Ida Bruun Kristensen

Muscle after spinal cord injury

The morphological and contractile changes of muscles below the level of the lesion after spinal cord injury (SCI) are dramatic. In humans with SCI, a fiber‐type transformation away from type I begins 4–7 months post‐SCI and reaches a new steady state with predominantly fast glycolytic IIX fibers years after the injury. There is a progressive drop in the proportion of slow myosin heavy chain (MHC) isoform fibers and a rise in the proportion of fibers that coexpress both the fast and slow MHC isoforms. The oxidative enzymatic activity starts to decline after the first few months post‐SCI. Muscles from individuals with chronic SCI show less resistance to fatigue, and the speed‐related contractile properties change, becoming faster. These findings are also present in animals. Future studies should longitudinally examine changes in muscles from early SCI until steady state is reached in order to determine optimal training protocols for maintaining skeletal muscle after paralysis. Muscle Nerve, 2009

Expression of osteoblast and osteoclast regulatory genes in the bone marrow microenvironment in multiple myeloma: only up-regulation of Wnt inhibitors SFRP3 and DKK1 is associated with lytic bone disease.

Abstract Multiple myeloma (MM) lytic bone disease (LBD) is caused by osteoclast activation and osteoblast inhibition. RANK/RANKL/OPG play central roles in osteoclast activation and Wnt inhibitor DKK1 in osteoblast inhibition. The role of other Wnt inhibitors is less clear. We evaluated gene expression of osteoclast regulators (RANK, RANKL, OPG, TRAIL, MIP1A), Wnt inhibitors (DKK1, SFRP2, SFRP3, sclerostin, WIF1) and osteoblast transcription factors (RUNX2, osterix) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the bone marrow (BM) microenvironment using snap-frozen BM biopsies, thereby achieving minimal post-sampling manipulation, and gene expression profiling (GEP) data, reflecting the in vivo situation. We analyzed 110 biopsies from newly diagnosed patients with MM and monoclonal gammopathy of unknown significance (MGUS) and healthy volunteers. LBD was evaluated using standard radiographs and the bone resorption marker CTX-1. Protein levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Among Wnt inhibitors, only SFRP3 and DKK1 were significantly overexpressed in advanced LBD, correlating with protein levels. SFRP3 correlated with CTX-1. Our findings support osteoblast inhibition as the driving force behind MM LBD.

Characterization of potential CD138 negative myeloma "stem cells".

Syndecan-1 is a heparan sulfate proteoglycan expressed by both normal and multiple myeloma (MM) plasma cells. Matsui et al. [1][1] reported the identification of a potential MM “stem cell” resembling less differentiated post germinal center B cells. These cells lacked expression of CD138 but

Decorin is down-regulated in multiple myeloma and MGUS bone marrow plasma and inhibits HGF-induced myeloma plasma cell viability and migration†

Decorin is a stromal‐produced small leucine‐rich proteoglycan known to attenuate tumour pro‐survival, migration, proliferation and angiogenic signalling pathways. Recent studies have shown that decorin interacts with the hepatocyte growth factor (HGF) receptor c‐Met, a potential key pathway in multiple myeloma (MM).

Hepatocyte growth factor pathway upregulation in the bone marrow microenvironment in multiple myeloma is associated with lytic bone disease.

Lytic bone disease (LBD) in multiple myeloma (MM) is caused by osteoclast hyperactivation and osteoblast inhibition. Based on in vitro studies, the hepatocyte growth factor (HGF) pathway is thought to be central in osteoblast inhibition. We evaluated the gene expression of the HGF pathway in vivo using bone marrow biopsies (BMBs) of patients with MM and monoclonal gammopathy of undetermined significance (MGUS), and healthy volunteers (HV). BMBs (N = 110) obtained at diagnosis were snap‐frozen and used to evaluate gene expression by quantitative reverse transcription polymerase chain reaction. LBD was evaluated using standard radiographs. Enzyme‐linked immunosorbent assay (ELISA) was performed on matched bone marrow plasma and immunohistochemistry on matched formalin‐fixed paraffin‐embedded biopsies. Gene expression of HGF, SDC1, and MET in BMBs were significantly altered in MM versus HV and MGUS, and HGF and MET correlated with the extent of LBD. A significant correlation between gene and protein expression levels was observed for SDC1 (Syndecan‐1) and HGF. The HGF bone marrow plasma level was significantly lower in MM patients with no/limited versus advanced LBD. Our novel approach using snap‐frozen BMBs seems generally applicable because it allows evaluation of gene expression independent of the extent of MM plasma‐cell infiltration. Our study highlights the importance of the HGF pathway in MM LBD.

Myeloma plasma cell expression of osteoblast regulatory genes: overexpression of SFRP3 correlates with clinical bone involvement at diagnosis

