The use of 19F spectroscopy and diffusion-weighted MRI to evaluate differences in gene-dependent enzyme prodrug therapies.

To evaluate noninvasive measures of gene expression and tumor response in a gene-dependent enzyme prodrug therapy (GDEPT), a bifunctional fusion gene between Saccharomyces cerevisiae cytosine deaminase (CD) and Haemophilus influenzae uracil phosphoribosyltransferase (UPRT) was constructed. CD deaminates 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), and UPRT subsequently converts 5FU to fluorouridine monophosphate, and both of these reactions can be monitored noninvasively in vitro and in vivo using 19F magnetic resonance spectroscopy (MRS). Following transient transfection the CD-UPRT fusion protein exhibited both UPRT and CD enzymatic activities as documented by 19F MRS. In addition, an increase in CD activity and thermal stability was witnessed for the fusion protein compared to native CD. Stable expression of CD-UPRT in 9L glioma cells increased both 5FC and 5FU sensitivity in vitro compared to CD-expressing and wild-type 9L cells. Noninvasive 19F MRS of both CD and UPRT gene function in vivo demonstrated that in animals bearing CD-expressing tumors there was limited conversion of 5FC to 5FU with no measurable accumulation of cytotoxic fluorinated nucleotides (F-nucs). In contrast, CD-UPRT-expressing tumors had increased CD gene activity with a threefold higher intratumoral accumulation of 5FU and significant generation of F-nucs. Finally, CD-UPRT yielded increased efficacy in an orthotopic animal model of high-grade glioma. More importantly, early changes in cellular water mobility, which are felt to reflect cellular death, as measured by diffusion-weighted MRI, were predictive of both durable response and increased animal survival. These results demonstrate the increased efficacy of the CD-UPRT GDEPT compared to CD alone both biochemically and in a preclinical model and validate both 19F MRS and diffusion-weighted MRI as tools to assess gene function and therapeutic efficacy.     

Effect of blockade of TNF-alpha and interleukin-1 action on bone resorption in early postmenopausal women.

After acute estrogen withdrawal in postmenopausal women, administration of anakinra or etanercept, specific blockers of IL-1 and TNF-alpha, respectively, reduced the rise in bone resorption markers to about one half of that in controls. This is consistent with an important role for these immune cytokines in mediating the effect of estrogen deficiency on bone. INTRODUCTION: Studies in rodents have implicated increased production of interleukin (IL)-1 beta and TNF-alpha as mediators of bone loss after ovariectomy, but their roles are unclear in humans whose immune system differs markedly from that of rodents. MATERIALS AND METHODS: We administered transdermal estradiol, 0.1 mg/d, for 60 days to 42 early postmenopausal women. Estrogen treatment was discontinued, and subjects were randomly assigned to intervention groups receiving 3 wk of injections with 0.9% saline, anakinra 100 mg/d, or etanercept 25 mg/twice weekly. Bone turnover was assessed by measuring serum carboxyl-terminal telopeptide of type 1 collagen (CTX) and amino-terminal telopeptide of type 1 collagen (NTX), markers for bone resorption, and serum amino-terminal propeptide of type 1 collagen (P1NP), a marker for bone formation. Results were expressed as percent change in markers from baseline (last 2 days of estrogen treatment and days 20 and 21 of intervention). RESULTS: The percent changes from baseline during intervention for serum CTX, urine NTX, and serum PINP, respectively, were 43.3 +/- 8.0%, 12.0 +/- 7.1%, and -41.0 +/- 2.5% for the control group; 25.9 +/- 6.3%, 9.5 +/- 4.0%, and -37.8 +/- 3.0% for the anakinra group; and 21.7 +/- 5.0%, 0.32 +/- 3.82%, and -34.5 +/- 3.9% for the etanercept group. Compared with the control group, the blunting of the increase in serum CTX fell just below the level of significance (p=0.10) after anakinra treatment, whereas the blunting of the increase in serum CTX (p=0.034) and in urine NTX (p=0.048) were significant after etanercept treatment. Other changes were not significant. CONCLUSIONS: The data are consistent with a role for TNF-alpha, and possibly for IL-1 beta, in mediating increased bone resorption during estrogen deficiency in women. Although either cytokine blocker reduced serum CTX by about one half, the effect of combined blockade could not be tested because of concerns about toxicity. The data do not exclude direct or indirect contributory roles for RANKL or for other cytokines.     

Changes to osteoporosis prevalence according to method of risk assessment.

