BK virus large T and VP-1 expression in infected human renal allografts

Objective. We investigated the expression of early and late phase BK virus (BKV) proteins and their interactions with host cell proteins in renal allografts, with ongoing polyomavirus associated nephropathy (PVAN), and correlated this with the nuclear and cell morphology. Methods. Frozen sections from three patients with renal allografts (two biopsies, one explant) with PVAN were analysed by indirect immunofluorescence using BKV specific anti-polyoma large T-antigen and anti-VP-1 antibodies, as well as anti-p53, anti-Ki67, anti-caspase-3, anti-bcl2 and anti-cytokeratin 22 antibodies. Nuclear morphology and size were estimated by DNA Hoechst staining. Results. In infected tubular cells the early and late phases of infection could be distinguished according to expression of large T-antigen or VP-1. The early phase revealed almost normal nuclear proportions, whereas in later phases nuclear size increased about 2 to 3 fold. Expression of large T-antigen was strongly associated with accumulation of p53 in the nucleus, accompanied by the activation of the cell cycle associated cell protein Ki67. In contrast, expression of BKV VP1 correlated only weakly with p53. Virus dependent cell lysis was due to necrosis, since neither caspase 3 nor nuclear nor cytoskeleton changes indicated apoptosis. Conclusion. In our selected patients with PVAN a clear distinction between early and late phases was possible, according to the protein expression patterns of BKV markers. Striking nuclear enlargement is only present in the late phase of infection. In the inflammatory setting of PVAN, BKV dependent effects appear to be mediated by the inhibition of p53, resulting in the activation of the cell cycle. We assume that in PVAN similar BKV mechanisms are operative as in certain in vitro system

C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques

Background. Detection of C4d along peritubular capillaries (PTC) in renal allograft biopsies is an independent prognostic marker of poor long-term graft survival. It is typically associated with circulating donor-specific antibodies. Since only little information is available on the best technique to stain C4d, we compared the two methods most often used for detecting C4d in renal allograft specimens. Methods. We investigated the expression of C4d along PTC in 64 renal allograft biopsies using a monoclonal antibody (Quidel) and immunofluorescence for frozen (F-IF) and a polyclonal antibody (Biomedica) and immunohistochemistry for formalin-fixed and paraffin-embedded (P-IHC) tissue samples. We compared the staining extent (diffuse, focal, minimal, no staining) in frozen and paraffin sections and evaluated the intra- and inter-observer concordance rates using kappa statistics. In addition, we determined the inter-observer concordance in 240 paraffin-embedded biopsies of a multi-centre study. Results. The inter- and intra-investigator concordance rate (κ = 0.9) of analysing the C4d expression by F-IF was excellent. In contrast, the detection of C4d by P-IHC demonstrated a substantially lower prevalence and extent of C4d expression with a lower intra- and inter-observer concordance rate (κ = 0.3). Only 69% of diffuse and 13% of focal C4d-expressing cases were in line classified by F-IF and P-IHC. On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by P-IHC than by F-IF. The inter-observer concordance rate in paraffin of the 64 renal biopsies and the multi-centre study was good, but not perfect (κ = 0.57 or 0.67). Conclusions. C4d staining determined on frozen tissue samples using F-IF with a monoclonal antibody appears to be better suited for diagnostic as well as research purposes. Future studies should correlate C4d staining patterns with circulating donor-specific antibodie

BK virus large T and VP-1 expression in infected human renal allografts

Objective. We investigated the expression of early and late phase BK virus (BKV) proteins and their interactions with host cell proteins in renal allografts, with ongoing polyomavirus associated nephropathy (PVAN), and correlated this with the nuclear and cell morphology

Digital pathology evaluation of complement C4d component deposition in the kidney allograft biopsies is a useful tool to improve reproducibility of the scoring

Complement C4d component deposition in kidney allograft biopsies is an established marker of antibody-mediated rejection. In the Banff 07 classification of renal allograft pathology, semi-quantitative evaluation of the proportion of C4d-positive peritubular capilaries (PTC) is used. We aimed to explore the potential of digital pathology tools to obtain quantitative and reproducible measure of C4d deposition in the renal allograft tissue

Expression of cathepsin K and MMP-1 indicate persistent osteodestruktive activity in longstanding ankylosing spondylitis

Abstract Background. Ankylosing spondylitis (AS) is a frequent largely genetically determined rheumatic disease that is characterised by spinal inflammation and new bone formation. However, the exact pathogenesis including pathology is still not clear. Objective. To analyse tissue obtained at spinal surgery by immunohistochemistry and compare the specimen of patients with AS to those with degenerative disc disease (DDD). Methods. Bony and soft tissue specimens of 30 AS and 20 DDD patients were obtained during spinal osteotomy from different anatomic regions including articular and spinous processes, interspinous ligaments and intervertebral disks. Immunohistolochemistry was performed with established markers for cathepsin-K, MMP-1, MMP-3 and RANK-ligand. Results. Cathepsin-K and MMP-1-positive cells were only observed in AS specimen. Cathepsin-K-positive multinucleated cells were detected at articular processes adjacent to fibrous tissues. MMP-1 was expressed in smaller mononuclear cells attached to bone, Invasion of bone by MMP-1 cells was seen at entheseal sites. In the intervertebral disks, most mononuclear cells were cathepsin-K-positive. Isolated cells expressing these matrix degrading enzymes found in DDD never showed signs of invasion. No differences were found for MMP-3 between AS and DDD. Clear expression of RANK ligand was only detected in one AS patient

