Ian Dick

Fear of falling in older women: a longitudinal study of incidence, persistence, and predictors.

OBJECTIVES: To determine longitudinal predictors of incident and persistent fear of falling (FOF) in older women.

Synergistic effect of CTLA-4 blockade and cancer chemotherapy in the induction of anti-tumor immunity.

Several chemotherapeutics exert immunomodulatory effects. One of these is the nucleoside analogue gemcitabine, which is widely used in patients with lung cancer, ovarian cancer, breast cancer, mesothelioma and several other types of cancer, but with limited efficacy. We hypothesized that the immunopotentiating effects of this drug are partly restrained by the inhibitory T cell molecule CTLA-4 and thus could be augmented by combining it with a blocking antibody against CTLA-4, which on its own has recently shown beneficial clinical effects in the treatment of patients with metastatic melanoma. Here we show, using two non-immunogenic murine tumor models, that treatment with gemcitabine chemotherapy in combination with CTLA-4 blockade results in the induction of a potent anti-tumor immune response. Depletion experiments demonstrated that both CD4(+) and CD8(+) T cells are required for optimal therapeutic effect. Mice treated with the combination exhibited tumor regression and long-term protective immunity. In addition, we show that the efficacy of the combination is moderated by the timing of administration of the two agents. Our results show that immune checkpoint blockade and cytotoxic chemotherapy can have a synergistic effect in the treatment of cancer. These results provide a basis to pursue combination therapies with anti-CTLA-4 and immunopotentiating chemotherapy and have important implications for future studies in cancer patients. Since both drugs are approved for use in patients our data can be immediately translated into clinical trials.

Determinants of Intact Parathyroid Hormone and Free 1,25-Dihydroxyvitamin D Levels in Mild and Moderate Renal Failure

Parameters of calcium and phosphate metabolism were measured in 27 patients with mild renal failure [glomerular filtration rate (GFR) 40-90 ml/min], 12 patients with moderate renal failure (GFR 20-39 ml/min) and in 12 healthy subjects. GFR was determined by technetium-99m diethylenetriamine penta-acetic acid clearance. Intact parathyroid hormone (PTH) was measured by a sensitive immunochemiluminometric assay and somatomedin-C was determined by radioimmunoassay. Both 1,25-dihydroxyvitamin D [1,25(OH)2D] and vitamin-D-binding protein were measured allowing calculation of the free 1,25(OH)2D index. By linear regression and multivariate analysis, PTH was negatively and independently correlated with GFR, plasma bicarbonate and 25-hydroxyvitamin D [25(OH)D] while free 1,25(OH)2D was positively correlated with GFR. Increased PTH secretion and reductions in 1,25(OH)2D were present in mild renal failure patients before any changes in plasma calcium, phosphate and bicarbonate were noted. Plasma alkaline phosphatase was significantly higher in mild chronic renal failure patients compared to normal subjects, possibly indicating early effects of the secondary hyperparathyroidism on the skeleton. Somatomedin-C did not correlate with the free 1,25(OH)2D index or a measure of 1,25(OH)2D production. It is concluded that the secondary hyperparathyroidism which occurs very early in the onset of chronic renal failure may be due to a reduction in the circulating concentration of 1,25(OH)2D consequent upon the renal failure. Low plasma bicarbonate and 25(OH)D also appear to be determinants of a raised PTH concentration. The compensatory increase in PTH presumably maintains extracellular calcium and phosphate levels constant but with possible deleterious effects on the skeleton.

Klotho gene polymorphisms are associated with osteocalcin levels but not bone density of aged postmenopausal women

Osteoporosis is known to have a strong genetic basis. It has been proposed that polymorphisms within the KL (klotho) gene have a significant effect on aging, in particular, the osteoblast defect of aging. The association between polymorphisms within this gene and biochemical markers of bone formation and resorption, bone structure, and fracture rates was studied in 1,190 postmenopausal women with a mean age of 75 years. Genotyping of these polymorphic sites was carried out using Matrix-Assisted Laser Desorption Ionization—Time of Flight (MALDI-ToF) mass spectrometry. The G allele of SNP c.1775G>A was associated with a lower osteocalcin level than the A allele (P = 0.004) in a codominant model. SNPs C-387T and IVS1+8262c>t both showed nonsignificant associations with osteocalcin (P values of 0.063 and 0.068, respectively), but a haplotype analysis of 2 of 5 haplotypes of the three SNPs with a frequency greater than 4% revealed a significant association with osteocalcin (P = 0.036). None of the individual polymorphisms or haplotypes analyzed showed any associations with a marker of bone resorption the deoxypyridinoline creatinine ratio, bone structure, or fracture data. Therefore, the G polymorphism within the c.1775G>A SNP site and a haplotype including this are associated with a reduced osteoblast product osteocalcin. These data suggest that variation in the KL gene product affects osteoblast activity independent of osteoclast activity but that this defect does not result in an effect on bone structure in this population, perhaps because of “rescue” by other genetic or environmental factors in this population.

