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Spherical and rod-shaped gold nanoparticles with surface poly (ethylene glycol) (PEG) chains were characterized for size, shape, charge, poly dispersity and surface plasmon resonance. The nanoparticles were injected intravenously to 6–8 weeks old female nu/nu mice bearing orthotopic ovarian tumors and their biodistribution in vital organs was compared. Gold nanorods were taken up to a lesser extent by the liver, had longer circulation time in the blood, and higher accumulation in the tumors, compared with their spherical counterparts. The cellular uptake of PEGylated gold nanoparticles by a murine macrophage-like cell line as a function of geometry was examined. Compared to nanospheres, PEGylated gold nanorods were taken up to a lesser extent by macrophages. These studies point to the importance of gold nanoparticle geometry and surface properties on transport across biological barriers.

The impact of feeding ractopamine hydrochloride (RAC) on growth performance and responses to handling and transport in heavy BW pigs was evaluated in a study performed as a split-plot design with a 3 × 3 factorial arrangement of treatments: 1) RAC level (0 vs. 5 vs. 7.5 mg/kg of feed) and 2) handling intensity (HI; gentle vs. moderate vs. aggressive); RAC level was the main plot and HI was the subplot. A total of 288 pigs housed in groups of 8 were used to evaluate growth performance over a 28-d RAC feeding period (98.5 ± 4.58 to 131.5 ± 7.45 kg BW). On d 29 of the study, the HI treatment was applied to 216 pigs (6/pen; 2/pen on each HI). This was followed by transportation for 1 h on a livestock trailer at the end of which pigs were subjected to a final handling procedure. Blood samples (to measure acid-base, cortisol, and catecholamine levels) were collected and rectal temperature was measured 2 h before the HI treatment (baseline) and after the final handling procedure (final). Feeding RAC (5 and 7.5 mg/kg) improved (0.01) ADG (9.9 and 9.0% for 5 and 7.5 mg/kg RAC, respectively) and G:F (8.8 and 11.8%, respectively) compared to controls, with no differences (0.05) between the 2 RAC levels. Increasing the intensity of handling decreased (0.001) final blood pH, bicarbonate, and base excess and increased (0.001) final blood lactate and plasma cortisol and norepinephrine levels. Aggressive compared to gentle handling increased (0.05) the incidence of pigs exhibiting open-mouth breathing and skin discoloration after the final handling procedure but had no effect (0.05) on the incidence on nonambulatory, noninjured pigs. There was no effect (0.05) of feeding RAC on final rectal temperature or blood acid-base measurements. Feeding 7.5, but not 5, compared to 0 mg/kg RAC increased (0.05) final plasma epinephrine levels and the incidence of nonambulatory, noninjured pigs. This study confirms the improved growth performance of pigs fed RAC and the negative effects of aggressive handling on physical, metabolic, and physiological responses of pigs. It also suggests that pigs fed 5 compared to 0 mg/kg RAC showed similar responses to transport and handling. However, pigs fed 7.5 mg/kg of RAC had a greater incidence of nonambulatory, noninjured pigs when subjected to the handling/transport model and this warrants further investigation.

Porcine relaxin has been reported to stimulate various human sperm functions. In this paper we report that human recombinant relaxin binds to human sperm with a high affinity (Kd = 6.5 x 10(-10)). The bound 125I-relaxin was not displaced by insulin, or human chorionic gonadotropin, however, it was displaced by unlabeled relaxin. In sperm function studies, recombinant human relaxin stimulated sperm motility, zona-free hamster egg penetration, and the acrosome reaction.

The effects of floor space on the trailer and journey time during transport from the farm to the packing plant on indicators of stress (open-mouth breathing, muscle tremors, and skin discoloration) and on the incidence of transport losses (dead on arrival, nonambulatory, noninjured, and nonambulatory, injured) were evaluated in a study involving 160 loads of market-weight pigs (BW 124.7 ± 4.38 kg) using a split-plot design with a 2 × 6 factorial arrangement of treatments: 1) journey time [main plot; short (1 h) and long (3 h)] and 2) floor space (subplot; 0.396, 0.415, 0.437, 0.462, 0.489, and 0.520 m(2)/pig, which is equivalent to 0.317, 0.332, 0.350, 0.370, 0.391, and 0.416 m(2)/100 kg of BW, respectively). Two consecutively loaded trailers were randomly allotted to journey time treatment. Floor space treatments were compared in the front 3 compartments on the top and bottom decks of the trailer and were created by varying the number of pigs per compartment, which confounds the effect of floor space with group size. Of the 17,652 pigs transported in 954 test compartments, 0.24% died or became nonambulatory. Neither journey time nor floor space had an effect (P0.05) on the incidence of dead and nonambulatory, injured pigs, or on total transport losses. There were interactions (P0.05) between journey time and floor space treatments for the incidences of nonambulatory, noninjured pigs and open-mouth breathing. For 2 of the smallest floor spaces (0.415 and 0.437 m(2)/pig), the incidence of nonambulatory, noninjured pigs was greater on short than on long journeys; for the other 4 floor spaces there was no effect (P0.05) of journey time. The incidence of open-mouth breathing for the 3 smallest floor spaces was greater (P0.05) for short than long journeys, whereas there was no effect (P0.05) of journey time for the 3 greatest floor spaces. The frequency of skin discoloration was greater (P0.001) for pigs transported at the 2 smallest floor spaces compared with the other 4 floor spaces. In summary, short journey time increased the frequency of indicators of stress after unloading at the plant for pigs transported at smaller floor spaces and also increased the incidence of nonambulatory, noninjured pigs at 2 of the 3 smallest floor spaces. However, neither transport floor space nor journey time had an effect on total losses.

