Deletion of the ferric uptake regulator Fur impairs the in vitro growth and virulence of Actinobacillus pleuropneumoniae

In order to investigate the role of the ferric uptake regulator Fur in the porcine lung pathogen Actinobacillus pleuropneumoniae, we constructed an isogenic in-frame deletion mutant, A. pleuropneumoniae Deltafur. This mutant showed constitutive expression of transferrin-binding proteins, growth deficiencies in vitro, and reduced virulence in an aerosol infection model.     

CD40 is functionally expressed on human keratinocytes

The CD40/gp39 pathway is known to be an important feature of B/T cell collaboration leading to T cell-dependent activation, proliferation or differentiation of B cells. Additionally, CD40 is involved in the regulation of B cell survival and apoptosis. Recently, CD40 has been shown to be expressed functionally on non-hematopoietic cells, i.e. endothelial cells. Here, we demonstrate that human keratinocytes (KC) cultured in vitro express CD40 constitutively. The surface expression of CD40 is markedly up-regulated following stimulation with interferon (IFN)-γ, but not with tumor necrosis factor-α or interleukin (IL)-1β. This process is regulated at the CD40 mRNA level as demonstrated by Northern blot analysis. Furthermore, ligation of CD40 via soluble gp39, the CD40 ligand, enhances intercellular adhesion molecule (ICAM)-1 and Bcl-x up-regulation on IFN-γ-stimulated KC, but not lymphocyte function-associated antigen (LFA)-3, B7-2, HLA-DR, or Fas expression. The release of IL-8 is also induced following CD40 ligation on KC. In psoriasis, a T cell-mediated inflammatory skin disease, KC have a markedly enhanced expression of CD40. This expression co-localizes with the expression of ICAM-1, Bcl-x, and an influx of CD3⁺ T cells. These findings suggest a functional role of CD40 on KC in inflammatory skin disorders such as psoriasis and could make a therapeutic intervention by disrupting the CD40/gp39 pathway an approach to consider in these inflammatory skin diseases.     

Collagen type I increases bone remodelling around hydroxyapatite implants in the rat tibia

The early interface reaction of cancellous bone to a nanocrystalline hydroxyapatite (HA) cement containing 3 wt% collagen type I (HA/Coll) with a setting under physiological temperature and pH was observed using immunohistochemical techniques. Pure HA served as a control. Cylinders with a diameter of 2 mm were implanted into the proximal tibia of 72 adult Wistar rats. Histological sections of 6 animals were prepared after 1, 2, 4, 6, 14 and 28 days. First, osteoblast-like cells as well as a marked reaction for osteonectin, osteopontin and its ligand CD44 were observed as early as 2 days after implantation at the interface around HA/Coll implants. Further, reactivity for ED1 and cathepsin D, both markers for phagocytotic cells, appeared earlier and stronger around HA/Coll. In cell counts, a significantly higher average number of ED1- and cathepsin D-positive phagocytotic cells was observed around the HA/Coll implants on days 6 (p < 0.01), 14 and 28 (p < 0.05). The number of osteopontin-positive cells was significantly higher around HA/Coll implants at days 6 and 14 (p < 0.05). Two weeks after the implantation, first islands of newly formed woven bone were observed around the HA/Coll implant, but not around the control implant. The amount of direct bone contact after 28 days averaged 28% around pure HA and 51% around HA/Coll implants (p < 0.05). While both implants displayed a good osteoconductivity, a higher bone remodelling activity was observed around collagen-containing HA implants compared to pure HA implants. It appears that the addition of collagen to HA implants can enhance both phagocytotic and osteogenic processes. This may result in an earlier acceptance and better osseointegration of the HA/Coll implants into the surrounding tissue.     

