[Evaluation of the influence of culturing on the intensity of biofilm formation by Klebsiella pneumoniae strains.] / Влияние условий культивирования на интенсивность биоплёнокообразования штаммами Klebsiella pneumoniaе.

Due to the prevalence of biofilm infections caused by Klebsiella pneumoniae, in laboratory diagnostic practice it has a great importance to obtain a standard model of Klebsiella biofilm for evaluating the bactericidal effect and effectiveness of antimicrobial drugs. Describes the method of Klebsiella biofilms formation in vitro. The intensity of biofilm formation was evaluated by the ability of bacteria to bind the crystal violet. The degree of film formation was measured by optical density. The presence of an intercellular matrix was confirmed by staining of Congo-red solution followed by light microscopy. The effect of exogenous and endogenous factors on biofilm formation by K. pneumoniae strains was investigated. The influence of the nutrient composition, the age of the culture («daily¼, «weekly¼), the presence of oxygen and the temperature conditions were studied. The nutrient composition of the medium significantly influenced on biofilm formation of K. pneumoniae: DMEM stimulated biofilm formation in most strains in vitro compared to TSB. The age of the culture (daily, weekly) did not significantly affect the biofilm formation of Klebsiella. At the same time, the temperature of culturing and the presence of oxygen can both stimulate and inhibit biofilm formation, depending on the strain under study. Most strains of Klebsiella better form a biofilm under aerobic conditions at 37º C.

[Evaluation of anti-candida activity of metabolites of enterococcal clinical isolates.]

When studying the effect of the metabolic products of clinical isolates of enterococci on the viability of Candida albicans, it was found that metabolites of all tested strains of Enterococcus faecium, E. faecalis had a fungistatic effect. At the same time a reliable fungicidal effect is a strain-specific feature. It is better to use the method of delayed antagonism on double-layer agar to assess the antifungal effect of enterococcal metabolism products.

Incorporation of monoclonal antibodies in living rat pheochromocytoma PC12 cells. Evidence for the intracellular formation of immune complex between the incorporated antibody and a target protein.

PC12 cells permeabilized with a low concentration of digitonin (5 microM) under controlled conditions were loaded with monoclonal antibodies (MoAb) against the regulatory subunit type II (RII) of cAMP-dependent protein kinase. After digitonin removal from the nutrient medium (DMEM) the loaded cells repaired within 20-30 min and recontinued growth. The inserted MoAb stayed in the repaired cells at least for several hours. MoAb inhibiting the cAMP binding activity of neural RII [Grozdova et al. (1992) Biochem. Int. 27, 811-822; Sveshnikova et al. (1996) Biochem. Int. 39, 1063-1070] were shown to bind the target antigen inside the cells and influence the properties of intracellular protein kinases.

Mass effect and death from severe acute stroke.

BACKGROUND: In severe acute stroke, the degree of midline cerebral displacement is related to level of consciousness but not to survival. Early identification of patients at high risk of death from mass effect would assist patient management decisions. METHODS: We measured lesion volume, horizontal pineal displacement (PD), and horizontal septum pellucidum displacement (SD) on axial CT of consecutive patients with severe (Canadian Neurological Scale score < or = 5) acute hemispheric stroke. We correlated CT measurements with the probability of 14-day survival. RESULTS: Forty-six (39%) of 118 patients died within 14 days and 72 (61%) died within 1 year following stroke. Crude risk factors for 14-day mortality were as follows: lesion volume > or = 400 ml, SD > or = 9 mm, PD > or = 4 mm, intraventricular hemorrhage, and coma on admission. Only SD (p = 0.001) and coma on admission (p = 0.019) remained significant in multivariate analysis, but PD was highly correlated with SD (r = 0.82). PD of > or = 4 mm on a scan performed within 48 hours of stroke onset identified patients with a low probability of 14-day survival (0.16; CI 0 to 0.32) with a specificity of 89% and a sensitivity of 46%. CONCLUSIONS: The degree of horizontal midline cerebral displacement correlates with the likelihood of death following stroke. Patients with > or = 4 mm PD on CT performed within 48 hours of stroke onset are at high risk for early death.

Properties of the regulatory subunit of cAMP-dependent protein kinase type II from human brain.

The regulatory subunit type II (RII) of cAMP-dependent protein kinase purified from human brain was represented by two proteins with apparent molecular masses of 51-52 kD and 54 kD. Dephosphorylation of human RII containing 3 mol phosphate/mol protein did not change the electrophoretic pattern. One-dimensional peptide mapping of 51-52 kD and 54 kD proteins after digestion with St. aureus V8 protease evidenced to their being distinct proteins. The data obtained permit to assume that human RII of neural type is represented by two isoforms.

The clinical value of methods to measure carotid stenosis.

