Quantitative comparison of immunohistochemical HER2-low detection in an interlaboratory study.

AIMS: Recently, human epidermal growth factor 2 (HER2)-low (i.e. HER2 score 1+ or 2+ without amplification) breast cancer patients became eligible for trastuzumab-deruxtecan treatment. To improve assay standardisation and detection of HER2-low in a quantitative manner, we conducted an external quality assessment-like study in the Netherlands. Dynamic range cell lines and immunohistochemistry (IHC) calibrators were used to quantify HER2 expression and to assess interlaboratory variability. METHODS AND RESULTS: Three blank slides with a dynamic range cell line and an IHC calibrator were stained with routine HER2 assays by 35 laboratories. Four different antibody clones were used: 19 (54.3%) 4B5, six (17.1%) A0485, five (14.3%) DG44 (HercepTest) and five (14.3%) SP3. Laboratories used two different detection kits for 4B5 assays: 14 (73.7%) ultraView and five (26.3%) OptiView. Variability of HER2 expression in cell lines, measured with artificial intelligence software, was median (min-max) = negative core 0.5% (0.0-57.0), 1+ core 4.3% (1.6-71.3), 2+ core 42.8% (30.4-92.6) and 3+ core 96.2% (91.8-98.8). The calibrators DG44 and 4B5 OptiView had the highest analytical sensitivity, closely followed by 4B5 ultraView. SP3 was the least sensitive. Calibrators of A0485 assays were not analysable due to background staining. CONCLUSIONS: As assays were validated for detecting HER2-amplified tumours, not all assays and antibodies proved suitable for HER2-low detection. Some tests showed distinct expression in the negative cell line. Dynamic range cell line controls and quantitative analysis using calibrators demonstrated more interlaboratory variability than commonly appreciated. Revalidation of HER2 tests by laboratories is needed to ensure clinical applicable HER2-low assays.

Glycemic control after switching to faster aspart in adults with type 1 diabetes.

AIMS: Post-prandial hyperglycemia remains an unmet need in the management of type 1 diabetes (T1D). In randomized trials, faster insulin aspart (FIA) showed modest but significant reductions of glycemic spikes after meals. Whether such benefit is evident in routine clinical practice is unclear. METHODS: We analyzed data of patients with T1D at the time they switched from a prior bolus insulin to FIA and at the first available follow-up. The primary endpoint was the change in the time spent in hyperglycemia > 250 mg/dl during daytime from flash glucose monitoring (FGM). Secondary outcomes included the change in HbA1c, body weight, insulin dose and other FGM metrics. RESULTS: We included 117 patients with T1D on multiple daily injections who switched to FIA, 57 of whom had data from FGM. Patients were 41-year-old, 51.3% men, with 19.3 years diabetes duration and a baseline HbA1c of 7.7% (60 mmol/mol). Mean observation time was 4.3 months. After switching to FIA, HbA1c declined by 0.1% (1 mmol/mol) only in patients with baseline HbA1c > 7.0% (53 mmol/mol). Time spent in hyperglycemia > 250 mg/dl during daytime was significantly reduced from 14.8 to 11.9% (p = 0.006). Time in range improved from 48.3 to 51.0% (p = 0.028). Results were consistent across various patient characteristics. CONCLUSIONS: Under routine care, patients with T1D who switched to FIA experienced a reduction in the time spent in hyperglycemia > 250 mg/dl during daytime and an increase in time in range. These improvements may be due to better control of post-prandial hyperglycemia, as observed in trials.

Serum magnesium in diabetic retinopathy: the association needs investigation.

