Jürgen Heesemann

Cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants.

Stage conversion between the tachyzoite and bradyzoite forms of the protozoan parasite Toxoplasma gondii is an important aspect in the pathogenesis of toxoplasmosis. In an initial investigation of molecular regulation of stage conversion in T. gondii, we describe the cloning and characterization of a bradyzoite‐specifically expressed gene (hsp30/bag1). Bradyzoite formation was induced in cell culture by alkaline pH, and this was followed by purification of this parasitic stage using magnetic cell sorting. A bradyzoite cDNA library was constructed by random amplification using the polymerase chain reaction. Screening with a bradyzoite‐specific monoclonal antibody identified a reactive clone. The amino acid sequence derived from the 687 bp open reading frame showed similarity to the conserved C‐terminal region of small heat‐shock proteins from plants. Stage‐specific expression of the naturally occurring 30kDa antigen in bradyzoites was confirmed by polyclonal antisera generated against the recombinant antigen, Immuno‐electron microscopy indicated a cytosolic location of this antigen in bradyzoites. The expression of HSP30/BAG1 seems to be regulated at the mRNA level, since reverse polymerase chain reaction using bradyzoite‐specific primers amplified transcripts in bradyzoites only, not in tachyzoites.

High incidence of serum antibodies to Escherichia coli O157 lipopolysaccharide in children with hemolytic-uremic syndrome

Because the classic hemolytic-uremic syndrome has been etiologically linked to intestinal infections by Escherichia coli O157 and other verotoxin-producing E. coli (VTEC), we examined 22 consecutive children with acute hemolytic-uremic syndrome for the presence of VTEC, using microbiologic methods, and for a specific immune response to O157 lipopolysaccharide in acute-phase and follow-up sera, using the indirect hemagglutination assay and the immunoblot procedure. Of 22 children with enteropathic hemolytic-uremic syndrome, 15 (68%) had evidence of VTEC infection by culture of the pathogen or detection of free verotoxin in the feces, or both. Significantly elevated titers of short-lived agglutinins and IgM class antibodies against the O157 lipopolysaccharide were found in 20 (91%) of 22 patients, but not in two of three patients with non-O157 E. coli isolates or in healthy children or children with diarrhea caused by other enteric pathogens (p less than 0.01). The combined microbiologic and serologic procedures provided evidence for VTEC infection in all 22 patients. The high incidence of anti-O157 lipopolysaccharide antibodies in these patients indicates the predominance and the pathogenic potential of this serogroup. Both serologic techniques proved to be valuable tools to further characterize this form of hemolytic-uremic syndrome. Future studies on the induction of protective immunity seem warranted.

In vivo neutralization of tumor necrosis factor-alpha and interferon-gamma abrogates resistance to Yersinia enterocolitica infection in mice

Cytokines are important mediators of the inflammatory host response against infectious agents. In this study, the role of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) in the elimination of a primary infection with highly virulent Yersinia enterocolitica serotype 0:8 strain WA-P has been investigated in C57BL/6 mice. The injection of anti-TNF-α or anti-IFN-γ antibodies (“serotherapy”) prior to the intravenous challenge of a sublethal dose of Y. enterocolitica caused an increased bacterial net-growth in the spleens, although this effect was more pronounced for anti-TNF-α treatment. The later treatment with anti-TNF-α or anti-IFN-γ antibodies on day 3 post infection likewise abrogated resistance to Y. enterocolitica and, subsequently, led to death from progressive infection. Our data demonstrate for the first time that the endogenous production of both the cytokines TNF-α and IFN-γ is required for the restriction of a primary Y. enterocolitica infection in mice.

Immunohistological Characterization of the Cellular Immune Response against Yersinia enterocolitica in Mice: Evidence for the Involvement of T Lymphocytes

Previous work from this laboratory has demonstrated that cloned T lymphocytes from spleens of Yersinia-infected mice can transfer immunity against Y. enterocolitica into naive animals. In this study, we investigated the cellular immune response to parenteral infection of Yersinia-resistant C57 BL/6 mice with the highly virulent Y. enterocolitica strain WA of serotype O:8 employing immunohistological methods. In the course of the infection the spleen and the liver were the organs most extensively affected. Histologically, three different patterns of inflammatory reactions could be observed: (i) small non-pyogenic granuloma-like lesions (in the liver only), (ii) microabscesses lacking a sharp outline, and (iii) larger abscesses disclosing a distinct cellular border (spleen and liver). Immunohistologically, Y. enterocolitica was detectable within abscesses but not in the small granuloma-like lesions present in the liver. CD11b/18 positive cells (= Mac-1-antigen expressed on macrophages, monocytes, granulocytes and NK-cells) could be shown in Yersinia-induced lesions. The number of these cells correlated with the extent of tissue alterations induced by Y. enterocolitica. More strikingly, we were able to demonstrate for the first time that both CD4 (helper) and CD8 (cytotoxic) T lymphocytes are present in Yersinia-induced lesions. In summary, we could demonstrate for the first time that granuloma-like lesions can be induced by Y. enterocolitica. Moreover, we supported our recent study suggesting that T lymphocytes are probably involved in the immune response against Y. enterocolitica in mice.

