Molecular Epidemiology of Hypervirulent K. pneumoniae and Problems of Health-Care Associated Infections.

The review describes virulence factors of hypervirulent K. pneumoniae (hvKp) including genes determining its virulence and discusses their role in the development of health-care associated infections. The contribution of individual virulence factors and their combination to the development of the hypervirulence and the prospects of using these factors as biomarkers and therapeutic targets are described. Virulence factors of hvKp and "classical" K. pneumoniae strains (cKp) with no hypervirulence genes were compared. The mechanisms of biofilm formation by hvKp and high incidence of its antibiotic resistance are of particular importance for in health care institutions. Therefore, the development of methods for hvKp identification allowing early prevention of severe hvKp infection and novel approaches to abrogate its spreading are new challenges for epidemiology, infection diseases, and critical care medicine. New technologies including bacteriological and molecular studies make it possible to develop innovative strategies to diagnose and treat infection caused by hvKp. These include monitoring of both genetic biomarkers of hvKp and resistance plasmid that carry of virulence genes and antibiotic resistance genes, creation of immunological agents for the prevention and therapy of hvKp (vaccines, monoclonal antibodies) as well as personalized hvKp-specific phage therapies and pharmaceuticals enhancing the effect of antibiotics. A variety of approaches can reliably prepare our medicine for a new challenge: spreading of life-threatening health-care associated infections caused by antibiotic-resistant hvKp strains.

[Generation of Antibiotic Tolerant Bacterial Persisters in Immunocompromized Patients with Hematologic and Malignant Diseases: A New Problem of Health-Care Associated Infections].

Background: Antibiotic tolerance (AT) represents one of the causes of the phenomenon of antibiotic resistance that allows escape of non-replicating metabolically inert microorganisms (persisters) from any antibiotics attack because molecular targets of antibiotics are lacking thereby creating the potential for chronic infections. Aims: Determine the heterogeneity of the strains of opportunistic pathogens E. coli and P. aeruginosa isolates from children with hematologic malignancies containing bacterial persisters that cause the AT phenomenon. Methods: Children with hematological malignancies were divided into 2 groups according to the intensity of antibiotic treatment of infectious complications. Ciprofloxacin-induced persisters were quantitatively determined in the biological materials obtained from sick children. Results: Within the clinical isolates of E. coli and P. aeruginosa, about a third of the strains belong to high-persisting. The numbers of persistent forms of bacteria did not correlate with a minimal inhibitory concentration values ciprofloxacin (r=0.148, n=25, p>0.05). Interestingly, higher level of formation of persistent E. coli and P. aeruginosa, is associated with higher frequencies of infection attacks, massive antibiotic use and unfavorable course of the disease in children. Conclusions: Therefore, detecting the persistent forms of bacterial pathogens including those associated with the health-care associated infection, specifically, in immunocompromised patients, should be included into the contemporary algorithms of microbiological observation and monitoring of patients and intrahospital environment.

[Microbial dormancy and prevention of healthcare-associated infections].

Healthcare-associated infections (HCAI) remain one of the most challenges of modern health care and assume increasing social and medical significance. The specific features of HCAI are frequent recurrences and inefficiency of antibiotic therapy, a reason for which is antibiotic resistance in microorganisms. The review discusses antibiotic resistance, a form of antibiotic tolerance (AT), and its role in the development of HCAI. It also describes essential differences between AT and antibiotic tolerance at the cellular and molecular genetic levels. Relationships between AT and dormancy of microorganisms, pathogens of HCAI, are discussed. The paper gives the data available in the literature on how AT occurs in HCAI pathogens and discusses the diagnosis of this condition. It also analyzes the literature data on pharmacological attempts to overcome AT and discusses novel approaches to antibiotic therapy for HCAI.

[Effect of Inherent Immunity Factors of Development of Antibiotic Tolerance and Survival of Bacterial Populations under Antibiotic Attack].