BACKGROUND: Magnesium has an essential role in glucose metabolism, and hypomagnesaemia is common in diabetes mellitus. However, the relationship between serum magnesium and diabetic retinopathy is poorly understood. AIM: To determine the association between serum magnesium levels and retinopathy in type 2 diabetic patients with normal renal function and to correlate it with severity of retinopathy. METHODS: This cross-sectional observational study was conducted in a semi-urban tertiary-care teaching hospital. Clinicodemographic profile and serum magnesium levels were determined in patients with type 2 diabetes mellitus (DM) with (group 1) and without (group 2) retinopathy. Serum magnesium levels were correlated with the presence and severity of retinopathy. RESULTS: Of 104 type 2 DM patients, 50 had retinopathy. Younger age, longer duration of disease and poorer glycaemic control (p < 0.05) were found to be associated with retinopathy. The mean serum magnesium levels in patients with retinopathy and those without retinopathy were 1.63 ± 0.30 mg/dL and 1.76 ± 0.22 mg/dL, respectively (p = 0.029). Reduced serum magnesium was associated with elevated fasting sugars (p = 0.019) and female gender (p = 0.037). On comparative analysis of patients with sight-threatening diabetic retinopathy (STDR), non-STDR and no retinopathy by ANOVA test, patients with STDR had significantly lower serum magnesium (1.55 ± 0.33 mg/dL) (p = 0.031). CONCLUSION: Serum magnesium levels were lower in patients with diabetic retinopathy. Patients with STDR had lower serum magnesium compared with those without STDR. SUMMARY: Serum magnesium, studied extensively for its role in glucose metabolism, was found to be lower in patients with diabetic retinopathy compared with those without retinopathy. Sight-threatening diabetic retinopathy had significantly lower levels of serum magnesium.

One-year caloric restriction and 12-week exercise training intervention in obese adults with type 2 diabetes: emphasis on metabolic control and resting metabolic rate.

PURPOSE: The effect of combined lifestyle interventions (LSI) including dietary and physical activity on metabolic health, energy metabolism and VO2max in diabetic patients has provided mixed results. We evaluated the impact of 1-year caloric restriction (CR), and 12-week supervised structured exercise training (SSET) on metabolic health, RMR and VO2max in obese adults with type 2 diabetes. METHODS: After 1-month education for LSI, 33 participants had anthropometric, biochemical and metabolic assessments. They then started CR based on RMR, and 3-month SSET during the months 1-3 (Early-SSET) or 4-6 (Late-SSET). Reassessments were planned after 3, 6 and 12 months. Using a per-protocol analysis, we evaluated parameter changes from baseline and their associations for the 23 participants (11 Early-SSET, 12 Late-SSET) who completed the study. RMR was adjusted (adjRMR) for age, sex, fat-free mass (FFM) and fat mass (FM). RESULTS: Compared with baseline, after 6 months we found significant increases in VO2max (+ 14%) and HDL-cholesterol (+ 13%), and reduction in body mass index (- 3%), FM (- 8%) and glycated hemoglobin (HbA1c, - 7%). Training-related caloric expenditure negatively correlated with changes in body weight (p < 0.001), FM (p < 0.001) and HbA1c (p = 0.006). These results were confirmed at the 12-month follow-up. Pooling together all follow-up data, adjRMR changes correlated with changes in glycemia (r = 0.29, p = 0.02), total-cholesterol (r = 0.29, p = 0.02) and VO2max (r = - 0.26,p = 0.02). No significant differences emerged between the Early- and Late-SSET groups. CONCLUSIONS: Combined intervention with SSET and CR improved metabolic control. Changes in metabolic health and fitness correlated with changes of adjRMR, which was reduced improving fitness, glycemia and cholesterolemia. CLINICAL TRIAL REGISTRY: Trial registration number: NCT03785379. URL of registration: http://clinicaltrials.gov .

Switching from twice-daily glargine or detemir to once-daily degludec improves glucose control in type 1 diabetes. An observational study.

BACKGROUND AND AIMS: Degludec is an ultralong-acting insulin analogue with a flat and reproducible pharmacodynamic profile. As some patients with type 1 diabetes (T1D) fail to achieve 24-h coverage with glargine or detemir despite twice-daily injections, we studied the effect of switching T1D patients from twice-daily glargine or detemir to degludec. METHODS AND RESULTS: In this prospective observational study, T1D patients on twice-daily glargine or detemir were enrolled. At baseline and 12 weeks after switching to degludec, we recorded HbA1c, insulin dose, 30-day blood glucose self monitoring (SMBG) or 14-day continuous glucose monitoring (CGM), treatment satisfaction (DTSQ), fear of hypoglycemia (FHS). We included 29 patients (mean age 34 ± 11 years; diabetes duration 18 ± 10 years). After switching to degludec, HbA1c decreased from 7.9 ± 0.6% (63 ± 6 mmol/mol) to 7.7 ± 0.6% (61 ± 6 mmol/mol; p = 0.028). SMBG showed significant reductions in the percent and number of blood glucose values <70 mg/dl and in the low blood glucose index (LBGI) during nighttime. CGM showed a significant reduction of time spent in hypoglycemia, an increase in daytime spent in target 70-180 mg/dl, and a reduction in glucose variability. Total insulin dose declined by 17% (p < 0.001), with 24% reduction in basal and 10% reduction in prandial insulin. DTSQ and FHS significantly improved. CONCLUSION: Switching from twice-daily glargine or detemir to once daily degludec improved HbA1c, glucose profile, hypoglycemia risk and treatment satisfaction, while insulin doses decreased. ClinicalTrials.govNCT02360254.

