Persistence of cell-mediated immunity three decades after vaccination with the live vaccine strain of Francisella tularensis.

The efficacy of many vaccines against intracellular bacteria depends on the generation of cell-mediated immunity, but studies to determine the duration of immunity are usually confounded by re-exposure. The causative agent of tularemia, Francisella tularensis, is rare in most areas and, therefore, tularemia vaccination is an interesting model for studies of the longevity of vaccine-induced cell-mediated immunity. Here, lymphocyte proliferation and cytokine production in response to F. tularensis were assayed in two groups of 16 individuals, vaccinated 1-3 or 27-34 years previously. As compared to naïve individuals, vaccinees of both groups showed higher proliferative responses and, out of 17 cytokines assayed, higher levels of MIP-1ß, IFN-γ, IL-10, and IL-5 in response to recall stimulation. The responses were very similar in the two groups of vaccinees. A statistical model was developed to predict the immune status of the individuals and by use of two parameters, proliferative responses and levels of IFN-γ, 91.1% of the individuals were correctly classified. Using flow cytometry analysis, we demonstrated that during recall stimulation, expression of IFN-γ by CD4(+) CCR7(+) , CD4(+) CD62L(+) , CD8(+) CCR7(+) , and CD8(+) CD62L(+) cells significantly increased in samples from vaccinated donors. In conclusion, cell-mediated immunity was found to persist three decades after tularemia vaccination without evidence of decline.

New approaches to diagnosis and therapy of tularemia.

Francisella tularensis is a potent pathogen and a cause of severe human disease. The outcome of tularemia will depend on rapid insertion of appropriate antibiotics. Until recently, effective clinical handling was hampered by shortcomings in laboratory diagnostics. No suitable direct methods were available and, because of risks and isolate recovery difficulties associated with laboratory work, culture has been rarely practiced. Due to achievements from work on modern technology, however, tularemia can now be rapidly and specifically diagnosed. Conventional PCR has been successfully applied on wound specimens of patients acquiring tularemia, and prospects for application on other human specimens are promising. Besides allowing diagnostics at high sensitivity and specificity, the PCR technology will also facilitate the identification of cases of tularemia presenting with aberrant signs and symptoms. Antibiotics for efficacious treatment of tularemia have been available for several decades. Although highly valuable, these drugs are afflicted with adverse effects and/or are available only for parenteral therapy. Recently, quinolones have been shown to afford a new valuable option for treatment of tularemia caused by F. tularensis subsp. holarctica (type B). Experience in treating more severe disease caused by F. tularensis subsp. tularensis (type A) is currently limited. In essence, the clinical handling of tularemia is currently facilitated by new achievements in molecular diagnostics and, at least with regard to type B tularemia, by the introduction of quinolones for therapy.

Revival of the case method: a way to retain student-centred learning in a post-PBL era.

BACKGROUND: In current renewal of medical education, problem-based learning (PBL) is the predominant approach. PBL is afflicted with limitations, which cause uncertainness about its future. A profoundly different approach is the case method, developed a century ago and today attracting much less interest in developmental work than PBL. AIM: To compare the characteristics of PBL and the case method and ask the question of whether the case method may serve as an alternative approach to student-centred learning. METHOD: The comparison was literature-based. RESULTS: PBL implicates fostering of self-directed learning and its prospects deal with depth and retention of knowledge and clinical reasoning skills. Problems are used to define learning goals and to stimulate students' interest in various aspects of an item, rather than just for problem-solving. In the small-group tutorials of a PBL curriculum, the teacher is assigned to facilitate the process of self-directed learning and needs not necessarily be a subject-matter expert. In spite of its exciting philosophy and an increased input of students' and teachers' time, the superiority of PBL as a mode of learning has not been convincingly demonstrated, either in terms of acquisition of knowledge or in clinical performance. Moreover, dysfunction is a well-recognized phenomenon. In some PBL tutorials, indifference towards the group discussion is encountered, including individual quietness or dominant behaviour and incomplete attendance. To cope with dysfunctional problems, efforts are recommended aiming to increase PBL tutors' and students' understanding of the group process. As opposed to PBL, the case method relies strongly on teacher-directed learning. Students are placed in a dilemma or a problem to be solved. After preparatory work, they meet for a discussion, lead by a subject-matter expert, who preferably has experienced the case in reality. As a chairperson, the teacher is supposed to stimulate the discussion and detect gaps and misunderstandings. Due to its teacher-dependent approach to learning, the case method is less susceptible to group dysfunction. The case method is also less resource consuming, primarily because it can be practised in groups several times larger than those of PBL. CONCLUSION: A revival of the case method seems warranted as an alternative means of interactive learning, which is simpler, easier to realize and less time-consuming with regard to both institutions and students.

