The modified Devine's procedure for the management of concealed penis in children: an experience of 131 cases.

OBJECTIVE: With the current study, we aimed at describing our experience of surgical management of concealed penis using the modified Devine's procedure. PATIENTS AND METHODS: Medical records of all patients undergoing surgical management of concealed penis using the modified Devine's procedure from January 2019 to June 2021 were retrieved. Penile length was measured before and after the procedure. Parental satisfaction on the penile size, morphology, voiding status and hygiene was measured using a 5-point Likert's scale. RESULTS: A total of 131 patients were included. The mean age of the patients was 8.56 ± 2.75 years. The mean BMI of the patients was 24.17 ± 1.78 kg/m2. The patients were followed up to a mean of 15 ± 3 months. The duration of the surgery was 89.93 ± 16.85 minutes. There was a statistically significant increase in penile length from 1.8 ± 0.64 cm to 5.02 ± 1.11 cm after the procedure (p < 0.01). There was a statistically significant improvement in satisfaction status of all domains at 6, 12 and 24 weeks as compared to pre-operative levels. CONCLUSIONS: The modified Devine's technique is a simple and effective surgical technique for management of concealed penis in children producing predictable results and excellent parental satisfaction. The low rate of complications and good cosmetic outcomes lend support to its use in clinical practice.

[Safety and efficacy of laparoscopic distal rectal transection by transanterior obturator nerve gateway].

Objective: To evaluate the safety and efficacy of distal rectal transection by using transanterior obturator nerve gateway (TANG) in laparoscopic radical resection for lower rectal cancers. Methods: A descriptive case series study was performed. Inclusion criteria: (1) patients with primary rectal adenocarcinoma, with the distance of 3-5 cm from tumor to anal verge, with normal anal function before surgery and a desire to preserve anus; (2) laparoscopic radical resection of rectal cancer was performed and the distal rectum was transected using TANG approach. Exclusion criteria: (1) patients with distant metastasis or receiving palliative surgery; (2) the distal rectum was transected using non-TANG approach; (3) patients receiving combined multiple organs resection; (4) patients complicated with other tumors requiring additional treatment during the study. Clinicopathological data of 50 patients with low rectal cancer undergoing laparoscopic resection using TANG approach between January 2019 and December 2020 in Peking University First Hospital were retrospectively collected. Perioperative conditions, length of specific pelvic lines, additional angle and postoperative short-term outcomes were observed and described. Additional angle was defined as the angle between the simulated stapling line with the traditional approach and the real stapling line with the TANG approach. Data following normal distribution were presented as Mean±SD, or M [quartile range (Q(R))] otherwise. Results: All the patients successfully completed laparoscopic surgery without transferring to open or transanal surgery. The median operative time was 193 (80) min and blood loss was 50 (58) ml. All tumors received R0 resection with the distance from the tumor to distal resection margin of 1.7 (0.4) cm and the anastomotic height of 2.0 (0.1) cm. Rectal transection was completed by one cartridge in 52.0% of the cases (26/50) and two cartridges in 48.0% (24/50). Length of the stapling line was 6.6 (1.5) cm. The time to construct the gateway was 8.0 (6.0) min. The vessel damage occurred in 4.0% of the cases (2/50) and none of the cases encountered obturator nerve damage. Inlets of the pelvis in TANG and traditional approach were (9.9±1.3) cm vs. (7.2±1.1) cm (t=24.781, P<0.001). Additional angle of TANG was (15±2) °. The transecting positions on the midline and right edge of the rectum specimen by TANG were 0.6 (0.2) cm and 1.0 (0.2) cm lower than those by the traditional approach. One case (2.0%) died of pulmonary infection on the 17th day after surgery, 2 cases (4.0%) received re-operation and 14 cases (28.0%) had postoperative complications, including anastomotic leakage (7/50, 14.0%), urinary retention (6/50, 12.0%), pelvic infection (2/50, 4.0%) and ileus (2/50, 4.0%). The median postoperative hospital stay was 12 (6) days. Conclusions: Laparoscopic distal rectal transection by using TANG approach is safe and effective in the treatment of low rectal cancer. As an alternative rectal transecting method, TANG has advantages especially for the obese and those with a contracted pelvis and ultralow rectal cancers.

[Analysis of risk factors of anastomotic leakage after laparoscopic intersphincteric resection for low rectal cancer and construction of a nomogram prediction model].

