Focal HDR brachytherapy boost to stereotactic radiotherapy (fBTsRT) for prostate cancer: a phase II randomized controlled trial.

BACKGROUND: For patients with a higher burden of localized prostate cancer, radiation dose escalation with brachytherapy boosts have improved cancer control outcomes at the cost of urinary toxicity. We hypothesize that a focal approach to brachytherapy boosts targeting only grossly visualized tumor volumes (GTV) combined with stereotactic radiotherapy will improve quality of life (QoL) outcomes without compromising cancer control. METHODS: 150 patients with intermediate or high-risk prostate cancer will be enrolled and randomized 1:1 in a cohort multiple randomized clinical trial phase 2 design. Patients are eligible if planned for standard-of-care (SOC) high dose rate (HDR) brachytherapy boost to radiotherapy (RT) with GTVs encompassing < 50% of the prostate gland. Those randomly selected will be offered the experimental treatment, consisting of focal HDR brachytherapy boost (fBT) of 13-15 Gy in 1 fraction followed by stereotactic radiotherapy (sRT) 36.25-40 Gy in 5 fractions to the prostate (+/- 25 Gy to the elective pelvis) delivered every other day. The primary endpoint is to determine if fBTsRT is superior to SOC by having fewer patients experience a minimally important decline (MID) in urinary function as measured by EPIC-26 at 1 and 2 years. Secondary endpoints include rates of toxicity measured by Common Terminology Criteria for Adverse Events (CTCAE), and failure-free survival outcomes. DISCUSSION: This study will determine whether a novel approach for the treatment of localized prostate cancer, fBTsRT, improves QoL and merits further evaluation. Trial registration This trial was prospectively registered in ClinicalTrials.gov as NCT04100174 as a companion to registry NCT03378856 on September 24, 2019.

Using fiducial markers in the prostate bed in postprostatectomy external beam radiation therapy improves accuracy over surgical clips.

BACKGROUND AND PURPOSE: The purpose of this work was to assess the stability of fiducial markers in the prostate bed and compared their use to surgical clips. PATIENTS AND METHODS: In this study, 3-4 gold fiducial markers were transrectally implanted in the prostate bed of 14 patients. The stability of the fiducial markers position (fiducial markers fixity) over an EBRT course was assessed. Furthermore, the advantages of the fiducial markers compared to the surgical clips were assessed and the interobserver variation between the two technologies was compared. RESULTS: The mean fiducial marker migration during a course of EBRT was small with 1.2 mm (SD ± 0.8 mm). Compared to fiducial markers, the matches with surgical clips were mismatched ≥ 2 mm in 68% of treatments. This discrepancy of > 2 mm was on average 3.7 ± 1.3 mm. There was less interobserver variability for matching of fiducial markers (0.8 ± 0.7 mm) than for surgical clips (2.0 ± 1.6 mm). CONCLUSION: Fiducial markers showed less interobserver variability in matching and less variation in position than surgical clips. Fiducial markers could ultimately help in reducing treatment margins.

Sci-Sat AM(2): Brachy-07: Tomosynthesis-based seed reconstruction in LDR prostate brachytherapy: A clinical study.

To develop a tomosynthesis-based dose assessment procedure that can be performed after an I-125 prostate seed implantation, while the patient is still under anaesthesia on the treatment table. Our seed detection procedure involves the reconstruction of a volume of interest based on the backprojection of 7 seed-only binary images acquired over an angle of 60° with an isocentric imaging system. A binary seed-only volume is generated by a simple thresholding of the volume of interest. Seeds positions are extracted from this volume with a 3D connected component analysis and a statistical classifier that determines the number of seeds in each cluster of connected voxels. A graphical user interface (GUI) allows to visualize the result and to introduce corrections, if needed. A phantom and a clinical study (24 patients) were carried out to validate the technique. A phantom study demonstrated a very good localization accuracy of (0.4+/-0.4) mm when compared to CT-based reconstruction. This leads to dosimetric error on D90 and V100 of respectively 0.5% and 0.1%. In a patient study with an average of 56 seeds per implant, the automatic tomosynthesis-based reconstruction yields a detection rate of 96% of the seeds and less than 1.5% of false-positives. With the help of the GUI, the user can achieve a 100% detection rate in an average of 3 minutes. This technique would allow to identify possible underdosage and to correct it by potentially reimplanting additional seeds. A more uniform dose coverage could then be achieved in LDR prostate brachytherapy.

Disruption of the neurokinin-3 receptor (NK3) in mice leads to cognitive deficits.

