A simple in vitro model to test the efficacy of antimicrobial agents released from dressings.

A new model for in vitro evaluation of an activity of antibacterial agents released from delivery systems containing antibiotics or antiseptics was developed. The model was composed of two layers of agar gel of two different concentrations in PBS. In the upper gel, the wells were cut and filled with polyurethane sponges saturated with bacterial broth culture. The sponges were then covered with tested dressings containing antibacterial agent. The model was tested by using two bacterial strains, Pseudomonas aeruginosa or Staphylococcus aureus and experimental collagen dressing with doxycyclin, amikacin, or with silver sulfadiazine. The number of colony-forming units (CFU) in the polyurethane sponges was significantly lower under the dressings with antibacterial agents as compared to the control (the same dressing without antimicrobials) during 3 days of observation. The new in vitro model can be recommended for examination of different antibacterial delivery systems instead of experimentally infected wounds in laboratory animals or instead of the other in vitro models.

Anti-pseudomonal activity of collagen sponge with liposomal polymyxin B.

An antibiotic delivery system has been developed using collagen sponge with liposome-encapsulated polymyxin B. Superficial, non-lethal infection was produced in mice by injecting Pseudomonas aeruginosa into the skin windows. Wounds were dressed with collagen sponge containing liposomal polymyxin B or containing empty liposomes (with PBS) as a control. Single dose of topically applied collagen sponge with encapsulated polymyxin B decreased bacterial cell number as compared to the control. Finally, after 8 days of experiment, the number of bacterial colonies dropped below 10(4) per biopsy. Presented polymyxin B delivery system offers potential clinical advantages.

The influence of Pseudomonas aeruginosa on liposomes.

The interaction of liposomes loaded with Ponceau red (used as a marker) with Pseudomonas aeruginosa cells was observed and it resulted in marker leakage. The marker leakage from liposomes was low in physiological fluids. The interaction was independent of secreted phospholipase C level and the serotype of the tested strain. Six of 37 examined isolates did not cause any release of the marker from the liposomes. Marker release of over 50% of total encapsulated material was observed only for ten of the strains tested.

Frequency of mitral annular calcification in patients on hemodialysis estimated by 2-dimensional echocardiography.

Mitral annular calcification (MAC) is a degenerative process associated with left ventricular hypertrophy (HLV) and progressive atherosclerosis, characteristic of the older age groups. Numerous investigations point to significantly earlier onset of atherosclerosis process in patients in final stage of chronic renal insufficiency. The aim of investigation was to determinate the MAC frequency in patients on hemodialysis and to try to find the correlation between MAC intensity and the duration of hemodialysis, age, sex, Ca/P, metabolism, level of parathormone and atherogenic factors. A group of 40 patients on hemodialysis (aged 20 to 67, 26 men and 24 women) were divided int two groups; group 1 without MAC, N = 17 (42.5%), X = 3.5, SD = 3.1; and group 2 with MAC, N = 23 (57.5%), X = 6.2, SD = 2.4. M-mode and 2-D echocardiography were performed in all patients. Group 2 was divided into three subgroups according to MAC quantitation: mild N = 16 (70%), severe, N = 4 (17%), moderate, N = 3 (13%). Study results showed positive correlation between MAC and serum values of Ca and P (p < 0.05). Increased values of HDL cholesterol, statistically significant at the level p < 0.05 were observed. Study results showed the correlation between MAC and time factor, i.e. duration of dialysis treatment to be statistically significant (p < 0.05). Cardiac calcified syndrome could be a sequela of MAC causing conduction disturbances, valvular stenosis or insufficiency, and arterial emboli or endocarditis.

Effects of free and liposome-encapsulated antibiotics on adherence of Pseudomonas aeruginosa to collagen type I.

The adherence of 27 clinical Pseudomonas aeruginosa strains to collagen type I was investigated by using a solid-phase assay. The influence of free antibiotics (amikacin, gentamicin, piperacillin, bacitracin, and polymyxin B) and liposome-entrapped antibiotics (amikacin and polymyxin B) on bacterial attachment to collagen type I was examined. The greatest inhibitory effect was shown for free and liposomal amikacin, which decreased the attachment of 74 and 100% of tested strains, respectively. The mean percent attachment (+/- standard deviation) in the presence of free amikacin was 65.7% (+/- 12.0%) as measured by solid-phase assay. In the presence of liposomal amikacin, the attachment ranged from 17.3% (+/- 6.0%) to 42.1% (+/- 9.4%), depending on the antibiotic solvent. In contrast, polymyxin B, even at a subinhibitory concentration, enhanced attachment of all P. aeruginosa isolates to collagen. Liposomal polymyxin B displayed a protective effect only when the encapsulated drug was of a low concentration. Application of liposome-encapsulated amikacin may be advantageous in injured tissues in which extracellular matrix structures become exposed.

Antibacterial activity of liposomal amikacin against Pseudomonas aeruginosa in vitro.