Objectives: To examine the risk factors of anastomotic leakage for low rectal cancers undergoing laparoscopic intersphincteric resection (ISR), and to construct a nomogram prediction model for it. Methods: The perioperative data of 302 low rectal cancer patients undergoing laparoscopic ISR by the same surgical team of Department of General Surgery, Peking University First Hospital between January 2012 and January 2019 were retrospectively reviewed. There were 190 males and 112 females, aging 60(14) years (range: 20 to 84 years). χ2 test, independent sample t test, U test and Logistic regression analysis were used to analyze the risk factors for anastomotic leakage. R software was used to complete the drawing of the nomogram prediction model, and the receiver operating characteristic curve was used to evaluate the predictive ability of the nomogram prediction model. Results: There were 24 patients (7.9%) had anastomotic leakage among the 302 patients enrolled, including 10 cases of grade A leakage, 9 cases of grade B leakage, and 5 cases of grade C leakage. Out of the 24 patients, 2 patients (8.3%) died, 3 patients (12.5%) received leakage-related reoperation. Median healing time of the anastomotic leakage was 74 (58) days (range: 14 to 180 days). Univariate analysis showed male gender (P=0.009), preoperative serum albumin concentration (P=0.004), neoadjuvant radiochemotherapy (P=0.017), preserving left colonic artery (P=0.002) and performing a diverting ileostomy (P=0.015) were significantly correlated with anastomotic leakage. Logistic multivariate analysis showed male gender (OR=6.052, 95%CI: 1.535 to 23.860, P=0.010), neoadjuvant radiochemotherapy (OR=4.098, 95%CI: 1.318 to 12.821, P=0.015), no preserving left colonic artery (OR=16.699, 95%CI: 3.051 to 91.406, P=0.001) and not performing a diverting ileostomy (OR=21.218, 95%CI: 4.341 to 103.710, P<0.01) were independent risk factors for anastomotic leakage. According to the results of multi-factor regression analysis, the nomogram prediction model was constructed. The area under the curve of the nomogram prediction model was 0.840 (95%CI: 0.766 to 0.914). After internal verification, the concordance index value of the model was 0.840. Conclusion: Male gender, neoadjuvant radiochemotherapy, no preserving left colonic artery and not performing a diverting ileostomy are independent risk factors for anastomotic leakage for low rectal cancers undergoing laparoscopic ISR.

[Mechanism of cross talk between tissue factor/active coagulation factor VII and epidermal growth factor receptor signalings in colon cancer cells in culture].

OBJECTIVE: To preliminarily verify the cross talk between tissue factor/active coagulation factor VII (TF/FVIIa) and epidermal growth factor receptor (EGFR) pathways in human colon cancer cells in culture. METHODS: FVIIa was treated to HT-29 (KRAS-wild type) and LoVo (KRAS-mutant) colon cancer cells to activate TF/FVIIa pathway, qRT-PCR and Western blot were used to detect the expressions of amphiregulin (AREG) and epiregulin (EREG), ligands of EGFR on mRNA and protein levels, respectively. After knocking down expression of TF by TF-targeted siRNA transfection, FVIIa was treated and mRNA expressions of AREG and EREG were detected to see whether the FVIIa-induced effects were dependent on TF. Expressions of mRNA of TF and FVII were detected by qRT-PCR following the activation of EGFR pathway by treatment with epidermal growth factor (EGF) to HT-29 and LoVo cells. RESULTS: After TF/FVIIa pathway was activated, for HT-29 cells, expressions of AREG (on mRNA level) and EREG (both on mRNA and protein level) were significantly down-regulated versus those of control group, gene expressions of AREG and EREG were 0.55±0.09 vs.0.99 ±0.09, 0.67±0.10 vs.1.02±0.02, protein expressions of EREG were 0.54±0.09 vs.1.04±0.13, all P<0.05. For LoVo cells, expressions of AREG (both on mRNA and protein level) and EREG (on protein level) were significantly up-regulated versus those of control group, gene expression of AREG were 1.87±0.39 vs. 0.93±0.23, protein expressions of AREG and EREG were 3.09±0.73 vs.1.11±0.21, 1.53±0.19 vs.0.97±0.23, all P<0.05. The regulating effect of AREG and EREG mRNA expression by FVIIa in HT-29 and LoVo cells could both be partly blocked by knocking down TF expression. For HT-29 cells, activation of EGFR pathway induced no significant TF mRNA expression, FVII mRNA expression was not detected. However,for LoVo cells, activation of EGFR pathway induced significantly higher mRNA expressions of both TF and FVII, expressions were 1.53±0.23 vs.1.00±0.23, 53.20±6.08 vs.1.00±0.15, all P<0.05. CONCLUSION: In colon cancer cell LoVo, when activated, TF/FVIIa pathway and EGFR pathway could interact through upregulating the other pathway's effectors, and mutant KRAS might play a critical role in the two pathways' cross talk.

