Exploring the impact of durvalumab on biliary tract cancer: insights from real-world clinical data.

INTRODUCTION: This study assesses the effectiveness of durvalumab with platinum and gemcitabine for biliary tract cancers (BTC). It aims to confirm the TOPAZ-1 trial results in a real-world context and explore the link between BTC molecular profiles and patient outcomes. METHODS: A retrospective analysis was conducted on 102 BTC patients treated with durvalumab, platinum, and gemcitabine at five cancer centers in Austria and one in Germany from 2022 to 2024. Molecular profiling used targeted DNA and RNA assays. Clinical endpoints, including progression-free survival (PFS) and overall survival (OS), were assessed using log-rank tests and Cox regression, with correlations to second-line molecular-targeted therapies. RESULTS: Among 102 patients, 60.8% had intrahepatic cholangiocarcinoma. The treatment achieved a disease control rate of 71.57% and an overall response rate of 35.11%. Median PFS was 6.51 months, and OS was 13.61 months. Patients under 65 had significantly better OS. Alterations in chromatin remodeling or homologous recombination repair genes were not predictive of survival benefit (HR: 0.45; p = 0.851 and HR: 1.63; p = 0.26, respectively). Patients with molecular-informed second-line therapy showed a trend toward survival benefit (HR: 0.23; p = 0.052). CONCLUSION: This study confirms the phase 3 trial results of durvalumab with platinum and gemcitabine, providing a substantial real-world dataset with detailed molecular characterization. No specific patient subgroup showed a markedly better response to durvalumab based on conventional NGS panels. Further research is needed to explore the link between immunotherapy responses and molecular subgroups.

Risk assessment in precapillary pulmonary hypertension: a comparative analysis.

BACKGROUND: Risk stratification is essential to assess mortality risk and guide treatment in patients with precapillary pulmonary hypertension (PH). We herein compared the accuracy of different currently used PH risk stratification tools and evaluated the significance of particular risk parameters. METHODS: We conducted a retrospective longitudinal observational cohort study evaluating seven different risk assessment approaches according to the current PH guidelines. A comprehensive assessment including multi-parametric risk stratification was performed at baseline and 4 yearly follow-up time-points. Multi-step Cox hazard analysis was used to analyse and refine risk prediction. RESULTS: Various available risk models effectively predicted mortality in patients with precapillary pulmonary hypertension. Right-heart catheter parameters were not essential for risk prediction. Contrary, non-invasive follow-up re-evaluations significantly improved the accuracy of risk estimations. A lack of accuracy of various risk models was found in the intermediate- and high-risk classes. For these patients, an additional evaluation step including assessment of age and right atrium area improved risk prediction significantly. DISCUSSION: Currently used abbreviated versions of the ESC/ERS risk assessment tool, as well as the REVEAL 2.0 and REVEAL Lite 2 based risk stratification, lack accuracy to predict mortality in intermediate- and high-risk precapillary pulmonary hypertension patients. An expanded non-invasive evaluation improves mortality risk prediction in these individuals.

[Assessment of Langerhans Cells in the Central Cornea as Tool for Monitoring Inflammatory Changes in Patients with Keratoconjunctivitis sicca under Topical Therapy with Cyclosporine A 0.05% Eye Drops]. / Messung der Dichte der Langerhans-Zellen als Verlaufsparameter bei Patienten mit Keratoconjunctivitis sicca unter topischer antiinflammatorischer Therapie mit Ciclosporin A 0,05% Augentropfen.