Effect of human inherent immunity factors of, a gene-encoded antibacterial peptide indolicidin (Ind) and a cytokine interleukin 1 (IL1) on formation of antibiotic-tolerant persister cells surviving in the presence of ciprofloxacin (Cpf, 100 µg/mL) and ampicillin (Amp, 100 µg/mL) in submerged bacterial cultures (Staphylococcus aureus FGA 209P, Escherichia coli K12, and Pseudomonas aeruginosa PAO1) was studied. While Ind in physiological concentrations (0.3 and 3.0 µg/mL) introduced to the lag- or exponential-phase cultures of test organisms exhibited no reliable effect on population growth, the number of persisters increased at 3.0 µg/mL. Bactericidal Ind concentrations (9 µg/mL) suppressed S. aureus growth (-0.1% of surviving cells) with subsequent recovery due to development of the more antibiotic-tolerant white variant. Treatment with Cpf after Ind addition resulted in mutual potentiation of their antimicrobial activity, with the number of S. aureus persisters 2 to 3 orders of magnitude lower than in the case of the antibiotic alone. IL1, another immunity factor, when introduced (0.1-1 ng/mL) to the exponentially growing S. aureus culture (but not to the lag phase culture) had a temporary growth-static effect, with the number of persisters surviving Cpf treatment (100 µg/mL) increasing by 1 to 2 orders of magnitude. Electron microscopy revealed significant alterations in the outer cell envelope layer of surviving S. aureus cells, which should be associated with their changed antigenic properties. Thus, the factors of human inherent immunity have a dose-dependent effect on the growth of bacterial populations. In combination with antibiotics, they exhibit synergism of antimicrobial action (indolicidin) and minimize (indolicidin) or increase (interleukin 1) the frequency of formation of persister cells responsible for survival of a population subjected to an antibiotic attack.

Delivery of Flt3 ligand (Flt3L) using a poloxamer-based formulation increases biological activity in mice.

Fms-like tyrosine kinase (Flt3L) is a potent stimulator of hematopoietic progenitor cell (HPC) expansion and mobilization; however, this requires 7-10 days of administration. We investigated whether sustained delivery of Flt3L using a poloxamer-based matrix (PG) could accelerate and/or improve the hematopoietic activity of Flt3L in mice. A single injection of PG-Flt3L stimulated significantly more rapid and greater HPC mobilization to the spleen and peripheral blood than the daily injection of Flt3L formulated in saline. Pharmacokinetic analysis demonstrated that the formulation of Flt3L in PG prolonged its elimination (Tbeta) half-life (2.3-fold) and increased its bioavailability (>two fold) and the time to maximum serum concentration (T(max)) (2.7-fold). Further, coadministration of G-CSF and PG-Flt3L allowed lower doses of Flt3L to be active, with significantly greater hematopoietic and mobilization activity, compared to the same total dose of G-CSF, Flt3L or G-CSF and Flt3L formulated in saline. These data demonstrate that formulation of Flt3L in PG significantly accelerates and increases HPC expansion and mobilization. The observation of increased bioactivity by PG-Flt3L in rodents suggests the potential for improved clinical efficacy of Flt3L by reducing the time required for HPC mobilization.

[Genetic aspects of the action of immunosuppressive agents]. / Geneticheskie aspekty deistviia razlichnykh immunodepressantov.

The data were obtained formerly that mice of certain strains essentially differ in the sensitivity to the immunodepressive and antiproliferative action of the alkylating agents (so-called opposite strains: DBA/2--highly sensitive, BALB/c--resistant). It is shown in the present work that with the use of the other non-alkylating immunodepressive agents (cytarabine, cyclosporin A, dexamethasone) that differ in the action mode, DBA/2 mice retain a high sensitivity whereas BALB/c mice a low sensitivity to all the immunodepressants. The sensitivity to the immunodepressive action in vivo directly correlates with that of the immunocompetent cells in vitro. Potential mechanisms determining the same type sensitivity to diverse immunodepressant in mice belonging to the above-indicated genotypes are under discussion.

[Ecological immunodeficiency: immunogenetic aspects of its etiology and correction]. / Ekologicheskii immunodefitsit: immunogeneticheskie aspekty ego razvitiia i korektsii.