Perforation predicts poor prognosis in patients with primary intestinal diffuse large B-cell lymphoma.

AIMS: To elucidate the clinicopathological features and prognostic factors of primary intestinal diffuse large B-cell lymphoma (PI-DLBL). METHODS AND RESULTS: Archival tissues from 30 tumours were used for tissue microarray construction, immunohistochemistry and interphase fluorescence in situ hybridization for chromosomal translocation. The M:F ratio was 1.7:1, with a median age of 60 years. The ileum and ileocaecum were most frequently involved (40% each). Fourteen (47%) were at stage I(E) disease, 15 (50%) at stage II(E). Five (17%) tumours were perforated at presentation. The tumours expressed Bcl-6 (73%), MUM1 (70%), Bcl-2 (67%) and CD10 (23%). Nine (30%) were classified as germinal centre B-cell (GCB) phenotype and 21 non-GCB. Eight of 30 (27%), 7/30 (23%) and 2/29 (7%) cases were positive for rearrangements involving IGH, BCL6, and C-MYC loci, respectively, whereas all cases were negative for BCL2 and CCND1 translocation. Perforation was a poor prognostic indicator, with a hazard ratio of tumour-related death at 8.75 (P = 0.001). The differentiation antigens, GCB versus non-GCB phenotype, or lymphoma-associated translocations were of no prognostic significance. CONCLUSIONS: We found a higher rate of perforation and lower frequency of GCB phenotype in PI-DLBL in Taiwan compared with other geographical areas; perforation is a poor prognostic indicator.

Activation of mitogen-activated protein kinases is essential for hydrogen peroxide -induced apoptosis in retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells are constantly exposed to oxidative injury while clearing byproducts of photoreceptor turnover, a circumstance thought to be responsible for degenerative retinal diseases. The mechanisms of hydrogen peroxide (H(2)O(2))-induced apoptosis in RPE cells are not fully understood. We studied signal transduction mechanisms of H(2)O(2)-induced apoptosis in the human RPE cell line ARPE-19. Activation of two stress kinases (JNK and p38) occurs during H(2)O(2) stimulation, and H(2)O(2)-mediated cell death was significantly reduced by their specific inhibition. Exposure to a lethal dose of H(2)O(2) elicited Bax translocation to the mitochondria and release of apoptosis-inducing factor (AIF) from the mitochondria, both of which were abolished by either JNK- or p38-specific inhibitors. Both H(2)O(2)-induced cell death and JNK/p38 phosphorylation were partially inhibited by C. difficile toxin B, inhibitor of Rho, Rac, and cdc42. Use of pull-down assays revealed that the small GTPase activated by H(2)O(2) is Rac1. This study is the first to demonstrate that H(2)O(2) induces a Rac1/JNK1/p38 signaling cascade, and that JNK and p38 activation is important for H(2)O(2)-induced apoptosis as well as AIF/Bax translocation of RPE cells.

Combined cord blood stem cells and gene therapy enhances angiogenesis and improves cardiac performance in mouse after acute myocardial infarction.

BACKGROUND: Gene and stem cell therapies hold promise for the treatment of ischaemic cardiovascular disease. However, combined stem cell and angiogenic growth factor gene therapy for acute ischaemic myocardium has not been previously reported. This study hypothesized that combined stem cell and gene therapy would not only augment new vessels formation but also improve myocardial function in acute ischaemic myocardium. METHODS: Human angiopoietin-1 (Ang1) cDNA and VEGF(165) cDNA were ligated into AAV vector. The purified CD34(+) cells were obtained from human umbilical cord blood samples. Cord blood CD34(+) cells were transduced with AAV vector encoding either the human Ang1 (AAV-Ang1) or VEGF(165) (AAV-VEGF) cDNA alone, or both (AAV-Ang1 plus VEGF). Immediately after ligation of the left anterior descending coronary artery in male SCID mice, culture-expanded CD34(+) cells transduced with AAV-Ang1, AAV-VEGF or AAV-Ang1 plus VEGF were injected intramyocardially at the left anterior free wall. RESULTS: Western blot showed that Ang1 and VEGF protein expressions were enhanced in the CD34(+)cells transduced with AAV-Ang1 and AAV-VEGF, respectively. Infarct size significantly decreased and capillary density significantly increased after treatment with CD34(+)/AAV-Ang1 plus VEGF when compared with treatment by CD34(+) only. Combined therapy with CD34(+) and AAV-Ang1, CD34(+) and AAV-VEGF, CD34(+) and AAV-Ang1 plus VEGF, all showed significantly higher cardiac performance in echocardiography than the therapy with CD34(+) alone 4 weeks after myocardial infarction. CONCLUSIONS: Combined therapy with human umbilical cord blood CD34(+) cells and both Ang1 and VEGF genes reduced infarct size, attenuated the progression of cardiac dysfunction and increased capillary density in acute myocardial infarction in mice.

Human Nopp140, which interacts with RNA polymerase I: implications for rRNA gene transcription and nucleolar structural organization.

Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity.

Droloxifene does not blunt bone anabolic effects of prostaglandin E2, but maintains prostaglandin E2-restored bone in aged, ovariectomized rats.

Droloxifene (DRO) is a selective estrogen receptor modulator that prevents bone loss by inhibition of bone turnover associated with estrogen deficiency in both growing and aged female rats. The purposes of this study were to test: (a) whether DRO can maintain prostaglandin E2 (PGE2)-restored bone after discontinuation of PGE2 in aged, ovariectomized (ovx) rats; (b) if an inhibition of bone turnover by DRO reduces bone anabolic effects of PGE2; and (c) whether bone mass restored by PGE2 plus DRO can be maintained after discontinuation of both agents. Female rats at 12 months of age were sham-operated (sham) or ovx. Three months postsurgery, ovx rats were treated with either PGE2 (3 mg/kg per day, subcutaneously [s.c.]) alone, or PGE2 plus DRO (10 mg/kg per day, per os [p.o.]) for 2 months. Thereafter, the PGE2 or PGE2 plus DRO treatment was withdrawn and the rats were then treated with either vehicle or DRO for another 1.5 months. Using dual-energy X-ray absorptiometry (DXA), total lumbar vertebral bone mineral density (LV-BMD) was determined in vivo at months 0, 3, 5, and 6.5. At the end of the study, the rats were autopsied, and BMD of total femur, femoral shaft, distal femoral metaphysis, and proximal femur was determined ex vivo by DXA. Standard static and dynamic bone histomorphometric parameters were determined on the fourth lumbar vertebral body (L-4). At 3, 5, or 6.5 months postsurgery, LV-BMD decreased significantly (-15%, -19%, and -19%, respectively) in the vehicle-treated ovx rats compared with sham. Beginning at 3 months post-ovx, PGE2 alone or in combination with DRO for 2 months completely restored LV-BMD back to the sham level. There was no difference in LV-BMD in PGE2 alone or PGE2 plus DRO. Upon cessation of PGE2 treatment, a significant decrease in LV-BMD was observed in the PGE2-alone group (-12%). On the other hand, when DRO treatment was given after discontinuation of PGE2, the PGE2-restored LV-BMD was completely maintained. In the PGE2 plus DRO group, no loss in LV-BMD was observed after cessation of either PGE2 alone or both PGE2 and DRO. However, treatment with DRO following 2 months of PGE2 plus DRO further increased LV-BMD (+10%). At the end of the study, ex vivo femoral BMD data confirmed the observation in lumbar vertebrae. Histomorphometric results of L-4 indicated that loss in bone mass after cessation of PGE2 in PGE2 alone group was associated with increased bone turnover. Treatment with DRO in the maintenance phase inhibited bone turnover and prevented bone loss induced by withdrawal of PGE2. Trabecular bone mass was maintained in the PGE2 plus DRO followed by vehicle group and further increased in the PGE2 plus DRO followed by DRO groups. We found that: (a) DRO is efficacious in maintaining PGE2-restored bone after discontinuation of PGE2; (b) DRO did not blunt the anabolic effects of PGE2; (c) bone loss occurred after cessation of treatment in the PGE2-alone group, whereas it was maintained after cessation of treatment in PGE2 plus DRO group; and (d) an additional anabolic effect was found in ovx rats treated with PGE2 plus DRO followed by DRO.