BACKGROUND: Keratoconjunctivitis sicca is one of the most common ocular diseases and is associated with a considerable decrease in the quality of life. It is accompanied by symptoms of discomfort, tear film instability, hyperosmolarity of the tear film, inflammation of the ocular surface and neurosensory abnormalities. It is still not clear yet if the density of Langerhans cells in the central cornea can be used for objective evaluation of the inflammation in the tear film and the ocular surface. PATIENTS AND METHODS: 47 patients (age 21 - 85 years, 13 male and 34 female patients) with moderate to severe keratoconjunctivitis sicca (according to the DEWS criteria severity level 2 - 3) were enrolled in this prospective, clinical trial. Patients were treated for 3 months with topical anti-inflammatory therapy with cyclosporine 0,05% eye drops twice daily. The following parameters were examined: OSDI Score, tear break-up time (TBUT), Schirmer test with anaesthesia, lid-parallel conjunctival folds (LIPCOF), in-vivo confocal microscopy with HRT II and RCM for evaluation of Langerhans cell density in the central cornea, visual acuity, tear film osmolarity and Meibomian gland dysfunction (MGD). RESULTS: After anti-inflammatory treatment, there was a significant reduction in the density of Langerhans cells (p ≤ 0.001**), an increase in TBUT (p ≤ 0.001**), as well as a significant decrease in tear film osmolarity (p ≤ 0.05*) and OSDI Score (p ≤ 0 001**). Visual acuity, MGD and Schirmer test did not change significantly. CONCLUSIONS: The results of this clinical trial show that topical anti-inflammatory therapy with cyclosporine A 0.05% eye drops in patients with moderate to severe keratoconjunctivitis sicca is effective in reducing inflammation of the ocular surface and the tear film, as well as in increasing the quality of life in these patients. Evaluation of Langerhans cell density in the central corneal epithelium by in vivo confocal microscopy is an effective objective diagnostic feature in monitoring anti-inflammatory therapy in patients with dry eye disease or other ocular surface pathologies.

Msb2 shedding protects Candida albicans against antimicrobial peptides.

Msb2 is a sensor protein in the plasma membrane of fungi. In the human fungal pathogen C. albicans Msb2 signals via the Cek1 MAP kinase pathway to maintain cell wall integrity and allow filamentous growth. Msb2 doubly epitope-tagged in its large extracellular and small cytoplasmic domain was efficiently cleaved during liquid and surface growth and the extracellular domain was almost quantitatively released into the growth medium. Msb2 cleavage was independent of proteases Sap9, Sap10 and Kex2. Secreted Msb2 was highly O-glycosylated by protein mannosyltransferases including Pmt1 resulting in an apparent molecular mass of >400 kDa. Deletion analyses revealed that the transmembrane region is required for Msb2 function, while the large N-terminal and the small cytoplasmic region function to downregulate Msb2 signaling or, respectively, allow its induction by tunicamycin. Purified extracellular Msb2 domain protected fungal and bacterial cells effectively from antimicrobial peptides (AMPs) histatin-5 and LL-37. AMP inactivation was not due to degradation but depended on the quantity and length of the Msb2 glycofragment. C. albicans msb2 mutants were supersensitive to LL-37 but not histatin-5, suggesting that secreted rather than cell-associated Msb2 determines AMP protection. Thus, in addition to its sensor function Msb2 has a second activity because shedding of its glycofragment generates AMP quorum resistance.

Outcome of surgical correction of medial patellar luxation in dogs weighing less than 10 kg.

BACKGROUND: Various trochleoplasty techniques, including trochlear wedge recession (TWR) and trochlear block recession (TBR), are used to treat dogs with medial patellar luxation (MPL). However, the objective outcomes of these surgical procedures are underreported. METHODS: Medical records were obtained for dogs weighing less than 10 kg that underwent either TWR or TBR and tibial tuberosity transposition to address grade I-III MPL. Long-term (at least 1 year after the last procedure) follow-up included orthopaedic and radiographic examinations, such as osteoarthritis score (OAS), ground reaction force (GRF) analysis and canine brief pain inventory (CBPI). RESULTS: Overall, 20 dogs (26 stifles) were followed up in the long term. Minor postoperative complications, medial patellar reluxation (MPR) and intermittent lameness occurred in 46.15%, 19.23% and 15% of the dogs, respectively. MPR occurred only in TWR-treated stifles, while mean OAS increased in all groups. Using the CBPI, the owners perceived an excellent or very good outcome in 95% of dogs. LIMITATIONS: The limitations of the study include its retrospective observational nature, a lack of randomisation and a small sample size. CONCLUSION: Surgical treatment resulted in a favourable outcome. GRF analysis could detect subtle differences in weight bearing in dogs treated for MPL, which might not be apparent clinically. There might be a higher risk for reluxation for TWR. However, a larger-scale prospective study would be required to find which treatment is superior.

