[INTERCONNECTION BETWEEN CELL MICROVESICULAR TRANSPORT AND PATHOGENS PERSISTENCE IN VITRO AND IN VIVO].

This review presents an information and proof evidence toward to the role of microvesicles, originating from the different sources pro- and eucaryotes in the initiation and development of persistence of several human and animal pathogens. Also an information about another properties of microvesicles, as well as the reference of role in the different somatic pathology, intercellular interaction and in the intracellular transport of biologically active macromolecules as well as life origin and evolutionary events.

Fatal Scopulariopsis infection in a lung transplant recipient: lessons of organ procurement.

Seventeen days after double lung transplantation, a 56-year-old patient with idiopathic pulmonary fibrosis developed respiratory distress. Imaging revealed bilateral pulmonary infiltrates with pleural effusions and physical examination demonstrated sternal instability. Broad-spectrum antibacterial and antifungal therapy was initiated and bilateral thoracotomy tubes were placed. Both right and left pleural cultures grew a mold subsequently identified as Scopulariopsis brumptii. The patient underwent pleural irrigation and sternal debridement three times but pleural and wound cultures continued to grow S. brumptii. Despite treatment with five antifungal agents, the patient succumbed to his illness 67 days after transplantation. Autopsy confirmed the presence of markedly invasive fungal disease and pleural rind formation. The patient's organ donor had received bilateral thoracostomy tubes during resuscitation in a wilderness location. There were no visible pleural abnormalities at the time of transplantation. However, the patient's clinical course and the location of the infection, in addition to the lack of similar infection in other organ recipients, strongly suggest that Scopulariopsis was introduced into the pleural space during prehospital placement of thoracostomy tubes. This case of lethal infection transmitted through transplantation highlights the unique risk of using organs from donors who are resuscitated in an outdoor location.

Nonthermal plasma affects viability and morphology of Mycoplasma hominis and Acholeplasma laidlawii.

AIM: To study the effects exerted by argon microwave nonthermal plasma (NTP) on cell wall-lacking Mollicutes bacteria. METHODS AND RESULTS: 10(8) CFU ml(-1) agar plated Mycoplasma hominis and Acholeplasma laidlawii were treated with the nonthermal microwave argon plasma for 30-300 s. The maximal 10- and 100-fold drop was observed for A. laidlawii and Myc. hominis, respectively. Similarly treated Escherichia coli and Staphylococcus aureus demonstrated the 10(5) and 10(3) drop, respectively. Removal of cholesterol affected resistance of A. laidlawii. 10 mmol l(-1) antioxidant butylated hydroxytoluene decreased mortality by a factor of 25-200. UV radiation alone caused 25-85% mortality in comparison with the whole NTP. Exogenously added hydrogen peroxide H2O2 did not cause mortality. NTP treatment of Myc. hominis triggered growth of microcolonies, which were several tenfold smaller than a typical colony. CONCLUSIONS: Despite the lack of cell wall, A. laidlawii and Myc. hominis were more resistant to argon microwave NTP than other tested bacteria. Mycoplasma hominis formed microcolonies upon NTP treatment. A role of UV and active species was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The first study of NTP effects on Mollicutes revealed importance of a membrane composition for bacterial resistance to NTP. New specific Myc. hominis morphological forms were observed. The study confirmed importance of the concerted action of reactive oxygen species (ROS) with UV and other plasma bioactive agents for NTP bactericidal action.

[Effect of low temperature plasma on various mycoplasma species].

AIM: Study the influence of low temperature (cold) electrolyte plasma (CEP) on survivability of some mycoplasma strains growing in agar as well as mycoplasma that most frequently contaminate transplantable human cell lines of normal and malignant origin with the aim of decontamination. MATERIALS AND METHODS: Mycoplasma hominis, Mycoplasma arginini and Aholeplasma laidlawii grown in agar and mycoplasma that contaminated transplantable human cell lines of normal (MT4) and malignant (HeLa) origin. Plasma source--Plasmatom device that generates CEP at normal atmosphere pressure and environment temperature. Exposure to plasma was carried out with adherence to the same modes for all the variants of biological substrate. The duration of exposure was selected randomly from 15 to 300 seconds. RESULTS: A pronounced bactericidal effect of high doses of CEP on all the tested mycoplasma variants exposed immediately after seeding into agar was shown. However after a passage a residual number of survived colonies was registered. Passage of colonies exposed in grown state even to high doses of CEP also showed survival of a residual number of bacteria in all the tested mycoplasma species. Exposure of M. hominis immediately after seeding to low doses of CEP resulted in formation of unusual mini-colonies identical to those isolated from humans infected by the same mycoplasma. During microbiological seeding into agar of cultural fluid from 2 spontaneously contaminated strains of transplantable human cells and exposed to CEP growth ofmycoplasma was not detected. CONCLUSION: CEP has pronounced bactericidal properties on various mycoplasma strains growing in both agar and contaminating eukaryotic cells. However even at high doses of exposure to CEP an insignificant part of bacterial cells growing in agar still survives. This may indicate a high degree of heterogeneity and adaptation of mycoplasma subjected to even such hard exposure as cold plasma with plasma-chemical mechanism of destruction of biological substrate.