[A surgical treatment of early partial closure of the epiphysial plate].

OBJECTIVE: To introduce the technique of epiphysiolysis and epiphysis grafting in the treatment of early partial closure of the epiphysial plate in children, and evaluate its effect. METHODS: Retrospective study was performed in 10 cases of cubitus varus and valgus, or genu varum and valgum due to early partial closure of the epiphysial plate, 6 cases received simple cuneiform osteotomy, and 4 cases received epiphysioloysis and epiphysis grafting. RESULTS: Clinical outcoming after 6-month to 5-year's follow-up showed 1 case deformity of recurrence and 2 cases of non-isometric limb after simple osteotomy, while no recurrence and isometric limb after epiphysiolysis and epiphysis grafting. CONCLUSION: Epiphysiolysis and epiphysis grafting may effectively prevent the recurrence of postoperative deformity, and restore the longitudinal growth of limb.

Effects of CP-336,156, a new, nonsteroidal estrogen agonist/antagonist, on bone, serum cholesterol, uterus and body composition in rat models.

We have discovered a new, nonsteroidal, potent estrogen agonist/antagonist, CP-336,156. CP-336,156 binds selectively and with high affinity to the human estrogen receptor-alpha with a half-inhibition concentration of 1.5 nM, which is similar to that seen with estradiol (4.8 nM). When given orally to immature (3-week-old) female Sprague-Dawley rats for 3 days at doses of 0.1, 1.0, 10, or 100 microg/kg x day, unlike 17alpha-ethynyl estradiol, CP-336,156 had no effect on uterine wet or dry weight. Similarly, no uterine hypertrophy was observed in aged (17-month-old) female rats treated (p.o.) with CP-336,156 at 10 or 100 microg/kg x day for 28 days. We also found that CP-336,156 decreased total serum cholesterol and fat body mass and had no effect on lean body mass in these aged female rats. In 5-month-old ovariectomized (OVX) Sprague-Dawley female rats, CP-336,156 completely prevented OVX-induced increases in body weight gain, total serum cholesterol, and serum osteocalcin at doses between 10 and 1000 microg/kg x day after 4 weeks. At these doses, CP-336,156 completely prevented OVX-induced bone loss and inhibited the increased bone turnover associated with estrogen deficiency in lumbar vertebrae, proximal tibiae, and distal femora. Similar to estrogen, CP-336,156 induced apoptosis and p53 expression with a concomitant decrease in the number of tartrate-resistant acid phosphatase-positive multinuclear cells in rat bone marrow cell cultures in vitro, suggesting that the induction of apoptosis may be a mechanism for the estrogenic activities of CP-336,156 in bone. In summary, CP-336,156 is a new, orally active, nonsteroidal, potent estrogen agonist/antagonist that has similar effects in bone as estradiol but without the uterine-stimulating effects associated with estradiol in rats.

The nucleolar phosphoprotein P130 is a GTPase/ATPase with intrinsic property to form large complexes triggered by F- and Mg2+.

We have previously identified a human nucleolar phosphoprotein p130 whose alterations during mitosis are correlated well with the nucleolar disassembly and reassembly. Further studies found that p130 in the cell lysates or after being purified by immunoprecipitation was able to form large complexes triggered by F- and Mg2+. These sodium dodecyl sulfate-insoluble p130 molecules were readily dissociated by adding EDTA to the complexes. It is known that F- and Mg2+ act on many GTPases and ATPases through the induction of a conformational transition mimicking the nucleoside triphosphate-bound state. These initial observations led us to discover that p130 functions as a GTP/ATP binding protein with intrinsic GTPase/ATPase activities. The rate of GTP hydrolysis by purified p130 under our experimental conditions was 0.8 mol/min/mol of p130. These results imply that p130, a novel nucleolar GTPase/ATPase, may switch its conformation in a nucleotide-dependent manner.

Comparative effects of droloxifene, tamoxifen, and estrogen on bone, serum cholesterol, and uterine histology in the ovariectomized rat model.

