Genetic Sharing with Cardiovascular Disease Risk Factors and Diabetes Reveals Novel Bone Mineral Density Loci

Bone Mineral Density (BMD) is a highly heritable trait, but genome-wide association studies have identified few genetic risk factors. Epidemiological studies suggest associations between BMD and several traits and diseases, but the nature of the suggestive comorbidity is still unknown. We used a novel genetic pleiotropy-informed conditional False Discovery Rate (FDR) method to identify single nucleotide polymorphisms (SNPs) associated with BMD by leveraging cardiovascular disease (CVD) associated disorders and metabolic traits. By conditioning on SNPs associated with the CVD-related phenotypes, type 1 diabetes, type 2 diabetes, systolic blood pressure, diastolic blood pressure, high density lipoprotein, low density lipoprotein, triglycerides and waist hip ratio, we identified 65 novel independent BMD loci (26 with femoral neck BMD and 47 with lumbar spine BMD) at conditional FDR < 0.01. Many of the loci were confirmed in genetic expression studies. Genes validated at the mRNA levels were characteristic for the osteoblast/osteocyte lineage, Wnt signaling pathway and bone metabolism. The results provide new insight into genetic mechanisms of variability in BMD, and a better understanding of the genetic underpinnings of clinical comorbidity.

A phase 2 study of high-activity 186Re-HEDP with autologous peripheral blood stem cell transplant in progressive hormone-refractory prostate cancer metastatic to bone

Purpose: We investigated the potential for improvement in disease control by use of autologous peripheral blood stem cell transplant (PBSCT) to permit administration of high activities of 186Re-hydroxyethylidene diphosphonate (HEDP) in patients with progressive hormone-refractory prostate cancer (HRPC).

Methods: Eligible patients had progressive HRPC metastatic to bone, good performance status and minimal soft tissue disease. Patients received 5,000 MBq of 186Re-HEDP i.v., followed 14 days later by PBSCT. Response was assessed using PSA, survival, pain scores and quality of life.

Results: Thirty-eight patients with a median age of 67 years (range 50–77) and a median PSA of 57 ng/ml (range 4–3,628) received a median activity of 4,978 MBq 186Re-HEDP (range 4,770–5,100 MBq). The most serious toxicity was short-lived grade 3 thrombocytopenia in 8 (21%) patients. The median survival of the group is 21 months (95%CI 18–24 months) with Kaplan-Meier estimated 1- and 2-year survival rates of 83% and 40% respectively. Thirty-one patients (81%, 95% CI 66–90%) had stable or reduced PSA levels 3 months post therapy while 11 (29%, 95% CI 15–49%) had PSA reductions of >50% lasting >4 weeks. Quality of life measures were stable or improved in 27 (66%) at 3 months.

Conclusion: We have shown that it is feasible and safe to deliver high-activity radioisotope therapy with PBSCT to men with metastatic HRPC. Response rates and survival data are encouraging; however, further research is needed to define optimal role of this treatment approach.

The Pro and Con of Measles Virus in Paget's Disease: Con

This is a joint study with Stuart Ralson's group on Paget's disease. For the last decade I have played a leading role in the evaluation of the postulated role of paramyxoviruses in Paget's Disease of Bone (PDB).

Bioreactor Expansion of Human Adult Bone Marrow-Derived Mesenchymal Stem Cells

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell transplantation (HSCT) alleviates complications such as graft-versus-host disease, leading to a speedy recovery of hematopoiesis. To meet such clinical demand, a fast MSCs expansion method is required. In the present study, we examined the feasibility of expanding MSCs from the isolated bone marrow mononuclear cells using a rotary bioreactor system. The cells were cultured in a rotary bioreactor with Myelocult� medium containing a combination of supplementary factors, including stem cell factor (SCF), interleukin 3 and 6 (IL-3, IL-6). After 8 days of culture, total cell numbers, Stro-1+CD44+CD34- MSCs and CD34+CD44+Stro-1- HSCs were increased 9, 29, and 8 folds respectively. Colony forming efficiency-fibroblast per day (CFE-F/day) of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSCs markers endoglin (SH2) and vimentin, whereas markers associated with lineage differentiation including osteocalcin (osteogenesis), Type II collagen (chondrogenesis) and C/EBPα (adipogenesis) were not detected. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocytes and adipocytes. Taken together, we conclude that the rotary bioreactor with the modified Myelocult� medium reported in this study may be used to rapidly expand MSCs.