Role of the small protein Mco6 in the mitochondrial sorting and assembly machinery.

The majority of mitochondrial precursor proteins are imported through the Tom40 ß-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for ß-barrel membrane protein insertion into the outer membrane and thus required for the assembly of the TOM complex. Here, we demonstrate that the α-helical outer membrane protein Mco6 co-assembles with the mitochondrial distribution and morphology protein Mdm10 as part of the SAM machinery. MCO6 and MDM10 display a negative genetic interaction, and a mco6-mdm10 yeast double mutant displays reduced levels of the TOM complex. Cells lacking Mco6 affect the levels of Mdm10 and show assembly defects of the TOM complex. Thus, this work uncovers a role of the SAMMco6 complex for the biogenesis of the mitochondrial outer membrane.

Testosterone replacement effectively inhibits the development of experimental autoimmune orchitis in rats: evidence for a direct role of testosterone on regulatory T cell expansion.

Despite the immune-privileged status of the male genital tract, infection and inflammation of the male genital tract are important etiological factors in male infertility. A common observation in clinical and experimental orchitis as well as in systemic infection and inflammation are decreased levels of testosterone. Emerging data point to an immunosuppressive role of testosterone. In our study, we substituted testosterone levels in experimental autoimmune orchitis (EAO) in rat by s.c. testosterone implants. EAO development was reduced to 17% when animals were treated with low-dose testosterone implants (3 cm long, EAO+T3) and to 33% when rats were supplied with high-dose testosterone implants (24 cm, EAO+T24) compared with 80% of animals developing disease in the EAO control group. In the testis, testosterone replacement in EAO animals prevented the accumulation of macrophages and significantly reduced the number of CD4(+) T cells with a strong concomitant increase in the number of regulatory T cells (CD4(+)CD25(+)Foxp3(+)) compared with EAO control. In vitro testosterone treatment of naive T cells led to an expansion of the regulatory T cell subset with suppressive activity and ameliorated MCP-1-stimulated chemotaxis of T lymphocytes in a Transwell assay. Moreover, expression of proinflammatory mediators such as MCP-1, TNF-α, and IL-6 in the testis and secretion of Th1 cytokines such as IFN-γ and IL-2 by mononuclear cells isolated from testicular draining lymph nodes were decreased in the EAO+T3 and EAO+T24 groups. Thus, our study shows an immunomodulatory and protective effect of testosterone substitution in the pathogenesis of EAO and suggests androgens as a new factor in the differentiation of regulatory T cells.

Target specificity of the Candida albicans Efg1 regulator.

Efg1 is a central transcriptional regulator of morphogenesis and metabolism in Candida albicans. In vivo genome-wide ChIP chip and in vitro footprint analyses revealed the Efg1 recognition sequence (EGR-box) TATGCATA in the yeast growth form of this human fungal pathogen. Upstream regions of EFG1 and genes encoding transcriptional regulators of hyphal growth including TCC1, CZF1, TEC1, DEF1 and NRG1 contained EGR- and/or EGR-like boxes. Unexpectedly, after brief hyphal induction the genome-wide Efg1 binding pattern was completely altered and new binding sites of yet unknown specificity had appeared. Hyphal induction abolished Efg1 accumulation on EFG1 and TCC1 promoters and led to rapid decline of both transcripts, although the Efg1 protein persisted in cells. While EFG1 promoter activity in the yeast growth form did not depend on bound Efg1, its downregulation under hyphal induction depended on the presence of Efg1 and the protein kinase A isoform Tpk2. Deletion analyses of the EFG1 upstream region revealed that none of its resident EGR-boxes is uniquely responsible for EFG1 promoter downregulation. These results suggest different binding specificities of Efg1 in yeast growth and in hyphal induction and suggest a brief time window following hyphal induction, in which Efg1 exerts its repressive effect on target promoters.

Measuring stress in clinical and nonclinical subjects using a German adaptation of the Perceived Stress Scale.