The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages.

Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-gamma)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-gamma-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-gamma after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS-/-) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47phox-/-) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-L-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO-, FeTPPS, completely reversed the IFN-gamma-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO-. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-gamma-induced macrophage killing of F. tularensis LVS. ONOO- is likely to be a major mediator of the killing.

Advantages of using the multiple case method at the clinical stage of medical education.

The multiple case method is an uncomplicated form of discussion learning. Its primary objective is to make students familiar with knowledge already presented to them in lectures and written material. For each session, a large number of situations within a theme are described in handouts. After individual preparatory work, a group of 20-40 (-60) students cooperates to reach understanding and acceptable handling. The main tasks of the teacher are to stimulate the discussion and detect misunderstandings. In a course on infectious diseases, we have successfully used this method of teaching on a large scale for more than a decade. During 24 consecutive runs of the course, with a total of approximately 500 students over a 6-year period, the method was consistently rated high by the students. The long-term persistence of this method can be attributed not only to its learning value but also to the fact that it is simple and easy to practise with relatively large groups of students, thereby decreasing the risk of teachers' burnout.

Tularaemia in Europe: an epidemiological overview.

Tularaemia exists endemically in most European countries. In some areas, such as Finland and Sweden, outbreaks comprising hundreds of cases are recorded at least once a decade. In other areas, outbreaks of such a magnitude occur only occasionally, except in times of war. Between outbreaks, the natural reservoir of the causative agent, Francisella tularensis, is unknown. The organism replicates intracellularly in protozoans. An association of tularaemia to natural water may be of significance in locating the reservoir. Epidemiological work has to date been slow, but is now facilitated by the development of new molecular methods. Due to a variation in numbers of short sequence-tandem repeats in the bacterial genome, individual strains of F. tularensis can today be distinguished.

Specific antibodies contribute to the host protection against strains of Francisella tularensis subspecies holarctica.

T cells are crucial to the control and eradication of the facultative intracellular bacterium Francisella tularensis. A contributory role of humoral antibodies in the host defence remains to be assessed. We used B-cell-deficient mice to study the possible contribution of antibodies to the defence against the live vaccine strain (LVS) or a clinical isolate of F. tularensis, both belonging to the subspecies holarctica (type B). When B-cell-deficient (Igmu(-/-)) mice of the C57BL/10 background were administered immune serum one day before intradermal injection of LVS, they developed lower bacterial numbers in skin, liver, and spleen than did mice receiving normal serum, and survived a challenge inoculum that was lethal for mice given normal serum. Administration of immune serum to C57BL/10 mice afforded protection also against infection with the clinical isolate of F. tularensis subsp. holarctica. Five days after intradermal inoculation of bacteria of the isolate, animals receiving immune serum showed 4log10 lower bacterial counts in liver and spleen than mice administered normal serum. In mice primed by LVS infection, T-cell immunity and host protection were strong and only a marginal contribution of immune serum against a secondary intradermal infection was demonstrated. Together, these findings show that specific antibodies contribute to the host defence of mice against F. tularensis subsp. holarctica.

Francisella tularensis inhibits Toll-like receptor-mediated activation of intracellular signalling and secretion of TNF-alpha and IL-1 from murine macrophages.

