Lars Morawietz

Synovitis score: discrimination between chronic low-grade and high-grade synovitis

Aims : To standardize the histopathological assessment of synovial membrane specimens in order to contribute to the diagnostics of rheumatic and non‐rheumatic joint diseases.

Proposal for a histopathological consensus classification of the periprosthetic interface membrane

Aims: The introduction of clearly defined histopathological criteria for a standardised evaluation of the periprosthetic membrane, which can appear in cases of total joint arthroplasty revision surgery. Methods: Based on histomorphological criteria, four types of periprosthetic membrane were defined: wear particle induced type (detection of foreign body particles; macrophages and multinucleated giant cells occupy at least 20% of the area; type I); infectious type (granulation tissue with neutrophilic granulocytes, plasma cells and few, if any, wear particles; type II); combined type (aspects of type I and type II occur simultaneously; type III); and indeterminate type (neither criteria for type I nor type II are fulfilled; type IV). The periprosthetic membranes of 370 patients (217 women, 153 men; mean age 67.6 years, mean period until revision surgery 7.4 years) were analysed according to the defined criteria. Results: Frequency of histopathological membrane types was: type I 54.3%, type II 19.7%, type III 5.4%, type IV 15.4%, and not assessable 5.1%. The mean period between primary arthroplasty and revision surgery was 10.1 years for type I, 3.2 years for type II, 4.5 years for type III and 5.4 years for type IV. The correlation between histopathological and microbiological diagnosis was high (89.7%), and the inter-observer reproducibility sufficient (85%). Conclusion: The classification proposed enables standardised typing of periprosthetic membranes and may serve as a tool for further research on the pathogenesis of the loosening of total joint replacement. The study highlights the importance of non-infectious, non-particle induced loosening of prosthetic devices in orthopaedic surgery (membrane type IV), which was observed in 15.4% of patients.

Immunization with Glucose-6-Phosphate Isomerase Induces T Cell-Dependent Peripheral Polyarthritis in Genetically Unaltered Mice

Rheumatoid arthritis is a chronic inflammatory disease primarily affecting the joints. The search for arthritogenic autoantigens that trigger autoimmune responses in rheumatoid arthritis has largely focused on cartilage- or joint-specific Ags. In this study, we show that immunization with the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (G6PI) induces severe peripheral symmetric polyarthritis in normal mice. In genetically unaltered mice, T cells are indispensable for both the induction and the effector phase of G6PI-induced arthritis. Arthritis is cured by depletion of CD4+ cells. In contrast, Abs and FcγR+ effector cells are necessary but not sufficient for G6PI-induced arthritis in genetically unaltered mice. Thus, the complex pathogenesis of G6PI-induced arthritis in normal mice differs strongly from the spontaneously occurring arthritis in the transgenic K/B × N model where Abs against G6PI alone suffice to induce the disease. G6PI-induced arthritis demonstrates for the first time the induction of organ-specific disease by systemic autoimmunity in genetically unaltered mice. Both the induction and effector phase of arthritis induced by a systemic autoimmune response can be dissected and preventive and therapeutic strategies evaluated in this model.

Formation of cartilage repair tissue in articular cartilage defects pretreated with microfracture and covered with cell-free polymer-based implants.

The aim of our study was to evaluate the mid‐term outcome of a cell‐free polymer‐based cartilage repair approach in a sheep cartilage defect model in comparison to microfracture treatment. Cell‐free, freeze‐dried implants (chondrotissue®) made of a poly‐glycolic acid (PGA) scaffold and hyaluronan were immersed in autologous serum and used for covering microfractured full‐thickness articular cartilage defects of the sheep (n = 4). Defects treated with microfracture only served as controls (n = 4). Six months after implantation, cartilage implants and controls were analyzed by immunohistochemical staining of type II collagen, histological staining of proteoglycans, and histological scoring. Histological analysis showed the formation of a cartilaginous repair tissue rich in proteoglycans. Histological scoring documented significant improvement of repair tissue formation when the defects were covered with the cell‐free implant, compared to controls treated with microfracture. Immunohistochemistry showed that the cell‐free implant induced cartilaginous repair tissue and type II collagen. Controls treated with microfracture showed marginal formation of a mixed‐type repair tissue consisting of cartilaginous tissue and fibro‐cartilage. Covering of microfractured defects with the cell‐free polymer‐based cartilage implant is suggested to be a promising treatment option for cartilage defects and improves the regeneration of articular cartilage.

