Ismini Dontas

Stimulation of innate immunity by susceptible and multidrug-resistant Pseudomonas aeruginosa: an in vitro and in vivo study

In attempt to investigate the stimulatory effect of Pseudomonas aeruginosa on innate immunity and to correlate it to its level of resistance to antimicrobials, 20 isolates were applied; 8 isolates were susceptible and 12 multidrug‐resistant. Genetic diversity was defined by PFGE. Human monocytes of two healthy volunteers were in vitro stimulated by the isolates for the production of pro‐inflammatory (TNF‐α, IL‐1β, IL‐6, IL‐8 and IL‐12) and anti‐inflammatory cytokines (IL‐10), of malondialdehyde and of procalcitonin. Cytokines were estimated by EIA, malondialdehyde by the thiobarbiturate assay and procalcitonin by an immunochemiluminometric assay. Survival of 48 Wistar rats was recorded after induction of sepsis by the intraperitoneal injection of three susceptible and three multidrug‐resistant isolates. To test whether comparative effect of the latter isolates on survival correlates with any difference of monocyte‐mediated release of pro‐inflammatory mediators, monocytes of two rats were in vitro stimulated for the production of TNF‐α and of malondialdehyde. In vitro stimulation of human monocytes by the susceptible isolates elicited elevated production of malondiadeheyde, of IL‐1β and of IL‐6 compared to stimulation by multidrug‐resistant isolates. Similar differences were found for TNF‐α and IL‐8, but they were not statistically significant. Production of IL‐10 and IL‐12 was not detected after stimulation with any isolate. Levels of procalcitonin were similar after induction with either susceptible or multidrug‐resistant isolates. Mean survival of animals was 7·56, 21·80 and 55·20 h, respectively, after challenge by the susceptible isolates and 28·89, 61·8 and more than 120 h, respectively, after challenge by the multidrug‐resistant isolates. Differences of survival were accompanied by greater rodent monocyte‐release of TNF‐α and malondialdehyde after stimulation by the susceptible isolates compared to multidrug‐resistant ones. It is concluded that considerable differences are encountered on the stimulation of human monocytes by susceptible and resistant isolates of Pseudomonas aeruginosa. These results correlate with in vivo evidence and might influence decision on therapeutics.

Local Treatment of Experimental Pseudomonas aeruginosa Osteomyelitis with a Biodegradable Dilactide Polymer Releasing Ciprofloxacin

ABSTRACT A biodegradable system of poly-d,l-dilactide releasing ciprofloxacin was assessed in a Pseudomonas aeruginosa osteomyelitis model after inoculation of the test pathogen into the left tibia of 76 New Zealand White rabbits; 31 were controls (group A), and 45 were implanted with the polymer at the infection site (group B). The rabbits were killed on a weekly basis, and cancellous bone was harvested for histopathology and for estimation of bacterial growth and the concentrations of ciprofloxacin. Tibial X ray was performed immediately before the animals were killed. The total number of fistulas with purulent discharge that developed after inoculation of the pathogen was counted, and fistulas with purulent discharge were found in 16 animals in group A (51.6%) and 3 animals in group B (6.7%) (P < 0.0001). The animals in group A had a profound loss of body weight compared to the animals in group B. The main radiological finding was the presence of sequestra in 25 animals (80.6%) in group A and 6 animals in group B (13.3%) (P < 0.0001). The bacterial load in group B was significantly reduced compared to that in group A, possibly due to the prolonged local antibiotic release at concentrations exceeding even 80 times the MIC for the test pathogen. The histology of animals killed after week 49 revealed a mild inflammatory reaction accompanied by diffuse fibrosis and new bone formation in group A animals and the presence of small polymer particles in group B animals. It is concluded that the system described achieved eradication of the pathogen, accompanied by clinical and radiologically confirmed benefits, so this treatment may be a candidate for the management of difficult orthopedic infections.

Lipid peroxidation by Pseudomonas aeruginosa in the pathogenesis of nosocomial sepsis

BACKGROUND To study whether Pseudomonas aeruginosa may directly trigger peroxidation of polyunsaturated fatty acids, since lipid peroxidation is a mechanism involved in the pathogenesis of sepsis. METHODS Gamma-linolenic acid (GLA) was administered intravenously at a dose of 25mg/kg in an infusion time of 10 minutes to seven male rabbits. Blood samples were collected from the hepatic veins and from the carotid artery at regular time intervals. One clinical isolate was ex vivo incubated with the serum derived from the latter samples and concentrations of malondialdehyde (MDA) were determined during incubation in the growth medium by the thiobarbiturate assay. RESULTS Elevated concentrations of MDA compared to their basal levels were found over the first three hours of incubation in the presence of samples collected 30 to 60 minutes after the end of the infusion of GLA. After infusion of GLA concentrations of arachidonic acid in the serum increased to concentrations comparable to those detected in sepsis. CONCLUSION Direct triggering of lipid peroxidation by nosocomial isolates might be proposed as a pathogenetic mechanism of sepsis.