The examinations of auto-transport Kazakh drivers have indicated that there is a significant reduction in some immunological parameters HLA-B5 and HLA-DR5-proliferative responses of lymphocytes to mitogens, production of interleukin-1 and interleukin-2, activity of NK and LAK-cells. It is suggested that these impairments occur with their long-term exposure to automobile transport effluents (ATE), since the same changes in immunological parameters were found previously in the experiments with animals exposed to ATE for a long time. Some of the detected immunoresponsive disorders are associated with the availability of definite HLA antigens, such as HLA-B5 and HLA-P5. The new immunomodulating agents thymohexin (TH) and phyto-extraction drugs C4 and C6 used in vitro substantially restored the lower functional activity of immunocompetent cells and production of cytokines (thymohexin was particularly effective). The most marked recovery was observed in the drivers with the phenotype HLA-B6+ and HLA-P5+, i.e. in persons with maximally ATE-reduced immunological parameters.

[Mechanisms of inhibiting production of tumor necrosis factor alpha by the synthetic hexapeptide--immunophane]. / Mekhanizmy ingibitsii porduktsii faktora nekroza opukhloi alph sinteticheskim geksapeptidom--immunofanom.

The authors examined the human blood mononuclear-induced tumor necrosis factor production using the new drug--the synthetic hexapeptide Immunophan. The levels of tumor necrosis factor in the supernatant liquid were measured by the enzyme immunoassay and the cytotoxic test using L-929 fibroblastoid cells. Following 2-8 hours of short-term incubation of mononuclear cells with Immunophan, there was a reduction in spontaneous or lipopolysaccharide - or ionophore A23187-induced production of tumor necrosis factor. As high as 5-20% of plastic-nonadherent cells treated with Immunophan in a concentration of 0.25 mu/ml were found to produce the same effect. Two-four hours after Immunophan activation, the cells produced into the supernatant liquid soluble factors with a molecular weight of 70-85 kD that suppressed the production and activity of tumor necrosis factor. Thus, the modulating effect of Immunophan against tumor necrosis factor production is associated with the induction of regulatory cells producing the soluble receptors of tumor necrosis factor. It is suggested that extrabody pharmacological induction of the cells that regulate the production of tumor necrosis factor, followed by their subsequent administration into the autologic organism might be used while developing new variants of extracorporeal treatments of the diseases which are characterized by the pathogenetically significant hyperproduction of the inflammatory cytokins,--tumor necrosis factor, interleukin-1, interleukin-6.

[Tumor necrosis factor in patients with suppurative-septic complications in terminal states and the possibilities for correcting its production]. / Faktor nekroza opukholi u bol'nykh s gnoino-septicheskimi oslozhneniiami terminal'nykh sostoianii i vozmozhnosti korrektsii ego produktsii.

The authors review the results of study of tumor necrosis factor (TNF) production by mononuclears in 37 patients recovered after critical conditions of various genesis. Relationships between TNF production, condition severity and complications development have been established. Use of a new correcting agent thymogexin ensured a modulating effect and reduced an excessively high TNF production both in vitro and in patients with pyoseptic complications.

[Tumor necrosis factor and possibilities of correction of its production in patients with suppurative-septic complications in terminal states]. / Faktor nekroza opukholi i vozmozhnosti korrektsii ego produktsii u bol'nykh s gnoino-septicheskimi oslozhneniiami terminal'nykh sostoianii.

The production of tumor necrosis factor (TNF) by mononuclears was studied in 37 patients after critical states of various origins. A relationship was revealed between the TNF production and the patient's condition severity and the development of pyoseptic complications. Use of thymohexin, a new drug for correction, provided a modulating effect and helped reduce the excessively increased TNF production both in vitro and in the patients with pyoseptic complications.

[Study of the phenomenon of specific suppression of the immune response in an adoptive transfer system]. / Izuchenie fenomena spetsificheskoi supressii immunnogo otveta v sisteme adoptivnogo perenosa.

It was revealed that the administration of the spleen cells (SC) of syngeneic animals immunized with a high dose of sheep red blood cells (SRBC) to intact mice led to a marked specific suppression of the recipients' immune response. The donors' SC obtained on the 14th day after the intraperitoneal injection of SRBC had the greatest suppressive activity. The SC of intact animals and mice given rat erythrocytes preliminarily failed to influence the immune response of the intact recipients in their SRBC immunization. Treatment of immune SC with the anti-T-serum (ATS) or the anti-B-globulin (ABG) and the complement considerably decreased or completely eliminated the suppressive activity. Administration of a mixture of two immune SC suspensions one of which was ATS- and another ABC-treated did not produce any suppression of the immune response in the intact recipients. It is supposed that the suppressor cells in the given model were T-lymphocytes expressing the antigens, common of cross-reacting with the B-cells.

