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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-1β, 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.

Lymphocytes purified from human blood responded poorly to phytohaemagglutinin (PHA). The response was increased by addition of red cell membranes but not by soluble products from red cells. PHA-coated red cell membranes stimulated purified lymphocytes. The response obtained was higher than that caused by an optimal concentration of PHA in soluble form. The results indicate that a complex of PHA and red cell membrane has higher lymphocyte stimulating activity than has free PHA.

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.

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.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.The comparison was literature-based.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.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.

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 bacteriumFrancisella tularensis. When intradermally infected with the live vaccine strainF. tularensisLVS, mice deficient in production of RNS (iNOS−/−mice) or in production of ROS by the phagocyte oxidase (p47phox−/−mice) showed compromised resistance to infection. The 50% lethal dose (LD50) for iNOS−/−mice was 50for p47phox−/−mice was 4,400 CFU, compared to an LD50of >500,000 CFU for wild-type mice. The iNOS−/−mice survived for 26.4 ± 1.8 days, and the p47phox−/−mice survived for 10.1 ± 1.3 days. During the course of infection, the serum levels of gamma interferon (IFN-γ) and interleukin-6 were higher in iNOS−/−and p47phox−/−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-γ compared to cells obtained from wild-type mice. In summary, mice lacking expression of RNS or ROS showed extreme susceptibility to infection withF. tularensisLVS. 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.

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.

In humans, expansion of circulating Vγ9Vδ2 T cells seems to be a pathophysiological denominator shared by protozoan and intracellular bacterial diseases. The assumption was tested here on legionellosis, a condition conforming to the category but not yet described with respect to γδ T cells. Levels of Vγ9Vδ2 T cells in peripheral blood were measured at various intervals in 14 subjects undergoing a Pontiac fever-like disease, shown by serological investigation to be caused byLegionella micdadei. In samples obtained 4 to 6 days after the onset of the disease, the mean percentage (± the standard deviation) of Vγ9Vδ2+T cells among CD3+cells was 1.0% ± 0.5%, compared to 5.0% ± 3.9% in healthy control subjects (P< 0.001). Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset, mean peak levels were as high as ≈15%. During the next 6 months, values slowly declined, although without reaching the normal range. Percentages of γδ+T cells expressing tumor necrosis factor alpha or gamma interferon in response to phorbol myristate acetate were assayed in vitro. At 14 to 16 days after the onset of disease, the expression of both cytokines was increased (P< 0.01), whereas at 5 to 7 weeks, the expression of tumor necrosis factor alpha was decreased (P< 0.05), possibly reflecting modulation of an inflammatory response. In conclusion, Pontiac fever was found to be associated with a pronounced and long-lasting expansion of Vγ9Vδ2 T cells, implying that the subset may also be pathophysiologically important in a mild and transient form of intracellular bacterial diseases. Surprisingly, the expansion was preceded by a depletion of circulatory Vγ9Vδ2 T cells. Possibly, Vγ9Vδ2 T cells are initially recruited to a site of infection before they expand in response to antigen and occur in high numbers in blood.

Children with tularemia are, irrespective of severity of disease, usually subjected to parenteral treatment with aminoglycosides. Based on available susceptibility testing, quinolones might be effective oral alternatives of parenteral therapy. These drugs cause arthropathy in immature animals, but this risk is currently regarded to be low in humans.In 12 patients (median age, 4 years; range, 1 to 10) with ulceroglandular tularemia, a 10- to 14-day course of oral ciprofloxacin, 15 to 20 mg/kg daily in 2 divided doses, was prescribed. Microbiologic investigations included identification of the infectious agent by PCR and culture of wound specimens, as well as determination of antibiotic susceptibility of isolates of Francisella tularensis.Defervescence occurred within 4 days of institution of oral ciprofloxacin in all patients. After a median period of 4.5 days (range, 2 to 24), the patients were capable of outdoor activities. In 2 cases, treatment was withdrawn after 3 and 7 days because of rash. In both cases a second episode of fever occurred. All children recovered without complications. In 7 cases F. tularensis was successfully cultured from ulcer specimens and tested for susceptibility to ciprofloxacin. MIC values for all isolates were 0.03 mg/l.In our sample of 12 patients ciprofloxacin was satisfactory for outpatient treatment of tularemia in children.