Osteolyüc bone disease (OBD) in mulüple myeloma (MM) is not fuUy understood. Microarray analysis of isolated plasma cells from patients with MM revealed overexpression of the osteoblast-regulating Wnt inhibitor DBCKl, which was associated with the degree of radiographie OBD [1]. In our recent findings in sorted MM plasma cells, only DKKl expression, but not expression of any of the examined osteoclast-acüvating factors, correlated with OBD [2]. Because we only found expression of DKKl in 72/171 of MM plasma cell samples, we further explored the expression of other osteoblast-inhibitory factors: secreted frizzled-related protein 3 (SFRP3), another Wnt inhibitor known to be secreted by myeloma plasma cells [3], and genes in the hepatocyte growth factor (HGF) pathway. Full-length HGF is known to be produced by myeloma plasma cells in vivo, and is among the 70 most up-regulated genes in MM [4]. Funcüonally, HGF inhibits osteoblastogenesis in vitro [5] and promotes migraüon of MM plasma cells [6]. Thus far, studies of HGF and its impact in MM have focused on measuring full-length or all isoforms of HGF expression. However, naturally occurring shorter isoforms of HGF (NKl and NK2) are known to work as parüal inhibitors of fuU-length HGF via binding to the HGF receptor MET [7]. Syndecan-1 (SDCl) is a low affinity co-receptor for HGF, and is known to modulate HGF interacüon with MET [8]. HGF bound to SDCl modulates the effect of HGF and may act as a carrier of HGF, thus infiuencing OBD [9]. We analyzed the expression levels of HGF and HGF transcript variants, MET, SDCl and SFRP3, in highly purified MM plasma cells from patients with MM and healthy volunteers using a sensitive and specific quanütaüve reverse transcripüon polymerase chain reacüon (qRT-PCR) protocol. Gene expression data were compared with convenüonal radiographicaUy assessed bone disease. Bone marrow (BM) samples from 171 newly diagnosed, untreated patients with MM and eight healthy volunteers were assessed. The median age of patients with MM was 62 years (range: 33-82), and the male/female raüo was 1.25:1. Thirty percent had no bone lesions, 28% had limited bone disease and 42% had advanced bone disease. Bone disease was divided into three subcategories (no OBD, limited or advanced) based on findings of convenüonal X-ray by senior specialists in radiology, as described by Haaber et ai [2], vñth limited OBD defined as one or > 2 osteolytic lesion(s) in a single anatomic region and advanced OBD as > 2 osteolyüc lesions in > 2 anatomic regions. Bone marrow samples were obtained from the iliac crest at the time of diagnosis following informed consent, and the study was approved by the Ethical Committee of Copenhagen Coimty and Region of Southern Denmark and was performed in accordance with the Declaration of Helsinki. Staining protocols and ñuorescence-activated cell sorting (FACS) were performed as previously described [2]. Brieñy, plasma cells were sorted directly into PCR tubes, resulüng in > 96% purity. We used an approach developed by our group for global amplification with sequence-independent primers [10]. For quantification of genes, we used Primer Express software (Applied Biosystems) to design reproducible qRTPCR assays (using SYBR green and hydrolysis prohes; see Table I for primers and probes). SYBR green assays showed only one peak, and the melüng point was verified as consistent in all samples. Serial diluüons determined the efficiency of the designed assays to be above 0.9. The quantitaüve gene expression for each paüent sample was examined in triplicate following the same protocol as previously described [2] and analyzed as described earlier [10]. The relative mRNA gene expression for each gene, ACT, was normalized to ß-actin (BA) expression. /Ul gene expression was determined with a fixed cut-off level (ioCtgene-ctBA= ;̂ QACt> ^Q-S) to define a sample as either posiüve or negative. Non-parametric tests for trends among ordered groups were used to compare differences in gene expression levels between > 2 groups. For comparison between two groups, two-sample Kolmogorov-Smith tests were used. Spearmans

Upregulation of Syndecan-1 in the bone marrow microenvironment in multiple myeloma is associated with angiogenesis.

Syndecan‐1 (SDC1), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and interleukin‐6 (IL6) are expressed by malignant plasma cells and cells in the bone marrow microenvironment and may be involved in the angiogenic process in multiple myeloma (MM).

Clinicopathological features of plasmablastic multiple myeloma: a population-based cohort

Multiple myeloma (MM) is a common malignant hematological disease displaying considerable heterogeneity. Historical data indicate a prognostic significance of plasmablastic morphology, proliferation, and adverse cytogenetics, but there is little knowledge on the degree of interdependency of these parameters. The aim of this study was to study the degree of overlap between these variables. In a consecutive population‐based cohort of 194 untreated MM patients, morphology, and proliferation index, using immunohistochemical double staining for Ki‐67 and CD138, was analyzed. In addition, cytogenetic changes were studied by karyotyping and fluorescence in situ hybridization (FISH). Plasmablastic morphology correlated with unfavorable clinical features, high proliferation index, high percentage of plasma cell infiltration in the bone marrow, abnormal karyotype, and del(13q) detected by karyotyping, which indicates that plasmablastic morphology reflects advanced and highly proliferative disease. However, plasmablastic morphology did not correlate with established adverse prognostic cytogenetics identified by FISH, for example, t(4;14), t(14;16) and del(17p).

Myeloid sarcoma developing in pre-existing pyoderma gangrenosum.

We report here a case of pyoderma gangrenosum in a patient with myelodysplastic syndrome developing into myeloid sarcoma as a sign of transformation to acute leukaemia. The patient was treated successfully with intensive chemotherapy and achieved complete remission, and her otherwise expanding ulcers started to heal. This is the first reported case of secondary blastic infiltration in pyoderma gangrenosum, and it underlines the importance of performing re-biopsy of non-healing ulcers, especially in patients with an underlying haematological disease.

Abnormal IGF-Binding Protein Profile in the Bone Marrow of Multiple Myeloma Patients

Insulin-like growth factor (IGF) signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM). In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6), leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17), monoclonal gammopathy of undetermined significance (MGUS) (n = 37), and control individuals (n = 15), using ELISA (IGFs) and 125I-IGF1 Western Ligand Blotting (IGFBPs). MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5-3.8 fold) and decrease in intact IGFBP-3 (0.6-0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration.