The Region of Difference Four is a Robust Genetic Marker for Subtyping Mycobacterium caprae Isolates and is Linked to Spatial Distribution of Three Subtypes.

Alpine Mycobacterium caprae isolates found in cattle and red deer display at least three genetic variations in the region of difference four (RD4) that can be used for further differentiation of the isolates into the subtypes 'Allgäu', 'Karwendel' and 'Lechtal'. Each genomic subtype is thereby characterized by a specific nucleotide deletion pattern in the 12.7-kb RD4 region. Even though M. caprae infections are frequently documented in cattle and red deer, little is known about the transmission routes. Hence, robust markers for M. caprae subtyping are needed to gain insight into the molecular epidemiology. For this reason, a rapid and robust multiplex PCR was developed for the simultaneous detection of three M. caprae RD4 subtypes and was used to subtype a total number of 241 M. caprae isolates from animals (145 cattle, 95 red deer and one fox) from Bavaria and Austria. All three subtypes occur spatially distributed and are found in cattle and in red deer suggesting transmission between the two species. As subtypes are genetically stable in both species it is hypothesized that the described genetic variations developed within the host due to 'within-host replication'. The results of this study recommend the genomic RD4 region as a reliable diagnostic marker for M. caprae subtype differentiation.

Improvement of detection of bacterial pathogens in normally sterile body sites with a focus on orthopedic samples by use of a commercial 16S rRNA broad-range PCR and sequence analysis.

A new commercially available universal 16S and 18S rRNA gene PCR test, which is followed by sequence analysis of amplicons (SepsiTest), was evaluated for rapid identification of pathogens in the diagnosis of bone and joint infections. Eighty-three orthopedic samples and 21 specimens from other normally sterile body sites collected from 84 patients were analyzed in parallel by culture and PCR for detection of bacteria and fungi. Compared to culture, the diagnostic sensitivity and specificity of PCR were 88.5% and 83.5%, respectively. The detection rate of PCR (34.6%) was higher than that of bacterial culture (25.0%) as a consequence of the presence of fastidious and noncultivable species in samples and antibiotic treatment of patients. Thirteen culture-negative infections were identified by PCR, and PCR was able to detect culture-proven polymicrobial infections. On the other hand, three samples were culture positive but PCR negative. SepsiTest was demonstrated to be a valuable supplemental tool in the rapid detection of bacteria, especially for fastidious and noncultivable organisms, allowing earlier initiation of pathogen-adapted therapy in patients with bone and joint infections.

Mycobacterium pinnipedii: transmission from South American sea lion (Otaria byronia) to Bactrian camel (Camelus bactrianus bactrianus) and Malayan tapirs (Tapirus indicus).

Tuberculosis infections caused by Mycobacterium (M.) pinnipedii in a South American sea lion, Bactrian camel, and Malayan tapirs kept in two zoological gardens spanning a time period of 5 years are reported. The zoos were linked by the transfer of one tapir. Conventional bacteriological and molecular methods were applied to detect the pathogen. Spoligotyping and MIRU/VNTR-typing performed to assess the genetic similarity revealed identical molecular characteristics of the isolates from all animals involved. Anti-tuberculosis antibodies were detected using ELISA and a recently developed serological rapid test. The study shows that: (i) using molecular methods, the assessment of the genetic relationship of infectious agents helps to confirm the routes of infection, and that (ii) immunological tests may help to detect tuberculosis infections ante mortem more reliably and early. This would prevent the transfer of tuberculosis by asymptomatic animals.

Outbreak of tuberculosis caused by Mycobacterium caprae in a zoological garden.

In the autumn of 2004, tuberculosis caused by Mycobacterium caprae occurred in a zoo in Slovenia. A dromedary camel (Camelus dromedarius) was killed after a history of progressive emaciation. Necropsy findings indicated disseminated tuberculosis, which was confirmed by cultivation of M. caprae. Consequently, a tuberculin skin test was performed in all epidemiologically linked animals and another dromedary camel and six bison (Bison bison) were positive and killed. Mycobacterium caprae was isolated from two bison while M. scrofulaceum and Mycobacterium spp. were found in two other bison, respectively. The second dromedary camel was found to be negative for mycobacteria under both microscopic and culture tests. The isolates were investigated with commercial identification kits, IS6110 PCR, IS6110 restriction fragment length polymorphism analysis, spoligotyping and mycobacterial interspersed repetitive units typing. Genotyping results revealed that the dromedary camel and the two bison were infected by the same M. caprae.