Background/Objective: Stress is perceived differently across individuals, which might be particularly true for nonclinical and clinical subjects. For this reason, we tested a German adaption of the 10-item Perceived Stress Scale (PSS-10) for model fit and measurement invariance in a big nonclinical and clinical sample. Method: We (1) conducted multiple confirmatory factor analysis (CFA) in 1,248 nonclinical subjects and 575 outpatients, (2) measurement invariance with multigroup CFA, (3) assessed correlations with relevant constructs and (4) calculated internal consistencies for overall stress and the subscales Helplessness and Self-efficacy. Results: In both samples, CFA revealed a robust two-factorial structure with an excellent model fit. Group comparisons revealed strict measurement invariance. Correlations with associated measures support validity. Internal consistencies were good to very good. Conclusions: We show highly satisfactory psychometric properties of the German PSS-10 for nonclinical and clinical individuals. Measurement invariance analyses demonstrated that varying stress levels of people with a different mental health status are due to true interindividual differences.

Antecedentes/Objetivo: El estrés se percibe de manera diferente entre los individuos, lo que podría ser particularmente cierto para los sujetos no clínicos y clínicos. Por esta razón, probamos una adaptación alemana de la Perceived Stress Scale de 10 ítems (PSS-10) para el ajuste del modelo y la invarianza de la medición en una gran muestra clínica y no clínica. Método: Realizamos (1) un análisis factorial confirmatorio múltiple (CFA) en 1.248 sujetos no clínicos y 575 pacientes ambulatorios, (2) invarianza de medición con CFA multigrupo, (3) correlaciones con constructos relevantes y (4) cálculos de la consistencia interna para la escala general y las subescalas Desvalidez y Autoeficacia. Resultados: En ambas muestras, el CFA reveló una estructura robusta de dos factores con un excelente ajuste del modelo. Las comparaciones grupales indicaron invarianza estricta. Las correlaciones con las medidas asociadas respaldan la validez. Los coeficientes de consistencia interna fueron buenos a muy buenos. Conclusión: Mostramos propiedades psicométricas altamente satisfactorias de la version alemana de la PSS-10 para individuos no clínicos y clínicos. Los análisis de invarianza de medición demostraron que los niveles variables de estrés de las personas con un estado de salud mental diferente se deben a diferencias interindividuales verdaderas.

Altered neural responses to social fairness in bipolar disorder.

Bipolar Disorder (BD) has a debilitating impact on psychosocial functioning and social decision-making. Recent evidence using the Ultimatum Game (UG) has shown increased rejection of moderately unfair offers in BD, suggesting impaired processing of ambiguous social information related to fairness. The present study builds upon this finding to investigate the neural substrates of fairness processing in BD. During functional magnetic resonance imaging scanning, euthymic BD patients (n = 41) and matched healthy controls (HC; n = 41) accepted or rejected very unfair, moderately unfair, or fair offers in the UG. Acceptance rates of moderately unfair offers were significantly lower in BD patients. This aberrant behavior co-occurred with abnormal brain responses to moderately unfair offers. Compared to HC, BD patients exhibited hypoactivation of right anterior insula in response to moderately unfair offers suggesting impaired integration of affective and contextual information. BD patients also displayed stronger deactivation of posterior and middle insula in response to moderately unfair offers reflecting impaired processing of the contextual aspects of fairness. The level of impulsivity of BD patients positively correlated with the abnormal deactivation of posterior and middle insula. A separate analysis revealed increased activation of dorsal ACC and left ventrolateral PFC in response to rejected compared to accepted offers in BD patients. Taken together, our findings suggest impaired processing of ambiguous social information in euthymic BD patients which is associated with increased rejection of moderately unfair offers. This impairment may reflect a failure to integrate contextual information and may be related to increased trait impulsivity.

Expression of co-stimulatory molecules, chemokine receptors and proinflammatory cytokines in dendritic cells from normal and chronically inflamed rat testis.