Homology-based inference sets the bar high for protein function prediction

Background: Any method that de novo predicts protein function should do better than random. More challenging, it also ought to outperform simple homology-based inference. Methods: Here, we describe a few methods that predict protein function exclusively through homology. Together, they set the bar or lower limit for future improvements. Results and conclusions: During the development of these methods, we faced two surprises. Firstly, our most successful implementation for the baseline ranked very high at CAFA1. In fact, our best combination of homology-based methods fared only slightly worse than the top-of-the-line prediction method from the Jones group. Secondly, although the concept of homology-based inference is simple, this work revealed that the precise details of the implementation are crucial: not only did the methods span from top to bottom performers at CAFA, but also the reasons for these differences were unexpected. In this work, we also propose a new rigorous measure to compare predicted and experimental annotations. It puts more emphasis on the details of protein function than the other measures employed by CAFA and may best reflect the expectations of users. Clearly, the definition of proper goals remains one major objective for CAFA

C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques

BACKGROUND: Detection of C4d along peritubular capillaries (PTC) in renal allograft biopsies is an independent prognostic marker of poor long-term graft survival. It is typically associated with circulating donor-specific antibodies. Since only little information is available on the best technique to stain C4d, we compared the two methods most often used for detecting C4d in renal allograft specimens. METHODS: We investigated the expression of C4d along PTC in 64 renal allograft biopsies using a monoclonal antibody (Quidel) and immunofluorescence for frozen (F-IF) and a polyclonal antibody (Biomedica) and immunohistochemistry for formalin-fixed and paraffin-embedded (P-IHC) tissue samples. We compared the staining extent (diffuse, focal, minimal, no staining) in frozen and paraffin sections and evaluated the intra- and inter-observer concordance rates using kappa statistics. In addition, we determined the inter-observer concordance in 240 paraffin-embedded biopsies of a multi-centre study. RESULTS: The inter- and intra-investigator concordance rate (kappa = 0.9) of analysing the C4d expression by F-IF was excellent. In contrast, the detection of C4d by P-IHC demonstrated a substantially lower prevalence and extent of C4d expression with a lower intra- and inter-observer concordance rate (kappa = 0.3). Only 69% of diffuse and 13% of focal C4d-expressing cases were in line classified by F-IF and P-IHC. On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by P-IHC than by F-IF. The inter-observer concordance rate in paraffin of the 64 renal biopsies and the multi-centre study was good, but not perfect (kappa = 0.57 or 0.67). CONCLUSIONS: C4d staining determined on frozen tissue samples using F-IF with a monoclonal antibody appears to be better suited for diagnostic as well as research purposes. Future studies should correlate C4d staining patterns with circulating donor-specific antibodies

Dexamethasone-containing PLGA superparamagnetic microparticles as carriers for the local treatment of arthritis

Superparamagnetic iron oxide nanoparticles (SPIONs) are attractive materials that have been widely used in medicine for diagnostic imaging and therapeutic applications. In our study, SPIONs and the corticosteroid dexamethasone acetate (DXM) are co-encapsulated into PLGA microparticles for the aim of locally treating inflammatory conditions such as arthritis. The magnetic properties conferred by the SPIONs could help to maintain the microparticles in the joint with an external magnet. The aim of this study was to investigate the interaction between magnetic microparticles and human synovial fibroblasts in terms of microparticle uptake (FACS, confocal and optical microscopy), internalization mechanism (Prussian Blue staining, TEM, immunofluorescence), cell toxicity (MTT) and tissue reaction after intra-articular injection (histology). The results show that the microparticles have an excellent biocompatibility with synoviocytes and that they are internalized through a phagocytic process, as demonstrated by fluorescence-activated cell sorting and morphological analyses of cells exposed to microparticles. Histological analysis showed that the prepared microparticles did not induce any inflammatory reaction in the joint. This type of carrier could represent a suitable magnetically retainable intra-articular drug delivery system for treating joint diseases such as arthritis or osteoarthritis

Enhanced Th1 and Th17 responses and arthritis severity in mice with a deficiency of myeloid cell-specific interleukin-1 receptor antagonist

The balance between interleukin-1 (IL-1) and its specific inhibitor, the IL-1 receptor antagonist (IL-1Ra), plays a major role in the development of arthritis. The purpose of this study was to investigate the role of IL-1Ra produced specifically by myeloid cells in the control of collagen-induced arthritis (CIA) by using myeloid cell-specific IL-1Ra-deficient mice (IL-1Ra(DeltaM))