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Diabetes is a metabolic disorder caused by insulin resistance and/or deficiency and impairs bone quality and bone healing due to altered gene expression, reduced vascularization, and prolonged inflammation. No effective treatments for diabetic bone healing are currently available, and most existing treatments do not directly address the diabetic complications that impair bone healing. We recently demonstrated that sustained and localized delivery of salicylic acid (SA) via an SA-based polymer provides a low-cost approach to enhance diabetic bone regeneration. Herein, we report mechanistic studies that delve into the biological action and local pharmacokinetics of SA-releasing polymers shown to enhance diabetic bone regeneration. The results suggest that low SA concentrations were locally maintained at the bone defect site for more than 1 month. As a result of the sustained SA release, a significantly reduced inflammation was observed in diabetic animals, which in turn, yielded reduced osteoclast density and activity, as well as increased osteoblastogenesis. Based upon these results, localized and sustained SA delivery from the SA-based polymer effectively improved bone regeneration in diabetic animals by affecting both osteoclasts and osteoblasts, thereby providing a positive basis for clinical treatments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2595-2603, 2016.

Congenital combined pituitary hormone deficiency (CPHD) may present with cholestasis in the neonate or during early infancy. However, its precise mechanism is unknown. A 3-mo-old boy presented with cryptorchidism and hypoplastic scrotum after birth. Neonatal jaundice was noted but temporarily improved with phototherapy. Jaundice recurred at 2 mo of age. Elevated direct bilirubin (D-Bil) and liver dysfunction were found but cholangiography showed no signs of biliary atresia (BA). Liver biopsy findings showed giant cell formation of hepatocytes with hypoplastic bile ducts. Subsequent magnetic resonance imaging (MRI) of the head revealed a hypoplastic pituitary gland with an ectopic posterior lobe, and the patient was diagnosed with congenital CPHD based on decreased secretion of cortisol and GH by the pituitary anterior lobe load test. D-Bil levels promptly improved after hydrocortisone (HDC) replacement. We subsequently began replacement with levothyroxine (L-T4) and GH, and liver histology showed normal interlobular bile ducts at 8 mo old. This is the first case report of proven histological improvement after hormone replacement therapy. This suggested that pituitary-mediated hormones, especially cortisol, might be involved in the development of the bile ducts.

Diabetes mellitus (DM) involves metabolic changes that can impair bone repair, including a prolonged inflammatory response. A salicylic acid-based poly(anhydride-ester) (SA-PAE) provides controlled and sustained release of salicylic acid (SA) that locally resolves inflammation. This study investigates the effect of polymer-controlled SA release on bone regeneration in diabetic rats where enhanced inflammation is expected. Fifty-six Sprague-Dawley rats were randomly assigned to two groups: diabetic group induced by streptozotocin (STZ) injection or normoglycemic controls injected with citrate buffer alone. Three weeks after hyperglycemia development or vehicle injection, 5 mm critical sized defects were created at the rat mandibular angle and treated with SA-PAE/bone graft mixture or bone graft alone. Rats were euthanized 4 and 12 weeks after surgery, then bone fill percentage in the defect region was assessed by micro-computed tomography (CT) and histomorphometry. It was observed that bone fill increased significantly at 4 and 12 weeks in SA-PAE/bone graft-treated diabetic rats compared to diabetic rats receiving bone graft alone. Accelerated bone formation in normoglycemic rats caused by SA-PAE/bone graft treatment was observed at 4 weeks but not at 12 weeks. This study shows that treatment with SA-PAE enhances bone regeneration in diabetic rats and accelerates bone regeneration in normoglycemic animals.