The impact of clinical risk factor-based absolute risk methods on the prevalence of high risk for osteoporotic fracture is unknown. We applied absolute risk methods to 6646 subjects and found that the prevalence of elderly women deemed to be at high risk increased substantially, whereas the overall prevalence was highly dependent on the threshold used to designate high risk. INTRODUCTION: Many groups have advocated using absolute risk methods that incorporate clinical risk factors to target patients for osteoporosis therapy. We examined how the application of such absolute risk classification systems influences the prevalence of those considered to be at high risk for osteoporotic fracture and compared these systems to one based solely on BMD. MATERIALS AND METHODS: Using 6646 subjects from the Canadian Multicentre Osteoporosis Study (CaMos), a prospective, randomly selected, population-based cohort, we assessed three different systems for determining prevalence of high risk for osteoporotic fracture: a BMD-based system; a simplified risk factor system incorporating age, sex, BMD, and two clinical risk factors; and a comprehensive system, incorporating age, sex, BMD, and seven clinical risk factors. The 10-year absolute risks of incident fragility fracture were compared across systems using three different high-risk thresholds. RESULTS: The prevalence of a T score < or = -2.5 was 18.8% (95% CI: 17.7-19.9%) in women and 3.9% (95% CI: 3.0-4.7%) in men. Using a 15% 10-year risk of fracture threshold, the prevalence of women at high risk increased to 46.9% (95% CI: 45.4-48.4) and 42.5% (95% CI: 41.1-43.9) when the comprehensive and simplified risk factor classification systems were used, respectively. Using a 25% 10-year absolute risk threshold, the prevalence of high risk was similar to that of the BMD-based system, whereas the 20% threshold gave intermediate rates. All thresholds analyzed resulted in an increased prevalence of older women at high risk for fracture, whereas only the 15% 10-year risk of fracture threshold resulted in an increase in the prevalence of men at high risk. CONCLUSIONS: The application of risk factor-based systems results in an increased prevalence of older women at high risk. The prevalence of individuals at high risk may increase with changes to the methods used to determine those who are eligible for therapy. These data have important implications for the pattern of care and costs of treating osteoporotic fractures.     

Effects of sex and age on bone microstructure at the ultradistal radius: a population-based noninvasive in vivo assessment.

In a population-based cross-sectional study, we examined effects of sex and age on bone microstructure at the wrist using high-resolution 3-D pQCT. Compared with women, men had thicker trabeculae in young adulthood and had less microstructural damage with aging. These findings may contribute to the virtual immunity of men to age-related increases in wrist fractures. INTRODUCTION: Although changes in bone microstructure contribute to fracture risk independently of BMD, it has not heretofore been possible to assess this noninvasively in population-based studies. MATERIALS AND METHODS: We used high-resolution 3-D pQCT imaging (voxel size, 89 mum) to define, in a random sample of women (n = 324) and men (n = 278) 21-97 years of age, sex and age effects on bone microstructure at the wrist. RESULTS: Relative to young women (age, 20-29 years), young men had greater trabecular bone volume/tissue volume (BV/TV; by 26%, p = 0.001) and trabecular thickness (TbTh; by 28%, p < 0.001) but similar values for trabecular number (TbN) and trabecular separation (TbSp). Between ages 20 and 90 years, cross-sectional decreases in BV/TV were similar in women (-27%) and in men (-26%), but whereas women had significant decreases in TbN (-13%) and increases in TbSp (+24%), these parameters had little net change over life in men (+7% and -2% for TbN and TbSp, respectively; p < 0.001 versus women). However, TbTh decreased to a greater extent in men (-24%) than in women (-18%; p = 0.010 versus men). CONCLUSIONS: Whereas decreases with age in trabecular BV/TV are similar in men and women, the structural basis for the decrease in trabecular volume is quite different between the sexes. Thus, over life, women undergo loss of trabeculae with an increase in TbSp, whereas men begin young adult life with thicker trabeculae and primarily sustain trabecular thinning with no net change in TbN or TbSp. Because decreases in TbN have been shown to have a much greater impact on bone strength compared with decreases in TbTh, these findings may help explain the lower life-long risk of fractures in men, and specifically, their virtual immunity to age-related increases in distal forearm fractures.     

Frozen section analysis of esophageal endoscopic mucosal resection specimens in the real-time management of Barrett's esophagus.

