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1.  Preanalytical variables and performance of diagnostic RNA-based gene expression analysis in breast cancer 
Virchows Archiv  2014;465(4):409-417.
Prognostic multigene expression assays have become widely available to provide additional information to standard clinical parameters and to support clinicians in treatment decisions. In this study, we analyzed the impact of variations in tissue handling on the diagnostic EndoPredict test results. EndoPredict is a quantitative reverse transcription PCR assay conducted on RNA from formalin-fixed, paraffin-embedded (FFPE) tissue that predicts the likelihood of distant recurrence in patients with ER-positive/HER2-negative breast cancer. In this study, we performed a total of 138 EndoPredict assays to study the effects of preanalytical variables such as time to fixation, fixation time, tumor cell content, and section storage time on the EndoPredict test results. A time to fixation of up to 12 h and fixation of up to 5 days did not affect the results of the gene expression test. Paired samples of FFPE sections with tumor cell content ranging from 15 to 95 % and tumor-enriched samples showed a correlation coefficient of 0.97. Test results of tissue sections that have been stored for 12 months at +4 or +20 °C showed a correlation of 0.99 when compared to results of nonstored sections. In conclusion, preanalytical tissue handling is not a critical factor for diagnostic gene expression analysis with the EndoPredict assay. The test can therefore be easily integrated into the standard workflow of molecular pathology.
PMCID: PMC4180906  PMID: 25218890
Breast cancer; Preanalytical; EndoPredict; Molecular pathology; Gene expression
2.  Multicentric Giant Cell Tumor of Bone: Synchronous and Metachronous Presentation 
Case Reports in Orthopedics  2013;2013:756723.
A 27-year-old man treated 2.5 years ago for synchronous multicentric giant cell tumor of bone located at the right proximal humerus and the right 5th finger presented now with complaints of pain in his right hip and wrist of two-month duration. Radiology and magnetic resonance revealed multicentric giant cell tumor lesions of the right proximal femur, the left ileum, the right distal radius, and the left distal tibia. The patient has an eighteen-year history of a healed osteosarcoma of the right tibia that was treated with chemotherapy, resection, and allograft reconstruction. A literature review establishes this as the first reported case of a patient with synchronous and metachronous multicentric giant cell tumor who also has a history of osteosarcoma.
PMCID: PMC3784266  PMID: 24106628
3.  BMP-2 Dependent Increase of Soft Tissue Density in Arthrofibrotic TKA 
Arthrofibrosis after total knee arthroplasty (TKA) is difficult to treat, as its aetiology remains unclear. In a previous study, we established a connection between the BMP-2 concentration in the synovial fluid and arthrofibrosis after TKA. The hypothesis of the present study was, therefore, that the limited range of motion in arthrofibrosis is caused by BMP-2 induced heterotopic ossifications, the quantity of which is dependent on the BMP-2 concentration in the synovial fluid.
Eight patients with arthrofibrosis after TKA were included. The concentration of BMP-2 in the synovial fluid from each patient was determined by ELISA. Radiologically, digital radiographs were evaluated and the grey scale values were determined as a measure of the tissue density of defined areas. Apart from air, cutis, subcutis and muscle, the soft-tissue density in the area of the capsule of the suprapatellar pouch was determined. The connection between the BMP-2 concentration and the soft-tissue density was then investigated.
The average BMP-2 concentration in the synovial fluid was 24.3 ± 6.9 pg/ml. The density of the anterior knee capsule was on average 136 ± 35 grey scale values. A linear correlation was shown between the BMP-2 concentration in the synovial fluid and the radiological density of the anterior joint capsule (R=0.84, p = 0.009).
We were able to show that there is a connection between BMP-2 concentration and soft-tissue density in arthrofibrosis after TKA. This opens up the possibility of conducting a prophylaxis against arthrofibrosis in risk patients by influencing the BMP-2 pathway.
PMCID: PMC3358793  PMID: 22629292
Arthrofibrosis; BMP-2; heterotopic ossifications; inflammation; stiff knee; total knee arthroplasty.