Steroid receptors for estrogen, progestogen and androgen in human ovarian cancer were characterized. 159,800xg supernatant was used as cytosol. In the cytosol, sucrose density gradients showed 5S and 8S binding peaks for each receptor. Steroid specificity studies showed that estrogen for [3H]-estradiol-receptor binding, progestogen for [3H]-R5020-receptor binding and androgen for [3H]-DHT-receptor binding were specific inhibitors. Equilibrium studies showed that the dissociation constant(Kd) was (15.3±3.1)x10-10M(n=8) for [3H]-estradiol receptor binding, (2.3, 4.6)x10-10M(n=2) for [3H]-R5020 receptor binding and (6.6, 26.0)x10-10M(n=2) for [3H]-DHT receptor binding.

Tritium-labeled steroids [progesterone, medroxyprogesterone acetate, ORG 2058 [16α-ethyl-21-hydroxy-19-nor-4-pregnene-3, 20-dione), R5020 (17, 21-dimethyl-19-norpregna-4, 9-diene-3, 20-dione), norethindrone and R1881 (17β-hydroxy-17α-methylestra-4, 9, 11-trien-3-one)] were evaluated and discussed for progesterone receptor assay in the cytosol of human uterine endometrium. Each labeled steroid bound to the receptor proteins for progesterone, possibly estrogen or androgen, and occasionally the proteins in the globulin fraction. It is suggested that it is better not to use a tritium-labeled steroid with the same steroid as a competitor in order to get a non-specific binder for the measurement of progesterone receptor assay. The value of the dissociation constant was between 10-9M and 10-10M whatever tritium-labeled or unlabeled steroid was used.

Human endometrial estrogen and progestogen receptor levels in the mid-secretory phase were determined in various types of endometrium to contribute to a better understanding of luteal insufficiency. The concentrations of both total estrogen and progestogen receptor sites (both cytosol and nuclear; both occupied and unoccupied) decreased in the out-of-phase endometrium (p

Recently, minimally invasive surgery based on a tissue engineering has become possible. We used an animal model to evaluate a method for bone regeneration by maxillary sinus floor augmentation that uses mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) instead of an autologous bone graft. The MSCs were isolated from rabbit iliac crest marrow aspirates, and PRP was obtained from peripheral blood. MSCs and PRP complexes were used for grafting. In the control group, autogenous particulate cancellous bone and marrow (PCBM) was collected and mixed with PRP. Histological examination showed that new bone formation was good in both groups at 2 and 4 weeks. At 8 weeks, lamellar bone had formed, but fatty marrow was also observed in both groups.The present results suggest that MSCs/PRP complexes may be used in place of autogenous bone to promote bone regeneration after maxillary sinus floor augmentation.

1. Gestrinone has been used for treatment of pelvic endometriosis, in doses of 2.5 mg twice a week. This study is designed to clarify it from the dynamics of sex steroid receptors in rabbit uterus. 2. Four different regimens were scheduled, namely daily 1 microgram estradiol-17 beta (E2, consistent with endogenous level of women) for 3 days, and together with either 30, 60 (consistent with 2.5 microgram of clinical dose) or 120 microgram(s) gestrinone, in single dose or with 20 microgram(s) gestrinone daily (divided dose in single 60 microgram(s) gestrinone administration) for 3 days. Receptors for estrogen (ER, type I and II) or progestin (PR) were determined by charcoal adsorption in cytosol and KCl extract, or sedimentation in non-KCl extractable fraction, using [3H]E2 or [3H]promegestone respectively. 3. Gestrinone decreased total ER (type I and II) levels, dose-dependently, except the case of 20 micrograms gestrinone daily in ER type II. Total ER type I level did not return to the pre-level until 3 days only after 120 microgram(s) gestrinone administration. Total ER type II level was decreasing in 3 days after 30, 60 or 120 micrograms gestrinone therapy. Total PR level was decreased in the order of strength: 120 micrograms gestrinone greater than 60 micrograms gestrinone greater than 30 microgram gestrinone greater than 20 micrograms gestrinone in group, and the recovery was not obtained until 72 hr only after 120 micrograms gestrinone therapy. The uterine weight was decreasing with the same strength in 3 days of the therapy independently upon the regimen. 4. In conclusion, gestrinone dose of 60 micrograms (2.5 mg of clinical dose) every 3 days is considered to be effective for anti-steroid action in sex steroid receptor dynamics in the rabbit uterus, contributing to the rationale for gestrinone treatment of pelvic endometriosis.