RATIONALE: The structurally related neuropeptides, substance P, neurokinin A, and neurokinin B, belong to a family of molecules termed tachykinins and are widely distributed in the central and peripheral nervous systems. These peptides mediate their effects through three G protein coupled receptor subtypes, the neurokinin-1, neurokinin-2 and neurokinin-3 receptors, respectively. OBJECTIVE: To study the physiological functions of NK3, a line of NK3 knockout mice were generated and characterized in a broad spectrum of well-established behavioral tests. RESULTS: In several tests, including spontaneous locomotor activity, elevated plus maze, forced swim, and hot plate, wild-type and knockout mice performed similarly. However, in several cognition tests, including passive avoidance, acquisition of conditioned avoidance responding (CAR), and the Morris water maze, NK3 knockout mice displayed deficits compared to wild-type mice. Although NK3 wild-type and knockout mice performed similarly in the training phase of the passive avoidance test, knockout mice had shorter latencies to enter the dark compartment on days 3 and 4, suggesting impaired retention. In the acquisition phase of the conditioned avoidance responding assay, NK3 knockout mice acquired the CAR task at a slower rate than wild-type mice. Once the CAR test was acquired, both NK3 wild-type and knockout mice responded similarly to clozapine and risperidone, drugs which suppress responding in this test. In the Morris water maze, NK3 knockout mice showed increased latencies to find the escape platform on day 3 of training, suggesting a modest, but significant delay in acquisition compared to wild-type mice. CONCLUSION: These studies suggest a role for NK3 in learning and memory in mice.

Effects of ambient air particles on nitric oxide production in macrophage cell lines.

We assessed the in vitro toxicity of various particles on three murine macrophage cell lines, (J774A.1, WR19M.1, RAW264.7). The cells were exposed to aqueous suspensions (0-100 microg/30 mm2 well) of urban particulate matter (SRM-1648, SRM-1649, EHC-93), fine particulate matter (PM2.5), titanium dioxide (SRM-154b), and respirable cristobalite (SRM-1879) for 2 h and were then stimulated with lipopolysaccharide (LPS, 100 ng/ml) and recombinant interferon-gamma (IFN, 100 U/ml). After overnight incubation with the particles and LPS/IFN, nitric oxide production was estimated from culture supernatant nitrite. Cell viability was determined by monitoring the rate of AlamarBlue reduction. The dose-effect relationships for nitrite and viability were modeled as a power function (Fold change = [Dose+1]beta), where beta represents the slope of the dose-response curve. Potency was defined as the rate of change in nitrite production corrected for cell viability (beta(POTENCY) = beta(NITRITE) - beta(VIABILITY)). Overall, the urban particles decreased nitric oxide production (beta(POTENCY) < 0), while exposure of the cells to fine particulate matter or cristobalite increased the production of nitric oxide (beta(POTENCY) > 0). Titanium dioxide (TiO2) was essentially inactive (beta(POTENCY) approximately to 0). The decrease in nitric oxide production seen in cells exposed to the urban particles was directly correlated to a decrease in the expression of inducible nitric oxide (iNOS) as determined by Western blot analysis. The results indicate that particles are modulators of nitric oxide production in murine macrophages and may directly disrupt expression of iNOS during concomitant pathogen exposure. Pathways leading to enhanced NO production causing cell injury, and to decreased NO release resulting in lower bacterial clearance, may both be relevant to the health effects of ambient particles.

Evaluation for Hsp70 as a biomarker of effect of pollutants on the earthworm Lumbricus terrestris.