[Prospects for the use of low-temperature gas plasma as an antimicrobial agent].

Results of application of LTP at atmospheric pressure as an antibacterial agent during the last decade are considered with reference to physicochemical mechanisms of its bactericidal action. The principles of designing modern LTP sources are described in conjunction with the results of LTP application against pathogenic bacteria in vitro and in biofilms. The possibility to destroy biofilm matrix by LTP is estimated along with the results of its testing for the treatment of acute and chronic wound surfaces. Prospects for the development of "plasma medicine" in this country and abroad are discussed with special emphasis on its advantages, such as the absence of long-acting toxic compounds, small probability of spontaneous mutations accounting for resistance to LTP, relatively low cost of LTP sources, independence of LTP effect of the surface relief, painless application.

[The atypical forms of mycoplasmas persisting in the organism of mycoplasma's infected persons].

The antigens, DNA and RNA of mycoplasmas are preset in the blood serum of persons infected with urogenital mycoplasmas. The planting of patients' tests of blood serum containing antigen M. hominis on the artificial growth mediums resulted in the growth of mini-colonies of mini-cells (20-50 nm). The colonies subcultured hardly but sometimes formed solid bacterial lawn though never acquired "fried-egg" classical mycoplasma form. The proofs of identity of these colonies to M. hominis are presented. The mini-cells possessed infectiousness and ability to persist on a long-run in the internal organs of experimentally infected mice. Apparently, mini-cells are formed under impact of stress factors of the host immune defense and they are one of forms of mycoplasma's persistence in human organism.

[Influence of low frequency magnetic field on characteristic of Pseudomonas aeruginosa].

AIM: To assess the level of bacteriostatic effect of low frequency magnetic field (LFMF) on Gram-negative bacteria able to form biofilms (Pseudomonas aeruginosa) compared to able to aggregation oligotrophes Caulobactor crescentus, Arcicella aquatica and Verrucomicrobium spinosum. MATERIALS AND METHODS: Frequencies 0.001-100 Hz with magnetic induction value 450 mcT1 together with various variants of time, duration and conditions of cultivation of bacteria were used. Bacteriostatic effectwas assessed by optic methods. RESULTS: Decrease of bacterial growth activity with efficacy coefficient Keef = 0.79 +/- 0.03 was demonstrated. CONCLUSION: LFMF moderately decreases growth of tested bacterial species.

[Mechanisms of interaction between HIV and Mycoplasma arginini in vitro].

AIM: Until now, the problem of effective therapy of HIV-infection is not resolved due to integrative type of interaction of HIV virus with target cell - T-lymphocyte. The study was aimed on search of method of deletion of HIV DNA-provirus from cell's genome. MATERIALS AND METHODS: Non-pathogenic for humans Mycoplasma arginini was used for coinfection of HIV-infected cells in model systems in vitro. RESULTS: Complex of mechanisms was documented leading to: blocking up to 50 - 60% of extracellular virus (according to titration results), cancel of apoptosis in infected cells stained on Hoechst, formation of defective vif(-) virions, which together with arginine-desaminase of M. arginini arrange permissive conditions for activation of cellular APOBEC3G with subsequent disruption of DNA- provirus and blocking of viral infection. As studies of ultrastructure showed, listed events resulted from direct interaction of HIV with mycoplasma. CONCLUSION: The elimination of HIV DNA-provirus is possible by co-infection of T-lymphocytes with M. arginini.

Interlaboratory comparison of cytomegalovirus viral load assays.

To assess interlaboratory variability in qualitative and quantitative cytomegalovirus (CMV) viral load (VL) testing, we distributed a panel of samples to 33 laboratories in the USA, Canada and Europe who performed testing using commercial reagents (n = 17) or laboratory-developed assays (n = 18). The panel included two negatives, seven samples constructed from purified CMV nucleocapsids in plasma (2.0-6.0 log(10) copies/mL) and three clinical plasma samples. Interlaboratory variation was observed in both actual (range, 2.0-4.0 log(10) copies/mL) and self-reported lower limits of detection (range, 1.0-4.0 log(10) copies/mL). Variation observed in reported results for individual samples ranged from 2.0 log(10) (minimum) to 4.3 log(10) (maximum)(.) Variation was greatest at low VLs. Assuming +/- 0.5 log(10) relative to the expected result represents an acceptable result, 57.6% of results fell within this range. Use of commercially available reagents and procedures was associated with less variability compared with laboratory-developed assays. Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). The significant interlaboratory variability in CMV VL observed may be impacting patient care and limiting interinstitutional comparisons. The creation of an international reference standard for CMV VL assay calibration would be an important step in quality improvement of this laboratory tool.

