The relationship between activity clusters detected by an automatic activity monitor and endocrine changes during the periestrous period in lactating dairy cows.

The aim of this study was to determine the relationship between observed estrous-related behavior, activity clusters (AC; detected by automatic activity monitor), endocrine profiles, and ovulation time. Twenty-one cows in estrus (after 2 cloprostenol treatments, 11 d apart) and 12 nonsynchronized cows, to establish Heatime (SCR Engineers Ltd., Netanya, Israel) herd baseline activity, were enrolled. Cows had Heatime monitors applied 3 wk before the trial to establish their own baseline activity level. Cows in standing estrus had ultrasonography and phlebotomy carried out every 4 h to determine dominant follicle size, endocrine profiles, and ovulation time. After ovulation, these procedures were repeated once on d 3 to 6. Heatime alerted estrus in 90% of cows, and incorrectly alerted 17% of AC. The mean±SEM duration for standing estrus was 9±1 and 13±1 h for estrous-related behavior. Estrous-related behavior began after the start of the proestrous estradiol-17ß (E2) increase (59±6.5 h). Cows with longer durations of raised proestrous E2 had longer intervals from its onset to the start of standing estrus and AC. The AC duration increased with longer durations of estrous-related behavior. Higher peak E2 occurred with longer standing estrus and estrous-related behavior. As E2 concentration decreased after the peak, 90% of cows still had estrous-related behavior. Duration of estrous-related behavior increased with higher average E2 concentration during the last 8 h before the start of the LH surge. During this surge 90% of cows had all of their standing estrus. As yields increased, so did the magnitude of the preovulatory FSH surges. Higher surges occurred with shorter standing estrus and estrous-related behavior. Cows with shorter LH surges had longer standing estrus. Peak LH preceded the AC peak (6.6±0.8 h). Duration of overlap between the AC start and the LH surge end ranged between 0 and 14 h; 1 cow had none. No association was found between the AC characteristics with the E2, LH, or FSH profiles. In conclusion, the relationship between the timing of the E2 increase and estrous activity may be mediated by other factors (GnRH surge). Estrous-related behavior, but not endocrine profiles, was related to AC duration. Timing of standing estrus during the LH surge ensures that mating allows sperm maturation before ovulation. Based on the interval from the start of an AC to ovulation (27±1 h), the optimum time to artificial insemination is, on average, between 9 and 15 h after the AC start.

Early pregnancy diagnosis on days 18 to 21 postinsemination using high-resolution imaging in lactating dairy cows.

The aim was to assess the ability of corpus luteum (CL) and uterine ultrasound characteristics on d 18 to 21 to predict pregnancy status in lactating dairy cows. Ultrasound examinations were carried out on cows (n = 164) on d 18 to 21 following artificial insemination (AI). Images of the uterus and CL were captured using a Voluson i ultrasound device (General Electric Healthcare Systems, Vienna, Austria) equipped with a 12-MHz, multi frequency, linear array probe. Serum concentrations of progesterone were determined from blood samples collected at each ultrasound examination. Images of the CL were captured and stored for calculation of CL tissue area and echotexture. Images of the CL and associated blood flow area were captured and stored for analysis of luteal blood flow ratio. Longitudinal B-mode images of the uterine horns were stored for analysis of echotexture. Diagnosis of pregnancy was made at each ultrasound examination based on CL blood flow, CL size, and uterine echotexture. Pregnancy was confirmed by ultrasonography on d 30 after AI. The relationship between ultrasound measures and pregnancy outcome, as well as the accuracy of the pregnancy diagnosis made at each ultrasound examination was assessed. Progesterone concentrations and CL tissue area were greater in pregnant compared with nonpregnant cows on all days. The CL blood flow ratio was higher in pregnant compared with nonpregnant cows on d 20 and 21 after AI. Echotexture measures of the CL and uterus were not different between pregnant and nonpregnant cows on any day of examination. The best logistic regression model to predict pregnancy included scores for CL blood flow, CL size, and uterine echotexture on d 21 following AI. Accuracy of pregnancy diagnosis was highest on d 21, with sensitivity and specificity being 97.6 and 97.5%, respectively. Uterine echotexture scores were similar for pregnant and nonpregnant cows from d 18 to 20. On d 21, pregnant cows had higher uterine echotexture scores compared with nonpregnant cows. The logistic regression equation most likely to provide a correct pregnancy diagnosis in lactating dairy cows included the visual score for CL blood flow, CL size, and uterine echotexture on d 21 after AI. In support of this finding, the diagnostic accuracy for visual scores of CL blood flow, CL size, and uterine echotexture were also highest on d 21.