Induction of heat shock proteins (Hsps) is often associated with a cellular response to a harmful stress or to adverse life conditions. The main aims of the present study were (1) to assess if stress-induced Hsp70 could be used to monitor exposure of the earthworm species Lumbricus terrestris to various soil pollutants, (2) to assess the specificity of pollutants in their tissue targeting and in Hsp70 induction, and (3) to evaluate if dose-response relationships could be established and if the stress-response observed was specific. The midgut/intestinal tissues of L. terrestris are shown to express an inducible member of the Hsp70 family after heat shock treatment in vitro and exposures to different soil toxicants in vivo (re: artificial soil). Short-term (24-72 hours) and long-term (14-16 days) exposures to the chemical standards chloroacetamide and pentachlorophenol and to heavy metals (Pb++, Cd++, Cu++, and Hg++) also affected the earthworms, and Hsp70 was induced in their midgut/intestinal tissues. After a 3-day exposure to heavy metals, the level of Hsp70 induction in the midgut/intestinal tissues appears to correlate well with the reported in vivo and in vitro toxicity data. Comparatively, in proximal and midbody wall muscle tissues of animals exposed to the heavy metals, a decrease in expression of Hsp70 was sometimes detected. Thus Hsp analysis by Western blot in L. terrestris tissues and particularly in the midgut/intestine proved to be a suitable and sensitive assay for adverse effects in earthworms and showed a good level of reproducibility despite some individual variations. The use of pristine/nonexposed animals transposed into contaminated environments as in the present study should therefore be of high ecological relevance. Induction of Hsp70 in earthworms should represent not only a good wide-spectrum biomarker of exposure but also a biomarker of effect since known toxicants altered gene expression in tissues of these animals, as contrasted with a simple accumulation of Hsp. Hence, the detection of Hsp70 in earthworms can constitute an early-warning marker for the presence of potentially deleterious agents in soils, with L. terrestris in particular and earthworms in general acting as potential sentinel animal species.

Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): implication of lysosomal proteases.

The poly(ADP-ribose) polymerase (PARP-1), a 113 kDa nuclear enzyme, is cleaved in fragments of 89 and 24 kDa during apoptosis. This cleavage has become a useful hallmark of apoptosis and has been shown to be done by DEVD-ase caspases, a family of proteases activated during apoptosis. Interestingly, PARP-1 is also processed during necrosis but a major fragment of 50 kDa is observed. This event is not inhibited by zVAD-fmk, a broad spectrum caspase inhibitor, suggesting that these proteases are not implicated in the necrotic cleavage of PARP-1. Since lysosomes release their content into the cytosol during necrosis, the proteases liberated could produce the cleavage of PARP-1. We therefore isolated lysosomal rich-fractions from Jurkat T cells. Our results reveal that the in vitro lysosomal proteolytic cleavage of affinity purified bovine PARP-1 is composed of fragments corresponding, in apparent molecular weight and function, to those found in Jurkat T cells treated with necrotic inducers like 0.1% H2O2, 10% EtOH or 100 microM HgCl2. Moreover, we used purified lysosomal proteases (cathepsins B, D and G) in an in vitro cleavage assay and found that cathepsins B and G cleaved PARP-1 in fragments also found with the lysosomal rich-fractions. These findings suggest that the necrotic cleavage of PARP-1 is caused in part or in totality by lysosomal proteases released during necrosis.

Potent and selective nonpeptide inhibitors of caspases 3 and 7.

5-Dialkylaminosulfonylisatins have been identified as potent, nonpeptide inhibitors of caspases 3 and 7. The most active compound within this series (34) inhibited caspases 3 and 7 in the 2-6 nM range and exhibited approximately 1000-fold selectivity for caspases 3 and 7 versus a panel of five other caspases (1, 2, 4, 6, and 8) and was at least 20-fold more selective versus caspase 9. Sequence alignments of the active site residues of the caspases strongly suggest that the basis of this selectivity is due to binding in the S2 subsite comprised of residues Tyr204, Trp206, and Phe256 which are unique to caspases 3 and 7. These compounds inhibit apoptosis in three cell-based models: human Jurkat T cells, human chondrocytes, and mouse bone marrow neutrophils.

Antagonism of oestrogen action in human breast and endometrial cells in vitro: potential novel antitumour agents.

PURPOSE: There is a need to find novel oestrogen receptor (ER) ligands that antagonize oestrogen action in the reproductive tissues and would therefore have therapeutic potential in oestrogen-dependent tumours. We tested novel ER ligands in both breast and endometrial cells to profile agonism/antagonism in these oestrogen target reproductive tissues. METHODS: Novel analogues of the ER antagonist ICI 182,780 were synthesized and tested for their ability to inhibit gene expression dependent on oestrogen response elements (ERE) in human breast (MCF-7) and endometrial (Ishikawa) cell lines. This activity was correlated with inhibition of oestrogen-induced cell proliferation and ER binding. RESULTS: The sulphide analogue (compound 1) and sulphone analogue (compound 2) had no intrinsic ERE-dependent agonism in either breast cancer or endometrial cells in culture. All three compounds dose-dependently inhibited ERE-mediated oestrogen agonism. Moreover, these ER ligands inhibited oestrogen-stimulated proliferation of breast cancer and endometrial cells. ICI 182,780, compound 1 and compound 2 were all able to bind both isoforms of the ER (ER alpha and ER beta). In endometrial cells, the relative binding to ER beta correlated with the ERE-dependent antioestrogenic effect of these ligands, suggesting that in this tissue this receptor is the predominant isoform that determines antioestrogenic activity. CONCLUSIONS: The ability of these analogues of ICI 182,780 to inhibit oestrogen-stimulated transcriptional activity and cell proliferation suggests that these agents, in particular the sulphone analogue, have therapeutic potential in the treatment of breast cancer without exhibiting the unwanted oestrogenic effects in the endometrium.