BACKGROUND & AIMS: The aim of this study was to assess the validity of frozen section analysis of endoscopic mucosal resection (EMR) specimens from Barrett's esophagus as compared with permanent sections for the detection of neoplasia. Frozen sections help to give immediate feedback for surgical procedures. It has not been determined whether EMR can be adequately interpreted by using frozen sections to aid endoscopists in completely resecting neoplastic lesions. METHODS: EMR specimens from Barrett's esophagus with high-grade dysplasia (HGD) and/or carcinoma were tested by frozen section. Pathologists evaluated EMR specimens for the depth of invasion as well as the appearance of clear margins of resection. The kappa statistic was calculated to assess the degree of agreement between the frozen section and permanent section diagnoses. RESULTS: Twenty-three consecutive patients underwent 30 EMRs with frozen section diagnosis. Frozen section revealed a carcinoma in 7 specimens (23%) and dysplasia in 20 (66%). Permanent sections found carcinoma in 8 specimens (26%), dysplasia in 19 specimens (63%), and normal or nondysplastic Barrett's esophagus in the remainder. The kappa statistic for the depth of invasion of EMR specimens was 0.93 (near perfect agreement). The kappa statistic for the margins of the EMR specimens was 0.80 (excellent agreement). CONCLUSIONS: This study indicated that frozen section analysis of esophageal EMR specimens is valid as compared with permanent section. This technique might allow rapid evaluation about the degree and depth of involvement of cancers. This allows physicians to make decisions regarding further therapy if margins are involved or decrease the use of EMR for histologically benign-appearing lesions.     

Evaluation of Class I ART restorations in Brazilian schoolchildren: three-year results

Atraumetic Restorative Treatment (ART) has been adopted around the world to avoid unnecessary extractions, especially in non-industrialized countries The development of specific glass ionomer cements marketed for the ART technique has contributed to the technical success rate. In this study. Ketac-Molar^a (3M ESPE. Dental Medzn Germany) was used to restore 150 Class I cavities in 118 Brazilian public school children, aged from 7–12 years. At baseline and at subsequent recalls. CPI probes with a ball-end of 0.5 millimeters (mm) were used to assess loss of restorative material, and photographic color transparencies of restorations were made. After six months. 83 patients returned for follow-up examinations, with 71.8% of their restorations designated as acceptable. After three years. 49 patients with 57 ART-restorations were evaluated, with 21.0% of these restorations graded as acceptable Another 29.8% of their restorations had been replaced by more permanent materials. The main objective of the ART technique is tooth retention; this was achieved for 94.7% of the restored teeth in a high caries risk population who returned for recalls.     

Histologic changes mimicking biliary disease in liver biopsies with venous outflow impairment.

Impairment of venous outflow from the liver manifests as zone 3 sinusoidal dilatation and congestion (SDC) in liver biopsy. The spectrum of histologic changes in portal tracts has not been described. We studied liver biopsies from 34 patients with a confirmed diagnosis of venous outflow impairment (VOI). Liver transplant recipients and biopsies with cirrhosis and hepatic neoplasms were excluded. Clinical records were reviewed for laboratory tests and radiographic findings. In all, 19 patients had right heart disease, 13 had classic Budd-Chiari syndrome and two had veno-occlusive disease. Liver chemistry tests showed elevated liver transaminases (n=21; 61.8%), elevated alkaline phosphatase (n=31; 91.2%) and GGT (all 13 cases tested). The elevation in ALT and AST was mild (below 200 U/l in all cases), while alkaline phosphatase (ALP) was elevated above 500 U/l in nine (26.5%) patients and above 1000 U/l in three cases. On biopsy, all cases showed SDC. The portal tracts showed (a) portal expansion with bile ductular proliferation (n=16; 47.1%) accompanied by lymphoplasmacytic infiltrate (n=10), lymphocytic cholangitis (n=3) and portal or periportal fibrosis (n=11), (b) Portal and/or periportal fibrosis without ductular proliferation (n=3; 8.8%) or (c) Normal portal tracts (n=15; 44.1%). The combination of elevated ALP and bile ductular changes on biopsy suggested chronic bile duct disease. Ultrasound/CT scan evaluation of bile ducts in 26 patients showed no biliary tree abnormality. Antimitochondrial antibody testing in eight cases also yielded negative results. In conclusion, bile ductular proliferation, portal inflammation and portal-based fibrosis are commonly seen in liver biopsies of patients with VOI even in the absence of bile duct disease. These changes are often accompanied by elevated ALP and GGT and can lead to the suspicion of chronic biliary disease. In the absence of demonstrable abnormalities in the biliary tree, these changes can be attributed to venous outflow impairment.     