Head-to-head comparison between flash and continuous glucose monitoring systems in outpatients with type 1 diabetes.

PURPOSE: Continuous glucose monitoring (CGM) is being increasingly used in clinical practice. The flash glucose monitoring (FGM) and CGM are different systems of interstitial glucose recording. We aimed to determine the agreement between the factory-calibrated FGM FreeStyle Libre (FSL) and the gold-standard CGM Dexcom G4 Platinum (DG4P). METHODS: We analyzed data from n = 8 outpatients with type 1 diabetes, who wore the FSL and DG4P for up to 14 days during their habitual life. We aligned FSL and DG4P recordings to obtain paired glucose measures. We calculated correlation coefficients, mean absolute relative difference (MARD), percentages in Clarke error grid areas, time spent in hyperglycaemia, target glycaemia, or hypoglycaemia, as well as glucose variability with both sensors. Comparison with self-monitoring of blood glucose (SMBG) was also performed. RESULTS: Patients varied in terms of age, diabetes duration, and HbA1c (from 5.9 to 9.6 %). In the pooled analysis of 10,020 paired values, there was a good correlation between FSL and DG4P (r 2 = 0.76; MARD = 18.1 ± 14.8 %) with wide variability among patients. The MARD was significantly higher during days 11-14 than in days 1-10, and during hypoglycaemia (19 %), than in normoglycaemia (16 %) or hyperglycaemia (13 %). Average glucose profiles and MARD versus SMBG were similar between the two sensors. Time spent in normo-, hyper-, or hypoglycaemia, and indexes of glucose variability was similarly estimated by the two sensors. CONCLUSIONS: In outpatients with type 1 diabetes, we found good agreement between the FSL and DG4P. No significant difference was detected in the estimation of clinical diagnostic parameters.

Safety and effectiveness of insulin detemir in combination with oral antidiabetic agents in an outpatient specialist setting: results of the Italian SOLVE™ observational study.

AIM: The addition of basal insulin to oral antidiabetics (OADs) is described by a large number of guidelines and commonly used in clinical practice as a way to start insulin therapy in patients with type 2 diabetes mellitus in order to maximize compliance and minimise the impact of side effects (mainly hypoglycemia and body weight increase). METHODS: SOLVE™ was a 24-week international observational study conducted in 10 countries (including Italy) for the evaluation of the safety and effectiveness of once-daily insulin detemir as add-on therapy in patients with type 2 diabetes mellitus (T2DM) already treated with one or more OADs. The Italian arm of the Solve™ Study aimed to evaluate the safety and the effectiveness of once-daily insulin detemir in combination with OAD agents for the treatment of patients with T2DM in the Italian outpatient specialist setting. The primary endpoint was to assess the incidence of serious adverse drug reactions (SADRs) including in the specific major hypoglycemic events during 24 weeks of once-daily insulin detemir treatment. RESULTS: A total of 4625 patients were enrolled in the study by 223 Italian centres for diabetes care. At baseline the mean (±SD) demographic characteristics of the patients were: age 66.5 (±10.0) years, duration of diabetes 13.25 (±8.14) years, weight 78.95 (±15.86) kg and BMI 29.5 (±5.0) kg/m2. At the end of the study, 3 SADRs (of which 2 were major hypoglycemia) were reported in 2 patients (<0.1%). The percentage of patients with at least 1 minor hypoglycemic event during the 4 weeks preceding insulin initiation was 3.6%. Following insulin initiation, 5.7% (as recorded at baseline visit) had at least 1 minor hypoglycemic event, which decreased slightly by the end of the study compared to baseline (4.8%). In addition, before insulin initiation the mean (±SD) glycemic control values were: fasting plasma glucose (FPG) 11.43 (±3.2) mmol/L and HbA1c 9.16% (±1.46). At the end of the study, HbA1c was reduced by 1.35% (±1.57) (P<0.001), FPG was reduced by 3.34 mmol/L (P<0.001) and the percentage of patients with HbA1c<7% was 21.9%. A mean reduction of 0.52 kg of body weight (P<0.001) was observed compared to before insulin initiation; the body weight reduction was more pronounced in patients with higher BMI before insulin initiation (-1.0 kg for 30