The biodistribution profile of a series of linear N-(2-hydroxylpropyl)methacrylamide (HPMA) copolymers was compared with that of branched poly(amido amine) dendrimers containing surface hydroxyl groups (PAMAM-OH) in orthotopic ovarian-tumor-bearing mice. Below an average molecular weight (MW) of 29 kDa, the HPMA copolymers were smaller than the PAMAM-OH dendrimers of comparable molecular weight. In addition to molecular weight, hydrodynamic size and polymer architecture affected the biodistribution of these constructs. Biodistribution studies were performed by dosing mice with (125)iodine-labeled polymers and collecting all major organ systems, carcass, and excreta at defined time points. Radiolabeled polymers were detected in organ systems by measuring gamma emission of the (125)iodine radiolabel. The hyperbranched PAMAM dendrimer, hydroxyl-terminated, generation 5 (G5.0-OH), was retained in the kidney over 1 week, whereas the linear HPMA copolymer of comparable molecular weight was excreted into the urine and did not show persistent renal accumulation. PAMAM dendrimer, hydroxyl-terminated, generation 6.0 (G6.0-OH), was taken up by the liver to a higher extent, whereas the HPMA copolymer of comparable molecular weight was observed to have a plasma exposure three times that of this dendrimer. Tumor accumulation and plasma exposure were correlated with the hydrodynamic sizes of the polymers. PAMAM dendrimer, hydroxyl-terminated, generation 7.0 (G7.0-OH), showed extended plasma circulation, enhanced tumor accumulation, and prolonged retention with the highest tumor/blood ratio for the polymers under study. Head-to-head comparative study of HPMA copolymers and PAMAM dendrimers can guide the rational design and development of carriers based on these systems for the delivery of bioactive and imaging agents.

Blood samples were obtained from miniature swine maintained on 0, 2, or 6 g/kg/24 hr ethanol for 8 months (N= 6 in each group). Samples from drinking pigs were taken after 8 hr of ethanol abstinence and all were coded and sent for “blinded” analysis. A fluorigenic high performance liquid chromatographic assay was used to quantify whole blood-associated acetaldehyde, hemoglobin-associated acetaldehyde, plasma-associated acetaldehyde, platelet-associated acetaldehyde, and lymphocyte-associated acetaldehyde. Detectable levels of acetaldehyde were found in each sample in both drinking and nondrinking pigs. Analysis of whole blood-associated acetaldehyde was most discriminatory in distinguishing nondrinking from drinking pigs (mean 21.4 ± 1.0 μM for nondrinkers vs. 24.6 ± 1.5 so for the group consuming 2 g/kg ethanol, p= 0.001). Measurements of hemoglobin-associated acetaldehyde normalized to protein concentration (250 ± 47 nmoles/g vs. 203 ± 33 SD, p < 0.05 drinking vs. nondrinking pigs) and platelet-associated acetaldehyde (0.46 0.34 vs. 0.15 ± 0.16 nmoles/3 × 10° platelets, p= 0.05 drinking vs. nondrinking pigs) were also useful in discriminating drinking from nondrinking animals. Analysis of plasma-associated acetaldehyde and lymphocyte-associated acetaldehyde were not useful as markers of ethanol consumption. The miniature swine proved to be a useful model for the evaluation of these potential markers of ethanol intake. In this model, increased amounts of acetaldehyde can be found in several blood compartments eight hr after cessation of ethanol. Further work is warranted to define the relative kinetics of each measurement and the potential utility of each as a marker for ethanol consumption in man.