Increased cancellous bone and connectivity in coxarthrosis

Patients with coxarthrosis (cOA) have a reduced incidence of intracapsular femoral neck fracture, suggesting that cOA offers protection. The distribution of bone in the femoral neck was compared in cases of coxarthrosis and postmortem controls to assess the possibility that disease-associated changes might contribute to reduced fragility. Whole cross-section femoral neck biopsies were obtained from 17 patients with cOA and 22 age- and sex-matched cadaveric controls. Densitometry was performed using peripheral quantitated computed tomography (pQCT) and histomorphometry on 10-µm plastic-embedded sections. Cortical bone mass was not different between cases and controls (P > 0.23), but cancellous bone mass was increased by 75% in cOA (P = 0.014) and histomorphometric cancellous bone area by 71% (P <0.0001). This was principally the result of an increase of apparent density (mass/vol) of cancellous bone (+45%, P = 0.001). Whereas cortical porosity was increased in the cases (P <0.0001), trabecular width was also increased overall in the cases by 52% (P <0.001), as was cancellous connectivity measured by strut analysis (P <0.01). Where osteophytic bone was present (n = 9) there was a positive relationship between the amount of osteophyte and the percentage of cancellous area (P <0.05). Since cancellous bone buttresses and stiffens the cortex so reducing the risk of buckling, the increased cancellous bone mass and connectivity seen in cases of cOA probably explain, at least in part, the ability of patients with cOA to resist intracapsular fracture of the femoral neck during a fall.

Regulation and consequences of differential gene expression in diabetic kidney disease

DIN (diabetic nephropathy) is the leading cause of end-stage renal disease worldwide and develops in 25-40% of patients with Type 1 or Type 2 diabetes mellitus. Elevated blood glucose over long periods together with glomerular hypertension leads to progressive glomerulosclerosis and tubulointerstitial fibrosis in susceptible individuals. Central to the pathology of DIN are cytokines and growth factors such as TGF-beta (transforming growth factor beta) superfamily members, including BMPs (bone morphogenetic protein) and TGF-beta 1, which play key roles in fibrogenic responses of the kidney, including podocyte loss, mesangial cell hypertrophy, matrix accumulation and tubulointerstitial fibrosis. Many of these responses can be mimicked in in vitro models of cells cultured in high glucose. We have applied differential gene expression technologies to identify novel genes expressed in in vitro and in vivo models of DN and, importantly, in human renal tissue. By mining these datasets and probing the regulation of expression and actions of specific molecules, we have identified novel roles for molecules such as Gremlin, IHG-1 (induced in high glucose-1) and CTGF (connective tissue growth factor) in DIN and potential regulators of their bioactions.

