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Background: Assessments, such as summative structured examinations, aim to verify whether students have acquired the necessary competencies. It is important to familiarize students with the examination format prior to the assessment to ensure that true competency is measured. However, it is unclear whether students can demonstrate their true potential or possibly perform less effectively due to the unfamiliar examination format. Hence, we questioned whether a 10-min active familiarization in the form of simulation improved medical students´ OSCE performance. Next, we wanted to elucidate whether the effect depends on whether the familiarization procedure is active or passive., Methods: We implemented an intervention consisting of a 10-min active simulation to prepare the students for the OSCE setting. We compared the impact of this intervention on performance to no intervention in 5th-year medical students (n = 1284) from 2018 until 2022. Recently, a passive lecture, in which the OSCE setting is explained without active participation of the students, was introduced as a comparator group. Students who participated in neither the intervention nor the passive lecture group formed the control group. The OSCE performance between the groups and the impact of gender was assessed using X 2 , nonparametric tests and regression analysis (total n = 362)., Results: We found that active familiarization of students (n = 188) yields significantly better performance compared to the passive comparator (Cohen´s d = 0.857, p < 0.001, n = 52) and control group (Cohen´s d = 0.473, p < 0.001, n = 122). In multivariate regression analysis, active intervention remained the only significant variable with a 2.945-fold increase in the probability of passing the exam (p = 0.018)., Conclusions: A short 10-min active intervention to familiarize students with the OSCE setting significantly improved student performance. We suggest that curricula should include simulations on the exam setting in addition to courses that increase knowledge or skills to mitigate the negative effect of nonfamiliarity with the OSCE exam setting on the students., (© 2024. The Author(s).)

Aim: The Austrian Competence Level Catalogue for Medical Skills clearly states the importance of teaching communicative and social competence in the different subject areas of undergraduate medical and dental education. This paper aims to present an overview of the academic courses at the Medical University of Innsbruck that explicitly address the promotion of communication and social skills in medical students. Method: This paper focuses on educators' descriptions of how communicating with patients is taught. The Medical University's longitudinal curriculum on medical interviewing is presented in detail. The courses on ethical principles in the dissection course, palliative medicine, and gender medicine are also outlined as examples. In addition, lecturers (n=536) participated in an online survey to determine the teaching and testing content regarding patient communication and to measure the value attached to the associated teaching and learning methods. Results: The examples given by educators to illustrate learning objectives, educational content, and the teaching methods used to impart communicative and social competence provide an overview of the courses which focus on this topic or intentionally address it during the course. The results of the online survey offer a broad overview of the awareness of the topic at the university. Different testing formats are used to assess the skills being taught. Conclusion: Familiarity with the various teaching methods used in the different courses is important for developing communicative and social competence in medical education. Active networking is necessary to anchor communicative and social competency as a major thread throughout an entire medical curriculum., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Exenberger et al.)

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., (© 2015 Blackwell Verlag GmbH.)

The autoimmune renal disease deficient for complement factor H-related (CFHR) genes and autoantibody-positive form of hemolytic uremic syndrome is characterized by the presence of autoantibodies specific for the central complement regulator, factor H, combined with a homozygous deficiency, mostly in CFHR3 and CFHR1 Because FHR3 and FHR1 bind to C3d and inactivated C3b, which are ligands for complement receptor type 2 (CR2/CD21), the aim of the current study was to examine whether FHR3-C3d or FHR1-C3d complexes modulate B cell activation. Laser-scanning microscopy and automated image-based analysis showed that FHR3, but not FHR1 or factor H, blocked B cell activation by the BCR coreceptor complex (CD19/CD21/CD81). FHR3 bound to C3d, thereby inhibiting the interaction between C3d and CD21 and preventing colocalization of the coreceptor complex with the BCR. FHR3 neutralized the adjuvant effect of C3d on B cells, as shown by inhibited intracellular CD19 and Akt phosphorylation in Raji cells, as well as Ca(2+) release in peripheral B cells. In cases of CFHR3/CFHR1 deficiency, the FHR3 binding sites on C3d are occupied by factor H, which lacks B cell-inhibitory functions. These data provide evidence that FHR3, which is absent in patients with the autoimmune form of hemolytic uremic syndrome, is involved in B cell regulation., (Copyright © 2016 by The American Association of Immunologists, Inc.)

