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.

Flt3 ligand enhances the immunogenicity of a gag-based HIV-1 vaccine.

Liposomes and Flt3 ligand (Flt3L), a ligand for the fms-like tyrosine kinase receptor Flt3/ FLK2, can augment the immune response to an HIV peptide vaccine. The HGP-30 peptide used in these studies is a synthetic peptide that corresponds to a highly conserved region of HIV-1 p17 gag (amino acids 86-115). Mice were immunized with HGP-30 or HGP-30 conjugated to keyhole limpet hemocyanin (KLH) and delayed-type hypersensitivity (DTH) responses, antibody (IgG) amount and antigen-specific proliferative responses by spleen cells were used to monitor the immune response. Daily injections of Flt3L prior to HGP-30 administration enhanced significantly an antigen-specific lymphocyte proliferation response when compared with Flt3L, HGP-30 alone or HGP-30 containing liposomes. Intravenous administration of HGP-30 was superior to intramuscular (i.m.) immunization for the induction of DTH responses. The HGP-30/KLH containing liposomes enhanced both DTH and antibody responses, while liposomes containing HGP-30 peptide elicited only T cell responses. In these studies, either Flt3L or liposomes increased DTH responses compared with the i.m. injection of the HGP-30 vaccine alone.

In vivo and in vitro immunomodulation induced by the extract of the mycelium fungus Polyporus squamosus.

The mycelial mass of the fungus Polyporus Squamosus strain 64 (PS-64) was disintegrated by mechanical and ultrasound treatments. After centrifugation, the supernatant containing the disintegrate was dialyzed and lyophilized. The resultant PS-64 extract was subsequently investigated as an immunomodulator of IgE and IgG responses to ovalbumin (OA) in (CBAxC57BL/6)F1 mice using passive cutaneous anaphylaxis (PCA) and enzyme-linked immunosorbent assay (ELISA), respectively. Multiple injections of PS-64 extract in doses of 1.5, 15, and 150 mg/kg administered before the primary or secondary immunization of mice with OA resulted in a dose-dependent inhibition of both IgE and IgG antibody responses to OA. In contrast to the inhibition of the anti-OA IgE response noted during the entire 3-week observation period, the anti-OA IgG response was restored to control level by the third week of secondary immunization. The glass microfiber-based whole blood histamine release assay demonstrated that various concentrations of the PS-64 extract did not influence histamine release induced either by anti-IgE or by specific allergens from basophils derived from whole blood of allergen-sensitized patients. Using the hemolytic plaque assay, significant suppression of IgM-secreting cell formation was noted in (CBAxC57BL/6)F1 mice administered various doses of the PS-64 extract before immunization. The PS-64 extract inhibited the in vitro proliferation of human mononuclear cells upon stimulation with phytohemagglutinin (PHA). In a dose-dependent manner, the PS-64 extract also inhibited delayed-type hypersensitivity reaction and skin graft rejection, similar to the effect noted with usage of Cyclosporin A (CsA) in (CBAxC57BL/6)F1 mice. Our investigation suggests that the immunomodulatory effects of PS-64 should be studied further for potential clinical therapeutic utility.

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.

[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.

[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.

[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.

[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.

[Cells-boosters as the modulators of immunogenesis]. / Kletki-usiliteli kak moduliatory immunogeneza.

In the course of mouse immune response to ram erythrocytes, cells capable of enhancing immune and proliferative response (enhancing cells, or EC) are shown to appear in the spleen. The EC effect is antigen-unspecific. Cycloheximide (CH), an inhibitor of protein synthesis, speeds up EC development, which is associated with a greater immune response. In vitro CH treatment of mouse spleen cells (SC), for 3 hours at 37 degrees C, CH taken in a concentration of 1-3 micrograms/ml, resulted in in vitro induction of EC, capable of enhancing proliferative response to PHA and recombinant IL-2 in normal SC and increasing immune response to ram erythrocytes. Glutaric aldehyde fixation of EC was not reflected in their immunostimulating activity, either in vivo, or in vitro. The data obtained point to the significance of CH-induced changes in membrane-associated structures in mediating EC effect. It is assumed that the agents, altering the expression of functionally-significant structures (membrane-linked mediators and auxiliary intercellular interaction molecules) on cell surface, may be used for extracorporeal treatment of lymphoid cells to be eventually re-introduced into the body to control immunologic disorders.

[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.