Quantitative Ultrasound of the Calcaneus in Greek Women: Normative Data are Different From the Manufacturer's Normal Range

Quantitative ultrasound (QUS) is considered a useful method in evaluating bone status. The aim of the present study was to establish the reference data for the QUS measurements of the calcaneus in a Greek population. We measured a QUS parameter, stiffness index (SI), at the right calcaneus in 1500 women using the Achilles express Ultrasonometer (GE Lunar, Madison, WI). Participants were divided into 7 groups according to their age with a 10-yr span in each group. A progressive decline was found in the SI values after the age of 39 yr in the current study. When the SI values were compared between the age groups, high statistically significant differences were obvious, especially between 20-29 and 50- to 59-yr age groups and 60-69 and 70- to 79-yr age groups (p < 0.0005). Additionally, in the Greek normal range (GNR), the SI values of those aged 60-69 and 70-79 yr were significantly higher (81.84+/-16.14 and 77.45+/-17.65, respectively) than those in the manufacturers normal range (MNR; 75.84+/-16.14 and 69.10+/-17.65, p < 0.005, respectively). Using the manufacturers values, significantly fewer women were classified as normal (48% vs 67.3%), although those with T-score < or =-2.5 were more (15.7%) compared with our Greek value (1.5%), and classification of subjects into risk-of-fracture categories was significantly different (kappa: 0.459, 66.2%, p < 0.0005). Multiple regression analysis showed that weight was the most significant predictor for SI in the age groups 30-39 (beta = 0.280, p < 0.05), 40-49, 60-69, and 70-79 yr (beta = 0.185, p < 0.005; beta = 0.329, p < 0.0005; beta = 0.494, p<0.0005, respectively). Using conventional categories of risk, we report a different classification of our subjects from those proposed by the manufacturer, supporting the concept that data specific to the Greek population are necessary.

Effect of Rheumatoid Arthritis on Volumetric Bone Mineral Density and Bone Geometry, Assessed by Peripheral Quantitative Computed Tomography in Postmenopausal Women Treated with Bisphosphonates

Objective. To investigate the effect of rheumatoid arthritis (RA) on volumetric bone mineral density (vBMD) and bone geometry in postmenopausal women treated with bisphosphonates. Methods. Fifty-three postmenopausal women with RA and 87 control subjects, comparable in terms of age, body mass index, and years since menopause, underwent peripheral quantitative computed tomography (pQCT) of the nondominant tibia. Results. At 4% (trabecular site), trabecular bone mineral content (BMC) and vBMD (p < 0.001) were lower in the RA group, while trabecular area was comparable. At 38% (cortical site), cortical BMC (p < 0.01), area (p < 0.05), and thickness (p < 0.001) were lower in the RA group, whereas vBMD was comparable. Endosteal circumference was higher (p < 0.05), whereas periosteal circumference was comparable, indicating cancellization of cortical bone. In the RA group, muscle area was lower (p < 0.001), while at 14% polar stress strength index was significantly lower (p < 0.01) in patients with RA, indicating impairment of bone mechanical properties. Conclusion. RA is associated with negative effects on both cortical and cancellous bone in postmenopausal women treated with bisphosphonates. Cortical geometric properties are also adversely affected mainly by increased endosteal circumference, whereas trabecular geometric properties are generally preserved.

Response to: A role for thiazide diuretic therapy in preventing bone loss, fracture and nephrolithiasis in individuals with thalassemia and hypercalciuria?

Dear Editor, We would like to thank AdamMorton for his interesting comments on the possible value of thiazide diuretics in the management of bone loss, fractures, and nephrolithiasis in patients with thalassemia and calciuria [1, 2]. We agree with the comments, however, to our knowledge, there are no studies evaluating the effects of thiazide diuretics on these outcomes in patients with thalassemia. Thiazides have documented protective effect on nephrolithiasis [3] and indeed have been associated with positive effects on bone mineral density [4, 5] even though the effects are small and not invariably demonstrated [6]. Fracture data are based only on observational studies and are inconclusive showing potential beneficial [7] or negative effects [8], possibly depending on age. Recently, a Cochrane review has shown an overall reduction in the risk of hip fractures [9]. Due to the complex pathophysiology of thalassemia, welldesigned studies evaluating the effects of thiazide diuretics on calciuria, nephrolithiasis, and bone mineral density and aiming to identify subsets of patients who might benefit from such an intervention would be valuable.