Genetic merit for fertility traits in Holstein cows: V. Factors affecting circulating progesterone concentrations.

This study investigated the factors affecting circulating progesterone (P4) concentrations in cows with similar genetic merit for milk production traits, but with extremes of good (Fert+) or poor (Fert-) genetic merit for fertility traits. Study 1: 28 cows were enrolled in an ovulation synchronization protocol at 61±13 (±standard deviation) days postpartum, and data are presented for 13 Fert+ and 9 Fert- cows that remained in the study. Progesterone concentrations were determined from d 0 to 9 (d 0=estrus) and on d 7, corpus luteum (CL) volume and blood flow area (BFA) were measured by B-mode and Doppler ultrasonography, respectively. Cows were administered PGF2α on d 7 in the p.m. and d 8 in the a.m. to regress the CL, and 2 controlled internal drug release devices were inserted per vaginum on d 8 in the a.m. Liver biopsies were collected on d 9 and hepatic mRNA abundance of genes involved in P4 catabolism was determined. On d 10, the controlled internal drug release inserts were removed and frequent blood samples were collected to measure the rate of decline in circulating P4. The Fert+ cows tended to have greater dry matter intake compared with Fert- cows (+0.79kg of dry matter/d), but similar milk production (29.82kg/d). After synchronized ovulation, the rate of increase in circulating P4 concentrations was greater in Fert+ cows compared with Fert- cows. No effect of genotype on CL volume was detected, but BFA was 42% greater in Fert+ cows compared with Fert- cows. The Fert- cows had greater mRNA abundance of cytochrome P450, family 3, subfamily A (CYP3A) compared with Fert+ cows, but the mRNA abundance of aldo-keto reductase family 1, member C1 (AKR1C1), AKR1C3, AKR1C4, and cytochrome P450, family 2, subfamily C (CYP2C) were similar. The half-life and metabolic clearance rate of P4 were similar in Fert+ cows and Fert- cows. Study 2: 23 cows were enrolled in an ovulation synchronization protocol at 55±7 (±standard deviation) d postpartum, and data are presented for 13 Fert+ and 8 Fert- cows that remained in the study. On d 4, 7, 10, and 13 (d 0=estrus), CL volume and BFA were measured as in study 1. Progesterone concentrations were measured from d 1 to 13. Corpus luteum volume was 41% greater in Fert+ cows compared with Fert- cows but no effect of genotype on BFA was detected. Mean circulating P4 concentrations were 79% greater in Fert+ cows compared with Fert- cows. Milk yield was similar in both genotypes. The results indicate that greater circulating P4 concentrations were primarily due to greater CL P4 synthetic capacity rather than differences in P4 clearance in this lactating cow genetic model of fertility.

Small-molecule inhibition of inflammatory ß-cell death.

With the worldwide increase in diabetes prevalence there is a pressing unmet need for novel antidiabetic therapies. Insufficient insulin production due to impaired ß-cell function and apoptotic reduction of ß-cell mass is a common denominator in the pathogenesis of diabetes. Current treatments are directed at improving insulin sensitivity, and stimulating insulin secretion or replacing the hormone, but do not target progressive apoptotic ß-cell loss. Here we review the current development of small-molecule inhibitors designed to rescue ß-cells from apoptosis. Several distinct classes of small molecules have been identified that protect ß-cells from inflammatory, oxidative and/or metabolically induced apoptosis. Although none of these have yet reached the clinic, ß-cell protective small molecules alone or in combination with current therapies provide exciting opportunities for the development of novel treatments for diabetes.