The purpose of this study was to compare the effects of droloxifene (DRO), tamoxifen (TAM), and 17 alpha-ethynyl estradiol (EE) on bone mineral density, bone histomorphometry, total serum cholesterol, and uterine histology in the ovariectomized (ovx) rat model. Sprague-Dawley female rats at five months of age were sham-operated and treated orally with vehicle (n = 8), or ovx (n = 56) and treated (p.o.) with either vehicle, DRO at 0.1 or 1.0 mg/kg daily, TAM at 0.1 or 1 mg/kg daily, or EE at 3 or 30 micrograms/kg daily for 4 weeks. The uterine wet weight and uterine histologic parameters (cross-sectional tissue area, stromal thickness, and luminal epithelial thickness) were determined. Femoral and lumbar vertebral bone mineral density was determined ex vivo using dual energy x-ray absorptiometry. Static and dynamic cancellous bone histomorphometry was performed on double-labeled, undecalcified longitudinal sections from proximal tibial metaphyses. Furthermore, the changes in total serum cholesterol and body weight gain were also determined. Compared to sham controls, ovx for four weeks significantly decreased uterine weight (-72%), uterine cross-sectional tissue area (-74%), stromal thickness (-52%), and luminal epithelial thickness (-53%). ovx rats treated with EE at 30 micrograms/kg/day maintained these parameters at the levels of sham controls. Uterine weight and uterine cross-sectional tissue area in 3 micrograms/kg/day of EE treated ovx rats were higher than that of vehicle-treated ovx rats. In ovx rats treated with TAM at both 0.1 and 1 mg/kg/day, these parameters were significantly less than sham controls but significantly higher than ovx controls. DRO at 0.1 mg/kg/day had no effects on all above parameters. Uterine weight and cross-sectional tissue area in 1 mg/kg/day of DRO treated ovx rats was slightly but significantly higher than that in ovx controls. However, DRO at 1 mg/kg/day had no effects on uterine stromal thickness and luminal epithelial thickness compared to ovx controls. The ovx-induced decrease in femoral and lumbar vertebral bone mineral density was prevented by treatment with EE at 30 micrograms/kg/day, TAM at both 0.1 and 1 mg/kg/day, or DRO at 1 mg/kg/day. Similarly, the decrease in bone mass and the increase in bone resorption and bone turnover in proximal tibial metaphyses were prevented by treatment with EE at 30 micrograms/kg/day or TAM at both 0.1 and 1 mg/kg/day, or DRO at 1 mg/kg/day. Total serum cholesterol decreased significantly in ovx rats treated with either EE, DRO, or TAM at all dose levels compared to vehicle treated ovx controls (-32% to -56%). The ovx-induced body weight gain was completely prevented by EE at 30 micrograms/kg/day, and partially prevented by DRO at 1 mg/kg/day. TAM at both 0.1 and 1 mg doses caused a significant decrease in body weight compared to both sham and ovx controls. Our results indicated that DRO prevented ovx-induced bone loss and lowered total serum cholesterol with an ED50 less than 1 mg/kg/day. The bone protective and cholesterol lowering effects of DRO were comparable to those observed with TAM and EE. However, DRO differed from TAM and EE in its lack of significant estrogenic effects on uterine tissue at doses which were bone protective. These data suggest that DRO may be a significant alternative to EE and TAM for prevention and treatment of postmenopausal osteoporosis.

Short-term systemic insulin-like growth factor-1 is unable to prevent cyclosporin A-induced osteopenia in the rat.

Immunosuppression with cyclosporin A (CsA) is effective in a number of immune-mediated diseases and in preventing rejection following organ transplantation. We have repeatedly demonstrated that CsA in the rat model produces accelerated bone remodelling with net bone loss, best characterized in trabecular bone. IGF-I holds promise as a treatment for various osteopenic conditions. Although currently a subject of much controversy, various studies have suggested that in vivo it is anabolic to cortical as well as trabecular bone. The purpose of this study was, in part, to further characterize the effects of CsA and IGF-I on trabecular and cortical bone, and to see whether systemic IGF-I is able to modulate CsA's deleterious skeletal effects. Sixty 10 week-old, male, Sprague-Dawley rats were randomized to receive the following daily for 3 weeks: (1) CsA vehicle (veh) per os (po) + recombinant human (rh) IGF-1 veh subcutaneously (sc); (2) CsA 15 mg/kg po + rhIGF-I-veh; (3) CsA-veh + rhIGF-I 200 microg/kg sc; (4) CsA-veh + rhIGF-I 600 microg/kg sc; (5) CsA 15 mg/kg + rhIGF-I 200 microg/kg, and (6) CsA 15 mg/kg + rhIGF-I 600 microg/kg. Rats were weighed and venous blood was sampled serially for determination of glucose, ionized calcium (Ca2+), PTH, vitamin D, and osteocalcin. Following sacrifice on day 20, histomorphometry was performed on double calcein-labeled tibial metaphysis and diaphysis. All rats receiving CsA had elevated levels of blood glucose and osteocalcin by day 9 and vitamin D at day 20. PTH was similar in all groups, and Ca2+ was only raised in the CsA and CsA + IGF-I 200 microg/kg groups. Rats receiving IGF-I 200 microg/kg and IGF-I 600 microg/kg gained more weight than either vehicle- or CsA-treated animals, attesting to IGF-1's anabolic properties. CsA caused severe trabecular bone loss, not prevented by IGF-I; it even further increased the eroded surface. CsA and IGF-I had little effect on cortical bone volume or marrow area. IGF-I increased endocortical matrix synthesis, as evidenced by the increases in the percent endocortical osteoid perimeter, an effect negated by the addition of CsA. This experiment demonstrates that trabecular bone is more susceptible than cortical bone to the deleterious effects of CsA and indicates little role for IGF-1 in the pathophysiology or treatment of CsA-induced bone disease at the given doses and duration of treatment.