To estimate the prevalence of bovine tuberculosis in the Alpine region, we studied the epidemiology of Mycobacterium caprae in wildlife during the 2009-2012 hunting seasons. Free-ranging red deer (Cervus elaphus) were a maintenance host in a hot-spot area, mainly located in Austria.

Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex, causes tuberculosis (TB) in man and animals. Some features distinguish M. caprae from its epidemiological twin, Mycobacterium bovis: M. caprae is evolutionarily older, accounts for a smaller burden of zoonotic TB and is not globally distributed, but primarily restricted to European countries. M. caprae occurs only in a low proportion of human TB cases and this proportion may even decrease, if progress toward eradication of animal TB in Europe continues. So why bother, if M. caprae is not an enigma for diagnostic TB tests and if resistance against first-line drugs is a rarity with M. caprae? This 'European' pathogen of zoonotic TB asks interesting questions regarding the definition of a species. The latter, seemingly only an academic question, particularly requires and challenges the collaboration between human and veterinary medicine.

The lack of complete genome sequence information for Mycobacterium caprae complicates a robust differentiation of M. caprae and Mycobacterium bovis. In this study, the presence or absence of M. caprae-specific single nucleotide polymorphisms in lepA and gyrB genes was assessed. The region of difference 4 (RD4) was analyzed for the identification and characterization of M. caprae. Molecular characteristics were evaluated in 12 recent M. caprae isolates from livestock and wildlife collected over a 3-year period in Bavaria, Germany. Conventional PCR strategies, sequence analysis of PCR fragments, and data from a next-generation sequencing approach together with variable-number tandem-repeat genotyping were utilized. Single nucleotide polymorphisms in the lepA and gyrB genes indicating the presence of M. caprae were detected in all the isolates. At least three different RD4 variants were found for Alpine M. caprae isolates. The results demonstrate that the RD4 region is rather heterogeneous in M. caprae genomes. As assumed by others, the presence of RD4 is critical for PCR-based differentiation of M. caprae from M. bovis, but in addition, the observed variability of RD4 allows the identification of M. caprae genotypes and may be indicative of a geographical-type appearance.

Background: The Austrian State medical universities had to change their admission system in 2005. Until this year admission to medical studies was unrestricted. Innsbruck Medical University chose the Eignungstest für das Medizinstudium in der Schweiz (EMS) aptitude test for admission testing., Aims and Objectives: Did the implementation of a selection process affect the academic performance and drop-out rates of students according to gender?, Methods: Two groups of students were compared: 'open admission' (2002-2004), and 'selected' (2006-2009). Academic performance was tested according to results in the final exams after year 1 (SIP 1; SIP, summative integrierte Prüfung) and after year 3 (SIP 3). Drop-out rates were recorded using the registration system of the university., Results: Both, male and female 'selected' students had a higher passing rate regarding SIP 1 and they passed SIP 1 in shorter time and using fewer attempts than the open admission group. The percentage in passing SIP 3 did not change due to change in admission. The drop-out rates were significantly reduced for male and female students. 'Unselected' female students had a significantly higher drop-out ratio than 'unselected' male counterparts. After EMS testing, the drop-out ratios of female and male students were not significantly different., Conclusion: Selected applicants were more able and better motivated to study medicine.

A survey of 143 hunter-harvested red deer for tuberculosis was conducted in an Alpine area in Western Austria over two subsequent years. There, single tuberculosis cases caused by Mycobacterium caprae had been detected in cattle and red deer over the preceding decade. The area under investigation covered approximately 500 km(2), divided into five different hunting plots. Lymph nodes of red deer were examined grossly and microscopically for typical tuberculosis-like lesions and additionally by microbiological culturing. Executing a detailed hunting plan, nine M. caprae isolates were obtained. Six out of nine originated from one single hunting plot with the highest estimated prevalence of tuberculosis, that is, 23.1%. All isolates were genotyped by mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing of 24 standard loci plus VNTR 1982. All nine isolates belonged to a single cluster termed "Lechtal" which had been found in cattle and red deer in the region, demonstrating a remarkable dominance and stability over ten years. This is the first report on a systematic prospective study investigating the prevalence and strain variability of M. caprae infection in red deer in Austria and in the Alpine countries.