Mesenchymal stem cell-dependent formation of heterotopic tendon-bone insertions (osteotendinous junctions)

Ligament-to-bone and tendon-to-bone interfaces (entheses, osteotendinous junctions [OTJs]) serve to dissipate stress between soft tissue and bone. Surgical reconstruction of these interfaces is an issue of considerable importance as they are prone to injury and the integration of bone and tendon/ligament is in general not satisfactory. We report here the stem cell-dependent spontaneous formation of fibrocartilaginous and fibrous entheses in heterotopic locations of the mouse if progenitors possess a tenogenic and osteo-/chondrogenic capacity. This study followed the hypothesis that enhanced Bone Morphogenetic Protein (BMP)-signaling in adult mesenchymal stem cells that are induced for tendon formation may overcome the tendon-inherent interference with bone formation and may thus allow the stem cell-dependent formation of tendon-bone interfaces. The tenogenic and osteo-/chondrogenic competence was mediated by the adeno- and/or lentiviral expression of the biologically active Smad8 signaling mediator (Smad8ca) and of Bone Morphogenetic Protein 2 (BMP2). Modified mesenchymal progenitors were implanted in subcutaneous or intramuscular sites of the mouse. The stem cell-dependent enthesis formation was characterized histologically by immunohistological approaches and by in situ hybridization. Transplantation of modified murine stem cells resulted in the formation of tendinous and osseous structures exhibiting fibrocartilage-type OTJs, while, in contrast, the viral modification of primary human bone marrow-derived mesenchymal stromal/stem cells showed evidence of fibrous tendon-bone interface formation. Moreover, it could be demonstrated that Smad8ca expression alone was sufficient for the formation of tendon/ligament-like structures. These findings may contribute to the establishment of stem cell-dependent regenerative therapies involving tendon/ligaments and to the improvement of the insertion of tendon grafts at bony attachment sites, eventually.     

Molecular differentiation of Culicoides biting midges (Diptera: Ceratopogonidae) from the subgenus Culicoides Latreille in Denmark

Identification of Culicoides biting midges to species has attracted attention due to the recent outbreak of bluetongue disease in Northern Europe. Identification of Culicoides to species level has been based on morphological characters and is difficult as several species belonging to species complexes are hard to distinguish. We evaluated the use of the mitochondrial DNA cytochrome oxidase I gene (COI) barcode region in the identification of species within the subgenus Culicoides. COI barcode sequence divergence within species was <1%, whereas it ranged from 12.5% to 19.8% between subgenus Culicoides species. The divergence of subgenus Culicoides species to C. nubeculosus from the subgenus Monoculicoides ranged from 24.4% to 26.1%. Specimens were differentiated into eight unique clusters, including the four common Palaearctic species Culicoides punctatus, Culicoides pulicaris, Culicoides impunctatus, and Culicoides grisescens. Additionally, this study confirms the existence of Culicoides halophilus as a valid taxon and presents the first Culicoides deltus barcode sequences. Three additional groups of specimens were identified: Culicoides dk1 with a COI barcode diverging by 14.3% to 17.2% from other subgenus Culicoides species and Culicoides Kalix and Culicoides dk3, which diverged by 5.9% from each other and showed 12.5% to 17.6% divergence in COI barcode to subgenus Culicoides specimens.     

Serological reagents for the immunohistochemical analysis of melanoma metastases in sentinel lymph nodes

For the immunohistochemical analysis of melanoma, various serological reagents are available. Melanocyte differentiation markers are reactive with cells and tumors of melanocytic lineage. HMB45 to gp100 has been the most commonly used melanocyte differentiation marker. Recently it was complemented by reagents such as antibodies to Melan-A/MART-1 and tyrosinase. Other reagents, whose reactivity is not strictly confined to melanocyte differentiation antigens, are also commonly used. Among them, the most prominent is S100. Other reagents are D5 to MITF or PNL-2. The properties of these reagents are presented, and their usefulness as markers in the setting of metastatic melanoma in sentinel lymph nodes is discussed.     

T cell surveillance of oncogene-induced prostate cancer is impeded by T cell-derived TGF-β1 cytokine

Tolerance induction in T?cells takes place in most tumors and is thought to account for tumor evasion from immune eradication. Production of the cytokine TGF-β is implicated in immunosuppression, but the?cellular mechanism by which TGF-β induces T?cell dysfunction remains unclear. With a transgenic model of prostate cancer, we showed that tumor development was not suppressed by the adaptive immune system, which was associated with heightened TGF-β signaling in T?cells from the tumor-draining lymph nodes. Blockade of TGF-β signaling in T?cells enhanced tumor antigen-specific T?cell responses and inhibited tumor development. Surprisingly, T?cell- but not Treg cell-specific ablation of TGF-β1 was sufficient to augment T?cell cytotoxic activity and blocked tumor growth and metastases. These findings reveal that T?cell production of TGF-β1 is an essential requirement for tumors to evade immunosurveillance independent of TGF-β produced by tumors.     