PCR and culture were comparatively evaluated for their abilities to demonstrate Francisella tularensis in wound specimens from tularemia patients during an outbreak in Sweden in 1998. For transport of the specimens used for PCR, a buffer solution containing a nuclease inhibitor was used, and for transport of the specimens used for culture, a commercial transport system was selected after experimental comparison of various systems. Of 40 patients with culture- and/or serology-verified ulceroglandular tularemia, PCR detected F. tularensis DNA in 30 (75%) patients, whereas culture detected bacterial growth in 25 (62%) patients. Compared to data from a previous study, the present inclusion of a nuclease inhibitor in the transport medium did not improve the sensitivity of the PCR, whereas the sensitivity of the culture procedure was significantly increased by selection of the system used for transport. Among eight patients with clinically suspected tularemia but with negative serology and culture, specimens from four patients showed detectable DNA. In three of these patients the diagnosis was verified by the demonstration of an F. tularensis -specific T-cell response in vitro. In conclusion, PCR was more sensitive than culture for demonstration of F. tularensis in wound specimens. Besides, we showed that tularemia may proceed without development of serum antibodies, and in these patients, PCR may be of special importance for verification of the diagnosis.

We here studied the antibody response to a booster dose four years after the administration of one single dose of recombinant HB vaccine. Before receiving the booster dose, levels of protective antibodies (anti-HBs) were generally low and 24/41 (59%) individuals lacked detectable antibodies (1 IU/L). Within 14 d of booster vaccination, 36/38 (95%) vaccinees showed levels of antibodies100 IU/L. Notably, these levels were at least as high as those of a reference group 12 months after initiation of vaccination according to the standard three-dose vaccination at intervals of 0, 1 and 6 months. In conclusion, one single dose of HB vaccine seemed to confer on young healthy individuals a well preserved B cell memory, disclosed as a rapid and strong antibody response to a second dose four years later.

Bank voles (Clethrionomys glareolus) serve as the reservoir for Puumala (PUU) virus, the aetiologic agent of nephropathia epidemica. The animals are believed to be persistently infected and the occurrence of serum antibodies is usually taken as an evidence of active infection. We found serum antibodies to PUU virus in 42 of 299 wild bank voles captured in a PUU virus endemic area. PUU virus RNA was demonstrated in lung specimens of 11 of these 42 animals and in 2 of them antigen was also found. Thus in the lungs of 31 of 42 seropositive animals neither PUU virus RNA nor antigen was detected. In 2 of 257 seronegative animals, lung specimens showed presence of PUU virus antigen and RNA. Isolation of PUU virus from lung tissue was successful in all 4 antigen-positive bank voles but in none of 16 tested antigen-negative animals. In conclusion, only a minority of bank voles with serum antibodies to PUU virus showed evidence of current infection.

Puumala virus, the causative agent of nephropathia epidemica (NE), occurs endemically in Europe and is spread mainly by the bank vole (Clethrionomys glareolus). In the vicinity of each of four households afflicted with NE, we studied rodents with regard to population density and prevalence of Puumala virus-specific antibodies. For each case area, a control area was randomly selected 10 km away, without regard to the presence of human settlement. During 6,000 trap nights, 328 rodents were caught, of which 299 were C. glareolus. The mean rodent densities of case and control areas were 6.6 and 3.7 animals per 100 trap nights (P < 0.001). The prevalence of serum antibodies was 15.9% in case areas compared with 5.6% in control areas (P < 0.05). In three of the case areas, where NE had occurred 3-10 weeks before trapping, the rodent density and seroprevalence were much higher than in the fourth area, where NE occurred 38 weeks before trapping. In conclusion, C. glareolus seropositive for Puumala virus occurred more frequently near households afflicted with NE than in control areas 10 km away.