Heparin and cancer revisited: mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis.

Independent studies indicate that expression of sialylated fucosylated mucins by human carcinomas portends a poor prognosis because of enhanced metastatic spread of tumor cells, that carcinoma metastasis in mice is facilitated by formation of tumor cell complexes with blood platelets, and that metastasis can be attenuated by a background of P-selectin deficiency or by treatment with heparin. The effects of heparin are not primarily due to its anticoagulant action. Other explanations have been suggested but not proven. Here, we bring together all these unexplained and seemingly disparate observations, showing that heparin treatment attenuates tumor metastasis in mice by inhibiting P-selectin-mediated interactions of platelets with carcinoma cell-surface mucin ligands. Selective removal of tumor mucin P-selectin ligands, a single heparin dose, or a background of P-selectin deficiency each reduces tumor cell-platelet interactions in vitro and in vivo. Although each of these maneuvers reduced the in vivo interactions for only a few hours, all markedly reduce long-term organ colonization by tumor cells. Three-dimensional reconstructions by using volume-rendering software show that each situation interferes with formation of the platelet "cloak" around tumor cells while permitting an increased interaction of monocytes (macrophage precursors) with the malignant cells. Finally, we show that human P-selectin is even more sensitive to heparin than mouse P-selectin, giving significant inhibition at concentrations that are in the clinically acceptable range. We suggest that heparin therapy for metastasis prevention in humans be revisited, with these mechanistic paradigms in mind.

Role of caspases in human renal proximal tubular epithelial cell apoptosis.

In the present study, we have used an in vitro model of apoptosis using primary human renal proximal tubular epithelial (RPTE) cells to investigate the mechanisms involved in renal cell apoptosis. Treatment of RPTE cells with okadaic acid for 24-48 h induced apoptosis in a concentration-dependent manner. Apoptosis was accompanied by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspase-9, -3, and -7. The induction of caspase activity correlated with the proteolytic cleavage of beta-catenin, suggesting that beta-catenin is a caspase substrate. The caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), resulted in a dose-dependent inhibition of apoptosis and beta-catenin cleavage. These data suggest that okadaic acid-induced apoptosis is p38 MAPK and caspase-dependent and that proteolytic cleavage of beta-catenin by caspases is likely to be a downstream molecular event associated with the morphological and cytoskeletal changes induced during apoptosis.

Distinct mechanisms of action of selective estrogen receptor modulators in breast and osteoblastic cells.

Raloxifene and idoxifene are selective estrogen receptor modulators (SERMs) that exhibit tissue-specific agonist or antagonist properties via interactions with the estrogen receptor (ER). Both compounds are similarly osteoprotective in the ovariectomized rat in vivo as assessed by measurement of bone mineral density, urinary pyridinium cross-links, and serum osteocalcin, suggesting a similar mechanism of action. However, we have identified a fundamental difference in this mechanism via the estrogen response element (ERE) in osteoblast-like cells. With the use of ERE-luciferase reporter constructs, raloxifene, like the complete ER-antagonist ICI-182780, acts as an antagonist via the ERE in osteoblastic cells. In contrast, idoxifene, like 17beta-estrogen itself and 4-OH-tamoxifen, acts as an agonist in osteoblastic cells via an ER/ERE-mediated mechanism. Both ICI-182780 and raloxifene inhibited the ERE-dependent agonist activity of 17beta-estradiol and idoxifene in osteoblastic cells. In contrast, in breast cells, raloxifene, idoxifene, 4-OH-tamoxifen, and ICI-182780 had no agonist activity and, indeed, raloxifene and idoxifene were potent antagonists of ERE-mediated 17beta-estradiol action, indicating an ERE-dependent mode of action in these cells. Although these SERMs exhibit a similar antagonist activity profile in breast cells, they can be distinguished mechanistically in osteoblastic cells.

Androgen formation and metabolism in the pulmonary epithelial cell line A549: expression of 17beta-hydroxysteroid dehydrogenase type 5 and 3alpha-hydroxysteroid dehydrogenase type 3.