Diagnosis of periprosthetic infection following total hip arthroplasty – evaluation of the diagnostic values of pre- and intraoperative parameters and the associated strategy to preoperatively select patients with a high probability of joint infection

BackgroundThe correct diagnosis of a prosthetic joint infection (PJI) is crucial for adequate surgical treatment. The detection may be a challenge since presentation and preoperative tests are not always obvious and precise. This prospective study was performed to evaluate a variety of pre- and intraoperative investigations. Furthermore a detailed evaluation of concordance of each preoperative diagnosis was performed, together with a final diagnosis to assess the accuracy of the pre-operative assumption of PJI.MethodsBetween 01/2005 and 02/2007, a prospective analysis was performed in 50 patients, who had a two stage revision because of assumed PJI. Based on clinical presentation, radiography, haematological screening, or early failure, infection was assumed and a joint aspiration was performed. Depending upon these findings, a two stage revision was performed, with intra-operative samples for culture and histological evaluation obtained. Final diagnosis of infection was based upon the interpretation of the clinical presentation and the pre- and intraoperative findings.ResultsIn 37 patients a positive diagnosis of PJI could be made definitely. The histopathology yielded the highest accuracy (0.94) in identification of PJI and identified 35 of 37 infections (sensitivity 0.94, specificity 0.94, positive-/negative predictive value 0.97/0.86). Intra-operative cultures revealed sensitivities, specificities, positive-/negative predictive values and accuracy of 0.78, 0.92, 0.96, 0.63 and 0.82. These values for blood screening tests were 0.95, 0.62, 0.88, 0.80, and 0.86 respectively for the level of C-reactive protein, and 0.14, 0.92, 0.83, 0.29 and, 0.34 respectively for the white blood-cell count. The results of aspiration were 0.57, 0.5, 0.78, 0.29, and 0.54.ConclusionThe detection of PJI is still a challenge in clinical practice. The histopathological evaluation emerges as a highly practical diagnostic tool in detection of PJI. Furthermore, we found a discrepancy between the pre-operative suspicion of PJI and the final post-operative diagnosis, resulting in a slight uncertainty in whether loosening is due to bacterial infection or not. The variation in accuracy of the single tests may influence the detection of PJI. Level of Evidence: Diagnostic Level I.

Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study

BackgroundRheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease that leads to progressive cartilage destruction. Advances in the treatment of RA-related destruction of cartilage require profound insights into the molecular mechanisms involved in cartilage degradation. Until now, comprehensive data about the molecular RA-related dysfunction of chondrocytes have been limited. Hence, the objective of this study was to establish a standardized in vitro model to profile the key regulatory molecules of RA-related destruction of cartilage that are expressed by human chondrocytes.MethodsHuman chondrocytes were cultured three-dimensionally for 14 days in alginate beads and subsequently stimulated for 48 hours with supernatants from SV40 T-antigen immortalized human synovial fibroblasts (SF) derived from a normal donor (NDSF) and from a patient with RA (RASF), respectively. To identify RA-related factors released from SF, supernatants of RASF and NDSF were analyzed with antibody-based protein membrane arrays. Stimulated cartilage-like cultures were used for subsequent gene expression profiling with oligonucleotide microarrays. Affymetrix GeneChip Operating Software and Robust Multi-array Analysis (RMA) were used to identify differentially expressed genes. Expression of selected genes was verified by real-time RT-PCR.ResultsAntibody-based protein membrane arrays of synovial fibroblast supernatants identified RA-related soluble mediators (IL-6, CCL2, CXCL1–3, CXCL8) released from RASF. Genome-wide microarray analysis of RASF-stimulated chondrocytes disclosed a distinct expression profile related to cartilage destruction involving marker genes of inflammation (adenosine A2A receptor, cyclooxygenase-2), the NF-κB signaling pathway (toll-like receptor 2, spermine synthase, receptor-interacting serine-threonine kinase 2), cytokines/chemokines and receptors (CXCL1–3, CXCL8, CCL20, CXCR4, IL-1β, IL-6), cartilage degradation (matrix metalloproteinase (MMP)-10, MMP-12) and suppressed matrix synthesis (cartilage oligomeric matrix protein, chondroitin sulfate proteoglycan 2).ConclusionDifferential transcriptome profiling of stimulated human chondrocytes revealed a disturbed catabolic–anabolic homeostasis of chondrocyte function and disclosed relevant pharmacological target genes of cartilage destruction. This study provides comprehensive insight into molecular regulatory processes induced in human chondrocytes during RA-related destruction of cartilage. The established model may serve as a human in vitro disease model of RA-related destruction of cartilage and may help to elucidate the molecular effects of anti-rheumatic drugs on human chondrocyte gene expression.

Modified immunohistological staining allows detection of Ziehl-Neelsen-negative Mycobacterium tuberculosis organisms and their precise localization in human tissue