Extracapsular extension but not the tumour burden of lymph node metastases is an independent adverse risk factor in lymph node-positive bladder cancer

Seiler R, von Gunten M, Thalmann G N & Fleischmann A
(2011) Histopathology  58 , 571–578
Extracapsular extension but not the tumour burden of lymph node metastases is an independent adverse risk factor in lymph node‐positive bladder cancer Aims: To evaluate risk factors in lymph node‐positive bladder cancer. Methods and results: Lymph node‐positive bladder cancer patients (n = 162), preoperatively staged N0M0, underwent cystectomy and standardized extended lymphadenectomy. Five‐year overall survival of the cohort was 33%. In univariate analysis, tumour stage (P < 0.006), extracapsular extension of lymph node metastases (P < 0.001), total diameter of metastases (P < 0.04) and lymph node stage (P < 0.03) were significantly correlated with overall survival (OS), disease‐specific survival (DSS) and recurrence‐free survival (RFS). On multivariate analysis, only extracapsular extension (OS, P < 0.002; DSS, P < 0.02; RFS, P = 0.058) and primary tumour stage (OS, P = 0.058; DSS, P < 0.02; RFS, P < 0.02) added independent prognostic information. Extracapsular extension of lymph node metastases did not correlate with a specific recurrence pattern; patients with organ‐confined tumours (pT1/2) never had pelvic relapse. Conclusions: Extracapsular extension of lymph node metastases but not lymph node tumour burden adds independent prognostic information in lymph node‐positive bladder cancer. These biological differences in lymph node‐positive bladder cancer are not reflected in the sixth, and challenge future, TNM classification.     

IL-5 is essential for vaccine-induced protection and for resolution of primary infection in murine filariasis

The pathways conferring immunity to human filariases are not well known, in part because human-pathogenic filariae do not complete a full life cycle in laboratory mice. We have used the only fully permissive infection of mice with filariae, i.e., infection of BALB/c mice with the rodent filarial nematode Litomosoides sigmodontis. Our previous results showed that worm development is inversely correlated with Th2 cytokine production and eosinophilia. The scope of the present study was to directly elucidate the role of interleukin-5 (IL-5) and eosinophils in controlling the development of L. sigmodontis after vaccination and in primary infection. BALB/c mice immunized with irradiated third-stage larvae (L3) were confirmed to have elevated IL-5 levels as well as high subcutaneous eosinophilia and to attack and reduce incoming larvae within the first 2 days, resulting in 70% reduction of worm load. Treatment of vaccinated mice with anti-IL-5 antibody (TRFK-5) suppressed both blood and tissue eosinophilia and completely abolished protection. This demonstrates, for the first time in a fully permissive filarial infection, that IL-5 is essential for protection induced by irradiated L3 larvae. In contrast, in primary-infected mice, anti-IL-5 treatment did not modify filarial infection within the 1st month, most likely because during primary infection IL-5-dependent mechanisms such as subcutaneous eosinophilia are induced too late to disturb worm establishment. However, there is a role for IL-5 late in primary infection where neutrophil-dependent worm encapsulation is also under the control of IL-5. Received: 30 March 2000     

Antimycin A resistance in a mutant Leishmania tarentolae strain is correlated to a point mutation in the mitochondrial apocytochrome b gene

In this paper we report the first case of antimycin A resistance in a protozoan parasite that is attributable to a mutation in the mitochondrial apocytochrome b (CYb) gene. We selected for, and isolated, a mutant Leishmania tarentolae strain that is resistant to antimycin A. This resistance was evident at the levels of the in vitro growth and enzymatic activity of the cytochrome bc1 complex. Molecular characterisation of the mutant revealed a Ser35Ile mutation in the expected region of the CYb gene. In kinetoplastids, CYb and other structural genes of the mitochondrial genome are located on the maxicircle component of the mitochondrial DNA, which is present in 20–50 copies. Primer-extension analysis confirmed the presence of the mutation at the mRNA level. The phenotypic manifestation of the mutation implies that the CYb mRNA is edited and translated within the mitochondrion. Thus, this finding provides direct evidence that edited RNAs are translated in kinetoplastid mitochondria. Furthermore, a defined mutation conferring drug resistance to a mitochondrial gene product can be exploited for the development of mitochondrial transfection systems for trypanosomatids. Received: 6 October / Accepted: 17 December 1999