Changes in calcineurin message, enzyme activity and protein content in the spinal dorsal horn are associated with chronic constriction injury of the rat sciatic nerve.

Plasticity in the spinal dorsal horn is thought to underlie the development of neuropathic pain. Calcineurin (protein phosphatase 3) plays an important role in plasticity in the brain. Here we examined whether chronic constriction injury (CCI) of the sciatic nerve modifies calcineurin expression in the spinal dorsal horn. Male rats were assigned to control (uninjured), sham-operated or CCI groups. CCI animals exhibited both a shift in weight bearing and a reduction in paw withdrawal latencies as signs of pain behavior. At 3 days (3D) the pain behavior was associated with a significant increase in calcineurin gene expression, enzyme activity and content of its Aα isoform in the ipsilateral spinal dorsal horn. In contrast, while the pain behavior persisted at 7 days (7D) calcineurin gene expression returned to control levels and activity and protein content decreased. A single intrathecal injection of MK-801 15 min before the ligation attenuated both signs of pain behavior in 3D but not 7D CCI animals. The same pre-treatment also prevented the CCI-associated increases in calcineurin in these animals. These data suggested an involvement of calcineurin in CCI-elicited neuropathic pain. The time-dependent divergent changes in calcineurin expression may underlie the different phases of neuropathic pain development.

Expression of beta-dystroglycan is reduced or absent in many human carcinomas.

AIMS: Dystroglycan is an important structural and signalling protein that is expressed in most human cells. alpha-Dystroglycan has been investigated and found to be reduced in human cancers, but there is only one published study on the expression of beta-dystroglycan in human cancer and that was only on small numbers of breast and prostatic cancers. The aim was to conduct a comprehensive immunohistochemical survey of the expression of beta-dystroglycan in normal human tissues and common cancers. METHODS AND RESULTS: Triplicate tissue microarrays of 681 samples of normal human tissues and common cancers were stained using an antibody directed against the cytoplasmic component of beta-dystroglycan. beta-Dystroglycan was strongly expressed at the intercellular junctions and basement membranes of all normal human epithelia. Expression of beta-dystroglycan was absent or markedly reduced in 100% of oesophageal adenocarcinomas, 97% of colonic cancers, 100% of transitional cell carcinomas of the urothelium and 94% of breast cancers. In the breast cancers, the only tumours that showed any retention of beta-dystroglycan expression were small low-grade oestrogen receptor-positive tumours. The only cancers that showed retention of beta-dystroglycan expression were cutaneous basal cell carcinomas. CONCLUSIONS: There is loss or marked reduction of beta-dystroglycan expression (by immunohistochemistry) in the vast majority of human cancers surveyed. Since beta-dystroglycan is postulated to have a tumour suppressor effect, this loss may have important functional significance.

A case of chondromatosis indicates a synovial stem cell aetiology.

OBJECTIVE: To evaluate cell cultures derived from intrasynovial nodules from a patient with primary synovial chondromatosis (PSC) for aberrant numbers/differentiation of osteochondroprogenitor cells. METHODS: Cell cultures were established from PSC synovial nodules, or normal bovine or human osteoarthritis (OA) synovia (for comparison). Multi-lineage potential was determined using well-characterized in vitro culture systems to assess osteogenic, chondrogenic and adipogenic capability. RESULTS: Primary PSC cell cultures were typically fibroblastic but contained islands of dense cell clusters/nodules, some of which were isolated and cultured separately [putative osteochondroprogenitris (pOCP) cultures]. OA synovial cultures had barely detectable levels of alkaline phosphatase (AP) that increased (0.006+/-0.008 to 0.141+/-0.000 nmol p-nitrophenol/min/cm(2)) with dexamethasone treatment. AP activity was higher in primary PSC cell cultures and further enhanced by dexamethasone (from 0.076+/-0.022 to 5.735+/-0.000 nmol p-nitrophenol/min/cm(2)). Histochemically, AP was localized as discreet areas within PSC cultures. No AP activity was detected histochemically in OA or normal bovine synovial cultures. The pOCP cultures had high basal AP (5.036+/-0.439 nmol p-nitrophenol/min/cm(2)) and spontaneously formed mineralized nodules, which increased in number under standard osteogenic conditions. Under chondrogenic conditions, micromass or pellet-cultured pOCP cells spontaneously synthesized a matrix containing glycosaminoglycans and collagen II. In adipogenic medium, the number of lipid-containing cells was increased. CONCLUSIONS: Compared with cultures established from OA or normal synovia, cell cultures established from PSC synovial nodules were enriched in osteochondroprogenitors, which, unlike normal mesenchymal cells, differentiated along chondrogenic and osteogenic lineages in the absence of dexamethasone.

Expression of receptor activator of nuclear factor kappabeta ligand (RANKL) and tumour necrosis factor related, apoptosis inducing ligand (TRAIL) in breast cancer, and their relations with osteoprotegerin, oestrogen receptor, and clinicopathological variables.