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.

While our previous studies have demonstrated that complement activation induced by complement receptors type 2 (CR2/CD21) and 1 (CR1/CD35) results in C3-fragment deposition and membrane attack complex (MAC) formation in human B cells, the consequences of these events for B-cell functions remain unknown. In the present study, we show that CR2-induced complement activation results in membrane depolarization, as indicated by annexin V binding, with kinetics similar to those of C3-fragment deposition and different from those of MAC formation. On the other hand, like MAC formation, depolarization requires activation of complement via the alternative pathway, as indicated by total inhibition upon neutralization of factor D, and is abrogated by combined blockade of CR1 and CR2, but not of either receptor alone. The membrane depolarization is not associated with the apoptosis of B cells, as examined by co-staining with APO-2.7 or by the TdT-mediated biotin-dUTP nick-end labelling (TUNEL) assay. Confocal microscopy revealed that depolarization and C3 deposition, unlike MAC deposition, are limited to restricted areas on the B-cell surface. Double staining revealed a close association between the C3-fragment patches and membrane depolarization, as well as redistribution of lipid rafts to these areas. We propose that these events may play a role in the regulation of B-cell signalling and cross-talk with T cells.

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.

Genotyping has become an indispensable tool in medical microbiology and epidemiology. One of the first targets has been Mycobacterium tuberculosis. Over the past 15 years approximately 900 pertinent publications have substantiated the value of the genotyping approach for tuberculosis control. New insights into the understanding of the natural history of tuberculosis, especially regarding the frequencies of reactivation, reinfection or multiple infection entailed adaptations of pathophysiological concepts. However, assessment of recent transmission, outbreak analysis, and detection of laboratory contamination still form the genuine scope of genotyping. Detection of unsuspected clusters of cases can provide clues to search for further, undetected cases. Uncovering false positive cultures spares the risks and costs of unnecessary treatment and may reveal systematic laboratory weaknesses. Several European countries already profit from nationwide prospective fingerprinting. After providing genotyping results to public health officials, these were able to document epidemiological links for substantially more tuberculosis patients. On a global scale, strain families and particular strains have been identified, characterised and traced in their spread. The importation of Beijing-genotype multidrug-resistant M. tuberculosis into Central European countries will be described here as an example. The goal for further developments is the ability to compare isolates for epidemiological purposes in a single step that also comprises species determination, drug resistance testing and detection of pathogenicity factors.

Objectives: To compare M. tuberculosis complex genotypes from representative regions of the Czech Republic in order to estimate changes in strain prevalence and in the extent of imported drug-resistant tuberculosis., Methods: Primary M. tuberculosis complex isolates (n=155) and follow-up isolates (n=15) from 155 patients from the first half of 2004 (98 from Prague, 37 from South Moravia and 35 from the Moravian-Silesian region) were genotyped by IS6110-RFLP, spoligotyping, and partly by VNTR-genotyping., Results: Based on IS6110-RFLP, 110 of 155 (71%) primary isolates were unique. Forty-five isolates (29%) were found in 15 clusters comprising two to six patients and all but one cluster were also discriminated by MIRU-VNTR-genotyping. Four clusters comprised patients from different regions, and six were ongoing for several years. An indication of MDR-strain transmission was found in one instance. All four Beijing-type isolates with any resistance were associated with immigration from Eastern Europe., Conclusions: The molecular epidemiological data of this period-prevalence, population based study and its comparison to earlier investigations point to a low extent of clustering between M. tuberculosis complex isolates in representative regions of the Czech Republic. Few clusters extending geographically and/or over several years were identified, providing a means for an in-depth analysis of risk factors of transmission. Beijing genotype isolates were shown to increase in prevalence to reach 6.5%. Drug resistant isolates of this genotype were associated with immigration of from Eastern Europe, although direct transmission of a resistant isolate was probable only in one of eleven cases.

Using the one-bead one-compound (OBOC) combinatorial method, four heptapeptide ligands of CD21 receptor, a cell surface marker of malignant B cell lymphoma, were identified with an innovative two-step fluorescence screening method to overcome the limitation caused by autofluorescence of TentaGel resin. The binding affinities of selected peptides, YILIHRN (B1), PTLDPLP (B2), and LVLLTRE (B3), were in the micromolar region as determined by a fluorescence quenching assay. Peptide B1 was conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via spacers of different lengths, composed of one to four repeats of the 8-amino-3,6-dioxaoctanoic acid (A) group. The evaluation of the biorecognizability of HPMA copolymer-B1 conjugates by the CD21 receptor revealed that increasing the number of repeats of A in the spacer from one to three resulted in continuous improvements in the biorecognition by the CD21 receptor; the increase from three to four repeats showed no significant effect. This work showed the potential of the OBOC combinatorial approach to select peptide ligands as targeting moieties for CD21 specific polymeric drug carriers.

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.

Background: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database., Results: The fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network., Conclusion: Our results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.

Cancer targeting with peptides has become promising with the emergence of combinatorial peptide techniques such as phage display. Using phage display under stringent screening conditions, we selected five distinct peptides that specifically recognized the CD21 receptor, a cell surface marker of malignant B cell lymphoma. Two highly hydrophobic sequences were excluded (RLAYWCFSGLFLLVC and PVAAVSFVPYLVKTY). The binding affinity toward CD21 of the other three selected peptides (RMWPSSTVNLSAGRR, PNLDFSPTCSFRFGC, and GRVPSMFGGHFFFSR) was analyzed with fluorescence quenching. Their dissociation constants were determined to be within the micromolar range. On the basis of the results of phage ELISA, competitive phage ELISA, and fluorescence quenching, the binding sites of the three selected peptides were found to reside within the first four short consensus repeats of CD21 (SCR1-4). The peptide RMWPSSTVNLSAGRR (P1) was bound to the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, a potential drug carrier for chemotherapeutic agents, and the surface binding properties of HPMA copolymer-P1 conjugates were investigated. Specific interactions were observed between HPMA copolymer-P1 conjugates and surface-bound receptor. Binding of HPMA copolymer-P1 conjugates was directly related to the amount of surface (MaxiSorp plate) bound receptor, and the binding of the conjugates could be inhibited by the application of a 3-4 orders-of-magnitude excess of free peptide over the peptide concentration in conjugates. The enhanced binding of polymer-bound peptide was ascribed to multivalent interactions between the HPMA copolymer-P1 conjugate and the surface-bound CD21 receptor.

Mycotic aortic aneurysms are rare complications of systemic tuberculosis that affect very few patients. We report a case of a false aneurysm of the visceral segment of the aorta that was associated with tuberculous vertebral osteomyelitis. Both conditions were successfully treated with antituberculous chemotherapy and a combined surgical procedure, ie, aneurysm resection and homograft implantation, followed by orthopedic stabilization of the spine.

Mycobacterium caprae, a recently defined member of the Mycobacterium tuberculosis complex, causes tuberculosis among animals and, to a limited extent, in humans in several European countries. To characterize M. caprae in comparison with other Mycobacterium tuberculosis complex members and to evaluate genotyping methods for this species, we analyzed 232 M. caprae isolates by mycobacterial interspersed repetitive unit (MIRU) genotyping and by spoligotyping. The isolates originated from 128 distinct epidemiological settings in 10 countries, spanning a period of 25 years. We found 78 different MIRU patterns (53 unique types and 25 clusters with group sizes from 2 to 9) but only 17 spoligotypes, giving Hunter-Gaston discriminatory indices of 0.941 (MIRU typing) and 0.665 (spoligotyping). For a subset of 103 M. caprae isolates derived from outbreaks or endemic foci, MIRU genotyping and IS 6110 restriction fragment length polymorphism were compared and shown to provide similar results. MIRU loci 4, 26, and 31 were most discriminant in M. caprae, followed by loci 10 and 16, a combination which is different than those reported to discriminate M. bovis best. M. caprae MIRU patterns together with published data were used for phylogenetic inference analysis employing the neighbor-joining method. M. caprae isolates were grouped together, closely related to the branches of classical M. bovis, M. pinnipedii, M. microti, and ancestral M. tuberculosis, but apart from modern M. tuberculosis. The analysis did not reflect geographic patterns indicative of origin or spread of M. caprae. Altogether, our data confirm M. caprae as a distinct phylogenetic lineage within the Mycobacterium tuberculosis complex.