Surfactant synthesis within developing fetal lung type II cells is affected by testosterone and 5alpha-dihydrotestosterone (5alpha-DHT). The pulmonary epithelial cell line A549, isolated from a human lung carcinoma, like normal lung type II cell, produces disaturated phosphatidylcholines and has been widely used for studying the regulation of surfactant production. Androgen receptor has been detected in A549 cells; however, the capacity of these cells for androgen synthesis and metabolism has not been investigated at molecular level. This study was undertaken to identify the steroidogenic enzymes involved in the formation and metabolism of androgens from adrenal C19 steroid precursors in A549 cells. When cultured in the presence of normal FCS, A549 intact cells converted DHEA to androstenediol, androstenedione principally to testosterone, and 5alpha-DHT to 5alpha-androstane 3alpha,17beta-diol. High levels of 17beta-hydroxysteroid dehydrogenase (HSD) and 3alpha-HSD activities were detected in both cytosol and microsomes isolated from homogenates. Analysis of A549 RNA indicated the presence of 17beta-HSD type 4 and type 5, and of 3alpha-HSD type 3 messenger RNAs. Very low levels of 3beta-HSD type 1 and 5alpha-reductase type 1 messenger RNAs and activities were detected. With regard to active androgen formation, there was little or no capacity for the conversion of DHEA to 5alpha-DHT. In contrast, androstenedione was rapidly transformed to testosterone. The pattern of steroid metabolism was not affected by the use of charcoal-stripped FCS or by the synthetic glucocorticoid dexamethasone. Together, our findings show that A549 cells express a pattern of steroid metabolism in which 17beta-HSD type 5 and 3alpha-HSD type 3 are the predominant enzymes. The level of androgens is regulated at the level of catalysis in intact cells such that the intracellular level of testosterone is stabilized, whereas 5alpha-DHT is rapidly inactivated by reduction to 3alpha,17beta-diol. This pattern of androgen metabolism has implications for the relative importance of testosterone and 5alpha-DHT in normal lung development and surfactant production.

Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro.

Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

Identification and cloning of a human urea transporter HUT11, which is downregulated during adipogenesis of explant cultures of human bone.

Bipotential cells in human trabecular bone explant cultures that express osteoblast characteristics are able to undergo adipogenesis in the presence of 3-isobutyl-1-methylxanthine plus dexamethasone (Nuttall et al. [1998] J Bone Miner Res 13:371-382). The initial studies of these bipotential cells in explant cultures have been extended to examine differential gene expression during osteoblast/adipocyte transdifferentiation. Using differential display, we have identified a gene expressed in trabecular bone explant cultures that is downregulated as these cells differentiate from an osteoblast to an adipocyte phenotype. Homology searching identified this gene as the human urea transporter HUT11. The expression and downregulation of HUT11 have been observed in multiple patient bone explant cultures. The size of the bone explant-derived HUT11 mRNA is approximately 4.4 kb, which is identical to the largest splice variant reported. In this article, we report the cloning and sequencing of this gene from primary human osteoblasts. In addition, we report tissue distribution for the bone explant-derived form of HUT11 mRNA and show a reciprocal relationship between the expression of HUT11 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma 2, which is a marker of adipocyte differentiation. Because the control of osteoblast/adipocyte transdifferentiation is unknown, selective downregulation of HUT11 during adipogenesis suggests that HUT11 expression may be a marker of the switch from an osteoblast to an adipocyte phenotype. Understanding the role of HUT11 in osteoblasts may provide insights into the mechanism controlling osteoblast and adipocyte differentiation.

Idoxifene: a novel selective estrogen receptor modulator prevents bone loss and lowers cholesterol levels in ovariectomized rats and decreases uterine weight in intact rats.