The effects of estradiol (E2), progesterone (P), and danazol on the production of interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) by OK-432 (a streptococcal preparation)-stimulated monocytes were examined. E2 and P at physiologic concentrations enhanced IL-1 beta and TNF production by monocytes from donors with lower control levels (without steroids added) of IL-1 beta and TNF. However, E2 and P at physiologic concentrations did not affect IL-1 beta and TNF production by monocytes from donors with higher control levels of IL-1 beta and TNF. Danazol inhibited IL-1 beta and TNF production by monocytes in a dose-dependent manner from not only donors with lower control levels of IL-1 beta and TNF but also donors with higher control levels of IL-1 beta and TNF. Danazol at a concentration of 10(-6) M significantly suppressed IL-1 beta and TNF production in the presence of E2 and/or P at concentrations giving peak responses of IL-1 beta production. These findings suggest possible new mechanisms of action for danazol in the treatment of endometriosis and infertility associated with immune abnormalities.

Rabbit uterus contains type I and II oestrogen binding sites in the cytosol, and nuclear fractions. Oestrogen-stimulated increase in uterine weight was inhibited by concurrent treatment with progesterone or danazol. Oestrogen-induced type I binding sites were decreased by progesterone and to a lesser extent by danazol, while oestrogen-induced type II sites were decreased almost equally in the two groups. Neither progesterone nor danazol alone caused any detectable changes in uterine weight. These findings may suggest that the anti-oestrogenic effect on uterine weight is more correlated with the change in type II binding sites of oestrogen than that in type I sites. Thus, danazol may exert anti-oestrogenic actions through a pathway independent from that of progesterone.

We attempted to assess the association between hyperandrogenemia and inappropriate gonadotropin secretion in women with polycystic ovaries (PCO). Thirty-one patients diagnosed as PCO by ultrasonography were divided into two subgroups: 17 with high serum total testosterone (T) level (or = 0.5 ng/ml) and 14 with normal serum total T level (0.5 ng/ml). Both subgroups presented for the complaints of oligomenorrhea and/or hirsutism. The control group consisted of 15 women with regular ovulation for reference data collection. The PCO subjects with normal T, but not those with high T, revealed remarkable depletion of sex hormone binding globulin (SHBG), as compared with control. The PCO subject groups with high and normal serum T did not differ with respect to estrogen level, androgen level, follicle-stimulating hormone and prolactin levels, and SHBG concentration. Solely serum luteinizing hormone (LH) level was observed to be higher in those with high T, as typical features, than another subgroup or control. These data suggest that an increase in bioactive T as a result of decrease in serum SHBG or LH elevation may contribute to ovarian dysfunction in the patient with PCO.

Estrone (E1), a principal estrogen in postmenopausal women, may have profound consequences in the maintenance and progression of hormone-sensitive endometrial carcinoma. This study was designed to investigate the specific binding sites for E1 in the tumor and in the normal endometrium. The binding of [3H]E1 to the cytosolic fraction and KCl-soluble nuclear fraction from 3 endometrial carcinomas showed saturation kinetics with an apparent equilibrium dissociation constant of approximately 37 and 50 nM, respectively. The specific binding site of E1 was also found in 3 normal endometria. However, the binding capacity in the endometrial carcinoma increased to 1.5-fold of that in the normal endometrium. E2 did not affect [3H]E1 binding to any subcellular fraction from endometrial carcinoma specimens. These findings demonstrate the presence of specific binding sites for E1 in human endometrial carcinoma. The endometrial carcinoma growth may be mediated, at least in part, by E1 and its binding site interaction.