Coexistence of heterogeneous populations ofToxoplasma gondii parasites within parasitophorous vacuoles of murine macrophages as revealed by a bradyzoite-specific monoclonal antibody

The expression of bradyzoite-specific antigens (Bsa) ofToxoplasma gondii was studied in murine bonemarrow-derived macrophages that had previously been infected with tachyzoites. Growth conditions that allowed only restricted replication ofToxoplasma gondii resulted in heterogeneous populations; (1) Bsa-positive and Bsa-negative parasites could be observed within one parasitophorous vacuole (heterogeneous vacuole community), and (2) homogeneous Bsa-positive and homogeneous Bsa-negative vacuole communities coexisted within one macrophage host cell. These observations suggest that stage conversion does not seem to be a synchromous event for a vacuole community.

Influence of antibiotics on IgA and IgG response and persistence of Yersinia enterocolitica in patients with Yersinia-associated spondylarthropathy.

SummaryThe IgA and IgG antibody response to plasmid-encoded outer membrane proteins was studied in 59 patients with yersinia-associated spondylarthropathy during 15 months of follow-up. Initially, all patients had specific IgA and IgG antibodies to the 36 and 46 kDa and 30% also to the 26 and 58 kDa released proteins, which correlated with the finding of virulentYersinia bacilli in intestinal biopsies. IgA disappeared in 69% of untreated patients after nine months and persisted in 31% after one year. IgA disappeared within three to six months in 81% of the patients treated with antibiotics for four to six weeks and persisted in 6% after one year (p<0.002). IgG antibodies to the 36 and 46 kDa outer membrane proteins persisted in 80% of all patients. Disappearance of IgA was coupled with disappearance of yersinia from intestinal biopsies.ZusammenfassungBei 59 Patienten mit Yersiniaassoziierter Spondylarthropathie wurde während 15 Monaten der Verlauf der IgA- und IgG-Antikörperantwort auf plasmidkodierte, äußere Membranproteine untersucht. Anfangs hatten alle Patienten spezifische IgA- und IgG-Antikörper gegen die 36 und 46 kDa und auch gegen die 26 und 58 kDa-Proteine, und dies korrelierte mit dem Nachweis virulenter Yersinien in Darmbiopsien. Bei 69% der unbehandelten Patienten war IgA nach neun Monaten verschwunden, bei 31% fand sich nach einem Jahr noch eine Persistenz. Bei Patienten, die vier bis sechs Wochen lang mit Antibiotika behandelt wurden, waren IgA- Antikörper innerhalb drei bis sechs Monaten in 81% der Fälle verschwunden; Persistenz nach einem Jahr war in 6% der Fälle nachzuweisen (p<0,002). IgG-Antikörper gegen die 36 und 46 kDa-Proteine persistierten bei 80% aller Patienten. Wenn IgA verschwand, war Yersinia auch nicht mehr in Darmbiopsien nachzuweisen.

The Cellular Immune Response Against Yersinia enterocolitica in Different Inbred Strains of Mice: Evidence for an Important Role of T Lymphocytes

Resistance of mice against infection with Yersinia enterocolitica has been shown to be related neither to the Ity locus coding for resistance against infection with Salmonella typhimurium and other pathogens nor to the H-2 locus. From other mouse infection models, e.g., murine leishmaniasis, there is evidence that a different T cell-dependent regulation of the host immune response in various inbred strains of mice determines the susceptibility to the infectious agent. However, until recently, little was known about the cellular immune response against Y. enterocolitica. Thus, in a first approach we used the highly virulent Y. enterocolitica strain WA of serotype O:8 and different inbred strains of mice (C57 BL/6, Balb/c and athymic T cell-deficient C57 BL/6 nude mice) to investigate the cell-mediated immunity against parenteral infection. Comparison of the median lethal dose and of the net-bacterial growth in the spleens of infected mice indicated that Balb/c mice could be considered as Yersinia-susceptible whereas C57 BL/6 mice were relatively resistant. However, in contrast to normal C57 BL/6, athymic T cell-deficient C57 BL/6 nude mice have proved to be highly susceptible to Yersinia infection suggesting that T cells are required for the elimination of the pathogen. This conclusion was supported by histomorphological and immunohistological results indicating that T lymphocytes were present in Yersinia-induced tissue lesions. Moreover, the adoptive transfer of Yersinia-specific T cell lines and clones into naive animals mediated significant protection against the pathogen in both Yersinia-resistant C57 BL/6 and in Yersinia-susceptible Balb/c mice. These findings emphasize an important role of T lymphocytes in the host response against Y. enterocolitica infection.