BACKGROUND: The North American (NASCET) and European (ECST) carotid surgery trials have shown a surgical benefit for symptomatic stenosis greater than 70%. The Asymptomatic Carotid Artery Surgery (ACAS) trial have shown some benefit for the stenosis greater than 60%. Although the NASCET/ACAS angiographic methods were similar, these are discrepant from ECST and have technical limitations inherent to measurement of the distal internal carotid artery (ICA) or guessing the ICA bulb diameter. METHODS: Consecutive carotid angiograms were analyzed to verify the relationships between proximal and distal aspects of the common carotid artery (CCA) and ICA bulb. We then compared the NASCET and ECST methods and, two new techniques, the Common Carotid (CC) and Carotid Stenosis Index (CSI). The CC method is based on a direct comparison of the residual lumen to the distal CCA diameter adjacent to the bulb. The CSI is based on the known relationship between the proximal CCA and ICA (1.2 x CCA diameter = proximal CCA diameter). The normal ICA bulb diameter can therefore be calculated from direct measurement of the CCA. RESULTS: 125 consecutive carotid angiograms were evaluated (250 arteries). Technical applicability of NASCET was 89%, ECST 95%, CC/CSI 99%. The CCA/ICA diameter ratios were established: 1.23 +/- 0.23 (ICA bulb/distal CCA), and 1.27 +/- 0.2 (ICA bulb/proximal CCA). The CCA is enlarged at its distal end that such the distal CCA/proximal CCA ratio is 1.04 +/- 0.12. The CC and CSI methods were statistically different in 8 of 10 groups when these methods were compared per decile stenosis (p < 0.04). However, CC and CSI methods disagreed in classifying patients into mild (0-29%), moderate (30-69%), and severe (70-99%) only in 3%, 5%, and 8% of cases. Linear regression analysis shows excellent correlation between the methods (CC = 15.7 + 0.82 x CSI, r2 = 0.92). Lumen asymmetry is most common with mild-to-moderate stenoses which may affect accuracy and reproducibility of measurements. CONCLUSIONS: We have confirmed previous data on the relationships between the components of the carotid artery. Of the different angiographic techniques, CSI is the most reliable validated method of measuring carotid stenosis, and is proposed as a bridge between results of carotid surgery trials, and to validate noninvasive modalities against angiography.

Immunochemical studies on human, bovine and pig brain regulatory subunits of cAMP-dependent protein kinase type II.

Regulatory subunits type II (RII) purified from human, pig and bovine brains were compared using II monoclonal antibodies (MoAb) against bovine brain RII. Bovine RII has at least 5 antigenic sites located in the N-terminal 1-110 residues. Immunochemical difference detected between human and animal RII was more pronounced than between pig and bovine RII. Certain MoAb influenced R-cAMP binding and holoenzyme formation. RII of the three species responded to MoAb attachment in a similar fashion. The results suggest that anchoring of neural protein kinase via the N-terminal part of RII may influence the enzyme activity.

Carotid artery stenosis in peripheral vascular disease.

PURPOSE: The goal of the study was to assess the prevalence and severity of symptomatic and asymptomatic carotid artery disease in patients with peripheral vascular disease (PVD). METHODS: Consecutive patients with clinically and Doppler scanning-proven PVD (category 1 or greater) underwent prospective screening for the presence of carotid atherosclerosis with color-coded duplex ultrasonography. Preexisting risk factors were recorded with a standard questionnaire and included sex, age, diabetes mellitus, history of smoking, hypertension, prior stroke/transient ischemic attacks, and coronary artery disease. RESULTS: Three hundred seventy-three consecutive patients were studied over 2 years. The mean age of the patients was 70 +/- 10 years; there were 223 (60%) men and 150 (40%) women; 71% of the patients had a history of smoking, 47% had coronary artery disease, 43% had hypertension, and 21% had diabetes mellitus. Two hundred eleven (57%) patients had 30% or greater carotid artery stenosis detected by carotid artery duplex scanning. Sixty-seven (32%) of these had symptoms of ischemic cerebral events, of whom 22 had potentially operable carotid artery stenoses (70% to 99%), whereas 72 of the 144 symptom-free patients had 60% to 99% stenosis. An additional 34 patients would be eligible candidates for the ongoing carotid endarterectomy trials (North American Symptomatic Carotid Endarterectomy Trial and European Carotid Surgery Trial). Although all the risk factors were associated significantly with PVD and carotid artery disease (p < 0.002), male sex and prior stroke/transient ischemic attack were the strongest predictors. CONCLUSIONS: Routine carotid ultrasound screening of 373 consecutive patients with category I or greater PVD revealed that 22 patients with symptoms and 72 symptom-free patients were potential surgical candidates, representing 25% of the study cohort. An additional 34 patients were potential candidates for enrollment into the North American Symptomatic Carotid Endarterectomy Trial and European Carotid Surgery Trial.

Traumatic rupture of the thoracic aorta: should one always operate immediately?

Although the traditional therapy for blunt traumatic rupture of the thoracic aorta (TRA) is immediate operative repair, there may be a selective role for delayed repair, particularly in patients with head trauma, respiratory failure, or cardiac dysfunction. The present study examines the hypothesis that TRA can be managed by selective delayed operative repair. Clinical data were collected from 59 consecutive patients with TRA at a regional trauma unit. All TRAs were at the aortic isthmus. Patients were retrospectively classified into three groups: group I (n = 12) included patients who either arrived in extremis or rapidly became unstable during triage; group II (n = 3) included patients who had no contraindications to early repair and underwent repair at the time of diagnosis; and group III (n = 44) consisted of patients who because of concomitant injuries or sepsis required initial admission and management in the intensive care unit until their clinical status had improved sufficiently to allow for deliberate delayed operative repair of the TRA. The delay ranged from 1 day to 7 months. Eight patients have yet to undergo repair and remain well at follow-up from 1 to 4 years. Overall survival rates in groups I, II, and III were 17%, 100%, and 82%, respectively. The surgery-related mortality rate in group III was 10% (three patients). Only two (4.5%) patients in group III died as a result of a ruptured aorta within 72 hours of admission. In conclusion, contrary to surgical doctrine, TRA may not require immediate operative repair in all cases, but may instead be managed selectively depending on the patient's clinical status.