The presentation of self antigens by dendritic cells (DC) plays an important role in the initiation and maintenance of autoimmunity. In a model of experimental autoimmune orchitis (EAO), we have previously characterized dominant testicular autoantigens and shown an increase in DC numbers during the course of disease. In this study, we have developed a protocol for the isolation of a highly pure population of DC ( approximately 97%) from the testis of EAO and control rats to analyse the expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules (CD80, CD86), chemokine receptors (CCR2, CCR7) and cytokines (IL-10, IL-12p70, TNF-alpha). By flow cytometry, we observed similar percentage and intensity levels of MHC class II, CD80 and CD86 expression in testicular DC in all groups. Moreover, by real-time RT-PCR we have detected significantly higher CCR7 mRNA level in isolated testicular DC from rats with EAO compared to controls, whereas the expression of CCR2 was decreased in orchitis. Transcripts of IL-12p40 were observed in DC from all groups, whereas the expression of IL-10 and the rate limiting IL-12 subunit p35 were detectable exclusively in testicular DC from the inflamed testes. In co-culture experiments, testicular DC isolated from EAO animals significantly enhanced naïve T-cell proliferation compared with control DC. Taken together these results suggest that testicular DC in control testis is not mature and functionally tolerogenic, whereas in EAO testis, IL-12 expression and stimulation of T-cell proliferation points to a mature immunogenic state prior imminent migration to the lymph nodes to amplify immune responses against testicular antigens.

Mania risk is characterized by an aberrant optimistic update bias for positive life events.

BACKGROUND: Early cognitive models of mania posit that a cognitive triad consisting of unrealistically optimistic beliefs about the self, world and future may predispose vulnerable individuals to develop manic symptoms. Hypomanic personality traits (HYP) pose such a vulnerability factor in the etiopathogenesis of mania. METHODS: To test the cognitive tenet of overly optimistic views of the future, 24 individuals with high-HYP and 24 age- and sex-matched controls (low-HYP) performed a belief update paradigm, during which they estimated their personal chances to experience future positive and negative life events. Afterwards, they were presented with the statistical likelihood of each event occurring to a peer living in the same socio-cultural environment and given the chance to adjust their initial estimates. RESULTS: High-HYP individuals exhibited an asymmetric belief revision for positive events, reflected by an exaggerated incorporation of better-than-expected and an impaired integration of worse-than-expected information, relative to their low-HYP control counterparts. The strength of this optimistic update bias was linked to the trait sensitivity of the behavioral approach system. Furthermore, high-HYP individuals demonstrated a more optimistic initial prediction bias, characterized by greater overestimations of their likelihood to experience positive events, and reported enhanced trait optimism. LIMITATIONS: The cross-sectional study relied on an extreme-group design to define mania risk. CONCLUSIONS: Due to the crucial role of future-oriented beliefs in guiding decision-making and goal-directed behavior, this optimistic update bias for positive events may cognitively underpin the mania-typical engagement in highly pleasurable activities despite warnings for harmful consequences.

Management of a Central Incisor with Horizontal Root Fracture for Esthetic and Functional Rehabilitation.

Severely traumatized incisors in young adults are a very common problem, and one that is difficult to treat because healing of these teeth is not always predictable and implant placement has to be delayed until adulthood. This case report of a horizontal root fracture in an avulsed central incisor illustrates the ability to maintain crucial tooth structure. A lateral socket augmentation procedure after extraction of a root fragment combined with an endodontic and regenerative periodontal treatment approach could preserve natural esthetics and function, which are directly related to quality of life. The preservation of alveolar bone volume following partial root removal will eventually facilitate later placement of a dental implant with an improved esthetic and functional prosthodontic result in a patient with a high-risk esthetic profile.

Susceptibility-weighted MRI of extrapyramidal brain structures in Parkinsonian disorders.