[Repeated intestinal tract perforations secondary to cryoglobulinemia and cytomegalovirus infection: a case report]. / Perforations digestives itératives secondaires à une cryoglobulinémie essentielle et au cytomégalovirus : à propos d'un cas.

Cryoglobulinemia associated vasculitis is usually related to hepatitis C virus. Clinical features include purpura, glomerulonephritis, or peripheral neuropathy. Intestinal involvement in cryoglobulinemia is rare and has a poor prognosis. It often leads to a therapeutic with immunosupressive drugs. The related immunodepression exposes to a reactivation of cytomegalovirus (CMV) infection that may also be potentially responsible for intestinal injury. We report a 68-year-old man who presented with iterative intestinal perforations secondary to a cryoglobulinemia vasculitis and then to a CMV infection.

Hydrogel-mediated DNA delivery confers estrogenic response in nonresponsive osteoblast cells.

Oligo(polyethylene glycol) fumarate (OPF) hydrogel has been employed in musculoskeletal tissue engineering for photoencapsulation of chondrocytes and as a matrix for marrow stromal cells differentiation. In this study, we have studied the application of OPF hydrogel for coencapsulation of DNA and bone cells and examined whether coencapsulation can enhance gene transfer by maintaining the DNA within the cellular microenvironment. Our results showed that plasmid DNA encoding green fluorescence protein (GFP), coencapsulated with bone tumor cells, was capable of transfecting the cells, and the transfected tumor cells continuously expressed GFP protein over the time course of study (21 days). Furthermore, we have examined the coencapsulation of estrogen receptor (ER) encoding plasmid DNA and human fetal osteoblast cells (hFOB) that lack endogenous ER. Our results show that the transfected cells responded to estrogen as alkaline phosphatase (ALP), and estrogen response element (ERE)-directed luciferase enzyme activities increased with estrogen treatment. Taken together, these studies show that OPF hydrogel could be further explored for targeted gene delivery in bone and other tissues encapsulated within the hydrogels.

In Type 1 diabetic patients with good glycaemic control, blood glucose variability is lower during continuous subcutaneous insulin infusion than during multiple daily injections with insulin glargine.

AIMS: The superiority of continuous subcutaneous insulin infusion (CSII) over multiple daily injections (MDI) with glargine is uncertain. In this randomized cross-over study, we compared CSII and MDI with glargine in patients with Type 1 diabetes well controlled with CSII. The primary end-point was glucose variability. METHODS: Thirty-nine patients [38.1 +/- 9.3 years old (mean +/- sd), diabetes duration 16.6 +/- 8.2 years, glycated haemoglobin (HbA(1c)) 7.6 +/- 0.8%], already on CSII for at least 6 months, were randomly assigned to CSII with lispro or MDI with lispro and glargine. After 4 months they were switched to the alternative treatment. During the last month of each treatment blood glucose variability was analysed using glucose standard deviation, mean amplitude of glycaemic excursions (MAGE), lability index and average daily risk range (ADRR). As secondary end-points we analysed blood glucose profile, HbA(1c), number of episodes of hypo- and hyperglycaemia, lipid profile, free fatty acids (FFA), growth hormone and treatment satisfaction. RESULTS: During CSII, glucose variability was 5-12% lower than during MDI with glargine. The difference was significant only before breakfast considering glucose standard deviation (P = 0.011), significant overall using MAGE (P = 0.016) and lability index (P = 0.005) and not significant using ADRR. Although HbA(1c) was similar during both treatments, during CSII blood glucose levels were significantly lower, hyperglycaemic episodes were fewer, daily insulin dose was less, FFA were lower and treatment satisfaction was greater than during MDI with glargine. The frequency of hypoglycaemic episodes was similar during both treatments. CONCLUSIONS: During CSII, glucose variability is lower, glycaemic control better and treatment satisfaction higher than during MDI with glargine.