Modification of DNA topoisomerase I enzymatic activity with phosphotyrosyl protein phosphatase and alkaline phosphatase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea:Decapoda).

DNA topoisomerase I was partially purified from the hepatopancreas of the shrimp Penaeus japonicus. The specific activity of the final preparation was 7,000,000 units/mg of protein with SV40 viral DNA as substrate. SDD-polyacrylamide gel electrophoresis of the final preparation yielded two major bands of proteins with M(r) 70,000 and M(r) 67,000, as well as less intense bands of proteins with M, 64,000 and M(r) 56,000. Incubation of the partially purified enzyme fraction with rabbit antiserum against human DNA topoisomerase I, allowed all these proteins except that of M(r) 56,000, to be positively reacted. Treatment of the partially purified DNA topoisomerase I with tyrosine kinase p43v-abl resulted in phosphorylation of only the two major subunits. Phosphorylation by tyrosine kinase p43v-abl or dephosphorylation by phosphotyrosyl protein phosphatase resulted in a decrease of the enzymatic activity. The treatment with shrimp alkaline phosphatase abolished the enzymatic activity of the purified DNA topoisomerase I in a dose-dependent manner. Thus, the DNA topoisomerase I was apparently isolated from the hepatopancreas of the shrimp P. japonicus in a phosphorylated form, and this phosphorylation was essential for expression of enzymatic activity in vitro. The activity of DNA topoisomerase I is inhibited by ZnCl2, CuCl2 and Pb(NH3)3 at millimolar concentrations, but less inhibition was observed with CaCl2.

Effects of droloxifene on prevention of cancellous bone loss and bone turnover in the axial skeleton of aged, ovariectomized rats.

The purpose of this study was to determine the efficacy of droloxifene (DRO), an estrogen antagonist/agonist, in preventing ovariectomy (OVX)-induced lumbar vertebral cancellous bone loss and bone turnover in aged female rats. Fifty-three Sprague-Dawley female rats were OVX or sham-operated at 19 months of age, and divided into 6 groups: (I) sham-operated controls; (II) OVX vehicle controls; (III) OVX rats treated with E2 at 30 micrograms/kg/day; (IV)-(VI) OVX rats treated with DRO at either 2.5, 5, or 10 mg/kg p.o. daily. The treatment period was 8 weeks. Static and dynamic cancellous bone histomorphometric parameters were determined on 4 and 10 microns thick, undecalcified, double-fluorescent labeled sections of the fourth lumbar vertebral body. Changes in body weight, uterine weight, and total serum cholesterol were also determined. OVX for 8 weeks in 19-month-old female rats resulted in reduced trabecular bone volume (-18%) and trabecular width (-10%) and increased labeling perimeter (+52%), bone formation rate/bone surface referent (+60%), bone formation rate/bone volume referent (+77%), osteoclast number (+41%), and osteoclast perimeter (+41%). E2 treatment at 30 micrograms/kg/day for 8 weeks prevented OVX-induced cancellous bone loss and decreased bone resorption, bone formation, and bone turnover to the values of sham controls. DRO at 2.5-10 mg/kg/day completely prevented bone loss and bone turnover associated with estrogen deficiency. Osteoclast number and perimeter were significantly decreased in DRO-treated-OVX rats compared to both sham and OVX controls. Trabecular bone volume, trabecular width, labeling perimeter, bone formation rate/bone surface referent, and bone formation rate/bone volume referent showed no differences in DRO-treated OVX rats compared to those of E2-treated OVX rats and sham controls. These histomorphometric results indicated that DRO is an estrogen agonist on cancellous bone of lumbar vertebral bodies of aged, OVX rats. Further, E2 treatment prevented the OVX-induced increase in body weight gain and nonsignificantly reduced total serum cholesterol compared to OVX controls. Body weight gain and total serum cholesterol did not differ between OVX rats treated with E2 and sham controls. In OVX rats treated with DRO, body weight decreased significantly in a dose-response manner, and total serum cholesterol was significantly reduced by 65% to 70% compared to both sham and OVX controls. In addition, treatment with E2 increased uterine weight to the value of sham controls in OVX rats. However, DRO had no effect on uterine weight at either 2.5 or 10 mg/kg/day, while it only slightly but significantly increased uterine weight over OVX controls at 5 mg/kg/day. We conclude that DRO was efficacious in the prevention of lumbar vertebral cancellous bone loss and in the decline of total serum cholesterol but had no effect on uterine weight in the aged, OVX female rats. Our data suggest that DRO is a potentially useful agent for the prevention of vertebral bone loss leading to spinal fractures in postmenopausal women.