The groE operon of Francisella tularensis LVS, encoding the heat shock proteins chaperone-10 (Cpn10) and Cpn60, was sequenced and characterized, and the T-cell response of LVS-vaccinated individuals to the two proteins and the third major chaperone, Ft-DnaK, was assayed. The cpn10 and cpn60 genes were amplified by PCR with degenerate oligonucleotides derived from the N-terminal sequence of the two proteins. The sequence analysis revealed the expected two open reading frames, encoding proteins with estimated Mrs of 10,300 and 57,400. The deduced amino acid sequences closely resembled Cpn10 and Cpn60 proteins of other prokaryotes. The genes constituted a bicistronic operon, the cpn10 gene preceding the cpn60 gene. Upstream of the cpn10 gene, an inverted repeat and motifs similar to -35 and -10 sequences of sigma70-dependent but not of sigma32-dependent promoters of Escherichia coli were found. The inverted repeat of the operon resembled so-called hairpin loops identified in other characterized prokaryotic groE operons lacking sigma32-dependent promoters. Primer extension analysis disclosed one and the same transcription start, irrespective of the presence or absence of heat or oxidative stress. After separation of lysates of the F. tularensis LVS organism by two-dimensional gel electrophoresis, DnaK, Cpn60, and Cpn10 were extracted and used as antigens in T-cell tests. When compared to those from nonvaccinated individuals, T cells from individuals previously vaccinated with live F. tularensis LVS showed an increased proliferative response to DnaK and Cpn60 but not to Cpn10. The present data will facilitate further studies of the involvement of the heat shock proteins in protective immunity to tularemia.

Since 1931, when tularaemia was first recognized in Sweden, the annual incidence has varied widely. Except for a few cases, ulceroglandular and respiratory tularaemia have been the only forms of the disease observed. Here, cases from Sweden of oropharyngeal tularaemia and of tularaemia septicaemia and meningitis, are reviewed. Since the cases occurred outside manifest outbreaks, diagnostic difficulties were encountered and the diagnosis was reached more by chance than due to clinical suspicion. Possibly, cryptic cases of tularaemia may be more frequent than what appears from clinical reports.

In 15 consecutive subjects hospitalized with nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome, the plasma concentrations of nitrate plus nitrite, stable metabolites of nitric oxide, were determined. From day 3 of onset of disease the concentrations increased, peak levels being reached on days 5 to 7. Maximal plasma concentrations of nitrate plus nitrite were correlated to the degree of hypotension (r = -0.64, p = 0.02) and levels of tumor necrosis factor (TNF)-alpha (r = 0.51, p = 0.05) and soluble TNF receptors p55 and p75 (r = 0.58, p = 0.03 and r = 0.54, p = 0.04, respectively) but not to levels of interferon-gamma or interleukin-10 (p0.05). The results are compatible with the well-known capacity of TNF-alpha to enhance production of nitric oxide, and suggest that nitric oxide may be of physiologic importance in hemorrhagic fever with renal syndrome.

Cytokine mRNA expression was determined in the liver of mice subcutaneously inoculated with a lethal dose of the highly virulent strain FSC 041 of Francisella tularensis subvar, tularensis or a sublethal dose of the live vaccine strain of F. tularensis subvar. palaearctica. Expression of mRNA for TNF-alpha, IL-12, IFN-gamma, and IL-10 was demonstrated within 48 h of inoculation, the kinetics being similar irrespective of bacterial strain used. Thus, the expression of a cytokine response believed to be important in the early host defence against live vaccine strain seemed insufficient to prevent the lethality of a more virulent strain.

Plasma levels of cytokines were measured by EIA in 15 subjects hospitalized with nephropathia epidemica, a European form of hantavirus-induced hemorrhagic fever with renal syndrome. Concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 were increased in all patients at admission, and the concentration of IL-10 was increased in most. TNF-alpha concentrations were still increased 1 week after onset of disease; levels of IL-6 and IL-10 were normalized. TNF-alpha was undetectable by the WEHI cell assay in serum samples obtained throughout the acute phase of disease. Serum levels of the two soluble TNF receptors p55 and p75 correlated with levels of the cytokine, indicating that receptor binding may be the reason for lack of bioactivity in vitro. TNF-alpha is known to induce pathophysiologic and clinical changes similar to those seen in nephropathia epidemica and in diseases caused by other hantaviruses.