Acute hematogenous osteomyelitis (AHOM) of the acetabulum is a rare condition in children and usually caused by Staphylococcus aureus. We present an 11-year-old soccer athlete who suffered from acute osteomyelitis involving the acetabulum caused by S. capitis, a normal flora of the human skin but never reported in this condition. The disease was associated with repetitive skin injuries of the knee and potential osseous microtrauma of the hip joint by frequent rigorous exercise. This unusual case suggests that osseous microtrauma of the acetabulum, in addition to repetitive skin injuries, allowed normal skin flora to colonize to the ipsilateral acetabulum, which served as a favorable niche and subsequently led to AHOM.

1. 1. This study was designed to investigate the presence of estrogen type I (high affinity, low capacity) and type II (low affinity, high capacity) binding sites in human peripheral blood monocytes and the effects of danazol on these sites. 2. 2. These two types of estrogen binding sites existed in human peripheral blood monocytes. 3. 3. Danazol bound to these sites in high concentration (10−6 M, clinical serum concentration during danazol therapy) and decreased the number of both sites. 4. 4. It is suggested that danazol has an anti-estrogenic action to the monocytes through the competition and suppression of estrogen binding sites as seen in the estrogen target organ.

Objective To investigate the expression of interleukin-1 (IL-1) beta messenger ribonucleic acid (mRNA) and IL-1 receptor antagonist (IL-1ra) mRNA in peritoneal macrophages. Design, Setting Peritoneal fluid (PF) samples were collected from patients who underwent laparoscopy or laparotomy. Northern blot analysis was performed at the reproductive research laboratory. Patients Twenty-six patients with endometriosis, 10 patients with postinflammatory pelvic adhesion, and 12 control women with normal pelvis. Main Outcome Measure Polyadenylated RNA isolated from peritoneal macrophages was analyzed on Northern blots by using synthetic oligonucleotide probes. Results The level of IL-1 beta mRNA expression was elevated in the group with stage I endometriosis, whereas the increased expression of IL-1ra mRNA was observed in the group with stages III and IV endometriosis. The level of IL-1 beta mRNA showed a positive correlation with that of IL-1 beta in PF and a negative correlation with the level of IL-1ra mRNA. Conclusions Our results suggest that peritoneal macrophages express IL-1ra mRNA rather than IL-1 beta mRNA with the progress of endometriosis and that peritoneal macrophages may secrete IL-1ra protein that modulates the effects of IL-1.

The levels of interleukin 1 beta (IL-1 beta) and tumor necrosis factor (TNF) in peritoneal fluid (PF-IL-1 beta and PF-TNF) and production of IL-1 beta and TNF by peritoneal macrophages were determined in patients with benign gynecologic disease. The level of PF-IL-1 beta was elevated in the acute pelvic inflammatory disease (PID) and stages I and II endometriosis (E I/II) groups compared with the normal pelvis group, but not in the myoma of the uterus, ovarian cyst, and postinflammatory pelvic adhesion groups. The level of PF-TNF was elevated in the PID, EI/II and stages III and IV endometriosis (EIII/IV) groups. There was no correlation between the levels of PF-IL-1 beta and PF-TNF. Neither the level of PF-IL-1 beta nor that of PF-TNF was correlated with the concentration of peritoneal macrophages. Peritoneal macrophages produced IL-1 beta and TNF in vitro in the absence of stimulants. The levels of PF-IL-1 beta and PF-TNF are presumably linked to the activation of peritoneal macrophages.

This study was designed to clarify the effects of danazol on levels of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, progesterone (P), and 17 beta-estradiol and endometrial steroid receptors (for estrogen [ER], progestin [PR], and androgen [AR] ) during luteal-phase and long-term treatment. These levels were compared with midluteal-phase levels for a histologically in-phase endometrium. Danazol given during the luteal phase to patients with in-phase endometrium decreased endometrial steroid receptor levels (total ER and total PR), and decreased serum P, LH, and FSH levels. Ten of the 17 patients treated (59%) still had in-phase endometrium. Danazol (400 mg/day) given for 1 month or more to patients with pelvic endometriosis increased serum LH and FSH levels within the normal range and endometrial total ER and PR levels. It appears that the effects of short-term and long-term treatment with danazol on serum hormone and endometrial steroid receptor levels differ.