Interlaboratory comparison of epstein-barr virus viral load assays.

To assess interlaboratory variability in qualitative and quantitative Epstein-Barr virus (EBV) viral load (VL) testing, we distributed a panel of samples to 28 laboratories in the USA, Canada and Europe who performed testing using commercially available reagents (n = 12) or laboratory-developed assays (n = 18). The panel included two negatives, seven constructed samples using Namalwa and Molt-3 cell lines diluted in plasma (1.30-5.30 log(10) copies/mL) and three clinical plasma samples. Significant interlaboratory variation was observed for both actual (range 1.30-4.30 log(10) copies/mL) and self-reported (range, 1.70-3.30 log(10) copies/mL) lower limits of detection. The variation observed in reported results on individual samples ranged from 2.28 log(10) (minimum) to 4.14 log(10) (maximum). Variation was independent of dynamic range and use of commercial versus laboratory-developed assays. Overall, only 47.0% of all results fell within acceptable standards of variation: defined as the expected result +/- 0.50 log(10). Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). Kinetics of change in VL appears more relevant than absolute values and clinicians should understand the uncertainty associated with absolute VL values at their institutions. The creation of an international reference standard for EBV VL assay calibration would be an initial important step in quality improvement of this laboratory tool.

[A comparative study of the role of the yadA, invA, and psaA genes in the pathogenicity of Yersinia pseudotuberculosis].

The roles of yadA, invA, and psaA genes introduced into the genetic background of the Y. pseudotuberculosis strain possessing the large p VM82 plasmid in virulence and invasion capacity were studied. Isogenic single mutants as well as double and multiple mutants of these genes were constructed and used. LD50 was used as a measure of virulence and the estimation of the ability to invade mammalian cells and the effect of infection on the weight changes of infected mice were used as additional indicators of pathogenicity. It was shown that the YadA had a major effect on the bacterial virulence when compared with the effects of PsaA and InvA. InvA appears to mediate the main pathway of the cellular invasion. YadA is responsible for the weight loss after infection of mice with sublethal doses of Y. pseudotuberculosis. The effects of YadA on virulence and of InvA on bacterial invasion were independent of the expression of the other genes studied. To our knowledge, this study showed for the first time the direct involvement of YadA in the virulence of Y. pseudotuberculosis in mice. Further pathomorphological studies are required to reveal the differences in the pathogenesis of pseudotuberculosis caused by yadA mutants or yadA+ bacteria of Y. pseudotuberculosis.

Herpes simplex and varicella zoster viruses: forgotten but not gone.

Herpesvirus infections are common complications of organ transplantation. The most frequent herpesvirus infections are caused by cytomegalovirus (CMV), herpes simplex (HSV) and varicella zoster (VZV). Despite expansion of the therapeutic armamentarium, HSV and VZV continue to cause morbidity and occasional mortality in transplant recipients. Here we review the incidence and risk factors for HSV and VZV disease, their clinical presentation, effects of newer immunosuppressive regimens and prophylaxis for HSV and VZV in solid organ transplant recipients.

[Perspective technologies for drug design].

The review present the literature data and the authors findings on perspective technologies for design of active chemical and natural compounds based on small molecules for highly specific therapy and correction of a wide variety of pathophysiological conditions in humans, that show overlapping development processes and have common mediators. The following strategies are discussed: chemogenomics strategy using computer screening libraries for generating of small molecule compounds with advantageous properties; strategy of chemokine network that provide control of many processes, from immunosurveillance to inflammation and from viral infections to cancer; strategy of using cytochrome P450 enzymes that guarantee maximum bioavailability of drugs and prevent their interaction and toxic effect; strategy of using superoxide dismutase enzymes (SOD) correcting superoxide anions overproduction in tissue injury and inflammation, from ischemia, organ transplantation to AIDS and cancer; strategy of telomer maintenance directed to anticancer and antiviral therapy as well as to correction of the aging processes; and strategy of short interfering RNAs capable to induce intracellular immunity to antigens of various origin.

[HIV reproduction inhibition by amino acid and dipeptide derivatives of fullerene C60]. / Ingibirovanie reproduktsii VICH s pomoshch'iu aminokislotnykh i dipeptidnykh proizvodnykh Fullerena C60.