Neuropeptide Y (NPY) and NPY Y1 receptor in periodontal health and disease

Abstract
OBJECTIVES:
Neuropeptide Y (NPY) coordinates inflammation and bone metabolism which are central to the pathogenesis of periodontitis. The present study was designed to determine whether NPY was quantifiable in human gingival crevicular fluid (GCF) and to test the null hypothesis that GCF levels of NPY were the same in periodontal health and disease. A subsidiary aim was to determine the potential functionality of released NPY by detecting the presence of NPY Y1 receptors in gingival tissue.
DESIGN:
The periodontitis group consisted of 20 subjects (10 females and 10 males) mean age 41.4 (S.D. 9.6 years). The control group comprised 20 subjects (10 females and 10 males) mean age 37.4 (S.D. 11.7 years). NPY levels in GCF were measured in periodontal health and disease by radioimmunoassay. NPY Y1 receptor expression in gingival tissue was determined by Western blotting of membrane protein extracts from healthy and inflamed gum.
RESULTS:
Healthy sites from control subjects had significantly higher levels of NPY than diseased sites from periodontitis subjects. NPY Y1 receptor protein was detected in both healthy and inflamed gingival tissue by Western blotting.
CONCLUSIONS:
The significantly elevated levels of NPY in GCF from healthy compared with periodontitis sites suggests a tonic role for NPY, the functionality of which is indicated by the presence of NPY Y1 receptors in local gingival tissue.

Extracellular BMP-antagonist regulation in development and disease: tied up in knots

Developmental processes are regulated by the bone morphogenetic protein (BMP) family of secreted molecules. BMPs bind to serine/threonine kinase receptors and signal through the canonical Smad pathway and other intracellular effectors. Integral to the control of BMPs is a diverse group of secreted BMP antagonists that bind to BMPs and prevent engagement with their cognate receptors. Tight temporospatial regulation of both BMP and BMP-antagonist expression provides an exquisite control system for developing tissues. Additional facets of BMP-antagonist biology, such as crosstalk with Wnt and Sonic hedgehog signaling during development, have been revealed in recent years. In addition, previously unappreciated roles for the BMP antagonists in kidney fibrosis and cancer have been elucidated. This review provides a description of BMP-antagonist biology, together with highlights of recent novel insights into the role of these antagonists in development, signal transduction and human disease.

Allelic depletion of grem1 attenuates diabetic kidney disease.

OBJECTIVE: Gremlin (grem1) is an antagonist of the bone morphogenetic protein family that plays a key role in limb bud development and kidney formation. There is a growing appreciation that altered grem1 expression may regulate the homeostatic constraints on damage responses in diseases such as diabetic nephropathy. RESEARCH DESIGN AND METHODS: Here we explored whether knockout mice heterozygous for grem1 gene deletion (grem1(+/-)) exhibit protection from the progression of diabetic kidney disease in a streptozotocin-induced model of type 1 diabetes. RESULTS: A marked elevation in grem1 expression was detected in the kidneys and particularly in kidney tubules of diabetic wild-type mice compared with those of littermate controls. In contrast, diabetic grem1(+/-) mice displayed a significant attenuation in grem1 expression at 6 months of diabetes compared with that in age- and sex-matched wild-type controls. Whereas the onset and induction of diabetes were similar between grem1(+/-) and wild-type mice, several indicators of diabetes-associated kidney damage such as increased glomerular basement membrane thickening and microalbuminuria were attenuated in grem1(+/-) mice compared with those in wild-type controls. Markers of renal damage such as fibronectin and connective tissue growth factor were elevated in diabetic wild-type but not in grem1(+/-) kidneys. Levels of pSmad1/5/8 decreased in wild-type but not in grem1(+/-) diabetic kidneys, suggesting that bone morphogenetic protein signaling may be maintained in the absence of grem1. CONCLUSIONS: These data identify grem1 as a potential modifier of renal injury in the context of diabetic kidney disease.

Pim2 cooperates with PML-RAR alpha to induce acute myeloid leukemia in a bone marrow transplantation model

Although the potential role of Pim2 as a cooperative oncogene has been well described in lymphoma, its role in leukemia has remained largely unexplored. Here we show that high expression of Pim2 is observed in patients with acute promyelocytic leukemia (APL). To further characterize the cooperative role of Pim2 with promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha), we used a well-established PML-RAR alpha (PR alpha) mouse model. Pim2 coexpression in PR alpha-positive hematopoietic progenitor cells (HPCs) induces leukemia in recipient mice after a short latency. Pim2-PR alpha cells were able to repopulate mice in serial transplantations and to induce disease in all recipients. Neither Pim2 nor PR alpha alone was sufficient to induce leukemia upon transplantation in this model. The disease induced by Pim2 overexpression in PR alpha cells contained a slightly higher fraction of immature myeloid cells, compared with the previously described APL disease induced by PR alpha. However, it also clearly resembled an APL-like phenotype and showed signs of differentiation upon all-trans retinoic acid (ATRA) treatment in vitro. These results support the hypothesis that Pim2, which is also a known target of Flt3-ITD (another gene that cooperates with PML-RAR alpha), cooperates with PR alpha to induce APL-like disease. (Blood. 2010; 115(22): 4507-4516)