Synopsis The relationship between the histological grade and the steroid action mechanism was investigated in human endometrial cancer. The concentration and detection rate of cytosol estrogen (ER) and progestin receptor (PR) tended to decrease, and the rate of detected both ER and PR decreased as the tumor progressed from well differentiated to poorly differentiated grades. It appears that the mechanism of action of estrogen is impaired in undifferentiated cancer. The total quantity and detection rate in the nuclear binding of estrogen-ER complexes tended to increase and those of progestin-PR complexes tended to decrease as the tumor grade advanced. One may speculate that estrogen is biologically effective due to the increased nuclear binding sites of estrogen-ER complex instead of decreased cytosol receptor sites in undifferentiated tumors.

Steroid binding macromolecules were investigated in normal ovaries in human subjects. The 248,000g homogenate supernatant was used for cytosol. Estrogen, progestogen and androgen bindings sedimentation coefficients were mainly situated in the 4–5S region. All these steroid bindings near the 6–8S region were scanty in amount or degraded during sucrose gradient centrifugation. On the other hand, steroid binding proteins around the 4–5S region contained weak steroid binding proteins as well. Equilibrium studies showed that the dissociation constant of all steroid-macromolecule bindings existed between 10-9M and 10-10M. Ligand specificity for each macromolecule was determined. It is suggested that the characteristics of these macromolecules are compatible with steroid receptors. Follicular ovaries, corpus luteum, ovaries where corpora lutea were resected, polycystic ovaries and a parovarian cyst contained steroid binding macromolecules.

To understand the mechanism of action of danazol, the binding of danazol to multiple classes of intracellular steroid binding proteins was studied in the human uterine endometrium. Danazol bound to endometrial receptors for estrogen, progesterone, and androgen and seemed to bind to endometrial intracellular corticosteroid-binding globulin and sex-hormone-binding globulin. Danazol occupies almost all binding sites of steroids in the steroid target cells in spite of the presence of endogenous steroids. It is speculated that the binding behavior of danazol may be related to its therapeutic effect on endometriosis.Binding of danazol to multiple classes of intracellular steroid binding proteins was studied in the human uterine endometrium in an effort to understand danazol's mechanism of action. Danazol, which binds to human endometrial cytosol receptors for androgen (AR), progestin (PR), and estrogen (ER), also appears to interact with sex hormone binding globulin and corticosteroid binding globulin present in the cytosol preparations. 17 beta-hydroxy-17 alpha-methyl-estra-4, 9, 11-trine-3-one-[17 alpha-methyl-3h], 87 Ci/mmol (3H-R1881) binds to AR and PR in the human uterine endometirum. Thus, 3H-R1881/PR binding was abolished by the presence of progesterone. Previously reported studies have demonstrated that danazol binds with relatively high affinity to AR. Also, the danazol/AR complex can translocate to the nucleus. These findings are consistent with the observed androgenic effects of danazol in rats and women. Danazol binds with moderate affinity to PR. The danazol/PR complex is poorly translocated to the nucleus, suggesting that danazol is antiprogestational. Yet, a major metabolite of danazol, ethisterone, is progestational. The progestational vs. the antiprogestational effects of danazol remain controversial. Danazol displacement of 6, 7-3H-)stradial-17 beta (3H-E2) from ER is not parallel to E2 displacement of 3H-E2 when compared with that of PR or AR. This discrepancy may derive from an affinity difference. Danazol binds poorly to ER, yet circulating concentrations of danazol in vivo may lead to complete occupancy of the ER by danazol, which may interfere with normal endometrial ER dynamics, resulting in an antiestrogenic effect. Danazol occupies almost all binding sites of steroids in the steroid target cells despite the presence of endogenous steroids. The binding behavior of danazol may be related to its therapeutic effect on endometriosis.