Arne Tärnvik

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‐α or IL‐1β and mobilizing an antimicrobial activity in response to bacterial lipopeptide or Escherichia coli‐derived LPS. Inhibition of TNF‐α 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‐κB), and degradation of the in‐hibitor of NF‐κB, IκB, 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‐α 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.

New approaches to diagnosis and therapy of tularemia.

Abstract:  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.

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.

Ciprofloxacin for treatment of tularemia in children.

Background. 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. Patients and methods. 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. Results. 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. Conclusion. In our sample of 12 patients ciprofloxacin was satisfactory for outpatient treatment of tularemia in children.

Inhibition of PHA-induced lymphocyte stimulation by the pregnancy zone protein

Mother and child are genetically different and the fetus could be looked upon as an allograft. The reason why this allograft is accepted is unknown. A physiological barrier between mother and fetus [ 1 ], and the presence of blocking antibodies [2] and hormones [3] have been suggested. A depression of the maternal cellmediated immune reactivity [4-6] is another explanation supported by the report of Kasakura [7] that maternal plasma during pregnancy suppressed the mixed leucocyte reaction. The concentration in plasma of many proteins is altered during pregnancy but some proteins are produced in extensively increased amounts. The pregnancy zone protein (PZ) [8-16] is an a2-globulin which is absent or present in only small amounts in the sera of non-pregnant women. During pregnancy the serum level of this globulin is increased in most women from 0 4 rag/100 ml to values between 50-200 mg/100 ml. The maximum is generally reached in the third trimester and after delivery there is a rapid decrease to normal levels [ 17]. In women with early spontaneous abortion PZ is found rarely or in small amounts [18]. The PZ protein has been isolated and found to have a mol. wt. of 326 000 [19]. In the present study PZ was found to inhibit the phytohaemagglutinin (PHA)-induced lymphocyte stimulation, believed to be an in vitro correlate of cellmediated immunity. 2. Materials and methods

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

ABSTRACT 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 (p47phox−/− mice) showed compromised resistance to infection. The 50% lethal dose (LD50) for iNOS−/− mice was <20 CFU, and the LD50 for p47phox−/− mice was 4,400 CFU, compared to an LD50 of >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 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.

Pulmonary involvement in nephropathia epidemica as demonstrated by computed tomography

SummaryIn a prospective study 19 adult patients with nephropathia epidemica were examined in the acute phase of disease with computed tomography (CT) of the lungs and conventional chest radiography. Infiltrates and/or pleural effusions were seen in ten of 19 patients. In two of the patients, abnormalities were disclosed only by CT. Patients with pathologic radiography findings had a more pronounced inflammatory response, as measured by C-reactive protein and leukocyte count, than did those with normal radiography findings. It is concluded that radiological evidence of pulmonary involvement is a common finding early in the course of nephropathia epidemica. The possibility that the lung may be a site of viral replication merits further investigation.ZusammenfassungNeunzehn erwachsene Patienten in der akuten Phase von Nephropathia epidemica wurden mit computertomographischem (CT) und konventionellem Thoraxröntgen untersucht. Infiltrate und/oder Pleuraergüsse wurden bei 10/19 der Patienten beobachtet. Bei zwei Patienten konnten pathologische Befunde nur mit Hilfe der CT erhoben werden. Patienten mit pathologischen Radiogrammen wiesen, verglichen mit normalen radiographischen Befunden, eine ausgeprägtere Entzündungsreaktion gemessen durch C-reaktives Protein und Leukozytenzahlen auf. Unsere Resultate zeigen, daß in der Frühphase der Nephropathia epidemica röntgenologisch häufig eine Lungenbeteiligung nachgewiesen werden kann. Die Möglichkeit, der Lunge als eine Lokalisation der viralen Replikation sollte weiter untersucht werden.In a prospective study 19 adult patients with nephropathia epidemica were examined in the acute phase of disease with computed tomography (CT) of the lungs and conventional chest radiography. Infiltrates and/or pleural effusions were seen in ten of 19 patients. In two of the patients, abnormalities were disclosed only by CT. Patients with pathologic radiography findings had a more pronounced inflammatory response, as measured by C-reactive protein and leukocyte count, than did those with normal radiography findings. It is concluded that radiological evidence of pulmonary involvement is a common finding early in the course of nephropathia epidemica. The possibility that the lung may be a site of viral replication merits further investigation. Neunzehn erwachsene Patienten in der akuten Phase von Nephropathia epidemica wurden mit computertomographischem (CT) und konventionellem Thoraxröntgen untersucht. Infiltrate und/oder Pleuraergüsse wurden bei 10/19 der Patienten beobachtet. Bei zwei Patienten konnten pathologische Befunde nur mit Hilfe der CT erhoben werden. Patienten mit pathologischen Radiogrammen wiesen, verglichen mit normalen radiographischen Befunden, eine ausgeprägtere Entzündungsreaktion gemessen durch C-reaktives Protein und Leukozytenzahlen auf. Unsere Resultate zeigen, daß in der Frühphase der Nephropathia epidemica röntgenologisch häufig eine Lungenbeteiligung nachgewiesen werden kann. Die Möglichkeit, der Lunge als eine Lokalisation der viralen Replikation sollte weiter untersucht werden.

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.

Booster vaccination with recombinant hepatitis B vaccine four years after priming with one single dose

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 antibodies > 100 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.

Adjuvanticity of ISCOMs incorporating a T cell-reactive lipoprotein of the facultative intracellular pathogen Francisella tularensis.

Immunostimulating complexes (ISCOMs) are known to be highly effective adjuvants for envelope antigens of viral agents, but have not been evaluated for use with antigens of intracellular bacteria. Balb/c mice were subcutaneously immunized with ISCOMs into which the T cell-reactive membrane protein TUL4 of Francisella tularensis had been incorporated. Spleen cells from the immunized mice responded in vitro to TUL4 and to heat-killed F. tularensis live vaccine strain (LVS) with proliferation and production of gamma-interferon, whereas spleen cells from control mice immunized with TUL4 only did not respond to the antigens. When mice immunized with TUL4 ISCOMs were challenged with F. tularensis LVS, bacterial counts in spleen and liver were lower than in non-immunized mice. Again, TUL4 had no effect when used without ISCOMs. When proteins of a total membrane preparation of F. tularensis LVS were incorporated in ISCOMs and used for immunization, a decrease in bacterial counts was obtained which was similar in magnitude to that of TUL4 ISCOMs. Generally, the adjuvant effects demonstrated did not compare with the excellent protective effect of live tularaemia vaccine. Nonetheless, ISCOMs provide a means whereby protective antigens of F. tularensis can be tested.