Idoxifene, a novel selective estrogen receptor modulator, was tested for its effects on bone loss, serum cholesterol, and uterine wet weight and histology in the ovariectomized (Ovx) rat. Idoxifene (0.5 mg/kg x day) completely prevented loss of both lumbar and proximal tibial bone mineral density (BMD). In an intervention study, idoxifene (0.5 and 2.5 mg/kg x day) completely prevented further loss of both lumbar and proximal tibial BMD during a 2-month treatment period commencing 1 month after surgery, when significant loss of BMD had occurred in the Ovx control group. Idoxifene reduced total serum cholesterol, which was maximal at 0.5 mg/kg x day. Idoxifene alone displayed minimal uterotrophic activity in Ovx rats and inhibited the agonist activity of estrogen in intact rats. Histologically, myometrial and endometrial atrophy were observed in both idoxifene and vehicle-treated Ovx rats. In this report, we also provide molecular-based evidence to support the observations in vivo of a novel selective estrogen receptor modulator (SERM) mechanism of action in bone and endometrial cells. Idoxifene is an agonist through the estrogen response element (ERE) and exhibits similar postreceptor effects to estrogen in bone-forming osteoblasts. Idoxifene also stimulates osteoclast apoptosis, and these pleiotropic effects ultimately could contribute to the maintenance of bone homeostasis. However, idoxifene differs from estrogen in a tissue-specific manner. In human endometrial cells, where estrogen is a potent agonist through the ERE, idoxifene has negligible agonist activity. Moreover, idoxifene was able to block estrogen induced gene expression in endometrial cells, which is in agreement with the observation in the intact rat study. In the uterus, idoxifene has a pharmacologically favorable profile, lacking agonist and therefore growth-promoting activity. Together with its cholesterol lowering effect and lack of uterotrophic activity, these data suggest that idoxifene may be effective in the prevention of osteoporosis and other postmenopausal diseases without producing unwanted estrogenic effects on the endometrium.

Efficacy and safety of acarbose in metformin-treated patients with type 2 diabetes.

OBJECTIVE: To demonstrate the efficacy, tolerability, and safety of acarbose compared with placebo in patients with type 2 diabetes inadequately controlled with diet and metformin (2,000 or 2,500 mg/day in divided doses). RESEARCH DESIGN AND METHODS: This study had a multicenter randomized double-blind placebo-controlled parallel-group comparison design. The trial lasted 31 weeks and consisted of a 1-week screening period, a 6-week placebo pretreatment period, and a 24-week period of acarbose or placebo, with a forced titration from 25-50 mg t.i.d. and a titration of 50-100 mg tid that was based on glucose control. The primary efficacy variable was the mean change from baseline in HbA1c. Secondary efficacy variables included mean changes from baseline in fasting and postprandial plasma glucose, serum insulin, and triglyceride levels. RESULTS: The addition of acarbose to patients on background metformin and diet therapy showed a statistically significant reduction in mean HbA1c of 0.65%. There were statistically significant reductions in fasting and postprandial plasma glucose and serum insulin levels compared with placebo. Gastrointestinal side effects were more frequently reported in the acarbose-treated patients. No significant differences in liver transaminase elevations were observed between patients treated with acarbose and those treated with placebo. CONCLUSIONS: The results of this study demonstrate that the addition of acarbose to patients with type 2 diabetes who are inadequately controlled with metformin and diet is safe and generally well tolerated and that it significantly lowers HbA1c and fasting and postprandial glucose and insulin levels.

Isolation, purification and partial characterization of chloragocytes from the earthworm species Lumbricus terrestris.

Chloragocytes were isolated from the earthworm species Lumbricus terrestris. After mechanical dissociation and sedimentation through Percoll, a highly purified fraction of viable chloragocytes was obtained. The isolated chloragocytes accumulated the vital dye neutral red and reduced the tetrazolium dye MTT, thereby indicating cellular integrity. Time of flight flow cytometric analyses revealed a main population of large and highly granulated cells in the 30-33 microm size range. Hydrolase measurements showed that beta-D-N-acetyl-glucosaminidase and acid phosphatase exhibited the highest activities (146.6 and 24.9 mU/mg of protein, respectively), possibly indicating a major role for these 2 hydrolases in the physiological function of chloragocytes. In contrast, other acid hydrolases such as beta-D-galactosidase and beta-D-glucuronidase had specific activities of respectively 26 and 182 times lower than the glucosaminidase. The specific activity of the membrane-bound alkaline phosphatase was comparable to that of its acid counterpart (18.9 vs. 24.9 mU/mg of protein, respectively) and this level of activity may show an important trans-membrane activity in chloragocytes. The cytoplasmic and mitochondrial enzyme isocitrate dehydrogenase had a level of activity comparable to that of the exclusively cytoplasmic enzyme lactate dehydrogenase (6.6 vs. 8.1 mIU/mg of protein, respectively). When L. terrestris chloragocyte homogenates were separated on Percoll, results showed that hydrolases and dehydrogenases were mainly associated with the lighter materials that remained above the Percoll layer. Nonetheless, the detection of significant proportions (15-25%) of the total recovered activity of acid phosphatase and beta-galactosidase in the enriched chloragosome fraction supports the notion that some chloragosomes may be 'lysosome-like' organelles.