Susceptibility-weighted MRI (SWI) is sensitive to T2 effects and mineralization.We investigated differences in the extrapyramidal brain structures on SWI between Parkinson disease (PD) and postural instability gait disorder (PIGD) patients and correlated the SWI values with the degree of gait dysfunction.Forty patients diagnosed with PD and PIGD underwent 3 Tesla magnetic resonance imaging (MRI) brain study. An SWI sequence (TE/TR/FA 20/33/15) was used. Ten regions of interest were placed in the midbrain and basal ganglia by 2 independent raters blinded to subject data and quantitatively evaluated.The inter-rater reliability between the raters was excellent (interclass correlation coefficient >0.8). The SWI intensity values in all regions were on average lower in PIGD than in PD patients, with the lowest results found in globus pallidus.Multivariate analysis showed a lower SWI hypointensity in the putamen and globus pallidus in PIGD compared with PD patients, with a similar trend for the other basal ganglia nuclei. Pearson correlation analysis showed a statistically significant positive correlation between SWI putaminal hypointensity and the Tinetti total score (r = 0.39, P = 0.01) in both PD and PIGD.SWI putaminal hypointensity may be a useful imaging marker in prospective evaluation for clinical progression for Parkinsonian disorders.

Synthesis and DFT calculations of spirooxaphosphirane complexes.

In situ formed Li/Cl phosphinidenoid complexes [Li(12-crown-4)][M(CO)5(ClPC5Me5)] 3a-c (M = Cr, Mo, W) reacted with cyclobutanone (4), cyclopentanone (5) and cyclohexanone (6) in Et2O to yield the first P-C5Me5 substituted C(3)-spirofused oxaphosphirane complexes 7a-c, 8a and 9a,a'. In the case of cyclopentanone and 1a the outcome of the reaction in THF was different: here the formation of 8a along with (anionic) phosphinoate complexes 14a and 15a was observed, the latter possess an unusual ring-opened oxaphosphirane and 2-cyclopentylidenecyclopentanone as co-ligands to the lithium cation. NMR, IR and MS data as well as single-crystal X-ray structures in the case of 7a-c, 8a, 9a and 15a are reported. DFT calculations on the parent 1-oxa-2-phosphaspiro[2.n]alkane pentacarbonylchromium(0) complexes 10 (a: n = 2; b: n = 3; c: n = 4; d: n = 5) revealed that both ring strain energies and G(r) values decrease significantly as the spiroring size increases. This is caused by an increase in the exocyclic α bond angle at the oxaphosphirane C(3) atom, hence decreasing the s-character of the corresponding orbitals involved in endocyclic bonds at C(3) and thus becoming better suited for accommodation of small ring angles.

Upper bonded retainers.

OBJECTIVE: To assess the frequency and type of upper bonded retainer failure and to identify possible predisposing factors. MATERIALS AND METHODS: The records of 466 consecutive patients with upper bonded retainers were analyzed retrospectively with respect to retainer failures and failure type as well as timing of failure, differences among operators, and the number of defects of the multibracket appliances (MB) prior to the retention period. RESULTS: A total of 58.2% of all patients experienced retainer failures. The average failure odds were 1.26 failures per retainer. The odds were highest for 3-3 retainers (1.37) and lowest for the 1-1 retainer (0.54). The detachment and total loss rates were significantly influenced by operator experience-both rates were lower for experienced practitioners. Total retainer losses occurred more frequently in case of previous MB defects, while retainer fractures were seen more frequently when the retainer included the canines. CONCLUSIONS: Upper bonded retainer failures are a frequent problem during the retention period (58.2% of patients). Less operator experience correlated with higher failure rates. An increased number of total retainer losses must also be expected with a decreasing number of bonding sites and in cases involving previous MB defects.

Identification of a dendritic cell population in normal testis and in chronically inflamed testis of rats with autoimmune orchitis.

Experimental autoimmune orchitis (EAO) in the rat is the primary chronic animal model for the investigation of one of the main causes of male infertility, viz., testicular inflammation. Dendritic cells (DC) are potent antigen-presenting cells that play a fundamental role in autoimmune disease. We investigated the number of DC in normal testis and examined whether DC infiltrated the testis during the development of EAO. EAO was induced by active immunization with testis homogenate and adjuvants in two strains of rat (Wistar and Sprague Dawley). The presence of DC in testis was determined, 50 and 80 days after the first immunization, by immunohistochemical staining with specific antibodies (OX-62 and CD11c), and then the total number of DC was measured by stereological analysis. Labeled cells were found only in the interstitial compartment and within granulomas of EAO animals. The number of DC in EAO testes increased compared with control rats in both strains, whereas the number of OX-62+ and CD11c+ cells in adjuvant controls remained unchanged compared with untreated rats. Interspecies variations in the quantity of DC were found, with the total number of DC per testis in untreated and adjuvant control Sprague-Dawley rats being about three times higher than that seen in Wistar rats. Moreover, the increase in DC numbers at 80 days was less prominent in EAO testes of Sprague-Dawley rats than in the Wistar strain in which EAO was more severe and showed a higher number of granulomae. Thus, we have identified the DC population in normal and chronically inflamed testis. The increase in DC observed in EAO suggests that, under inflammatory conditions, the modified action(s) of these cells is a factor in the induction of the autoimmune response in testis.