The effect of lactation on post-partum uterine involution in Holstein dairy cows.

The objective was to examine the effect of lactation on uterine involution in post-partum dairy cows. Holstein primiparous cows were used (n = 19, mean age: 3.9 ± 0.1 years). At calving, cows were randomly assigned to one of two treatment groups, lactating (n = 11) or non-lactating (i.e. dried off at calving, n = 8). Examination of the reproductive tract was carried out by ultrasonography twice weekly until week 7 post-partum. Blood samples were collected twice weekly for the analysis of progesterone to indicate the resumption of cyclicity and metabolites indicative of energy status. Uterine involution was assessed in terms of size of the uterine horns, uterine body diameter and uterine fluid volume as assessed by the amount of non-echogenic material measured by ultrasound and position of the uterus. Vaginal mucous score was taken on day 28 post-partum for the assessment of uterine inflammation. Resumption of cyclicity (serum progesterone > 1 ng/ml) had occurred in both groups on average by day 21 post-partum. Concentrations of non-esterified fatty acids and beta-hydroxybutyrate were higher, whereas concentrations of glucose, insulin and IGF-1 were lower (p < 0.05) in lactating compared to non-lactating cows. Lactating cows had a smaller mean uterine body diameter (p < 0.05) than non-lactating cows from days 28 to 42 post-partum (day 28: 20.2 ± 1.3 vs 24.9 ± 1.5 mm, respectively) and had a lower mean uterine fluid volume up to day 49 (p < 0.05). By day 49, there was no difference in uterine diameter (15.2 ± 1.8 vs 15.2 ± 1.6 mm) or uterine fluid volume (0.11 ± 0.38 vs 0.18 ± 0.46) between lactating and non-lactating cows, respectively. Vaginal mucous score revealed no evidence of uterine inflammation in either group. In conclusion, while lactation induced significant alterations in metabolic status, it did not have a major effect on the rate of uterine involution as defined in this study.

Effect of follicular aspiration just before ovulation on corpus luteum characteristics, circulating progesterone concentrations and uterine receptivity in single-ovulating and superstimulated heifers.

The aim of this study was to investigate, in unstimulated and superstimulated heifers, the effect of follicle aspiration just before ovulation on corpus luteum (CL) development, circulating progesterone (P(4)) concentrations and the ability of the uterus to support embryo development. Following follicle aspiration or ovulation timed from GNRH administration, CL development was assessed by daily ultrasonography, and CL function was assessed in terms of the capacity to produce P(4) and the expression of genes involved in steroidogenesis in luteal tissue. The capacity of the uterine environment to support conceptus development was assessed following transfer and recovery of in vitro-produced embryos. Follicular aspiration just before the expected time of ovulation leads to a significant reduction in CL diameter, CL area and area of luteal tissue. This was associated with a decrease in circulating P(4) in both unstimulated and superstimulated heifers. Follicle aspiration leads to a reduction in conceptus length and area on day 14 in unstimulated heifers only. Follicle aspiration leads to a reduction in the expression of LHCGR in luteal tissue from unstimulated heifers compared with those in which the CL formed after ovulation. Superstimulation significantly reduced the expression of STAR in luteal tissue in both ovulated and follicle-aspirated heifers. In conclusion, in stimulated and unstimulated heifers, aspiration of the preovulatory dominant follicle(s) just before expected ovulation interferes with the subsequent formation and function of the CL, in terms of size and P(4) output and this, in turn, is associated with a reduced capacity of the uterus to support conceptus elongation in unstimulated heifers.

Effects of human chorionic gonadotrophin administration on day 5 after oestrus on corpus luteum characteristics, circulating progesterone and conceptus elongation in cattle.