A potentially deleterious role of IGFBP-2 on bone density in aging men and women.

The role of the IGFs and IGFBPs on age-related changes in BMD in adult men and women is not well understood. Studying an age-stratified community based sample of 344 men and 276 women, we found higher IGFBP-2 levels to be associated with lower BMD. IGFBP-2, which increases with age in both men and women, was the strongest, most consistent predictor of BMD among the IGF/IGFBPs studied. INTRODUCTION: Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are important regulators of tissue growth and metabolism, but their association with BMD in adult men and women is controversial. MATERIALS AND METHODS: In an age-stratified, random sample of the community population, we examined the role of serum levels of IGF-I, IGF-II, and IGFBP-1, -2, and -3 on BMD of the proximal femur (total hip), lateral spine, midshaft, and ultradistal radius as measured by DXA. We explored the association before and after adjustment for potential confounders, including age, bioavailable estradiol and testosterone, sex hormone binding globulin (SHBG), and measures of total fat and skeletal muscle mass. RESULTS: We studied 344 men (age, 23-90 years) and 276 women (age, 21-93 years; 166 postmenopausal) not on hormone replacement or oral contraceptives. In both men and women, IGF-I and IGFBP-3 levels fell with advancing age, whereas IGFBP-2 levels tended to rise with age. There was an inverse association of IGFBP-2 with BMD at most skeletal sites in men and both premenopausal and postmenopausal women, whereas lower IGF-I and IGFBP-3 were associated with lower BMD in men and postmenopausal women only. Lower IGF-II was associated with lower BMD in men only. There were no associations between IGFBP-1 and BMD in either sex. After adjustment for age, in most cases, we found no further associations between IGF-I, IGF-II, or IGFBP-3 and BMD. In contrast, after age adjustment, higher IGFBP-2 remained a predictor of lower BMD in men and postmenopausal women at all sites except for the lateral spine (for men: r = -0.21, -0.20, and -0.19, all p < 0.001; and for postmenopausal women: r = -0.34, -0.24, and -0.25, all p < 0.01, for the total hip, midshaft, and ultradistal radius, respectively). IGFBP-2 remained an independent negative predictor of BMD in men, postmenopausal women, and all women combined after additional adjustment for bioavailable sex steroids, but not at all sites after adjustment for SHBG and muscle mass. In premenopausal women, IGFBP-2 had similar associations as seen in postmenopausal women, but they were weaker and not statistically robust. CONCLUSIONS: Among the IGF/IGFBPs in our study, IGFBP-2 was a key negative predictor of BMD among men and women, particularly postmenopausal women. Our findings suggest a potential role of the IGF/IGFBP system in regulating bone loss in aging men and women and identify a previously under-recognized, potentially deleterious role for IGFBP-2, a known inhibitor of IGF action that increases with age in both sexes. Whether the action of the IGF/IGFBP system on bone metabolism is mediated partly through its effects on muscle mass or SHBG deserves further study.     

Effect of link protein concentration on articular cartilage proteoglycan aggregation

Previous work has shown that alterations in proteoglycan aggregates are among the first changes detected with aging, disuse, and degeneration of articular cartilage, yet the cause or causes of these alterations remain unknown. To determine if differences in link protein concentration can explain alterations in the assembly, size, and stability of articular cartilage proteoglycan aggregates, we isolated proteoglycan monomer (aggrecan) and link protein from adult bovine articular cartilage and then assembled proteoglycan aggregates from aggrecan and 0.8% hyaluronan relative to aggrecan weight, in the presence of 0, 2, 4, 6, 8, 10, 15, and 20% concentrations of link protein relative to aggrecan weight. We determined the amount, sedimentation coefficient, and stability of the aggregates by analytical ultracentrifugation and measured their dimensions by electron microscopy with use of the monolayer technique. Increased aggregate size, as determined by ultracentrifugation, was directly correlated with an increased number of aggrecans per aggregate and with increased hyaluronan length, as determined by electron microscopy. The concentration of link protein significantly influenced aggregation: concentrations of 6–8% produced maximum aggregation, aggregate stability, and uniformity of aggrecan spacing; concentrations greater than 10% led to the formation of superaggregates (aggregates with sedimentation velocities greater than 100 S that may result from linking two or more hyaluronan filaments) but decreased aggregate stability; and concentrations of less than 4% link protein significantly decreased aggregation, the size and stability of aggregates, and the regularity of aggrecan spacing. The latter observations suggest that a decline in the concentration of link protein could decrease the organization and stability of the articular cartilage matrix.