The influence of liposomal amikacin on Pseudomonas aeruginosa was studied. P. aeruginosa clinical isolates caused release of encapsulated amikacin from liposomes. The liposomal amikacin proved to be active as bactericidal agent after 3 h of incubation with P. aeruginosa. Incubation of P. aeruginosa with liposomal amikacin resulted in inhibition of the growth when equivalent of 2 MIC was added but not when equivalent of 1 MIC was added. Susceptibility of bacterial isolates to the liposomal amikacin varied with bacterial strain used, but generally encapsulation of amikacin did not enhance their antibacterial activity.

Antidietary antigen antibodies in the sera of patients with burns as a potential marker of gut mucosa integrity failure.

It was hypothesized that a failure of gut mucosa integrity after burn injury may lead to translocation of food proteins as well as bacteria and endotoxins. Certain, normally digested proteins may stimulate the humoral immune response as they enter the circulation. To test this hypothesis, serum samples from 16 patients with burns and 10 healthy donors were tested for the presence of antigliadin and anti-beta-lactoglobulin antibodies of immunoglobulin M (IgM), IgG, IgA, and IgE classes by means of enzyme-linked immunosorbent assay. Patients and donors remained on a standard diet that included wheat (gliadin) and milk products (beta-lactoglobulin). In the group of six patients with severe burns (mean total body surface area burn = 50%), antigliadin and anti-beta-lactoglobulin IgG and IgA antibodies were detected. In the control group and in 10 patients with small to moderate burns (mean total body surface area burn = 15%), these antibodies were not observed. There was a significant correlation between antigliadin IgG and IgA titers (r greater than 0.9) and anti-beta-lactoglobulin antibodies (r greater than 0.7) in patients with burns. Results suggest immunization by gliadin and beta-lactoglobulin in the group of patients with severe burns.

[Kinetics of leukocytes in patients on hemodialysis]. / Kinetika leukocita u bolesnika na hemodijalizi.

The total numbers of leukocytes and relevant fractions of granulocytes in 51 patients on chronic intermittent hemodialysis were determined. The leukocytes were counted before the beginning, during the third hour of and one to two hours after the hemodialysis. Along with the kinetics of leukocytes the levels of C3 and C4 components of the complement were tested. A decrease of total numbers of leukocytes was observed as well as that of all the fractions of granulocytes during the third hour of dialysis. The intensity of leukopenia depended on the structure of dializer membrane. Observing the components C3 and C4 along with the values of leukocytes there was no association between these two alterations. The authors concluded that leukopenia during the hemodialysis is not caused by complement activation in the contact of plasma with the membrane of the dializer but by the mechanical effect of adhesion or microaggregation of leukocytes on the membrane of the dializer.

Bioactive parathyroid hormone in canine progressive renal insufficiency.

Bioactive parathyroid hormone and hormonal actions were monitored as hyperparathyroidism evolved in a model of progressive canine renal failure. Circulating levels of bioactive and immunoreactive parathyroid hormone rose as renal insufficiency worsened, but elevations, especially in bioactivity, were most marked in the final stage of uremia. By gel filtration analysis, the major circulating bioactive moiety was similar to the major glandular form of parathyroid hormone, although a smaller-molecular-weight entity was seen in the final stage of renal failure. Renal phosphate threshold fell, urinary hydroxyproline corrected for glomerular filtration rose, and plasma 1,25-dihydroxyvitamin D fell but remained detectable, as renal function deteriorated. The results demonstrate a progressive rise in bioactive parathyroid hormone, show the appearance of a small-molecular-weight bioactive entity in severe renal disease, and correlate effects of the rising bioactive parathyroid hormone with changes in renal phosphate handling and with skeletal resorption.

Variations in the level of haematogenous antitumour immunity during progressive tumour growth and spontaneous blood-borne metastatic spread.

Inbred DBA2 mice bearing the syngeneic 1699 mammary tumour in the hind limb were challenged intravenously with 125IUdR-labelled single 1699 cell suspensions, and the amount of radioactivity in the lungs compared 20--24 h later with that in the lungs of normal mice or in those of mice bearing the antigenically unrelated syngeneic SaD2 tumour. An immunologically specific decrease in radioactivity was evident at variable periods of time after tumour induction, depending on the number of cells used to induce the leg tumours, but fell below that in normal mice as the leg tumours progressed beyond a weight of approximately 1 g. As assessed by microscopic scanning of serial histological sections of the same lungs the incidence of spontaneous metastases rose to between 80 and 100% immediately the amount of cell loss from the lungs of the tumour-bearing mice reached that of the normal controls. This extremely rapid series of events does not allow a definitive conclusion as to whether immunity failed and led to metastatic spread or vice versa, but does underline the strong association of immunity with the blood-borne dissemination of tumour cells in this tumour system. Following excision of tumours, in no instance was immunity detected 14 days later, and in a single experiment did not reach detectable limits until 25 days after excision, at a time when the lung metastases were observed mostly to have regressed spontaneously.