4.  Bonding the foe – NETting neutrophils immobilize the pro-inflammatory monosodium urate crystals 
In the presence of sodium, uric acid from purine metabolism precipitates as monosodium urate (MSU) needles and forms renal calculi or causes gouty arthritis in kidneys and joints, respectively. The latter is characterized by red, hot, and swollen arthritic joints. Here we report the in vitro effect of MSU crystals on blood granulocytes and analyze their contribution to granuloma formation and neutrophil extracellular traps (NETs) formation (NETosis) in synovial fluid of patients with gouty arthritis in vivo. We observed that MSU crystals induce NETosis in vitro in a reactive oxygen species (ROS)-dependent manner. Indeed, blocking ROS (e.g., the oxidative burst) by various anti-oxidants partially inhibited NETosis induced by MSU crystals. Analyses of synovial fluids and of tissue sections of patients suffering from gout revealed that NETs are also formed in vivo, especially during acute gouty flares and/or granuloma formation. Since prolonged exposure to NETs carries the risk for the development of chronic inflammation we also studied the opsonization of NETs, as a prerequisite for their clearance. The established dead cells’ opsonins C3b, galectin-9, and CRP decorated the residual dead cells’ corpses and opsonized these for disposal. Surprisingly, all three soluble pattern recognizing molecules spared the spread NET structures. We conclude that (i) MSU crystals are strong inducers of ROS-dependent NETosis and (ii) that the prolonged presence of NET-pathogen or NET-crystal aggregates observed in patients with systemic autoimmunity, especially in those with low serum DNase-1 activity, cannot be compensated by CRP, complement, and galectin-mediated phagocytic clearance.
PMCID: PMC3517988  PMID: 23233855
NETosis; NETs; MSU; opsonins; inflammation; ROS; gout
5.  Synovial tissues concentrate secreted APRIL 
Arthritis Research & Therapy  2009;11(5):R144.
A proliferation-inducing ligand (APRIL) from the TNF family, owing to its role in the generation and survival of plasma cells (PCs), is currently targeted for rheumatoid arthritis (RA) treatment. However, little is known about APRIL expression in RA lesions, hampering our understanding of the way APRIL may modulate this autoimmune disease.
We performed immunological staining of human normal, non-RA and RA synovial tissues with a pair of antibodies specifically recognizing APRIL-producing cells and secreted APRIL.
We detected significant amounts of secreted APRIL in normal synovium mostly concentrated around blood vessels and at the lining layer, but no cells producing APRIL. Meanwhile, we observed that blood neutrophils constitutively secrete APRIL, indicating that blood APRIL may diffuse into the synovium via its fenestrated vessels. Synovium from non-RA and RA patients retained similarly secreted APRIL, but in this case APRIL-producing cells, including neutrophils and macrophages, were present in the tissue. Notably, PCs - when present in RA synovium - accumulated in areas of APRIL retention, spreading from blood vessels towards the lining layer.
PCs accumulate in synovial zones rich in secreted APRIL, consistent with a pro-survival role of APRIL for PCs in RA. The concentration of APRIL by normal synovium indicates that this tissue may constitute a proper environment for PCs even before RA onset.
PMCID: PMC2787289  PMID: 19788740
6.  Autoregulation of Th1-mediated inflammation by twist1 
The Journal of Experimental Medicine  2008;205(8):1889-1901.
The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κB (NF-κB)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-κB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohn's disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-γ, IL-2, and tumor necrosis factor-α, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.
PMCID: PMC2525589  PMID: 18663125
7.  Decrease in expression of bone morphogenetic proteins 4 and 5 in synovial tissue of patients with osteoarthritis and rheumatoid arthritis 
Bone morphogenetic proteins (BMPs) have been identified as important morphogens with pleiotropic functions in regulating the development, homeostasis and repair of various tissues. The aim of this study was to characterize the expression of BMPs in synovial tissues under normal and arthritic conditions. Synovial tissue from normal donors (ND) and from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) were analyzed for BMP expression by using microarray hybridization. Differential expression of BMP-4 and BMP-5 was validated by semiquantitative RT-PCR, in situ hybridization and immunohistochemistry. Activity of arthritis was determined by routine parameters for systemic inflammation, by histological scoring of synovitis and by semiquantitative RT-PCR of IL-1β, TNF-α, stromelysin and collagenase I in synovial tissue. Expression of BMP-4 and BMP-5 mRNA was found to be significantly decreased in synovial tissue of patients with RA in comparison with ND by microarray analysis (p < 0.0083 and p < 0.0091). Validation by PCR confirmed these data in RA (p < 0.002) and also revealed a significant decrease in BMP-4 and BMP-5 expression in OA compared with ND (p < 0.015). Furthermore, histomorphological distribution of both morphogens as determined by in situ hybridization and immunohistochemistry showed a dominance in the lining layer of normal tissues, whereas chronically inflamed tissue from patients with RA revealed BMP expression mainly scattered across deeper layers. In OA, these changes were less pronounced with variable distribution of BMPs in the lining and sublining layer. BMP-4 and BMP-5 are expressed in normal synovial tissue and were found decreased in OA and RA. This may suggest a role of distinct BMPs in joint homeostasis that is disturbed in inflammatory and degenerative joint diseases. In comparison with previous reports, these data underline the complex impact of these factors on homeostasis and remodeling in joint physiology and pathology.