The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1 and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max)=109+/-27.2 x 10(7) l/mole) was ca. 9- and 6-fold greater, respectively, than those for CR1 or CR2 acting alone (K(a)=13.2+/-5.3 and 18.5+/-3.5 x 10(7) l/mole). The high avidity of the CR1-CR2 complex for C3i is consistent with its rates of C3i uptake and release being determined by CR1 and CR2, respectively.

Resting CD4(+) T cells in the lymphoid tissue (LT) are essential producers of virions at the beginning of HIV infection in vivo. We previously developed a model that allowed in vitro infection of non-prestimulated T lymphocytes in the presence of autologous B lymphocytes and complement. In this study, we try to clarify the mechanism(s) responsible for virus transmission in unstimulated autologous B cell/T cell co-cultures. Ex vivo analyses of patient plasma samples revealed that HIV was opsonized. Flow cytometry showed that opsonized virus preferentially bound to complement receptor (CR)-2 on B lymphocytes in primary B cell/T cell co-cultures. As indicated by cytokine measurements and transwell experiments, soluble factors seemed to play a minor role in enabling infection. Rather, direct interaction between B and T lymphocytes and direct binding of opsonized virus to CR2 on B cells turned out to be essential for productive infection. Antibodies blocking cell-cell adhesion inhibited p24 antigen production. An anti-CR2 antibody blocking C3d-CR2 binding also significantly reduced viral replication. Since the infection of unstimulated T cells by opsonized primary HIV isolates in the presence of B cells was highly efficient independent of the tropism of the virus, this mechanism may be critical in the pathogenesis of HIV.

Twelve cases of Mycobacterium bovis subsp. caprae infection have occurred in four humans, three cattle, and five red deer in western Austria since 1994. DNA-fingerprinting of the isolates suggested transmission in and between these species over several years. Contact with cattle, but not with goats, was found to be associated with three of four human cases.

Normal human B lymphocytes are known to activate the alternative pathway (AP) of complement, leading to C3-fragment deposition and membrane attack complex (MAC) formation. The process is mediated via complement receptor type 2 (CR2, CD21), with complement receptor type 1 (CR1, CD35) playing a subsidiary role. In this study, we examine the relative contributions of CR1 and CR2 to the deposition of C3 fragments and MAC on B lymphocytes under circumstances where all complement pathways are operational. C3-fragment deposition and MAC formation were assessed on human peripheral B lymphocytes in the presence of 30% autologous serum. Blocking the CR2 ligand-binding site with monoclonal antibody (mAb) FE8 resulted in significant reduction (37.9+/-11.9%) in C3-fragment deposition, whereas MAC formation was only marginally affected (12.1+/-22.2% reduction). Blocking the CR1 binding-site resulted in significant reduction of both C3-fragment deposition (22.0+/-14.5%) and MAC formation (47.4+/-13.8%). Both the lack of CR2 influence on MAC formation and the promotion of C3-fragment deposition by CR1 are in striking contrast to the situation where only the AP is operational. The presence of erythrocytes (E) bearing CR1, however, markedly reduced both C3-fragment deposition and MAC formation. Our data suggest that C3-fragment deposition and MAC formation on B lymphocytes in vivo may involve both AP and classical pathway activation, with CR1 contributing significantly to the latter. On the other hand, the presence of extrinsic CR1, on E, may serve to limit spontaneous MAC formation and thereby ensure cell survival in the circulation.

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.

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.

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.

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)

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.

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 (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 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.

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.

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.

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.

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.