A community outbreak of tuberculosis in Southern Austria: lessons learned for a targeted use of molecular epidemiological methods and tuberculin skin testing.

A cluster of 10 cases of tuberculosis disease (one of them extrapulmonary) occurred from July 2001 until November 2003 in a health district in Southern Austria. Eight patients were culture confirmed and shared an identical strain. One of these eight cases was identified as outbreak-related by molecular strain typing only. Due to public pressure, a further 600 persons received chest X-ray and clinical examinations. Apart from one case which could be excluded from the outbreak because of a different strain pattern, no outbreak-related case of active tuberculosis was detected by this non-targeted procedure. Tuberculin skin testing, not part of the Austrian routine protocol of contact investigation in adults, was initiated after diagnosis of case 8. Forty-nine latently infected contacts were detected. Population-based genotyping of all isolates, prioritization of contact investigations and early use of targeted tuberculin skin testing are critical for effective tuberculosis control in low-incidence countries.

CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes.

Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane-bound C3 convertase. We have investigated whether this might lead to the generation of a C5 convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4.4 mM MgCl2/20 mM EGTA, which abrogates the classical pathway of complement without affecting the alternative pathway. Blockade of the CR2 ligand-binding site with the monoclonal antibody FE8 resulted in 56 +/- 13% and 71 +/- 9% inhibition of the C3-fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade of the CR1 binding site with the monoclonal antibody 3D9 also resulted in a minor reduction in MAC deposition, while FE8 and 3D9, in combination, markedly reduced deposition of both C3 fragments (91 +/- 5%) and C9 (95 +/- 3%). The kinetics of C3-fragment and MAC deposition, as well as the dependence of both processes on CR2, indicate that MAC formation is a consequence of alternative pathway activation.

B cell-mediated infection of stimulated and unstimulated autologous T lymphocytes with HIV-1: role of complement.

In vivo, human immunodeficiency virus type 1 (HIV-1) is opsonized with complement fragments and virus-specific antibodies (Ab). Thus, HIV is able to interact with complement receptor (CR) - and Fc receptor (FcR) - positive cells such as B cells, follicular dendritic cells or macrophages. In this study we demonstrate that the interaction between B cells and HIV has an impact on autologous primary T cell infection in vitro. We confirmed the presence of complement-fragments and virus-specific Ab on serum-treated HIV using a virus-capture assay. In experiments with CR2-specific Ab we showed that the virus/B cell interaction was mainly dependent on CR2. In infection experiments immobilisation of HIV on stimulated tonsil B cells greatly enhanced the infection of interleukin (IL)-2-activated autologous tonsil T cells. Surprisingly, enhancement of T cell infection by B cell-HIV complexes was observed even in the absence of mitogenic stimuli such as PMA and was independent of the addition of exogenous IL-2. Taken together, these results indicate that primary B cells are able to efficiently transmit opsonised HIV to autologous primary T cells and induce a massive enhancement of infection. These in vitro experiments mimic the in vivo situation in the lymphoid tissue and suggest an alternative mechanism for the infection of primary T cells.

Detachment of human immunodeficiency virus type 1 from germinal centers by blocking complement receptor type 2.

After the transition from the acute to the chronic phase of human immunodeficiency virus (HIV) infection, complement mediates long-term storage of virions in germinal centers (GC) of lymphoid tissue. The contribution of particular complement receptors (CRs) to virus trapping in GC was studied on tonsillar specimens from HIV-infected individuals. CR2 (CD21) was identified as the main binding site for HIV in GC. Monoclonal antibodies (MAb) blocking the CR2-C3d interaction were shown to detach 62 to 77% of HIV type 1 from tonsillar cells of an individual in the presymptomatic stage. Although they did so at a lower efficiency, these antibodies were able to remove HIV from tonsillar cells of patients under highly active antiretroviral therapy, suggesting that the C3d-CR2 interaction remains a primary entrapment mechanism in treated patients as well. In contrast, removal of HIV was not observed with MAb blocking CR1 or CR3. Thus, targeting CR2 may facilitate new approaches toward a reduction of residual virus in GC.

C3 synthesis and CRs expression during differentiation of a murine stem cell line.