Apolipoprotein E4 Is Associated With Reduced Calcaneal Quantitative Ultrasound Measurements and Bone Mineral Density in Elderly Women

Some studies have reported an association between the apolipoprotein E4 (APOE4) allele and reduced bone density and increased propensity to fracture, but this remains controversial as other studies have not found an association between APOE4 and bone density or fracture. No information is available concerning the effect of the APOE4 allele on quantitative ultrasound (QUS) parameters. We therefore examined this issue in a population-based study of 1332 healthy elderly women, examining the effect of the APOE4 allele on QUS parameters at the calcaneus and comparing this to dual-energy X-ray absorptiometry (DEXA) bone mineral density (BMD) at the hip. In addition, we examined the effect of the APOE4 allele on fracture. Subjects who had at least one APOE4 allele (n = 308) had lower calcaneal QUS parameters and lower hip BMD at the total hip, trochanter, and intertrochanter, but not the femoral neck, compared to subjects without an APOE4 allele (n = 1024) after adjustment for age, body mass index (BMI), and smoking. The decrement in QUS parameters and BMD was approximately 2%. Those subjects having an APOE4 allele were also more likely to fall into a low bone density group, defined by a T score of <1 SD below the young normal range (odds ratio [OR] 1.55, 95% confidence interval [CI] 1.08-2.22). We compared both prevalent and incident nontraumatic fractures over 2 years in the APOE4-present group compared with the APOE4-absent group. There were 354 subjects who entered the study with a history of one or more prevalent fractures, and 104 subjects sustained a nontraumatic fracture during the study. These fractures were not associated with the presence of the APOE4 allele, but a 2% decrement in BMD was unlikely to be associated with a statistically observable increase in fractures in this study. The APOE4 allele was not associated with a difference in any biochemical measures of bone formation or resorption, or in estrogen concentration, nor was it associated with a difference in BMI. Therefore, we conclude that the APOE4 allele is associated with a consistent decrease in both QUS parameters at the calcaneus and BMD at the clinically important hip site, and that this is not associated with differences in biochemical measures of bone formation or resorption.

THE EFFECT OF ESTROGEN DEFICIENCY ON CALCIUM BALANCE IN MATURE RATS

Abstract. The role of estrogen in the regulation of calcium balance is still poorly understood. A calcium balance study was performed to examine the effects of estrogen status in relation to fecal calcium loss as a component of bone loss after oophorectomy (OOX) in the mature rat. The components of the classic calcium balance were compared with calcium balance estimates obtained from whole body bone density. Six month or older Sprague Dawley rats were allocated to either a sham-operated or OOX group and fed a 0.1% calcium diet. The bone mineral density (BMD) and bone mineral content (BMC) were measured at baseline, 6 weeks, and 9 weeks. A calcium balance was done for 6 days before and 6 weeks post OOX. The fall in BMD from baseline to 9 weeks in the OOX group was significantly greater than in the sham-operated group. The calcium balance was more negative at baseline than at 6 weeks in both groups of animals because they had not adapted to the low calcium diet. However, the increase in calcium balance was significantly less in the OOX animals than in the sham-operated animals. The greater the rise in calcium balance from the baseline to the 6 weeks balance the less the fall in the calcium content of the whole body (Spearman correlation: r = 0.604 P = 0.008). The fall in fecal calcium, but not urine calcium or calcium consumed, was negatively correlated with the change in whole body BMC (Spearman correlation: fecal calcium r =−0.763 P = 0.001). Thus, the primary effect of estrogen deficiency on calcium balance in the mature rat appears to be calcium flux in the bowel, rather than renal calcium handling.

The Effects of Dietary Calcium Deprivation on Serum Calcitriol Levels in Premenopausal and Postmenopausal Women

The effect of the postmenopausal state on serum calcitriol levels was studied in 13 normal premenopausal women and 10 normal postmenopausal women under basal conditions and during seven days of dietary calcium deprivation. Calcitriol levels and the free calcitriol index were lower in the postmenopausal women, and this difference persisted during calcium deprivation. One of the major actions of calcitriol is to increase gut calcium absorption. Relative calcitriol deficiency, despite retained renal synthetic reserve, would in part explain the increased dietary calcium requirement to maintain calcium balance in postmenopausal women.

