassay.

Salts as a source of metals in dialysis fluids: an assessment study by means of neutron activation analysis.

Thirty-nine trace metals were determined by neutron activation analysis (NAA) in nine salts (glucose, NaCl, KCl, CaCl2, MgCl2, Na-lactate, Na-acetate, NaHCO3 and NaOH), from different European producers, used in the preparation of dialysis fluids. Metal concentrations vary widely from less than 1 ng g-1 to several micrograms per gram. The data are used to assess the potential metal contribution of the salts to a hypothetical dialysis fluid. A comparison of these calculations with NAA of commercial haemodialysis solutions, and water used for their preparation, suggests that salts are the main source of metal contamination of the dialysate. Despite this, the chemical purity of the salts analyzed is considered to be good and it would be unrealistic to expect a clearly better chemical quality on a commercial scale. In order to protect the health of dialysis patients against abnormal metal exposure, toxicological research directed towards the estimation of the effective daily metal exposure and the determination of possible metal overloads in dialysis patients is necessary. A standardization of the dialysis systems actually used is also an important aspect if uncontrolled metal contamination of the dialysis fluid is to be avoided.

[Platelet aggregation in whole blood and prostacyclin in hyperlipemia types II and IV]. / Aggregazione piastrinica su sangue intero e prostaciclina nelle iperlipemie di tipo II e IV.

Platelets play an essential role in the pathogenesis of atherosclerosis. Prostacyclin is a strong physiological inhibitor of platelets aggregation; prostacyclin indeed is involved in the regulation of platelet interactions with vessel walls and is considered to play a major role in the homeostatic balance. The impedance aggregometry allows the evaluation of platelet aggregation in whole blood. We valued platelet aggregation in whole blood induced by ADP (10 microM) in 40 healthy subjects and in 40 type II and type IV hyperlipemic subjects. Meanwhile by radioimmunoassay we dosed 6-keto PGF1 alpha, a stable product of prostacyclin, in 7 healthy subjects and in 33 hyperlipemic subjects. The statistical investigation put in evidence that at higher plasmatic levels of cholesterol, triglycerides and LDL correspond a greater platelet sensitivity to the aggregating agent, while the opposite happens to higher levels of HDL. The dosage of 6-keto PGF1 alpha put in evidence an increase of this substance in hyperlipemic as to healthy subjects, probably as an answer to augmented platelet aggregation.

[Platelet aggregation in whole blood and thromboxane B2 in type II and IV hyperlipidemia]. / Aggregazione piastrinica su sangue intero e trombossano B2 nelle iperlipemie di tipo II e IV.

Platelets play an essential role in the pathogenesis of atherosclerosis; by impedance method we valued in whole blood platelet aggregation induced by collagen in 40 healthy subjects and in 40 type II and type IV hyperlipemic subjects. Meanwhile by radioimmunoassay we dosed thromboxane B2, a stable product of thromboxane A2, released by platelets during activation, in 7 healthy subjects and 25 hyperlipemic subjects. The statistical investigation put in evidence that at higher plasmatic levels of cholesterol, triglycerides and LDL correspond a greater platelet sensitivity to the aggregating agent, while the opposite happens to higher levels of HDL. The dosage of thromboxane B2 put in evidence a moderate increase in hyperlipemic as to healthy subjects, probably pointing to a state of platelet activity.

Cigarette smoking and platelet function.

To assess the influence of cigarette smoking on platelet activation, we studied the changes in intraplatelet and platelet-released serotonin (5-HT) and plasma levels and platelet-associated production of thromboxane B2 (TXB2), in 6 non smokers and 6 habitual smokers, before and after acute exposure to smoke. Before smoking, habitual smokers showed slightly higher, albeit not significantly, 5-HT platelet concentrations and TXB2 plasma levels, as well as lower TXB2 platelet production after collagen and even more after ADP stimulation (0.59 +/- 0.27 vs 1.35 +/- 0.46 and 0.99 +/- 0.47 vs 2.08 +/- 0.51 ng/10(8) platelets for habitual smokers vs controls, 4 and 10 min after ADP, p less than 0.02). No significant differences in platelet 5-HT release were observed. Acute smoking did not induce any significant change from baseline in either 5-HT or TXB2 for controls, while significantly reduced TXB2 production from ADP-challenged platelets from habitual smokers (0.30 +/- 0.15 vs 0.59 +/- 0.27 ng/10(8) platelets, immediately after smoking vs baseline, p less than 0.01). Ninety min after the completion of the smoking, the values had returned to baseline. Immediately after smoking, significant differences were found between habitual smokers and controls for TXB2 platelet production (2.76 +/- 1.78 vs 6.42 +/- 1.60, p less than 0.025 and 3.01 +/- 1.90 vs 6.44 +/- 2.26 ng/10(8) platelets, p less than 0.05, for habitual smokers vs controls, 4 and 10 min after the addition of collagen; 0.30 +/- 0.15 vs 1.20 +/- 0.84 and 0.79 +/- 0.50 vs 1.70 +/- 0.74 ng/10(8) platelets, p less than 0.05, after ADP stimulation). Differences were no longer significant 90 min after smoking. Our data indicate that cigarette smoking is associated with platelet dysfunction, which seems due to impairment of metabolic platelet capacity rather than increased platelet activation in vivo.

[Serum and cerebrospinal fluid antibodies against measles virus in patients with multiple sclerosis]. / Anticorpi sierici e liquorali verso il virus del morbillo in pazienti affetti da sclerosi multipla.

Measles c.f.a. and h.i.e. were titrated in sera and, when possible, in the cerebrospinal fluids of 183 SM patients and in sera from a control group of subjects. No significative difference was found by the h.i. test, whereas significantly higher was the frequence of positives by the c.f. test in the SM group, with no correlation with the therapy to which the patients were subjected. The presence of c.f.a. in 13 of 48 cerebrospinal fluids tested, together with a low value of the serum/CSF antibodies ratio and with the lack of CSF antibodies against other viral antigens tested (HSV, rubeola), could indicate an intra CNS production of measles antibodies. To sum up, our results suggest an involvement of measles virus at least in some of the MS patients considered.