C3 production, release and CRs expression during the neutrophilic differentiation of a murine non tumorigenic cell line is investigated. The murine non tumorigenic cell line 32DCl3(G) which undergoes terminal differentiation into polymorphonuclear granulocytes when cultured in presence of G-CSF was selected as a suitable in vitro model for this study. The results show that as the cells progress into the differentiation program, levels of C3 mRNA increase, accompanied by increased C3 production. As differentiation progresses the cells gradually express CRs on their surface; these are undetectable on the surface of undifferentiated cells. As a consequence of CRs appearance, cells become able to bind C3 through receptorial binding. Differences were found in the modality of C3 secretion: differentiated cells tend to store C3 in their intracellular compartments rather than secrete it continuously into the medium and they respond to membrane stimulation with increased secretion of C3. Treatment of 32DCl3(G) with TNF-alpha increased C3 production in a time- and dose-dependent fashion. Cell response to this stimulus progressively increases during the differentiation process suggesting that they acquire functionality in the signal transduction mechanisms.

Tracing uptake of C3dg-conjugated antigen into B cells via complement receptor type 2 (CR2, CD21).

Electron microscopy was used to study the internalization and delivery of ligands for complement receptor type 2 (CR2, CD21) to endocytic compartments of B-lymphoblastoid Raji cells. Opsonized antigen was mimicked with purified C3dg conjugated to colloidal gold. C3dg-gold bound specifically to the cell surface in a time-dependent manner, and preincubation of the cells with a monoclonal antibody blocking the CR2 ligand-binding site completely inhibited any C3dg-gold binding. Notably, the binding of C3d-gold was confined to cell surface protrusions, eg, microvilli. C3dg-gold was apparently internalized through coated pits located at the bases of microvilli and could be traced to different compartments of the endocytic pathway. The morphologic characteristics and intracellular distribution of these multivesicular or multilaminar structures were compatible with those of compartments known to harbor major histocompatibility complex (MHC) class II molecules. Immunolabeling showed that the internalized C3dg-gold colocalized with MHC class II in these structures. These data provide the first ultrastructural evidence that complement-coated antigens are endocytosed by antigen-nonspecific B cells by CR2 and are delivered to the compartments in which peptide loading for antigen presentation occurs. They support the notion that CR2 may play a role in antigen presentation by B cells regardless of B-cell receptor specificity. (Blood. 2000;95:2617-2623)

Simultaneous infection with two drug-susceptible Mycobacterium tuberculosis strains in an immunocompetent host.

An important assumption for DNA fingerprinting of Mycobacterium tuberculosis is that patients are infected with only one strain at a time. Nonetheless, we demonstrate a case of simultaneous infection with two drug-susceptible strains of M. tuberculosis in an immunocompetent patient by IS6110 restriction fragment length polymorphism and spoligotyping. Epidemiological data prove the patient's involvement in two independent clusters. Thus, double infections should be suspected with fingerprints showing divergent band intensities.

Epstein-Barr virus infection of human astrocyte cell lines.

Epstein-Barr virus (EBV) is implicated in different central nervous system syndromes. The major cellular receptor for EBV, complement receptor type 2 (CR2) (CD21), is expressed by different astrocyte cell lines and human fetal astrocytes, suggesting their susceptibility to EBV infection. We demonstrated the infection of two astrocyte cell lines, T98 and CB193, at low levels. As infection was mediated by CR2, we used two stable CR2 transfectant astrocyte cell lines (T98CR2 and CB193CR2) to achieve a more efficient infection. We have monitored EBV gene expression for 2 months and observed the transient infection of T98 and T98CR2 cells and persistent infection of CB193 and CB193CR2 cells. The detection of BZLF1, BALF2, and BcLF1 mRNA expression suggests that the lytic cycle is initiated at early time points postinfection. At later time points the pattern of mRNA expressed (EBER1, EBNA1, EBNA2, and LMP1) differs from latency type III in the absence of LMP2A transcription and in the expression of BALF2 and BcLF1 but not BZLF1. A reactivation of the lytic cycle was achieved in CB193CR2 cells by the addition of phorbol esters. These studies identify astrocyte cell lines as targets for EBV infection and suggest that this infection might play a role in the pathology of EBV in the brain.

C3 molecules internalize and enhance the growth of Lewis lung carcinoma cells.