A genome-wide association study for malignant mesothelioma risk

Malignant mesothelioma (MM) is a uniformly fatal tumour of mesothelial cells. MM is caused by exposure to asbestos however most individuals with documented asbestos exposure do not develop MM. Although MM appears to aggregate within families, the genetics of MM susceptibility is a relatively unexplored area. The aim of the current study was to identify genetic factors that contribute to MM risk. A genome-wide association analysis of 2,508,203 single nucleotide polymorphisms (SNPs) from 428 MM cases and 1269 controls from Western Australia was performed. Additional genotyping was performed on a sample of 778 asbestos-exposed Western Australian controls. Replication of the most strongly associated SNPs was undertaken in an independent case-control study of 392 asbestos-exposed cases and 367 asbestos-exposed controls from Italy. No SNPs achieved formal genome-wide statistical significance in the Western Australian study. However, suggestive results for MM risk were identified in the SDK1, CRTAM and RASGRF2 genes, and in the 2p12 chromosomal region. These findings were not replicated in the Italian study, although there was some evidence of replication in the region of SDK1. These suggestive associations will be further investigated in sequencing and functional studies.

Strong spontaneous tumor neoantigen responses induced by a natural human carcinogen

A key to improving cancer immunotherapy will be the identification of tumor-specific “neoantigens” that arise from mutations and augment the resultant host immune response. In this study we identified single nucleotide variants (SNVs) by RNA sequencing of asbestos-induced murine mesothelioma cell lines AB1 and AB1-HA. Using the NetMHCpan 2.8 algorithm, the theoretical binding affinity of predicted peptides arising from high-confidence, exonic, non-synonymous SNVs was determined for the BALB/c strain. The immunoreactivity to 20 candidate mutation-carrying peptides of increased affinity and the corresponding wild-type peptides was determined using interferon-γ ELISPOT assays and lymphoid organs of non-manipulated tumor-bearing mice. A strong endogenous immune response was demonstrated to one of the candidate neoantigens, Uqcrc2; this response was detected in the draining lymph node and spleen. Antigen reactive cells were not detected in non-tumor bearing mice. The magnitude of the response to the Uqcrc2 neoantigen was similar to that of the strong influenza hemagglutinin antigen, a model tumor neoantigen. This work confirms that the approach of RNAseq plus peptide prediction and ELISPOT testing is sufficient to identify natural tumor neoantigens.

In Vitro Kinetic Properties of the Thr201Met Variant of Human Aromatase Gene CYP19A1: Functional Responses to Substrate and Product Inhibition and Enzyme Inhibitors

CONTEXT The T(201)M variant (rs28757184) within exon 5 of the human aromatase gene CYP19A1, present in up to 20% of some populations, has been reported to reduce prostate cancer progression. OBJECTIVE We hypothesized that the T(201)M variant would alter the structure of the enzyme and thus would also affect function compared to wild-type human aromatase. DESIGN HEK293 cells were transiently transfected with CYP19A1 wild-type or T(201)M variant gene transcripts made by site-directed mutagenesis and enzyme activity measured using tritiated androstenedione as the substrate. The effects of differing concentrations of substrate and product (E1 and E2) and four aromatase inhibitors were assessed. RESULTS At all substrate concentrations tested, the T(201)M variant showed substantially increased activity compared to the wild-type (Vmax: variant, 738 +/- 36 pmol/h . mg; wild-type, 189 +/- 17 pmol/h . mg, P < 0.0001; Km: variant, 64.4 +/- 19.3 nm; wild-type, 46.6 +/- 9.1 nm, P = 0.04). Kinetic analysis showed evidence of substrate inhibition for the wild-type, but no product inhibition was demonstrated for either transcript. Formestane, chrysin, and letrozole had no differential inhibitory effect on the two transcripts, but aminoglutethimide inhibition was substantially reduced in the variant compared to wild-type (IC(50): wild-type, 1.3 +/- 0.2 nm; variant, 45 +/- 14.2 nm, P = 0.002; and Ki: wild-type, 0.7 +/- 0.2 nm; variant, 29.6 +/- 9.7 nm, P = 0.0001). CONCLUSIONS In addition to loss of function mutations previously described, a new naturally occurring relatively common alteration of enzyme structure at T(201)M increases enzyme activity and reduces the inhibitory effect of aminoglutethimide. These findings identify the T(201)M site, distant from the substrate-binding site and not previously considered to play a role in enzyme activity, as a functionally important area of the enzyme that may play a role in the propensity to disease. Common to other cytochrome P450 enzymes, wild-type aromatase demonstrates substrate but not product inhibition.