Nocturnal hypoglycaemia in Type 1 diabetic patients, assessed with continuous glucose monitoring: frequency, duration and associations.

AIMS: We quantified the occurrence and duration of nocturnal hypoglycaemia in individuals with Type 1 diabetes treated with continuous subcutaneous insulin infusion (CSII) or multiple-injection therapy (MIT) using a continuous subcutaneous glucose sensor. METHODS: A microdialysis sensor was worn at home by 24 patients on CSII (mean HbA(1c) 7.8 +/- 0.9%) and 33 patients on MIT (HbA(1c) 8.7 +/- 1.3%) for 48 h. Occurrence and duration of nocturnal hypoglycaemia were assessed and using multivariate regression analysis, the association between HbA(1c), diabetes duration, treatment type (CSII vs. MIT), fasting and bedtime blood glucose values, total daily insulin dose and mean nocturnal glucose concentrations, and hypoglycaemia occurrence and duration was investigated. RESULTS: Nocturnal hypoglycaemia < or = 3.9 mmol/l occurred in 33.3% of both the CSII- (8/24) and MIT-treated patients (11/33). Mean (+/- sd; median, interquartile range) duration of hypoglycaemia < or = 3.9 mmol/l was 78 (+/- 76; 57, 23-120) min per night for the CSII- and 98 (+/- 80; 81, 32-158) min per night for the MIT-treated group. Multivariate regression analysis showed that bedtime glucose value had the strongest association with the occurrence (P = 0.026) and duration (P = 0.032) of nocturnal hypoglycaemia. CONCLUSIONS: Microdialysis continuous glucose monitoring has enabled more precise quantification of nocturnal hypoglycaemia occurrence and duration in Type 1 diabetic patients. Occurrence and duration of nocturnal hypoglycaemia were mainly associated with bedtime glucose value.

Effects of stable transfection of human fetal osteoblast cells with estrogen receptor-alpha on regulation of gene expression by tibolone.

Tibolone is a synthetic steroid which undergoes tissue selective metabolism into several metabolites having estrogenic, progestogenic or androgenic activities. The effects of 3 alpha-hydroxy tibolone (Org 4094), 3 beta-hydroxy tibolone (Org 30126) and their sulfated metabolites were investigated on human fetal osteoblasts (hFOB). Tibolone had no effect on selected osteoblast marker proteins in estrogen-receptor negative hFOB cells. In contrast, 3 alpha-hydroxy and 3beta-hydroxy tibolone resulted in dose-dependent increases in alkaline phosphatase activity in estrogen receptor (ER) alpha-positive hFOB cells. The maximum increase for both metabolites was comparable to the effects of an optimal dose of 17beta-estradiol, and occurred at 10 muM. At 20 muM, both metabolites increased mRNA levels for alkaline phosphatase and type 1 collagen and protein levels for osteocalcin. Sulfated metabolites of tibolone also increased alkaline phosphatase activity. The estrogen receptor antagonist ICI 182, 780 inhibited stimulation of alkaline phosphatase activity by sulfated and non-sulfated tibolone metabolites, but was more potent on the former. Taken together, these results suggest that stable transfection of ER alpha into hFOB cells confers regulation by 3 alpha-hydroxy and 3beta-hydroxy tibolone metabolites of osteoblast metabolism.

2-methoxyestradiol inhibits differentiation and is cytotoxic to osteoclasts.