The diagnosis of mycobacterial infection depends on the Ziehl–Neelsen (ZN) stain, which detects mycobacteria because of their characteristic acid‐fast cell wall composition and structure. The histological diagnosis of tuberculosis (TB) comprises various aspects: (1) sensitive detection of mycobacteria; (2) precise localization of mycobacteria in the context of granulomatous lesions; (3) ‘staging’ of disease according to mycobacterial spread and granulomatous tissue integrity. Thus, detection of minute numbers of acid‐fast bacteria in tissue specimens is critical. The conventional ZN stain fails to identify mycobacteria in numbers less than 104 per ml. Hence many infections evade diagnosis. PCR is highly sensitive, but allows neither localization within tissues nor staging of mycobacterial disease, and positive findings frequently do not correlate with disease. In this study, an anti‐Mycobacterium bovis bacille Calmette–Guérin polyclonal antiserum (pAbBCG) was used to improve immunostaining, which was compared to the ZN stain in histological samples. Screening of tissue samples including lungs, pleural lesions, lymph nodes, bone marrow, and skin for mycobacterial infection revealed that pAbBCG staining detects infected macrophages harbouring intracellular mycobacteria or mycobacterial material as well as free mycobacteria that are present at low abundance and not detected by the ZN stain. The positive pAbBCG staining results were confirmed either by PCR analysis of microdissected stained tissue or by culture from tissue. This immunostaining approach allows precise localization of the pathogen in infected tissue. Copyright

Description of B lymphocytes and plasma cells, complement, and chemokines/receptors in acute liver allograft rejection.

Background. Although antibody mechanisms play a pathogenetic role in liver allograft rejection, no data exist on B lymphocytes, plasma cells, complement, and chemokines in rejected liver tissue. Methods. Liver biopsy specimens from 25 patients with acute allograft rejection (AR) (rejection activity index, RAI score: 1–9) were analyzed by immunohistochemistry (IH) and reverse transcriptase-polymerase chain reaction (RT-PCR) and compared with biopsy specimens taken prior to implantation (PI). The number of CD20+ and CD138+ cells was evaluated, and the presence and abundance of the chemokines macrophage inflammatory protein (MIP)-3&agr;, CXCL9, CXCL10, CXCL11, CXCL12, and their receptors CCR-6, CXCR3, and CXCR4 were examined. Complement depositions were visualized by C4d IH. Results. The numbers of B lymphocytes (P=0.002) and plasma cells (P=0.022) were significantly higher in AR biopsy specimens compared with PI biopsy specimens. MIP-3&agr;+ and CCR-6+ cells were detected in the portal fields of all AR biopsy specimens. IH double staining revealed a colocalization of MIP-3&agr;+/CD20+ cells; C4d deposits could be demonstrated along the portal capillaries. All examined chemokines and receptors could be detected in normal liver tissue and in AR biopsy specimens by RT-PCR and semiquantitative RT-PCR, demonstrating an overexpression of CXCL10 and -11. Conclusions. The significant increase of B lymphocytes and plasma cells during acute rejection, together with the lack of a significant increase of proliferating cells, indicates that the migration of B lymphocytes and plasma cells—promoted by the expression of B-cell activating chemokines/receptors—plays a key role in acute liver rejection. The C4d deposits along the portal capillaries indicate a humorally mediated alloresponse caused by the accumulated B and plasma cells.

Twenty-three neutrophil granulocytes in 10 high-power fields is the best histopathological threshold to differentiate between aseptic and septic endoprosthesis loosening

Aims: The histopathological diagnosis of infection in periprosthetic tissue from loose total joint endoprosthesis has been the subject of controversy. The aim was to define a histological criterion that would best differentiate between aseptic and septic endoprosthesis loosening.

Receptor activator of nuclear factor κB ligand is expressed in resident and inflammatory cells in aseptic and septic prosthesis loosening

Objective: The pathogenesis of periprosthetic bone loss in aseptic and septic prosthesis loosening is unclear. There is considerable evidence that macrophages and osteoclasts play a key role in focal bone erosion and osteolysis around the prosthesis. RANKL (receptor activator of nuclear factor κB ligand) was shown to be a potent osteoclastogenic factor, and to be involved in bone destruction of myeloma and rheumatoid arthritis patients. Osteoprotegerin (OPG) is the natural RANKL inhibitor and may prevent periprosthetic bone loss. Methods: The presence and distribution of RANKL, its receptor RANK and OPG in the periprosthetic interface of septically (n=5) and aseptically (n=6) loosened prostheses was examined by immunohistochemistry and immunoblotting. Additionally, the immunophenotype of the inflammatory infiltrate was determined [CD3, CD68, Ki‐67, tartrate‐resistant acid posphatase (TRAP)]. Results: Aseptic and septic cases revealed a different histopathologic pattern. However, in all cases RANKL and RANK could be demonstrated in macrophages and giant cells. In addition, RANKL detected by immunoblot analysis proved to have the same molecular weight as a recombinant RANKL used as a control (31 kD and approximately 48 kD). OPG was detected in aseptic loosening, where macrophages showed a strong staining, but multinucleated giant cells were only weakly stained. A weak OPG staining was also observed in septic loosening. Conclusion: The pathogenesis of bone loss in septic loosening remains unclear, because the septic membrane bears few macrophages and giant cells, and half of them express OPG. In aseptic loosening, macrophages might not be stimulated by RANKL as a result of OPG expression. But multinucleated giant cells may be activated, as they hardly express OPG. They might be responsible for periprosthetic bone loss in aseptic loosening as a result of their RANKL and RANK expression.