Possible reasons for failure of conventional tests for diagnosis of fatal congenital toxoplasmosis: Report of a case diagnosed by PCR and immunoblot

Diagnosis of subclinical congenital toxoplasmosis has to rely on serological methods or isolation of the parasite. We present a case of congenital toxoplasmosis, in which conventional tests failed to establish the diagnosis. It was shown that this infant developed an intrathecal antibody response that was directed only against one of twoToxoplasma gondii strains used for routine diagnosis. In contrast to conventional tests, the diagnosis of cerebral toxoplasmosis could be established by using immunoblot and polymerase chain reaction (PCR). We therefore suggest that in unclarified cases, PCR and immunoblot, using at least two different strains ofT. gondii, should be considered as additional tools for diagnosis of an infection withToxoplasma and that examination of cerebrospinal fluid may be critical. Die Diagnose der subklinischen kongenitalen Toxoplasmose basiert auf serologischen Methoden oder der Erregerisolierung. Wir stellen hier ein Neugeborenes mit kongenitaler Toxoplasmose vor, bei dem die konventionelle Diagnostik versagte. Es konnte gezeigt werden, daß dieses Kind intrathekal Antikörper bildete, die jedoch nur gegen einen von zwei routinemäßig verwendetenToxoplasma gondii Laborstämmen gerichtet waren. Im Gegensatz zu konventionellen Labormethoden konnte die Diagnose einer zerebralen Toxoplasmose mittels Immunoblot und Polymerase-Ketten-Reaktion (PCR) bestätigt werden. Wir schlagen deshalb vor, daß in unklaren Fällen die PCR und der Immunoblot mit mindestens zwei verschiedenenToxoplasma-Stämmen zusätzlich zur bestehenden Labordiagnostik angewandt werden sollte und daß die Untersuchung des Liquors entscheidend sein kann.SummaryDiagnosis of subclinical congenital toxoplasmosis has to rely on serological methods or isolation of the parasite. We present a case of congenital toxoplasmosis, in which conventional tests failed to establish the diagnosis. It was shown that this infant developed an intrathecal antibody response that was directed only against one of twoToxoplasma gondii strains used for routine diagnosis. In contrast to conventional tests, the diagnosis of cerebral toxoplasmosis could be established by using immunoblot and polymerase chain reaction (PCR). We therefore suggest that in unclarified cases, PCR and immunoblot, using at least two different strains ofT. gondii, should be considered as additional tools for diagnosis of an infection withToxoplasma and that examination of cerebrospinal fluid may be critical.ZusammenfassungDie Diagnose der subklinischen kongenitalen Toxoplasmose basiert auf serologischen Methoden oder der Erregerisolierung. Wir stellen hier ein Neugeborenes mit kongenitaler Toxoplasmose vor, bei dem die konventionelle Diagnostik versagte. Es konnte gezeigt werden, daß dieses Kind intrathekal Antikörper bildete, die jedoch nur gegen einen von zwei routinemäßig verwendetenToxoplasma gondii Laborstämmen gerichtet waren. Im Gegensatz zu konventionellen Labormethoden konnte die Diagnose einer zerebralen Toxoplasmose mittels Immunoblot und Polymerase-Ketten-Reaktion (PCR) bestätigt werden. Wir schlagen deshalb vor, daß in unklaren Fällen die PCR und der Immunoblot mit mindestens zwei verschiedenenToxoplasma-Stämmen zusätzlich zur bestehenden Labordiagnostik angewandt werden sollte und daß die Untersuchung des Liquors entscheidend sein kann.

Comparison of a commercial enzyme immunoassay and an immunoblot technique for detection of immunoglobulin A antibodies toToxoplasma gondii

A commercial enzyme immunoassay (Platelia-Toxo IgA) and an immunoblot technique were compared with regard to their ability to detect IgA antibodies to the major surface protein P30(SAG1) ofToxoplasma gondii in 105 serum samples from patients with suspected or proven acquired toxoplasmosis. Comparison of the IgA-EIA with the immunoblot technique showed a concordance of 81.0 %, with a sensitivity of 92.6 % and a specificity of 78.4 %. Due to its high sensitivity the IgA-EIA might detect IgA antibodies againstToxoplasma gondii at an early stage of infection, although excessive sensitivity could lead to detection of IgA antibodies for an extended period of time following the onset of infection.