[Supressor activity of spleen cells during medically induced immunologic tolerance]. / O supressornoi aktivnosti kletok selezenki pri lekarstvenno indutsirovannoi immunologicheskoi tolerantnosti.

SRBC tolerance was induced in mice (CBA X C57BL/6) F1 by single intraperitoneal injection of 6 X 10(9) SRBC and of cyclophosphamide (100-200 mg/kg) in 44-46 hours. Spleen cells of tolerant mice obtained at various periods after the tolerance induction (in 12-26 days) failed to decrease their immune response to SRBC after administration to intact syngeneic recipients. Contrary to intact mice, tolerant animals were incapable of producing suppressor cells after a single SRBC immunization. Only when 3 additional injections of high SRBC doses (6 X 10(9)) were given to tolerant mice the spleen cells in them acquired the capacity to inhibit the immune response after administration to normal mice. It is supposed that the absence of suppressor cells in induction of the immunological tolerance by means of cyclophosphane was caused by the processes of clone elimination. Suppressor cells can originate in tolerant animals under the effect of intensive antigenic stimulation, this leading to enhancement of the tolerance state as a result of additional SRBC injections.

[Nonspecific antigenic suppression of the formation of immunologic memory to heteroerythrocytes]. / Antigennespetsificheskaia supressia formirovaniia immunologicheskoi pamiati k chuzherodnym éritrotsitam.

Spleen cells (SC) of mice immunized with sheep red blood cells contain factor that suppresses primary immune response and the formation of immunologic memory. During studies of antigen-specific suppression of the immunogenesis, it has been discovered that suppressor factor of SC non-specifically blocks the development of immunologic memory for the other antigen (rat red cells) without affecting primary immune response to this antigen administration. It is assumed that at an early stage the cells responsible for the formation of immunologic memory are more sensitive to the non-specific effect of suppressor factor than those involved in the generation of primary immune response.

[Analysis of certain mechanisms of antigen-specific suppression of the immune response]. / Analiz nekotorykh mekhanizmov antigenspetsificheskoi supressii immunnogo otveta.

CBA mice were immunized with sheep red blood cells (SRBC) to obtain immune spleen cells (ISc) which were used to suppressor cells. Administration of ISC to intact syngeneic recipients on the immunization day led to a more powerful suppression of the immune response as compared to that seen one day after antigen injection. Four days after immunization the animals' immune response was not liable to be suppressed. ISC extract possessed similar effects with respect to the immune response of normal spleen cells which were transplanted to the cyclophosphamide-treated recipients. The immune response of spleen cells from mice immunized with SRBC in a dose of 10(6) was less liable to be suppressed. Hyperimmune spleen cells from donors immunized with SRC in a dose of 10(9) were insensitive to ISC or to the extract. Experiments with the use of adoptive transfer of a mixture of immune and intact T- and B-cells have disclosed that B-cells from hyperimmune donors were resistant to suppression. Therefore, B-lymphocytes are the most probable target cells exposed to T-suppressors in the given system. The mechanism is discussed of the selective effect of T-suppressors on B-cells in the course of the immune response development during immunization with high doses of antigen.

[Mechanism of the suppression of the formation of immunologic memory]. / O mekhanizme supressii formirovaniia immunologicheskoi pamiati.

An extract from splenocytes of mice immunized with sheep red blood cells contains suppressor factor (SF) that specifically inhibits the primary immune response. The system of adoptive transfer was studied for the action of the SF on the formation of immunologic memory for sheep and rat blood cells. It was established that the SF carrying a receptor for specific antigen causes nonspecific suppression of the generation of immunologic memory cells. Within the system in question radioresistant T helper cells act as suppression targets. It is suggested that the final effect of the SF lies in the inhibition of the release of transmitters promoting differentiation or proliferation of T memory cells.

[Action of diucifon-treated syngeneic splenic cells of mice on the magnitude of the immune response]. / Issledovanie deistviia singennykh kletok selezenki myshei, obrabotannykh diutsifonom, na velichinu immunnogo otveta.