Target-aimed synthesis of a new class of water soluble amino acid and dipeptide derivatives of fullurene (C60 - X) for inhibition of specific virus enzymes, i.e. protease and reverse transcriptase of HIV (P HIV and RT HIV) in cell culture lytic and chronic infections was performed. Out of 13 tested substances, 8 showed inhibitory activity and 5 were effective in pharmacological doses (ID50 varied within 0.46 to 1.0 mcm/ml with respect to the lytic infection and 5.0 to 12.5 mcm/ml with respect to the chronic infection). The activity of (1), (2), (6), (7) and (8) was comparable to that of azidothymidine, a nucleozide inhibitor of RT HIV in the cell culture lytic infection. The substances also showed marked virucidal action. The cytotoxicity (survival, antiproliferative effect) varied from low to very low with respect to the rapidly dividing cells MT4 and HTHIV27 (CD50 > 200-800) and was somewhat higher with respect to PBL (CD50 > 100). The selectivity index (SI = CD50/ID50) was equal to 165-2000 for various samples. The prototype derivatives (1) and (2) had a selective (competitive) inhibitory action on the recombinant protease of HIV with IC50 = 1.25-2.76 mcM, while derivatives (1), (la) and (2) had a noncompetitive inhibitory action on the recombinant reverse transcriptase of HIV (Ki = 7.9-12.1 mM). The pharmacokinetic study of the prototype derivative (1) on laboratory animals revealed no acute or chronic toxicity up to the terminal high concentrations. As for (1), its high interspecies (mice--rabbits) relative bioavailability equal to 110% was shown.

Functional analysis of iceA1, a CATG-recognizing restriction endonuclease gene in Helicobacter pylori.

iceA1 in Helicobacter pylori is a homolog of nlaIIIR, which encodes the CATG-specific restriction endonuclease NlaIII in Neisseria lactamica. Analysis of iceA1 sequences from 49 H.pylori strains shows that a full-length NlaIII-like ORF is present in 10 strains, including CH4, but in other strains, including strain 60190, the ORFs are truncated due to a variety of mutations. Our goal was to determine whether iceA1 can encode a NlaIII-like endonuclease. Overexpression in Escherichia coli of iceA1 from CH4, but not from 60190, yielded NlaIII-like activity, indicating that the full-length iceA1 is a functional endonuclease gene. Repair of the iceA1 frameshift mutation in strain 60190 and its expression in E.coli yielded functional NlaIII-like activity. We conclude that iceA1 in CH4 is a functional restriction endonuclease gene, while iceA1 in 60190 is not, due to a frameshift mutation, but that its repair restores its restriction endonuclease activity.

A three-step strategy for targeting drug carriers to human ovarian carcinoma cells in vitro.

To improve tumor-to-tissue ratios of anticancer agents in radioimmunotherapy, a three-step targeting approach was used to deliver biotinylated liposomes to human ovarian cancer cells (NIH:OVCAR-3, SK-OV-3) in vitro. Targeting was based upon the use of two antibodies specific for the CA-125 antigen that is highly expressed on NIH:OVCAR-3 cells but not expressed on SK-OV-3 cells. Briefly, the approach consists of prelabeling target cells with biotinylated anti-CA-125 antibody and FITC-labeled streptavidin (SAv) prior to administration of biotinylated liposomes containing a marker dye for visualization by confocal laser scanning microscopy (CLSM). In addition, the two anti-CA-125 antibodies (B27.1 and B43.13) were labeled with FITC and incubated with ovarian cancer cells at 37 degrees C from 30 min to 24 h to study binding and uptake kinetics. Shedding kinetics of bound antibody from tumor cells was performed using radiolabeled B27.1. Results demonstrated that both B27.1 and B43.13 specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells. Biotinylation, FITC-labeling and radiolabeling of the antibodies did not compromise immunoreactivity. Less than 6% of the bound B27.1 was shed from tumor cells by 4 h following incubation, and the antibody-antigen complex resided predominantly on the cell surface by 4 h at 37 degrees C with slow internalization by 12-24 h. Biotinylated, conventional liposomes were specifically and effectively delivered to OVCAR-3 cells prelabeled with biotinylated B27.1 and SAv. The slow internalization and shedding properties of these antibodies are useful for multistep pretargeting methods. Thus, a modified targeting strategy, utilizing a bispecific antibody and liposomes, may be feasible for radioimmunoliposomal therapy of ovarian cancer.

Search for x-ray induced acceleration of the decay of the 31-yr isomer of (178)Hf using synchrotron radiation.

Enhanced decay of the 31-yr isomer of (178)Hf induced by x-ray irradiation has been reported previously. Here we describe an attempt to reproduce this result with an intense "white" x-ray beam from the Advanced Photon Source. No induced decay was observed. The upper limits for the energy-integrated cross sections for such a process, over the range of energies of 20--60 keV x rays, are less than 2 x 10(-27) cm(2) keV, below the previously reported values by more than 5 orders of magnitude; at 8 keV the limit is 5 x 10(-26) cm(2) keV.