The emerging relevance of the cysteine protease Cathepsin S in disease

Cathepsin S is a lysosomal cysteine protease that has been shown to play a key role in MHC class II antigen presentation. Consequently, it has been extensively evaluated as a therapeutic target in autoimmune diseases, such as rheumatoid arthritis and psoriasis. Additionally, clinical and mechanistic evidence is emerging, revealing its inappropriate expression and secretion in a wide range of disease states including atherosclerosis and tumourigenesis. This review covers the known role and consequences of cathepsin S activity in these pathological disorders, highlighting various studies that have demonstrated its utility as a therapeutic target. This review also examines challenges that exist towards the development of agents that specifically target this protease and discusses the studies to date that have applied cathepsin S inhibitors in disease models.

Long-term bone marrow culture profiles in patients with myelodysplastic syndromes are not explicable by defective apoptosis

It has been suggested that increased intramedullary apoptosis may explain the paradox between peripheral blood cytopenias and the hyper- or normo-cellular bone marrow observed in the myelodysplastic syndromes (MDS). We wished to see if culture performance could be related to the presence of apoptotic cells in a group of patients with MDS (12 patients) and other patients with peripheral blood cytopenias (six patients) which caused diagnostic difficulty. There was no correlation between LTBMC or adherent cell growth and the presence of apoptotic cells in the original marrow sample. A variable degree of apoptosis was observed in both groups of patients. LTBMC profiles correlated well with diagnosis but were unrelated to the extent of intramedullary apoptosis. This suggests that apoptosis is a much more ubiquitous process in disease than previously thought. (C) 1998 Elsevier Science Ltd. All rights reserved.

Lead contamination and associated disease in captive and reintroduced red kites Milvus milvus in England

Since 1989, a red kite Milvus milvus reintroduction programme has been underway in the United Kingdom, with 4-6 week old nestlings brought into captivity and held for 6-8 weeks before reintroduction. As scavengers, red kites may consume unretrieved game, and ingest shot or lead (Pb) fragments in their prey's flesh. We evaluated exposure to Pb in captive and wild red kites by taking blood samples from 125 captive young red kites prior to release, through analysing 264 pellets (regurgitated by wild birds) collected from under a roost site, and analysing Pb concentrations in livers and/or bones of 87 red kites found dead between 1995 and 2003. Lead isotope analyses of livers were also conducted in an effort to identify Pb exposure routes. Forty-six (36.8%) kites sampled prior to release had elevated blood Pb concentrations (201-3340 microg l(-1)). The source of this Pb was probably small fragments of lead ammunition in the carcasses of birds or mammals either fed to the nestlings by their parents or, more likely, subsequently whilst in captivity. Once released, kites were also exposed to lead shot in their food, and a minimum of 1.5-2.3% of regurgitated pellets contained Pb gunshot. Seven of 44 red kites found dead or that were captured sick and died within a few days had elevated (>6 mg kg(-1) dry weight [d.w.]) liver Pb concentrations, and six of these (14%) had concentrations of >15 mg kg(-1) d.w., compatible with fatal Pb poisoning. Post-mortem analyses indicated that two of these birds had died of other causes (poisoning by rodenticide and a banned agricultural pesticide); the remaining four (9%) probably died of Pb poisoning. Bone samples from 86 red kites showed a skewed distribution of Pb concentration, and 18 samples (21%) had Pb concentrations >20 mg kg(-1) d.w., indicating elevated exposure to Pb at some stage in the birds' life. Lead isotopic signatures (Pb (208/206); Pb (206/207)) in liver samples of the majority of kites were compatible with those found in lead shot extracted from regurgitated pellets. Lead isotope ratios found in the livers of kites with very low Pb concentrations were distinct from UK petrol Pb isotopic signatures, indicating that birds were exposed to little residual petrol Pb. We conclude that the primary source of Pb to which red kites are exposed is lead ammunition (shotgun pellets or rifle bullets), or fragments thereof, in their food sources; in some cases exposure appears sufficient to be fatal. We make recommendations to reduce Pb poisoning in both captive and wild red kites and other scavenging species.