C3 molecules from normal murine serum are mainly bound to Lewis lung carcinoma cells (3LL) that do not express CRs, mainly through covalent binding as determined by the appearance of bands stained with anti-C3 and larger than 190 kD in immunoblots of proteins in whole cell extracts. Methylamine-treated, or zymosan-treated normal mouse serum, heat inactivated, or EDTA-treated murine serum resulted in low C3 deposition on 3LL cells, as indicated by fluorescence tests and immunoblotting. Cytofluorimetric studies showed that C3 molecules bound to 3LL cells were internalized in a time- and temperature-dependent process. This was confirmed by electronmicroscopic studies. The conditions allowing C3 fixation to acceptor sites and subsequent internalization increased cell proliferation. This was also true, when serum from mice genetically deficient in C5 was used which stresses the role of C3 in contrast to effects of membrane attack complex formation.

Complement receptor type two (CR2,CR21): a target for influencing the humoral immune response and antigen-trapping.

Cellular receptors for complement C3 fragments deposited on antigens are important bricks in the wall defending against microbial pathogens. The part of complement receptor type 2 (CR2; CD21) deals with enhancing humoral immune responses and with long-term trapping of C3d-coated antigen by follicular dendritic cells. CR2 is also pivotal for Epstein-Barr virus (EBV) infection. Here, the current understanding, how CR2 interacts with its ligands C3d, EBV, and CD23 is summarized. The potential to target CR2 for clinical therapy or immunization purposes are discussed.

Characterization of C3dg binding to a recess formed between short consensus repeats 1 and 2 of complement receptor type 2 (CR2; CD21).

To allow for a better characterization of the ligand binding structures of human complement receptor type 2 (CR2; CD21), we have established an IgG1 kappa mouse mAb, FE8, that interferes efficiently with binding of C3dg and EBV to CR2. In contrast to mAb OKB7, the only well-characterized mAb with similar specificity, mAb FE8 blocked binding of soluble C3dg or particles carrying multiple copies of surface-bound C3dg to CR2 or induced complete removal of these ligands from the receptor. In vitro EBV infection of B lymphocytes, on the other hand, was abrogated by mAbs FE8 and OKB7 with similar dose-response characteristics. As FE8 was shown to recognize a discontinuous epitope, a series of overlapping peptides derived from SCR1 and -2 and immobilized on cellulose was screened with FE8. The results suggest that up to five discontinuous sequences contributed to the epitope. The sequence 63-EYFNKYS-69, located between the two SCR units, reacted most intensively. Two other sequences, 16-YYSTPI-21 and 105-NGNKSVWCQANN-116, are located between Cys1 and Cys2 of SCR1 and around Cys3 of SCR2, respectively. Based on the solution structure for two factor H SCRs, a three-dimensional model of SCR1 and -2 was generated. The FE8 binding peptide sequences were located in relative proximity to each other, bounding the recess formed between SCR1 and -2. This potential of mAb FE8 is currently unique and may be exploited for interfering with conditions of unwanted recognition of C3dg-coated structures by the immune system.

Study of beta-lactam resistance in ceftazidime-resistant clinical isolates of Enterobacteriaceae.

Mechanisms and transferability of beta-lactam resistance in 50 ceftazidime resistant strains of Enterobacteriaceae was studied. These strains were selected from 1991 E. coli, 1035 Enterobacter spp., 168 Citrobacter spp. and 1371 Klebsiella spp., isolated from patients hospitalized in ICUs and in the pediatric and urology departments of six hospitals in Bratislava during the years 1994-1996. The selected strains expressed the resistance not only to ceftazidime (50/50) but also to ampicillin (50/50), ceftriaxone (50/50), cefotaxime (49/50) and cefoxitin (45/50). The mechanism of resistance in all 50 strains was the production of beta-lactamases by conjugation, using either ceftazidime or cefotaxime for the selection of transconjugants and by isolation of R-plasmids ranging from to 55-87 kb from donor strains and from transconjugants. A total of 21 isolates possessed chromosomally encoded resistance and 25 clinical isolates and their transconjugants expressed ESBL sensitive to clavulanate. Selected E. coli and Klebsiella pneumoniae isolates expressed the presence of TEM and SHV enzymes determined by isoelectric focusing. The possible trends in the development of antimicrobial resistance in Slovakia in the future are indicated.

The C-terminus of factor H: monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor H-related proteins.