PMCID: PMC1526630  PMID: 16542506
8.  Perspectives and limitations of gene expression profiling in rheumatology: new molecular strategies 
Arthritis Research & Therapy  2004;6(4):140-146.
The deciphering of the sequence of the human genome has raised the expectation of unravelling the specific role of each gene in physiology and pathology. High-throughput technologies for gene expression profiling provide the first practical basis for applying this information. In rheumatology, with its many diseases of unknown pathogenesis and puzzling inflammatory aspects, these advances appear to promise a significant advance towards the identification of leading mechanisms of pathology. Expression patterns reflect the complexity of the molecular processes and are expected to provide the molecular basis for specific diagnosis, therapeutic stratification, long-term monitoring and prognostic evaluation. Identification of the molecular networks will help in the discovery of appropriate drug targets, and permit focusing on the most effective and least toxic compounds. Current limitations in screening technologies, experimental strategies and bioinformatic interpretation will shortly be overcome by the rapid development in this field. However, gene expression profiling, by its nature, will not provide biochemical information on functional activities of proteins and might only in part reflect underlying genetic dysfunction. Genomic and proteomic technologies will therefore be complementary in their scientific and clinical application.
PMCID: PMC464885  PMID: 15225356
expression profiling; genomics; molecular strategies; pathway models; signatures
9.  Infection of Synovial Fibroblasts in Culture by Yersinia enterocolitica and Salmonella enterica Serovar Enteritidis: Ultrastructural Investigation with Respect to the Pathogenesis of Reactive Arthritis 
Infection and Immunity  2001;69(12):7915-7921.
Synovial fibroblasts were infected with Yersinia enterocolitica or Salmonella enterica serovar Enteritidis and analyzed by electron microscopy and fluorescence in situ hybridization. Intracellular bacterial replication was followed by degradation leading to “ghosts” possessing lipopolysaccharides but not DNA. However, single bacteria survived for more than 2 weeks. Therefore, transient intra-articular infection might be the missing link between initial intestinal infection and late synovial inflammation in the pathogenesis of reactive arthritis.
PMCID: PMC98891  PMID: 11705977
10.  IgVH genes from different anatomical regions, with different histopathological patterns, of a rheumatoid arthritis patient suggest cyclic re-entry of mature synovial B-cells in the hypermutation process 
Arthritis Research  2000;2(4):303-314.
In the present study 55 IgVH genes amplified from three different anatomical regions of a rheumatoid arthritis (RA) patient were analyzed, adding further information on synovial B-cell maturation and recirculation in RA. This analysis demonstrated somatically mutated IgVh genes in all regions studied, with amino acid deletions and mixed IgVh molecules, suggesting the existence of a novel pathway to generate (auto) antibody specificities. Comparison of amino acid sequences of amplified genes that belong to the VH1 family (with predominantly the same germline counterpart) exhibited strong homology, indicating an apparently conserved mutational pattern. This suggests that the number of antigens that activate B cells in different locations is restricted. The most striking result was the finding of clonally related sequences in different anatomical regions, indicating a recirculation of activated B cells between the different affected joints.
Although IgV genes in rheumatoid B cells have been intensively analyzed, many questions concerning antigen driven B-cell maturation and recirculation remain unanswered. It would be interesting to know whether B-cell maturation in rheumatoid tissue is different from that in secondary lymphatic organs. Moreover, it would be interesting to know whether there exists a restricted number of antigens that act on the lesions of different anatomical sites of the RA patient, and whether B cells recirculate between the different joints.
RNA and genomic DNA were prepared from tissue sections from three different anatomical sites, with different histopathologies and different onsets (left and right peroneal tendons and cubita synovial membrane), of a RA patient. Genomic DNA was amplified by seminested polymerase chain reaction (PCR), and the cDNA corresponding to the RNA was amplified by PCR using primers specific for each IgVH family. The obtained sequences were compared with their germline counterparts on the V-Base data Bank [1]. An immunohisto-chemical analysis of the infiltrate and the clinical data of local disease activity were also included.
In the locations with longer disease duration (right peroneal tendon 5 months, left peroneal tendon 2 months) a very intense inflammatory infiltrate with germinal centers containing Ki-M4-positive follicular dendritic cells (FDC) was observed. In the location with shorter disease duration (right cubita 2 weeks) a low, diffuse and nonfollicular infiltration with marked oedema was detected. From the 55 analyzed clones seven expressed nonfunctional rearrangements (pseudogenes) with stop codons, and 48 were found to express functional genes. Among the 48 clones that expressed functional genes, there were two that had amino acid deletions on their complementarity determining region (CDR)2 - clones K194/1 and K194/111 - similar to the ones described by Wilson et al [2] and Goossens et al [3]. Two types of mixed molecules were found. Mixed molecules of the first type (k194/57, k194/67 and k194/109) are composed of rearrangements of two different IgV genes. Mixed molecules of the second type (k194/126, k194/119, k194/30 and k194/99) are composed of a IgV gene rearrangement that is fragmented by insertions of small random sequences. These insertions are different from the ones described by Wilson et al [2] since they are not duplicates or parts of IgV genes. The ratio of replacement mutations to silent mutations (R/S ratio) increased with disease duration. There was strong heterogeneity among the CDR3 segments.