The aim of the present study was to test the hypothesis that elevated concentrations of progesterone (P4) resulting from the induction of an accessory corpus luteum (CL) by human chorionic gonadotrophin (hCG) administration on day 5 after oestrus would lead to advanced conceptus elongation on day 14 following embryo transfer on day 7. The oestrous cycles of cross-bred beef heifers were synchronised and animals were randomly assigned to receive either of two treatments: (1) intramuscular injection of 3000 IU hCG on day 5 after oestrus (n=14); or (2) intramuscular injection of saline on day 5 after oestrus (n=13). Ovaries were scanned daily by transrectal ultrasonography to assess CL development. Serum concentrations of P4 were determined from daily blood samples collected from the jugular vein. In vitro-produced bovine blastocysts were transferred to synchronised recipients on day 7 after oestrus (n=15 blastocysts per recipient). Heifers were killed on day 14 after oestrus and the uterus was flushed to recover the embryos. Injection of hCG on day 5 induced ovulation of the dominant follicle in all treated heifers and increased the total area of luteal tissue on the ovary, which was associated with a significant increase (P<0.001) in serum concentrations of P4 from day 7 to day 14. Positive associations were detected between circulating P4 with CL area (within-day correlations ranging from r=0.45 to r=0.67) and total area of luteal tissue (within-day correlations ranging from r=0.65 to r=0.86) Administration of hCG did not affect the proportion of day 14 conceptuses recovered. However, compared with the control group, hCG-treated heifers had increased conceptus length (3.91±1.23 vs. 5.57±1.02 mm, respectively; P=0.06), width (1.00±0.06 vs. 1.45±0.05 mm, respectively; P=0.002) and area (5.71±0.97 vs. 8.31±0.83, respectively; P=0.02). Although numerically greater, mean interferon-τ (IFNT) production in vitro did not differ significantly (P=0.54) between embryos recovered from hCG-treated and control heifers. In contrast, there was a strong positive correlation between individual embryo length (r=0.76; P<0.001) and individual embryo area (r=0.72; P<0.001) and IFNT production. In conclusion, administration of hCG on day 5 after oestrus resulted in the formation of an accessory CL and hypertrophy of the original CL, the result of which was an increase in P4 concentrations from day 7 onwards. These elevated P4 concentrations were associated with an increased conceptus area. Furthermore, conceptus size was highly correlated with IFNT secretion in vitro.

Chondro/osteoblastic and cardiovascular gene modulation in human artery smooth muscle cells that calcify in the presence of phosphate and calcitriol or paricalcitol.

Vitamin D sterol administration, a traditional treatment for secondary hyperparathyroidism, may increase serum calcium and phosphorus, and has been associated with increased vascular calcification (VC). In vitro studies suggest that in the presence of uremic concentrations of phosphorus, vitamin D sterols regulate gene expression associated with trans-differentiation of smooth muscle cells (SMCs) to a chondro/osteoblastic cell type. This study examined effects of vitamin D sterols on gene expression profiles associated with phosphate-enhanced human coronary artery SMC (CASMC) calcification. Cultured CASMCs were exposed to phosphate-containing differentiation medium (DM) with and without calcitriol, paricalcitol, or the calcimimetic R-568 (10(-11)-10(-7) M) for 7 days. Calcification of CASMCs, determined using colorimetry following acid extraction, was dose dependently increased (1.6- to 1.9-fold) by vitamin D sterols + DM. In contrast, R-568 did not increase calcification. Microarray analysis demonstrated that, compared with DM, calcitriol (10(-8) M) + DM or paricalcitol (10(-8) M) + DM similarly and significantly (P < 0.05) regulated genes of various pathways including: metabolism, CYP24A1; mineralization, ENPP1; apoptosis, GIP3; osteo/chondrogenesis, OPG, TGFB2, Dkk1, BMP4, BMP6; cardiovascular, HGF, DSP1, TNC; cell cycle, MAPK13; and ion channels, SLC22A3 KCNK3. R-568 had no effect on CASMC gene expression. Thus, SMC calcification observed in response to vitamin D sterol + DM may be partially mediated through targeting mineralization, apoptotic, osteo/chondrocytic, and cardiovascular pathway genes, although some gene changes may protect against calcification. Further studies to determine precise roles of these genes in development of, or protection against VC and cardiovascular disease are required.

Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis.

High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG(-/-) mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG(-/-) mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG(-/-) mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-kappaB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG(-/-) mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.

Calcium exchange and ionized cytoplasmic calcium in resting and activated human monocytes.

