Tatyana Radoucheva

Protective activity of Lentinan in experimental tuberculosis.

Protective effects of Lentinan (Ajinomoto, Japan) against Mycobacterium tuberculosis infection were studied by in vitro and in vivo mouse models. The effectiveness of Lentinan administrated intraperitoneally (i.p.) before infection at a dose of 1 mg/kg three times at 2-day intervals was monitored in vivo by several parameters (body temperature; spleen weight; CFU counts of M. tuberculosis in spleen, liver and lung; and histomorphological observations). Peritoneal macrophages obtained from animals treated with Lentinan were greatly stimulated, as assayed by establishing their number, acid phosphatase activity, H2O2 production and killing ability against M. tuberculosis in vitro. The in vivo model demonstrated that administration of Lentinan before infection can mobilize host defense potential and reduce mycobacterial infection.

Effects of intraperitoneal and intranasal application of Lentinan on cellular response in rats.

Lentinan (Ajinomoto, Japan) was administrated intraperitoneally (i.p.) and intranasally (i.n.) at different doses (1, 5 and 10 mg/kg) to rats. Effectiveness of Lentinan treatment was evaluated by comparative testing of cell activation (establishing the number, glycolytic and acid phosphatase activity, H2O2 production and killing ability against Salmonella enteritidis and Staphylococcus aureus) at two different compartments--peritoneal and broncho-alveolar cavities. The results indicated that Lentinan induced high-grade activation of peritoneal cells (PCs) and especially of broncho-alveolar cells (BACs) with markedly enhanced effector function (killing ability against S. aureus). Generally, Lentinan, known usually with its parenteral routes of application, can be successful to stimulate the host cell response in the respiratory tract by intranasal route of administration.

Cell wall-deficient forms (L-forms) of Listeria monocytogenes in experimentally infected rats

Experimental infections were induced with different bacterial forms of Listeria monocytogenes: parental (S-forms), protoplastic (L-forms) and combined inoculum of both forms by i.p. injection of rats. The parental bacterial forms (S-forms) were isolated up to 7 days after challenge from the peritoneal cavity and the liver, while the L-forms were isolated up to 60 days from the peritoneal cavity. Continuous adhesion of L-forms on the peritoneal macrophage surface was found by scanning-electron microscopy. Erythrocyte and leucocyte count as well as some clinical chemistry parameters were measured during infections. They showed different dynamics in the three experimental groups. Histomorphological changes in the liver (microabscesses and mononuclear cellular granulomas) of infected animals were observed. They were less intensive and appeared later in rats infected with L-forms. The experiments demonstrated that infections caused by parental bacterial forms and by combined inoculum took an acute course, while the infection caused by L-forms could be distinguished as a prolonged and persistent one.

Exhibition of persistent and drug-tolerant L-form habit of Mycobacterium tuberculosis during infection in rats

A model for studying mycobacterial L-form formation in vivo was established to demonstrate the ability of M. tuberculosis to behave as a drug-tolerant L-form persister. Rats were infected by intranasal (i.n.) and intraperitoneal (i.p.) routes with 1×108 cells/ml of M. tuberculosis. At weekly intervals during a period of five weeks, samples from lung, spleen, liver, kidney, mesenterial and inguinal lymph nodes, broncho-alveolar and peritoneal lavage liquid were plated simultaneously on Löwenstein-Jensen (LJ) medium or inoculated into specially supplemented for L-forms Dubos broth (drug-free and drug-containing variants). The use of liquid media enabled isolation of mycobacterial L-form cultures during the whole period of experiment including the last two weeks, when tubercle bacilli were not isolated on LJ medium. An unique feature of mycobacterial L-forms was their ability to grow faster than the classical tubercle bacilli. Isolation and growth of L-form cultures in primary drug-containing media demonstrated their drug-tolerant properties. Electron microscopy of liquid media isolates showed that they consisted of morphologically heterogenous populations of membrane-bound and of variable sized L-bodies that completely lack cell walls. The identity of the isolated non-acid fast and morphologically modified L-forms as M. tuberculosis was verified by specific spoligotyping test. The results contribute to special aspects concerning the importance of mycobacterial L-form phenomenon for persistence and latency in tuberculosis, phenotypic drug tolerance, as well as for diagnosis of difficult to identify morphologically changed tubercle bacilli which are often mistaken for contaminants.

Killing of Pseudomonas pseudomallei by polymorphonuclear leukocytes and peritoneal macrophages from chicken, sheep, swine and rabbits.

Differences in the kinetics of Pseudomonas pseudomallei killing by peritoneal macrophages (PM) and polymorphonuclear leucocytes (PMNL) from chickens, sheep, swine and rabbits were found. P. pseudomallei was rapidly killed by porcine PM and PMNL. However the bacterial killing by ovine and lapine PM and PMNL proceeded at a slower rate. In contrast, chicken PM and PMNL ingested and killed the lowest number of P. pseudomallei bacteria. The differences in the bactericidal activity of PM and PMNL from different animal species correlated with the level of their acid phosphatase and glycolytic activity.

Behaviour of Salmonella dublin in mice and rats upon intraperitoneal infection

The fate of Salmonella dublin in mice and rats after intraperitoneal infection was studied in relation to the bacterial net growth rate in the peritoneal cavity, liver and spleen, and to the corresponding inflammatory cellular response in the peritoneal cavity. Rats showed a better host defence, expressed by a greater cellular inflammatory response followed by suppressed bacterial growth. In contrast, the cellular response of mice to the higher bacterial growth was relatively low. These results may explain some mechanisms of natural resistance to salmonellosis in different hosts.