We have generated monoclonal antibodies (mAbs) specific for the C-terminus of factor H that can be used as inhibitory antibodies for heparin binding and for the specific detection of factor H and factor H-related proteins (FHRs) in plasma and triacylglycerol-rich lipoproteins. Four distinct mAbs were established: IXF9 (IgG1), VD3 (IgG2a), VIG8 (IgG1) and IIC5 (IgG1). Each reacts specifically with FHR-1 and factor H (and also with FHR-2 in the case of VIG8), but none binds to the related FHR-3 and FHR-4 proteins nor to factor H-like protein 1. By the use of deletion mutants of factor H and by comparing the reactivity with FHR-1 and FHR-2, the binding epitopes of the mAbs were identified and localized to different short consensus repeats (SCRs): mAbs IXF9 and VD3 bind to related or even identical sites within SCR18 (factor H) and SCR3 (FHR-1) respectively. mAbs VIG8 and IIC5 bind to different epitopes located within SCRs 19 to 20 of factor H and SCRs 4 to 5 of FHR-1 respectively. Only mAb VIG8 reacts with the corresponding SCRs 3 to 4 of FHR-2. These antibodies are useful for the detection of the corresponding proteins in biological specimens such as fractions of lipoproteins. In addition, mAb VIG8 has the unique feature of inhibiting binding of factor H to heparin. Given the recent identification of a heparin- and a C3b-binding domain within the C-terminus of factor H, these mAbs should provide useful tools for functional analysis and for the precise localization of the domain(s) required for this interaction.

A novel mechanism of alternative pathway complement activation accounts for the deposition of C3 fragments on CR2-expressing homologous cells.

Complement receptor type 2 (CD21, CR2), the receptor for the C3 fragment C3dg, activates complement via the alternative pathway and also serves as a preferential acceptor site for C3 fragments. The molecular basis for this phenomenon, which has recently been demonstrated for B lymphocytes in vivo, is currently not understood. Here we present a model for this CR2-dependent complement activation. The inactive C3 (iC3), which forms spontaneously in serum in low amounts by reaction of native C3 with H2O, binds noncovalently to the N-terminal part of CR2. Subsequent association of properdin and factor B, and cleavage of factor B by factor D lead to formation of a C3 convertase associated with CR2, thus focussing covalent C3 deposition to CR2 itself. This model is supported by the following experimental findings. 1) By FACS analysis and radioreceptor assays we showed that iC3, properdin, and factor B bound to CR2 on Raji B cells, MT2 T cells, and peripheral blood B cells. 2) Both binding of these proteins and complement activation by CR2-expressing cells were reduced in parallel by Abs against CR2. 3) 125I-labeled C3b was covalently deposited on CR2, when hemolytically active 125I-labeled C3 was added to Raji cells preincubated with iC3, factor B, properdin, and factor D, thus proving functionality of CR2-bound C3 convertase. This model of C3 convertase activity formed on CR2 domains inaccessible for decay-accelerating factor offers an explanation for the deposition of C3 found on CR2-expressing cells.

Differential chemotactic activities of sensory neuropeptides for human peripheral blood mononuclear cells.

We studied the chemotactic effects of calcitonin gene-related peptide, vasoactive intestinal peptide, substance P (SP), and secretoneurin on PBMC and PBL using micropore filter assays. All four peptides induced migration of PBMC, whereas only calcitonin gene-related peptide, vasoactive intestinal peptide, and SP were chemotactic for PBL. Secretoneurin, known to induce monocyte chemotaxis, was unable to affect lymphocyte migration. Effects of SP on PBL were characterized by checkerboard analyses and represented true chemotaxis. Both T and B cells responded chemotactically to SP, the functional activity of SP residing in its C-terminal amino acid sequence. Involvement of neurokinin (NK) receptors was supported by inhibition of SP-induced migration of PBL with an NK1 receptor antagonist and induction of migration with [Sar9, Met(O2)11]SP and [PyrGlu6, Pro9]SP(6-11), two specific agonists for NK1 receptors, but not with [beta-Ala8]NK A(4-10), an agonist for NK2 receptors. PBL chemotaxis to SP was abolished by inhibition of tyrosin kinase but not by that of protein kinase C. Preincubation of PBL with pertussis or cholera toxin inhibited SP chemotaxis, indicating that in PBL, NK receptors for chemotaxis probably are coupled with G protein and involve a tyrosin kinase signaling pathway. We conclude that, together with calcitonin gene-related peptide and vasoactive intestinal peptide, SP is a lymphocyte chemoattractant, whereas secretoneurin, which is coreleased from sensory nerve endings, is not.