The amino acid sequences that belonged to the VH1-family obtained from the three anatomical regions were primarily compared with the amino acid sequences of their closest germline counterparts (Fig. 1a). One result from this comparison was the heterogeneity in the CDR3 rearrangements. Moreover, sequences k194/58 and k194/82 are clonally related (confirmed at nucleotide level). Then, the 21 amino acid sequences were compared with the most widely used germline counterpart IgHV1-18*01 (Fig. 1b). All of these VH1 sequences had mainly conservative mutations in the framework region (FR) and nonconservative mutations in the CDR. Also, there was an almost overall conservation of the mutational cold spots and 'structural cold spots' [4] among the 19 VH1 segments. The replacement (11 from 19 replacements resulted in a proline residue) in position 34 of CDR2 could be interpreted as an antigen-selected mutational hotspot.
The comparison of the five sequences belonging to IgHV4-30-1/4-31*02 resulted in two types of clonal relation (Fig. 2a). The first type of clonal relation, between sequences k194/100 and k194/101 (Fig. 2b), suggests that both sequences are derived from a single progenitor cell. The second type of clonal relation is between sequences k194/23, k194/102 and k194/103 (Fig. 2c). It suggests that an unmutated progenitor cell gave rise to k194/23 (left peroneal tendon), from which k194/103 (right cubita) derived and later generated k194/102 (right cubita).
The analysis of the 55IgVH sequences corroborates the findings of other groups that studied a singlelocation and RA B-cell hybridomas [5,6,7,8,9,10] and adds further information on B-cell distribution and activation in RA. First, amino acid deletions and mixed molecules could be interpreted as novel pathways to generate antibody specificities, leading, for instance, to autoreactive antibodies that could contribute to the local and systemic tissue destruction. Second, an apparently conserved mutational pattern among the 19 amino acid VH1 segments suggests that in all three RA lesions of this patient the synovial B cells are dealing with a restricted number of antigens. Third, the existence of clonally related B cells in the cubita and left peroneal tendon leaves no doubt that in this patient there is a cyclic re-entry of mutated B cells in the hypermutation process [11]. The already mutated B cells from the early RA lesions sequentially colonize new germinal centers in secondary lymphatic organs as proposed by Kepler et al [12]. These reactivated B-cells then invade new anatomical regions, leading to the perpetuation of the chronic inflammation in RA.
PMCID: PMC17813  PMID: 11056671
IgVH genes; rheumatoid arthritis; synovial B-cell recirculation
11.  Amyloid arthropathy associated with multiple myeloma: polyarthritis without synovial infiltration of CD20+ or CD38+ cells 
Amyloid  2013;21(1):28-34.
To describe histological, immunohistochemical and ultrastructural features of synovial biopsies of amyloid arthropathy associated with multiple myeloma (MM).
Synovial biopsies from affected joints of two patients with MM and amyloid arthropathy were examined with light and electron microscopy, and immunohistochemically for expression of CD3, CD8, CD20, CD38, CD68, Ki-67 and vWF. Results were compared to values from osteoarthritis (OA, n = 26), rheumatoid arthritis (RA, n = 24) and normal (n = 15) synovial membranes.
There was no or only mild lining hyperplasia. Vascular density was not elevated, and there were few Ki-67+ proliferating cells in the stroma. The Krenn synovitis score classified one specimen as “low-grade” and one as “high-grade” synovitis. CD68+ and CD3+ cells were the predominant mononuclear inflammatory cells, whereas CD20+ and CD38+ cells were absent from both synovial membrane and synovial fluid sediment. Electron microscopy demonstrated amyloid phagocytosis by synovial macrophages. In hierarchical clustering the two amyloid arthropathy specimens were more closely related to OA than to RA or normal synovium.
This first detailed immunohistological analysis of MM-associated amyloid arthropathy suggests that it is a chronic synovitis that evolves despite the loss of humoral immunity seen in advanced MM. Instead, amyloid phagocytosis by synovial macrophages likely triggers and perpetuates local disease.
PMCID: PMC3956491  PMID: 24286442
Amyloidosis; arthritis; B cells; multiple myeloma; plasma cells; synovitis

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