2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, is highly cytotoxic to a wide range of tumor cells but is harmless to most normal cells. However, 2-ME prevented bone loss in ovariectomized rats, suggesting it inhibits bone resorption. These studies were performed to determine the direct effects of 2-ME on cultured osteoclasts. 2-ME (2 microM) reduced osteoclast number by more than 95% and induced apoptosis in three cultured osteoclast model systems (RAW 264.7 cells cultured with RANKL, marrow cells co-cultured with stromal support cells, and spleen cells cultured without support cells in media supplemented with RANKL and macrophage colony stimulating factor (M-CSF)). The 2-ME-mediated effect was ligand specific; 2-hydroxyestradiol (2-OHE), the immediate precursor to 2-ME, exhibited less cytotoxicity; and 2-methoxyestrone (2-MEOE1) the estrone analog of 2-ME, was not cytotoxic. Co-treatment with ICI 182,780 did not antagonize 2-ME, suggesting that the cytotoxicity was not estrogen receptor-dependent. 2-ME-induced cell death in RAW 264.7 cells coincided with an increase in gene expression of cytokines implicated in inhibition of differentiation and induction of apoptosis. In addition, the 2-ME-mediated decrease in cell survival was partially inhibited by anti-lymphotoxin(LT)beta antibodies, suggesting that 2-ME-dependent effects involve LTbeta. These results suggest that 2-ME could be useful for treating skeletal diseases in which bone resorption is increased, such as postmenopausal osteoporosis and cancer metastasis to bone.

Role of anti-granulocyte Fab' fragment antibody scintigraphy (LeukoScan) in evaluating bone infection: acquisition protocol, interpretation criteria and clinical results.

Scintigraphy using anti-granulocyte Fab' fragment (LeukoScan) was performed in a series of 220 consecutive patients with suspected bone infection referred to our centre between September 1999 and June 2002. Two protocols were compared for interpreting scans: (1) evaluation of early 4 h imaging alone (protocol A), and (2) evaluation both of early and delayed 24 h imaging (protocol B). Protocol A and protocol B showed equal values of sensitivity (91.9% in patients with diabetic foot and 84.2% in patients with joint prosthesis/peripheral bone implants). Conversely, specificity was higher adopting protocol B than protocol A: 87.5% vs 75.0% in patients with diabetic foot, and 85.7% vs 76.2% in patients with joint prosthesis/peripheral bone implants, respectively. In particular, an improvement in specificity using protocol B was found in those patients with infection and with only a mild LeukoScan uptake in the early 4 h imaging: in these patients an increasing uptake intensity pattern observed in the delayed 24 h imaging was indicative of infection while a decreasing pattern suggested a negative result. Instead, the evidence of a high uptake intensity in the early LeukoScan imaging was a strong indicator of infection and delayed imaging in these cases did not further improve specificity. In conclusion, in our experience, LeukoScan showed high sensitivity in diagnosing bone infection in patients with diabetic foot and joint prosthesis or other peripheral bone implants. Moreover, in patients with an early high LeukoScan uptake intensity further delayed images appears unnecessary for the purpose of diagnosing infection. In contrast in patients with an early mild LeukoScan uptake intensity only, delayed imaging appears to be recommendable for improving specificity.

Unanticipated changes in steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase in rat tibiae.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels are commonly used as an internal control to normalize gene expression data based on the belief that this gene is constitutively expressed. However, GAPDH mRNA levels increased by more than 2.5-fold in tibiae of hindlimb unloaded female rats compared to L32 mRNA levels. Similarly, GAPDH mRNA levels increased compared to 18S ribosomal RNA. Treatment with growth hormone and alcohol show no disparity in GAPDH mRNA levels whereas in some experiments, parathyroid hormone and 17beta-estradiol increased GAPDH mRNA levels. Taken together, these findings indicate that it is essential to demonstrate that GAPDH expression is not altered prior to using the gene as an internal control.

A microdialysis technique for continuous subcutaneous glucose monitoring in diabetic patients (part 2).

The aim of this study was to evaluate the reproducibility, the accuracy and the reliability of a continuous subcutaneous glucose measuring system. The GlucoDay system (A. Menarini I.F.R. S.r.l.-Florence, Italy) is a portable instrument provided with a micro-pump and a biosensor, coupled to a microdialysis system (see part 1). This instrument has demonstrated high reliability coupled with a low degree of invasivity. The profiles of glucose monitoring allow to achieve an excellent knowledge of the real variation of glucose in diabetic patients. The reproducibility study showed a bias lower than 10% between instruments. The accuracy study showed a difference from the reference method lower than 15%.

Measurement of gene expression following cryogenic mu-CT scanning of human iliac crest biopsies.