Droloxifene inhibits cortical bone turnover associated with estrogen deficiency in rats.

Droloxifene (DRO), an estrogen antagonist/agonist, has been shown to possess estrogen-like effects in inhibiting bone turnover leading to cancellous bone loss in ovariectomized (OVX) rats. The purpose of this study was to determine the effects of DRO on cortical bone turnover in OVX rats. Sprague-Dawley female rats at 5 months of age were sham-operated (sham, n = 8) and orally treated with vehicle, or OVX (n = 56) and orally treated with either vehicle, DRO at 0.1, 1, 5, or 10 mg/kg/day, or 17 alpha-ethynyl estradiol (EE) at 3 or 30 micrograms/kg/day for 4 weeks. Static and dynamic cortical bone histomorphometry was performed on double fluorescent labeled, undecalcified cross sections of tibial diaphyses (proximal to the tibiofibular junction). There were no significant differences in tibial diaphyseal cross sectional area, marrow cavity area, and cortical bone area between groups after 4 weeks of administration. Periosteal mineralizing surface, mineral apposition rate, and bone formation rate-surface referent and endocortical eroded surface increased significantly, while endocortical mineral apposition rate and bone formation rate-surface referent increased nonsignificantly in OVX controls compared to sham controls. Treatment with DRO at doses of 0.1 to 10 mg/kg/day dose-dependently attenuated the OVX-induced higher bone formation indices in both the periosteal and endocortical surfaces and higher bone resorption index in the endocortical surface. At the highest dose (10 mg/kg/day), DRO completely inhibited the increases in bone formation and resorption indices in OVX rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Intermittent treatment of prostaglandin E2 with risedronate is more anabolic than prostaglandin E2 alone in the proximal tibial metaphysis of ovariectomized rats.

This study is designed to test how intermittent application of prostaglandin E2 (PGE2) and risedronate (Ris) alone or in combination acts on the cancellous bone mass in estrogen-deficient rats. Sprague-Dawley rats were ovariectomized (ovx) or sham-ovx'd at 6 months of age. PGE2 (6mg/kg/d), Ris (5 micrograms/kg/twice a week) or PGE2 plus Ris were given for 60 days to ovx rats immediately after operation and followed by 60 days without treatment. The drugs were then reapplied for another 60 days. Static histomorphometry was performed on the secondary spongiosa of proximal tibial metaphysis (PTM). Sixty days of ovx lost trabecular bone and number, Ris prevented ovx-induced bone loss. PGE2 added 48% extra cancellous bone, but the new bone was completely lost after 60 days of withdrawal. Another 60 days of PGE2 treatment only partially restored the trabecular bone, the bone mass was still -42% lower than that of sham-ovx controls. Co-treatment of PGE2 with Ris added the same amount of bone as PGE2 alone after the first 60 days treatment period, but differed from PGE2 alone in that the new bone lost less during the 60 days withdrawal period. Re-application of co-treatment for another 60 days added more extra bone. We concluded that intermittent co-treatment with anabolic and anti-resorptive agents is more effective than anabolic agent alone in long-term therapy of cancellous bone in estrogen-deficient rats.