Bone-targeting radiopharmaceuticals including radium-223

ABSTRACT: Bone-seeking radionuclides including samarium-153 ethylene diamine tetramethylene phosphonate and strontium-89 have been used for decades in the palliation of pain from bone metastases especially from prostate cancer. Emerging evidence of improved survival in metastatic castration-resistant prostate cancer (CRPC) with the first-in-class a-radionuclide, radium-223 (Ra) has rekindled interest in the role of bone-seeking radionuclide therapy.We review the literature for randomized controlled trials of bone-seeking radionuclides and explore some of the issues regarding the optimal use of these agents. In particular, we discuss dose, dose rate, radiobiology, and quality of radiation and postulate on potential future directions in particular combination schedules. ß-Emitting, bone-seeking radionuclides have proven ability to control pain in prostate cancer metastatic to bone with pain response rates in the order of 60% to 70% when used as single agents. Most of the published trials were underpowered to detect differences in survival; however, there is evidence of the potential for disease modification when these agents are used in combination with chemotherapy or in multiple cycles.Data from the recent phase III ALSYMPCA trial that compared Ra to placebo in symptomatic CRPC demonstrate a significant improvement in median overall survival of 3.6 months for patients with symptomatic CRPC metastatic to bone treated with 6 cycles of the a-emitting radionuclide Ra compared with placebo. The success of Ra in improving survival in CRPC will lead this agent to become part of the treatment paradigm for this disease, and with such an excellent safety profile, Ra has huge potential in combination strategies as well as for use earlier in the natural history of metastatic prostate cancer.

Serial chimerism analyses indicate that mixed haemopoietic chimerism influences the probability of graft rejection and disease recurrence following allogeneic stem cell transplantation (SCT) for severe aplastic anaemia (SAA): indication for routine assessment of chimerism post SCT for SAA

Ninety-one patients were studied serially for chimeric status following allogeneic stem cell transplantation (SCT) for severe aplastic anaemia (SAA) or Fanconi Anaemia (FA). Short tandem repeat polymerase chain reaction (STR-PCR) was used to stratify patients into five groups: (A) complete donor chimeras (n = 39), (B) transient mixed chimeras (n = 15) (C) stable mixed chimeras (n = 18), (D) progressive mixed chimeras (n = 14) (E) recipient chimeras with early graft rejection (n = 5). As serial sampling was not possible in Group E, serial chimerism results for 86 patients were available for analysis. The following factors were analysed for association with chimeric status: age, sex match, donor type, aetiology of aplasia, source of stem cells, number of cells engrafted, conditioning regimen, graft-versus-host disease (GvHD) prophylaxis, occurrence of acute and chronic GvHD and survival. Progressive mixed chimeras (PMCs) were at high risk of late graft rejection (n = 10, P <0.0001). Seven of these patients lost their graft during withdrawal of immunosuppressive therapy. STR-PCR indicated an inverse correlation between detection of recipient cells post-SCT and occurrence of acute GvHD (P = 0.008). PMC was a bad prognostic indicator of survival (P = 0.003). Monitoring of chimeric status during cyclosporin withdrawal may facilitate therapeutic intervention to prevent late graft rejection in patients transplanted for SAA.