We have performed a comprehensive study of calcium tracer flux, distribution, content, and ionized cytoplasmic calcium during monocyte activation. A model of monocyte calcium was developed from 45Ca uptake and exodus curves which indicated that cell calcium was partitioned between three compartments. The magnitude of the time constants for each pool lead us to propose cellular locations for these three compartments: a surface plasma membrane pool, a cytoplasmic pool, and an organelle pool. 45Ca uptake and exodus experiments were analyzed using a nonlinear least squares fit of compartmental exchange rates and sizes. The production of superoxide was used as a reflection of the state of activation of the monocytes treated with Concanavalin A (Con A). We found that Con A-treated monocytes have an increase in the calcium exchange rate with the cytoplasmic pool from 0.04 to 0.07/min (P less than 0.05), and an increase in the size of the cytoplasmic pool from 0.08 to 0.13 pmol/cell (P less than 0.05). There were no significant changes in the exchange rates or sizes associated with either of the other two compartments. The cytoplasmic ionized calcium was measured with the fluorescent probe, Quin 2, which indicated a resting level of 83 nM free calcium in unadhered monocytes. Con A stimulation caused a doubling of the cytoplasmic free calcium to 163 nM within 45 s. This increment in cytoplasmic free calcium preceded the onset of superoxide following Con A treatment. These studies indicate that Con A binding to the plasma membrane increases the monocyte plasma membrane permeability to calcium. External calcium enters the cell at an increased rate and contributes to both internally bound and free calcium. The magnitude of the increase in free calcium is proportional to the concentration of Con A and stimulates calcium extrusion via the calcium transport ATPase. Moreover, there is an increased concentration of ionized cytoplasmic calcium which has the potential to interact with other cellular regulators that modulate cell activation and superoxide production.

Relationship of superoxide production to cytoplasmic free calcium in human monocytes.

Calcium has been proposed as an intracellular second messenger for activation of secretion, phagocytosis, and the oxidative burst of neutrophils. We have examined the role of calcium in human monocyte activation. Concanavalin A (Con A)-stimulated monocytes displayed an increment in cytoplasmic ionized calcium at 31 +/- 6 s and the onset of superoxide production at 61 +/- 9 s. The increase in cytoplasmic calcium invariably preceded the onset of superoxide production. If the external calcium concentration was reduced to less than 28 nM by the addition of 10 mM EGTA, superoxide production was not diminished at 5 min; however, superoxide production decreased thereafter. The Con A-evoked increment in cytoplasmic ionized calcium was blunted upon the addition of EGTA and decreased further with time. Both the production of superoxide and the Con A-evoked increment in cytoplasmic ionized calcium displayed a 50% inhibition after 15 min of calcium depletion and were completely inhibited after 60 min. Total cell calcium fell from 0.7 to 0.5 fmol/cell, and the basal level of ionized calcium fell from 83 to 30 nM after 60 min. Histidine, a strong chelator of divalent cations other than calcium and magnesium, had no effect on monocyte superoxide production or on ionized calcium concentrations, indicating that EGTA inhibition was due to cell calcium depletion. In calcium-depleted cells, Con A did not evoke superoxide production until calcium was restored to the incubation medium. The restoration of calcium to Con A-treated, calcium-depleted monocytes permitted a rapid rise in the cytoplasmic ionized calcium, and the production of superoxide within 9 s. These data suggest that an increase in ionized cytoplasmic calcium is necessary for the activation of monocyte superoxide production by Con A. The rise in ionized calcium in response to Con A results, in part, from an internal redistribution of calcium, which is sufficient to permit superoxide generation.

FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation.