BACKGROUND: Receptor activator of nuclear factor kappabeta ligand (RANKL) has an important role in bone remodelling, and tumour necrosis factor related, apoptosis inducing ligand (TRAIL) can induce apoptosis in cancer cells. Their functions are linked by their interactions with osteoprotegerin (OPG). OBJECTIVE: To investigate the expression of RANKL and TRAIL in a large series of unselected breast cancers and to analyse the relations between these expressions and the expression of OPG, oestrogen receptor, and clinicopathological variables. METHODS: 395 breast cancers were sampled into tissue microarrays and immunohistochemistry undertaken for RANKL and TRAIL. RESULTS: There was strong expression of RANKL in 14% of the cancers and strong expression of TRAIL in 30%. Expression of RANKL had a negative association with expression of oestrogen receptor (p = 0.036). Expression of TRAIL had a negative association with the Nottingham Prognostic Index (p = 0.021). There was a significant negative relation between expression of RANKL and TRAIL (p<0.005). Unsupervised cluster analysis produced a dendrogram that showed a clear division into two groups, and the expression of oestrogen receptor was significantly higher in one of those groups (p = 0.012). CONCLUSIONS: There is apparent loss of expression of RANKL in 86% of breast cancers; those tumours that retain expression tend to be oestrogen receptor negative and of a high histological grade. There is strong expression of TRAIL in 30% of breast cancers and these tend to be of better prognostic type. These results may be important in the processes of metastasis to bone and the apoptotic cell death pathway in cancer.

Colonic dendritic cells, intestinal inflammation, and T cell-mediated bone destruction are modulated by recombinant osteoprotegerin.

Autoimmune associated bone disease and intestinal inflammation are closely linked with deregulation and hyperactivation of autoreactive CD4 T cells. How these T cells are activated and mediate disease is not clear. Here we show that in the Interleukin 2-deficient mouse model of autoimmunity spontaneous osteopenia and colitis are caused by increased production of the ligand for receptor activator of NFkappaB (RANKL). RANKL acting via its receptor, receptor activator of NFkappaB (RANK), increases bone turnover and promotes intestinal dendritic cell (DC) survival in vivo. Modulation of RANKL-RANK interactions with exogenous recombinant osteoprotegerin (Fc-OPG) reverses skeletal abnormalities and reduces colitis by decreasing colonic DC numbers. This study identifies a common causal link between bone disease and intestinal inflammation and establishes the importance of DC in mediating colonic inflammation in vivo.

Osteoprotegerin inhibits the development of osteolytic bone disease in multiple myeloma.

Multiple myeloma is a B-cell malignancy characterized by the accumulation of plasma cells in the bone marrow and the development of osteolytic bone disease. The present study demonstrates that myeloma cells express the critical osteoclastogenic factor RANKL (the ligand for receptor activator of NF-kappa B). Injection of 5T2MM myeloma cells into C57BL/KaLwRij mice resulted in the development of bone disease characterized by a significant decrease in cancellous bone volume in the tibial and femoral metaphyses, an increase in osteoclast formation, and radiologic evidence of osteolytic bone lesions. Dual-energy x-ray absorptiometry demonstrated a decrease in bone mineral density (BMD) at each of these sites. Treatment of mice with established myeloma with recombinant osteoprotegerin (OPG) protein, the soluble decoy receptor for RANKL, prevented the development of lytic bone lesions. OPG treatment was associated with preservation of cancellous bone volume and inhibition of osteoclast formation. OPG also promoted an increase in femoral, tibial, and vertebral BMD. These data suggest that the RANKL/RANK/OPG system may play a critical role in the development of osteolytic bone disease in multiple myeloma and that targeting this system may have therapeutic potential.

The potent bisphosphonate ibandronate does not induce myeloma cell apoptosis in a murine model of established multiple myeloma.

Bisphosphonates are effective in the management of bone disease in patients with multiple myeloma and recent reports have suggested that they may also have an anti-tumour activity. In support of this, we have previously demonstrated that bisphosphonates can induce myeloma cell apoptosis in vitro; however, it remains unclear whether this occurs in vivo. We have therefore investigated the effect of the potent bisphosphonate ibandronate in the 5T2MM murine model of established multiple myeloma. Short-term treatment with a high dose of ibandronate had no effect on either myeloma cell number or the proportion of myeloma cells undergoing apoptosis. These observations suggest that although bisphosphonates induce apoptosis in myeloma cells in vitro, they may not have the same anti-tumour effects in vivo.

Trihalomethanes as initiators and promoters of carcinogenesis.

Chloroform and other trihalomethanes are contaminants of drinking water that have been demonstrated to be carcinogenic in laboratory animals. Determination of the mechanism of carcinogenicity of chloroform is required so that the animal data can be extrapolated to estimate the human health hazard. The extent of the binding of chloroform to rat liver and kidney DNA was approximately 0.1% the level of binding found for dimethylnitrosamine. Neither chloroform nor bromoform, in contrast to diethylnitrosamine-initiated GGTase-positive foci in either intact or partial hepatectomized rats, promoted with phenobarbital. Tumor-promoting activity of chloroform was indicated by the slight significant increase, compared to untreated controls, in the incidence of GGTase-positive foci in rats initiated with diethylnitrosamine (DENA) followed by the administration of chloroform twice weekly for a total of 15 doses. In this study, rats administered only the DENA or the chloroform did not contain an increased incidence of GGTase-positive foci compared to untreated controls. However, the incidence of foci in the group that received DENA followed by chloroform was not statistically different from that in either the group that received only the DENA or only the chloroform. In conclusion, we were unable to demonstrate tumor-initiating activity for chloroform, and the tumor-promoting activity of chloroform indicated by our results requires further confirmation.