Objectives: To investigate the specificity of three anti-CD68 monoclonal antibodies (mAbs) for macrophages (Mw) in immunohistochemistry (IHC) and flow cytometry (FACS). Methods: IHC was performed on cryostat sections of rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes using the anti-CD68 mAbs KP1, EBM11, and PGM1, and the fibroblast (FB) markers CD90 and prolyl 4-hydroxylase. Expression of CD68 was also analysed by FACS on the monocytic cell lines THP-1 and U937, as well as on synovial fibroblasts (SFB), skin FB, and gingival FB (both surface and intracellular staining). Results: In IHC, there was an overlap between CD68 (mAbs KP1 and EBM11) and the FB markers CD90/ prolyl 4-hydroxylase in the lining layer, diffuse infiltrates, and stroma of RA and OA synovial membranes. In FACS analysis of THP-1 and U937 cells, the percentage of cells positive for the anti-CD68 mAbs KP1 and EBM11 progressively increased from surface staining of unfixed cells, to surface staining of pre-fixed cells, to intracellular staining of the cells. Upon intracellular FACS of different FB, nearly all cells were positive for KP1 and EBM11, but only a small percentage for PGM1. In surface staining FACS, a small percentage of FB were positive for all three anti-CD68 mAbs. Conclusion: An overlap between CD68 (mAbs KP1 or EBM11) and the FB markers CD90 or prolyl 4-hydroxylase may prevent unequivocal identification of Mw in synovial tissue by IHC or in monocytic cells and FB upon intracellular FACS. This may be due to sharing of common markers by completely different cell lineages.

KeY is a tool that provides facilities for formal specification and verification of programs within a commercial platform for UML based software development. Using the KeY tool, formal methods and object-oriented development techniques are applied in an integrated manner. Formal specification is performed using the Object Constraint Language (OCL), which is part of the UML standard. KeY provides support for the authoring and formal analysis of OCL constraints. The target language of KeY based development is Java Card DL, a proper subset of Java for smart card applications and embedded systems. KeY uses a dynamic logic for Java Card DL to express proof obligations, and provides a state-of-the-art theorem prover for interactive and automated verification. Apart from its integration into UML based software development, a characteristic feature of KeY is that formal specification and verification can be introduced incrementally.

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All tissues of the joint are affected in some way in osteoarthritis because the joint is an interactively functioning unit. Our goal was to investigate the combined responses of articular cartilage and subchondral bone to altered loading conditions to improve our understanding of the physiology of these two components and, ultimately, the pathophysiology of osteoarthritis. A group of 20 female beagle dogs were divided pairwise into runners (n = 10) and controls (n = 10). The running training on a treadmill started at the age of 15 weeks, and during the following 40 weeks the running distance was gradually increased to 40 km/day with a 15 degree uphill inclination. With this daily running distance the beagles ran another 15 weeks. The samples for histology were taken from 11 different locations of the knee joint. Subchondral bone and articular cartilage histomorphometry was carried out in three different regions of the specimens (central, middle, and peripheral regions) using an image-analyzing system and an eyepiece graticule. In all regions of the articular cartilage, both the uncalcified and calcified cartilage showed slightly increased thickness in the runner dogs. The change was more evident in the peripheral and the central areas. The thickness of the subchondral bone plate tended to be higher in runners, too. Bone histomorphometric parameters showed significant signs of increased remodeling. The most notable change was the enlargement of the bone formation surface. The most intense remodeling was usually observed either centrally or peripherally in the articular surface. The strongest increase in trabecular bone volume and thickness of the cartilage was recorded in the femoropatellar area.(ABSTRACT TRUNCATED AT 250 WORDS)

BackgroundElective knee and hip arthroplasty is followed by infections in currently about 0.5–2.0 % of cases – a figure which is on the increase due to the rise in primary implants. Correct diagnosis early on is essential so that appropriate therapy can be administered. This work presents a retrospective analysis of the diagnoses of patients suffering infections after total hip or knee arthroplasty.Methods320 patients with prosthetic joint infection (PJI) following knee or hip arthroplasty were identified. They comprised a) 172 patients with an infection after total hip arthroplasty (THA): 56 % females (n = 96) and 44 % males (n = 76) with a mean age of 70.9 (39–92) years; and 148 patients with an infection after total knee arthroplasty (TKA): 55 % females (n = 82) and 45 % males (n = 66) with a mean age of 70.7 (15–87) years.ResultsAlthough significantly more TKA than THA patients reported pain, erythema, a burning sensation and swelling, no differences between the two groups were observed with respect to dysfunction, fever or fatigue. However, significant differences were noted in the diagnosis of loosening (THA 55 %, TKA 31 %, p < 0.001) and suspected infection using conventional X-rays (THA 61 %, TKA 29 %, p < 0.001). FDG-PET-CT produced very good results in nearly 95 % of cases. There were no differences between THA and TKA patients regarding levels of inflammation markers. Histological evaluation proved to be significantly better than microbiological analysis.SummaryThe clinical picture may be non-specific and not show typical inflammatory symptoms for a long time, particularly in PJI of the hip. As imaging only provides reliable conclusions after the symptoms have persisted for a long time, morphological imaging is not suitable for the detection of early infections. FDG-PT-CT proved to be the most successful technique and is likely to be used more frequently in future. Nevertheless, there are currently no laboratory parameters which are suitable for the reliable primary diagnosis of PJI. Diagnosis requires arthrocentesis, and the fluid obtained should always be examined both microbiologically and histologically.

To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1β. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

Controlling large particle systems in collective dynamics by a few agents is a subject of high practical importance, e.g., in evacuation dynamics. In this paper we study an instantaneous control approach to steer an interacting particle system into a certain spatial region by repulsive forces from a few external agents, which might be interpreted as shepherd dogs leading sheep to their home. We introduce an appropriate mathematical model and the corresponding optimization problem. In particular, we are interested in the interaction of numerous particles, which can be approximated by a mean-field equation. Due to the high-dimensional phase space this will require a tailored optimization strategy. The arising control problems are solved using adjoint information to compute the descent directions. Numerical results on the microscopic and the macroscopic level indicate the convergence of optimal controls and optimal states in the mean-field limit,i.e., for an increasing number of particles.

Richter's transformation (RT) is an aggressive lymphoma which occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL-phase involving TP53, CDKN2A, MYC, and NOTCH1, however a significant proportion of RT cases lack CLL-phase associated events. Here, we report that high levels of AKT phosphorylation occurs both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in RT patients. Genetic over-activation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and DLBCL. Multi-omics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed a S100-protein-defined subcluster of highly aggressive lymphoma cells, which developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation towards aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.