It was demonstrated previously that treatment of lymphocytes with the immunostimulant diucifon leads to the secretion of a substance having the biological activity of T cell growth factor. The present work demonstrates that injection into mice on the day of immunization of spleen syngeneic cells treated with diucifon increases the immune response 3-5-fold as compared to the action of untreated cells. Injection of spleen cells incubated without diucifon on day 3 after immunization significantly increases the immune response as compared with control. The cells treated with diucifon and injected at the same time reduce the immune response as compared with the action of spleen cells incubated without diucifon. The data obtained can be used during immunostimulant therapy.

[Resistance of the suppressor function of T-cells to agents possessing an antiproliferative action]. / Rezistentnost' supressornoi funktsii T-kletok k agentam, obladaiushchim antiproliferativnym deistviem.

Administration of cyclophosphamide in a dose of 50 to 400 mg/kg to mice immunized with sheep red blood cells failed to decrease significantly the capacity of the splenic cells of these mice to suppress the primary immune response in transplantation to intact syngeneic recipients. Irradiation of the donors of immune splenic cells (ISC) in a dose of 900 r or treatment of ISC in vitro with mitomycin C failed to influence their suppressor activity. Supernatant obtained after the ultracentrifugation of ISC treated with ultrasound inhibited the primary immune response of intact mice. A conclusion was drawn that the suppressor effect of ISC was caused by the factor produced by T-cells. Active proliferation of these cells was not necessary for the realization of its action.

[Properties of an antigen-specific suppressor factor of immune spleen cells]. / Svoistva antigenspetsificheskogo supressornogo faktora immunnykh kletok selezenki.

Splenocytes of mice immunized with sheep red blood cells (SRBC) were destroyed by ultrasound and ultracentrifuged at 20.000g for 30 min. The supernatant (SN) was used as a source for suppressor factors. It was shown that suppression in the given system was antigen-specific since the immune response of normal splenocytes (NS) transplanted along with SN into cyclophosphamide-treated (200 mg/kg) recipients considerably diminished in the course of immunization with SRBC rather than with rat red blood cells (RRBC). Absorption of SN with SRBC rather than with RRBC, mouse or human red blood cells completely abolished its suppressor activity. The suppressor factor was absorbed both by syngeneic and allogeneic splenocytes rather than by hepatocytes. The suppressor activity of SN considerably decreased after exposure to pronase and on heating at 56 degrees C for 1 h. The activity of immune serum against SRBC remained unchanged both on heating at 56 degrees C and after absorption with splenocytes. It is assumed that antigen-specific suppressor factor is a protein of non-immunoglobulin nature, bearing receptors for antigen and syngeneic or allogeneic splenocytes. A possibility of specific suppression of the immune response is discussed, not requiring restriction according to H-2 complex.

Intracellular metabolism and action of acyclic nucleoside phosphonates on DNA replication.

9-(2-phosphonylmethoxyethyl)guanine (PMEG) is an acyclic nucleoside phosphonate derivative that has demonstrated significant anticancer activity in a number of in vitro and in vivo animal model systems. In this study, we compared the cellular metabolism of PMEG and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a clinically active anti-HIV and antihepatitis agent, and the inhibitory activities of their putative active diphosphate derivatives, PMEGpp and PMEApp, respectively, toward human cellular DNA polymerases. PMEG was significantly more cytotoxic than PMEA against a panel of human leukemic cells. The diphosphate derivatives were the major metabolites formed in cells on both these agents, with PMEGpp reaching cellular concentration approximately 4-fold higher than that achieved for PMEApp. These differences in cellular accumulation of the diphosphate derivatives were not, however, sufficient to account for the 30-fold difference in cytotoxicity between the two analogs. PMEGpp was also at least a 7-fold more effective inhibitor of in vitro simian vacuolating virus 40 DNA replication system than that of PMEApp (IC50 = 4.6 microM). Studies with a defined primed DNA template showed that PMEGpp was a potent inhibitor of both human polymerases alpha and delta, two key enzymes involved in cellular DNA replication, whereas PMEApp inhibited these enzymes relatively poorly. From these studies, we can conclude that the factors that contribute to the enhanced antileukemic activity of PMEG derives both from its increased anabolic phosphorylation and the increased potency of the diphosphate derivative to target the cellular replicative DNA polymerases.