The fibroblast growth factors (FGFs) play key roles in controlling tissue growth, morphogenesis, and repair in animals. We have cloned a novel member of the FGF family, designated FGF-18, that is expressed primarily in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain. Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members. FGF-18 has a typical signal sequence and was glycosylated and secreted when it was transfected into 293-EBNA cells. Recombinant murine FGF-18 protein (rMuFGF-18) stimulated proliferation in the fibroblast cell line NIH 3T3 in vitro in a heparan sulfate-dependent manner. To examine its biological activity in vivo, rMuFGF-18 was injected into normal mice and ectopically overexpressed in transgenic mice by using a liver-specific promoter. Injection of rMuFGF-18 induced proliferation in a wide variety of tissues, including tissues of both epithelial and mesenchymal origin. The two tissues which appeared to be the primary targets of FGF-18 were the liver and small intestine, both of which exhibited histologic evidence of proliferation and showed significant gains in organ weight following 7 (sometimes 3) days of FGF-18 treatment. Transgenic mice that overexpressed FGF-18 in the liver also exhibited an increase in liver weight and hepatocellular proliferation. These results suggest that FGF-18 is a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine.

Establishment of critical timing of progesterone supplementation on corpus luteum and embryo development in beef heifers.

Optimal concentrations of progesterone (P4) during early pregnancy are a major determinant of embryo survival in cattle. This study examined the effects of P4 supplementation, following a period of induced low P4, on corpus luteum (CL) development, circulating P4 concentrations and embryo development. A total of 107 beef heifers from one herd were used in the study. Following AI (Day 0) at a synchronized oestrus, heifers were randomly assigned to 1 of 5 treatment groups: (1) no subsequent treatment (control; n=15); (2) administration of a synthetic prostaglandin F2α analogue (PG) on Days 3, 3.5 and 4 to induce low P4 and no further treatment (LP4; n=23); administration of PG as above followed by P4 supplementation via insertion of a Controlled Internal Drug Release device from (3) Day 4-7 (P4 d4-7; n=22); (4) Day 4-10 (P4 d4-10; n=23); or (5) Day 7-10 (P4 d7-10; n=24). Embryo and CL characteristics were determined following slaughter on Day 16. Embryo size was greater in heifers administered P4 d4-7 and P4 d4-10 in comparison with LP4. Corpus luteum area was significantly reduced in heifers administered P4 d4-7 and P4 d4-10 up to Day 7, and Day 10 (P4 d4-10), in comparison with LP4 heifers. Initiation of supplemental P4 on Day 7 had no effect on CL area or embryo size in comparison with LP4 heifers. Concentrations of P4 were less in P4 d4-7, P4 d7-10 and P4 d4-10 on Day 15 in comparison with LP4 heifers. The results of the study indicate an initiation and duration effect of supplemental P4 on circulating P4, embryo and CL development following induction of LP4 on Day 3 and 4.

Radiation therapy and hyperthermia improve the oxygenation of human soft tissue sarcomas.

The adverse prognostic impact of tumor hypoxia has been demonstrated in human malignancy. We report the effects of radiotherapy and hyperthermia (HT) on soft tissue sarcoma oxygenation and the relationship between treatment-induced changes in oxygenation and clinical treatment outcome. Patients receiving preoperative radiotherapy and HT underwent tumor oxygenation measurement pretreatment after the start of radiation/pre-HT and one day after the first HT treatment. The magnitude of improvement in tumor oxygenation after the first HT fraction relative to pretreatment baseline was positively correlated with the amount of necrosis seen in the resection specimen. Patients with <90% resection specimen necrosis experienced longer disease-free survival than those with > or = 90% necrosis. Increasing levels of tumor hypoxia were also correlated with diminished metabolic status as measured by P-31 magnetic resonance spectroscopy.

Osteoprotegerin prevents and reverses hypercalcemia in a murine model of humoral hypercalcemia of malignancy.

Osteoprotegerin (OPG), a novel, secreted tumor necrosis factor receptor family member that inhibits osteoclast formation and activity was examined for its activity in a syngeneic tumor model of humoral hypercalcemia of malignancy. Normal mice bearing Colon-26 tumors develop increases in both parathyroid hormone-related protein (PTHrP) expression and plasma PTHrP, marked hypercalcemia, and increased bone resorption. OPG, given either at the onset of hypercalcemia or after it had occurred, blocked tumor-induced increases in bone resorption and hypercalcemia and rapidly normalized blood ionized calcium. In tumor-bearing mice, OPG treatments reduced osteoclast activity from approximately 2-fold above normal into the subphysiological range but had no effects on tumor size, tumor-induced cachexia, or PTHrP levels. The potent effects of OPG in this humoral hypercalcemia of malignancy model suggest a potential therapeutic role for OPG in the prevention and treatment of this disorder.

Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma.

This study was performed to explore the relationship between tumor oxygenation and treatment outcome in human soft tissue sarcoma. Twenty-two patients with nonmestastatic, high-grade, soft tissue sarcomas underwent preoperative irradiation and hyperthermia and pretreatment measurement of tumor oxygenation. The 18-month actuarial disease-free survival was 70% for patients with tumor median oxygen pressure (pO2) values of >10 mm Hg but only 35% for those with median pO2 values of <10 mm Hg (P=0.01). There were eight treatment failures; the first site of recurrence was lung in all patients. Median pO2 was 7.5 mm Hg for metastasizing tumors versus 20 mm Hg for nonmetastasizing tumors (P=0.03). Potential mechanisms and implications for clinical trial design are discussed.

Role of surgical resection in pelvic Ewing's sarcoma.

PURPOSE: The improved survival in patients with Ewing's sarcoma over the past two decades has placed increased importance on achievement of local disease control. Ewing's sarcoma that arises in the pelvis has been recognized to have a worse prognosis than that in the appendicular skeleton, and the role of surgical resection in these cases remains controversial. The current study attempts to identify a benefit to surgical resection in these patients. METHODS: We retrospectively examined 39 patients who presented with Ewing's sarcoma in a pelvic location, all of whom were treated systemically with chemotherapy. Twenty patients received radiation only as a means of local control, and 19 underwent resection with or without radiation therapy. The patients were evaluated with end points of disease-free survival and overall survival for a minimum of 24 months and a mean of 58 months. RESULTS: There was an even distribution among patients who underwent surgical resection for local control as compared with those who received only radiation therapy with respect to age, site, date of treatment, and stage of disease. Despite uncontrolled biases including tumor size and response to chemotherapy that would be expected to favor patients who undergo resection, surgery in addition to or in substitution for radiation therapy did not result in a statistically significant increase in disease-free survival or overall survival. Local disease control was comparable between those who underwent resection and those who did not: three patients in each group developed a local recurrence. CONCLUSION: Currently, morbidity of surgical resection should be weighed against the efficacy and secondary complications of radiation therapy in the decision-making process for local disease control. The issue of whether overall survival and local disease control is improved in patients who undergo surgical resection remains controversial and may require a prospective randomized trial to be answered definitively.

The effect of Al2O3 barrier layers in TiO2/dye/CuSCN photovoltaic cells explored by recombination and DOS characterization using transient photovoltage measurements.

Solid-state dye-sensitized solar cells of the type TiO(2)/dye/CuSCN have been made with thin Al(2)O(3) barriers between the TiO(2) and the dye. The Al(2)O(3)-treated cells show improved voltages and fill factors but lower short-circuit currents. Transient photovoltage and photocurrent measurements have been used to find the pseudo-first-order recombination rate constant (k(pfo)) and capacitance as a function of potential. Results show that k(pfo) is dependent on V(oc) with the same form as in TiO(2)/dye/electrolyte cells. The added Al(2)O(3) layer acts as a "tunnel barrier", reducing the k(pfo) and thus increasing V(oc). The decrease in k(pfo) also results in an increased fill factor. Capacitance vs voltage plots show the same curvature (approximately 150 mV/decade) as found in TiO(2)/dye/electrolyte cells. The application of one Al(2)O(3) layer does not cause a significant shift in the shape or position of the capacitance curve, indicating that changes in band offset play a lesser role in the observed V(oc) increase. Cells made with P25 TiO(2) have, on average, 2.5 times slower recombination rate constants (longer lifetimes) than those made with colloidal TiO(2). The cells with P25 also show 2.3 times higher trap density (DOS), which results in little change in the V(oc) between the two types of TiO(2). It is further noted that the recombination current in these cells cannot be calculated from the total charge times the first order rate constant.

Ultrasound monitoring of blood flow and echotexture of the corpus luteum and uterus during early pregnancy of beef heifers.