Identification of immunodominant autoantigens in rat autoimmune orchitis.

Infection and inflammation of the genital tract are amongst the leading causes of male infertility. Experimental autoimmune orchitis (EAO) in the rat serves as a model for the investigation of inflammatory testicular impairment. In this study, experiments were conducted to identify the molecules that are responsible for eliciting the autoimmune attack on the testis. EAO was induced in in-bred Wistar rats by active immunization with testis homogenates (EAO group I). Development of disease was observed using histological techniques and a new non-invasive three-dimensional (3D) imaging technology for in vivo monitoring, termed flat-panel volumetric computed tomography (fpvCT). Examination of control and EAO testes demonstrated the superior image quality of high-resolution fpvCT. A proteomics approach using 2D SDS-PAGE and immunoblotting analysis with EAO sera identified 12 spots. Seven were subsequently identified by mass spectrometry as heat shock proteins 60 (Hsp60) and 70 (Hsp70), disulphide isomerase ER-60, alpha-1-anti-trypsin, heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), sperm outer dense fibre major protein 2 (ODF-2), and phosphoglycerate kinase 1. Hsp70, ODF-2, hnRNP H1, and ER-60 were identified by all EAO sera studied. To test the capacity of the identified proteins to elicit testicular autoimmune disease, recombinant proteins were used either individually or in combination to immunize rats (EAO group II). In all groups, the incidence of EAO was 25%. Inflammatory-type (ED1+) and resident (ED2+) macrophages, lymphocytes (CD45RA+), and dendritic cells (Ox-62+) were strongly increased in EAO group II animals, comparable to the testes of EAO I rats. Pre-immunization with a low dose of recombinant Hsp 70, hnRNP H1 or ODF-2 before induction of EAO with testis homogenate significantly delayed the onset of EAO but could not prevent disease. The identification of testicular autoantigens will allow a better understanding of disease pathogenesis and could provide a basis for the development of novel therapies for inflammation-based male infertility.

The early transcription factor GATA-2 is expressed in classical Hodgkin's lymphoma.

Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin's lymphoma (cHL) are thought to be derived from germinal centre B-cells in almost all cases. However, expression profiling has revealed that HRS cells do not show a germinal centre B-cell-like phenotype. Although the nature of this aberrant phenotype and the underlying molecular mechanisms remain largely unknown, it has been reported that the activity of NOTCH1 plays an important role in the growth and survival of HRS cells. In some leukaemic cell lines, the effect of Notch signalling is mediated by the early transcription factor GATA-2. This and the fact that HRS cells lack expression of PU.1, which can repress Gata-2, led to an investigation of GATA-2 expression in HRS cells. GATA-2 expression was found in all the cHL-derived cell lines studied, but not in a Burkitt lymphoma-derived cell line. In addition, 50% of biopsies from patients with cHL contained GATA-2-expressing HRS cells. In contrast, neither normal germinal centre B-cells nor malignant cells of nodular lymphocyte-predominant Hodgkin's lymphoma, Burkitt lymphoma or diffuse large B-cell lymphoma expressed GATA-2. Thus, GATA-2 expression was found specifically in HRS cells of cHL, suggesting that GATA-2 is important in establishing the abnormal B-cell phenotype of HRS cells.

Identification of the periplasmic cobalamin-binding protein BtuF of Escherichia coli.

Cells of Escherichia coli take up vitamin B(12) (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in Salmonella. The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His(6) tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.