An important consideration in interpreting indices of gene expression in human bone is relating mRNA levels to functional endpoints such as bone architecture. In the present study, a method was developed for quantitative measurement of gene expression and bone morphology in the same specimen. Three-dimensional images of iliac crest bone biopsies from healthy premenopausal women were obtained using a novel high resolution cryogenic mu-CT scanner. RNA was isolated from the biopsies and mRNA levels were measured for genes related to bone metabolism. The gene expression profile and variability of expression within iliac crest biopsies of women was similar to human osteoblastic cell lines and rat long bones. mRNA for alkaline phosphatase, bone matrix proteins, and selected cytokines and cytokine receptors were consistently detected in biopsies. As previously shown in rat bone, there was a tight correlation between mRNA levels for type 1 collagen and osteonectin, a weaker correlation between type 1 collagen and osteocalcin and no correlation between bone matrix proteins and alkaline phosphatase. The relative abundance of the mRNA for the three most prevalent transforming growth factor-beta (TGF-beta) isoforms in bone (TGF-beta(1)>> TGF-beta(3)> TGF-beta(2)) was the same as the known abundance of the corresponding TGF-beta peptides in bone matrix. The results demonstrate the feasibility of analyzing the three-dimensional architecture of a bone biopsy using cryogenic mu-CT imaging and then measuring expression of genes related to bone cell function within the same specimen following RNA extraction and analysis.

A telemedicine support for diabetes management: the T-IDDM project.

In the context of the EU funded Telematic Management of Insulin-Dependent Diabetes Mellitus (T-IDDM) project, we have designed, developed and evaluated a telemedicine system for insulin dependent diabetic patients management. The system relies on the integration of two modules, a Patient Unit (PU) and a Medical Unit (MU), able to communicate over the Internet and the Public Switched Telephone Network. Using the PU, patients are allowed to automatically download their monitoring data from the blood glucose monitoring device, and to send them to the hospital data-base; moreover, they are supported in their every day self monitoring activity. The MU provides physicians with a set of tools for data visualization, data analysis and decision support, and allows them to send messages and/or therapeutic advice to the patients. The T-IDDM service has been evaluated through the application of a formal methodology, and has been used by European patients and physicians for about 18 months. The results obtained during the project demonstration, even if obtained on a pilot study of 12 subjects, show the feasibility of the T-IDDM telemedicine service, and seem to substantiate the hypothesis that the use of the system could present an advantage in the management of insulin dependent diabetic patients, by improving communications and, potentially, clinical outcomes.

2-methoxyestradiol induces interferon gene expression and apoptosis in osteosarcoma cells.

2-Methoxyestradiol (2-ME), a naturally occurring mammalian metabolite of 17beta-estradiol, has been implicated as a physiological inhibitor of tumor cell proliferation. In this study, the effects of 2-ME on cultured osteosarcomatous cells were investigated. Dose-dependent growth inhibition was observed in MG63 and TE85 human osteosarcoma cells exposed to 2-ME. The cell killing by 2-ME was ligand-specific; the immediate precursor (2-hydroxyestradiol), the parent compound (17beta-estradiol), and the equivalent metabolite of estrone (2-methoxyestrone) exhibited less potency and efficacy. Furthermore, 2-ME was similarly effective at killing immortalized human fetal osteoblastic cells (hFOB) with and without estrogen receptor-alpha and -beta and rat osteosarcoma cells (ROS17/2.8). The cytotoxicity of 2-ME was selective to transformed and immortalized osteoblastic cells; 2-ME (2 microm) had no effect on the proliferation of primary cultures of human osteoblasts. Co-treatment with the potent estrogen receptor ligand, ICI-182,780, did not reduce 2-ME-induced osteosarcoma cell death, implying that this action is not mediated by conventional estrogen receptors. The expression levels of bone matrix protein genes, type 1 collagen and osteonectin, were transiently reduced after 2-ME treatment, suggesting that the surviving cells are capable of producing bone matrix. The 2-ME-mediated killing of osteosarcoma cells was due to the induction of apoptosis; treatment induced expression of interferon genes within 12 h and histological evidence of apoptosis within 48 h of 2-ME treatment. Thus, our results demonstrate that 2-ME is highly cytotoxic to osteosarcoma cells but not normal osteoblasts. These findings suggest that further study of 2-ME as a potential intervention for treatment of osteosarcoma is warranted.