The aim was to characterize changes in the ultrasound characteristics of the CL and uterus in pregnant, inseminated nonpregnant, and cyclic beef heifers and to correlate findings with systemic progesterone (P4) concentrations with the intention of identifying possible markers for early identification of pregnancy. Heifers were randomly selected for artificial insemination after estrus synchronization. Ultrasound examinations of the CL and uterus were carried out by transrectal ultrasonography using a high-resolution ultrasound scanner equipped with a 12 MHz linear array probe on Days 7, 11, 14, 16, and 18 after artificial insemination (Day 0; i.e., estrus). Cross-sectional B-mode images of the CL were captured for calculation of CL tissue area and echotexture. Images of the CL and associated blood flow were captured and stored for analysis of luteal blood flow area and ratio. Longitudinal B-mode images of the uterine horns were captured just beyond the bifurcation of the uterine horns and stored for analysis of contrast and homogeneity (MaZda v4.6; Technical University of Lodz, Institute of Electronics, Poland). A total of three images were captured for each structure of interest. Serum concentrations of P4 were determined from blood samples collected at each ultrasound examination. After pregnancy diagnosis by ultrasound, heifers were retrospectively allocated as being pregnant (embryonic heartbeat on Day 28; n = 14) or nonpregnant (interestrous interval 18-21 days; n = 8) and their data were compared with noninseminated cyclic heifers (n = 10). Corpus luteum tissue area did not appear to change between pregnant, nonpregnant, or cyclic control groups between Days 7 and 18 (P > 0.05). No significant differences in CL echotexture characteristics were found between groups at any time point. There were no significant differences between pregnant, nonpregnant, and cyclic control groups for CL blood flow area (P > 0.05). However, CL blood flow ratio decreased significantly (P < 0.05) in both inseminated nonpregnant and cyclic heifers between Days 14 and 18, whereas it remained unchanged in pregnant heifers (P > 0.05). Uterine homogeneity was not significantly different between groups at any time point (P > 0.05). However, uterine contrast was significantly greater (P < 0.05) in pregnant compared with cyclic control heifers on Days 16 and 18. Concentrations of P4 were lower (P < 0.05) in nonpregnant and control heifers than in pregnant heifers from Days 16 to 18. In conclusion, there were differences between nonpregnant and cyclic heifers compared with pregnant heifers in P4 concentrations from Day 16. On Day 18, the CL and uterine characteristics were different between the nonpregnant and pregnant heifers. Ultrasound measures of CL blood flow and uterine echotexture may be useful to establish pregnancy status. Further investigation is required to identify if pregnancy diagnosis can be made on Day 18 or at a later day postpartum.

Transforming growth factor beta 1 stimulates type II collagen expression in cultured periosteum-derived cells.

Chondrogenesis can occur during a bone repair process, which is related to several growth factors. Transforming growth factor beta 1 (TGF-beta 1) downregulates the expression of type II collagen by chondrocytes in vitro, but injection of TGF-beta 1 into the periosteum in vivo increases type II collagen mRNA levels and initiates chondrogenesis. We examined the effect of TGF-beta 1 on collagen gene expression in a bovine periosteum-derived cell culture system to evaluate its direct effect on the periosteum. Cultured cells expressed alkaline phosphatase and collagen pro alpha 1(I) and pro alpha 1(II) mRNAs. A low level of type II collagen synthesis was demonstrated by immunoprecipitation. TGF-beta 1 had no effect on periosteal cell proliferation. Expression of collagen pro alpha 1(I) mRNA did not change with TGF-beta 1 treatment, but alkaline phosphatase mRNA showed a dose-dependent decrease. Expression of collagen pro alpha 1(II) mRNA was stimulated 2.7-fold by TGF-beta 1. TGF-beta 1 also caused a 2.6-fold increase in type II collagen synthesis by immunoprecipitation. These findings indicate that TGF-beta 1 is an enhancer of the expression of the chondrocyte phenotype of the periosteal cells and suggest that TGF-beta 1 is important in initiating and promoting cartilage formation in vivo.