Microbial ligands, including lipopolysaccharide (LPS) and bacterial lipoproteins, activate Toll-like receptors (TLR) of mononuclear phagocytes, thereby inducing proinflammatory cytokines and antimicrobial activity. We show that Francisella tularensis, an intracellular pathogen, is capable of inhibiting this macrophage response. Infection with the live vaccine strain F. tularensis LVS rendered cells of the murine macrophage-like cell line J774A.1 incapable of secreting TNF-alpha or IL-1beta and mobilizing an antimicrobial activity in response to bacterial lipopeptide or Escherichia coli-derived LPS. Inhibition of TNF-alpha secretion occurred also when J774 cells were infected with F. tularensis LVS in the presence of chloramphenicol, but not when they were infected with a mutant of F. tularensis LVS defective in expression of a 23 kDa protein that is upregulated during intracellular infection. Purified F. tularensis LPS did not show an agonistic or antagonistic effect on the E. coli LPS-induced activation of the J774 cells. Francisella tularensis LVS suppressed the capability of the cells to respond to LPS or bacterial lipopeptide (BLP) with activation of nuclear factor kappa B (NF-kappaB), and degradation of the in-hibitor of NF-kappaB, IkappaB, was blocked during the infection. Also the LPS- or BLP-induced phosphorylation of the mitogen-activated protein kinase p38 and the transcription factor c-Jun was inhibited by F. tularensis LVS but not by the 23 kDa protein mutant. In conclusion, F. tularensis appears capable of abrogating the TNF-alpha and IL-1 responses of macrophages induced by E. coli LPS or BLP via a mechanism that involves suppression of several intracellular pathways and is dependent on expression of a bacterial 23 kDa protein.

Distinct roles of reactive nitrogen and oxygen species to control infection with the facultative intracellular bacterium Francisella tularensis.

Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are important mediators of the bactericidal host response. We investigated the contribution of these two mediators to the control of infection with the facultative intracellular bacterium Francisella tularensis. When intradermally infected with the live vaccine strain F. tularensis LVS, mice deficient in production of RNS (iNOS(-/-) mice) or in production of ROS by the phagocyte oxidase (p47(phox-/-) mice) showed compromised resistance to infection. The 50% lethal dose (LD(50)) for iNOS(-/-) mice was <20 CFU, and the LD(50) for p47(phox-/-) mice was 4,400 CFU, compared to an LD(50) of >500,000 CFU for wild-type mice. The iNOS(-/-) mice survived for 26.4 +/- 1.8 days, and the p47(phox-/-) mice survived for 10.1 +/- 1.3 days. During the course of infection, the serum levels of gamma interferon (IFN-gamma) and interleukin-6 were higher in iNOS(-/-) and p47(phox-/-) mice than in wild-type mice. Histological examination of livers of iNOS(-/-) mice revealed severe liver pathology. Splenocytes obtained 5 weeks after primary infection from antibiotic-treated iNOS(-/-) mice showed an in vitro recall response that was similar in magnitude and greater secretion of IFN-gamma compared to cells obtained from wild-type mice. In summary, mice lacking expression of RNS or ROS showed extreme susceptibility to infection with F. tularensis LVS. The roles of RNS and ROS seemed to be distinct since mice deficient in production of ROS showed dissemination of infection and died during the early phase of infection, whereas RNS deficiency led to severe liver pathology and a contracted course of infection.

In vitro susceptibility to quinolones of Francisella tularensis subspecies tularensis.

Francisella tularensis is a potent pathogen and a possible bioterrorism agent, for which quinolones offer promising new therapeutic options. There are, however, no data on the susceptibility to quinolones of natural isolates of F. tularensis tularensis, the highly virulent North American subspecies. In the present study, 8 isolates of F. tularensis tularensis, originating from 8 different states of the USA, and 16 US isolates of F. tularensis holarctica were tested. All 24 isolates showed MIC values < or = 0.125 mg/l to 6 different quinolones. Against ciprofloxacin, the predominant quinolone used to date in therapy against subspecies holarctica, MIC values were consistently < or = 0.064 mg/l. Thus quinolones seem to be promising options for the treatment of tularemia, including cases caused by the highly virulent subspecies F. tularensis tularensis.