This study demonstrates the selective tumor targeting and the antitumor efficacy of the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound mesochlorin e6 monoethylenediamine (Mce6) and HPMA copolymer-bound Adriamycin (ADR) in combination photodynamic therapy (PDT) and chemotherapy against human ovarian OVCAR-3 carcinoma xenografted in female athynmic mice. The concentrations of Mce6 and ADR in blood and tissues, in free or HPMA copolymer-bound form, were determined by fluorescence and high-performance liquid chromatography fluorescence assays, respectively. Xenograft responses to single and combination therapies were recorded. The peak concentration of HPMA copolymer-Mce6 conjugate in tumor was achieved 18 h after administration. For HPMA copolymer-bound drugs, the concentration ratios of liver and spleen versus muscle were significantly higher than those of free drugs. The HPMA copolymer-bound drugs demonstrated selective targeting and accumulation in the tumor, probably attributed to the enhanced permeability and retention effect. In vivo studies revealed that all tumors in the treatment groups showed significant responses after receiving any of the various types of therapy as compared with controls (P0.001). PDT with HPMA copolymer-Mce6 conjugate (PDTMC) at a dose of 13.4 mg/kg (1.5 mg/kg of Mce6 equivalent) and light doses of 110 J/cm2 at 12 and 18 h, respectively, resulted in significant suppression of the growth of OVCAR-3 tumors. Three courses of chemotherapy using 35 mg/kg (2.2 mg/kg of ADR equivalent) of HPMA copolymer-ADR conjugate (CHEMO) were effective in suppressing the growth of tumors. Single PDTMC plus multiple CHEMO exhibited significantly greater therapeutic efficacy than multiple CHEMO. In the group of mice receiving multiple PDTMC, tumor recurrence became obvious after day 20. However, 10 of 12 tumors exhibited complete responses in the group of mice receiving multiple PDTMC plus multiple CHEMO. The least to most effective treatments were ranked as follows: multiple CHEMOsingle PDTMC plus multiple CHEMOmultiple PDTMCmultiple PDTMC plus multiple CHEMO. The results clearly demonstrate that: (a) HPMA copolymer-bound drugs exhibited selective tumor accumulation contrary to free drugs; (b) PDT using HPMA copolymer-Mce6 conjugate with multiple light irradiations was a better therapy than that with single light irradiation; and (c) combination chemotherapy and photodynamic therapy with HPMA copolymer-ADR and HPMA copolymer-Mce6 conjugates was the most effective regimen.

Two isoforms of prostaglandin G/H synthase, PGS-1 and PGS-2, catalyze the formation of prostaglandins (PG). Nonselective PGS inhibitors, e.g., indomethacin, reduce the number of ovulations and PG levels in many animal models. This study evaluated the effects of the selective PGS-2 inhibitor NS-398, compared to indomethacin, on ovulation number and on PG and steroid production both in vivo and in vitro in the rat. NS-398 reduced the synthesis of PGE2 in isolated, LH-stimulated preovulatory follicles incubated in vitro. The inhibition by NS-398 was similar to that of indomethacin. Maximal inhibition was noted from 0.1 microM. Neither progesterone nor cAMP production was affected by NS-398 or indomethacin. The effect of in vivo administration of NS-398 (1, 3, or 10 mg/kg BW, s. c.) to proestrous rats 1 h after the injection of an ovulatory dose of hCG was monitored in follicles extirpated 10 h after hCG. These follicles were incubated in vitro, and NS-398 dose-dependently reduced PGE2 production. The synthesis of cAMP and progesterone was not altered. In separate experiments, the same doses of NS-398 were injected to determine their effect on ovulation in vivo. The number of ovulations was decreased by the highest dose of NS-398. In the in vitro ovarian perfusion model, NS-398 (10 microM) reduced the number of ovulations initiated by LH and isobutylmethylxanthine. Lower doses of NS-398 (0.1 and 1 microM) were less effective. The production of prostanoids (PGE2, PGF2alpha, and 6-keto-PGF1alpha) was reduced in a dose-dependent manner by NS-398. The secretion of steroids was not affected. This study demonstrates that selective inhibition of PGS-2 by NS-398 reduces LH/hCG-stimulated production of prostanoids and the number of ovulations both in vivo and in vitro. These results provide direct evidence to strengthen the role of the inducible, granulosa cell-expressed PGS-2 as one of the key regulators in the ovulatory process and also document that the elevated and perhaps sustained levels of PG are obligatory for ovulation.

Eicosanoids, the active metabolites of arachidonic acid, are grouped into cyclooxygenase products (prostaglandins [PGs] and thromboxanes) and lipoxygenase products (leukotrienes [LTs] and lipoxins). Numerous studies suggest a role for the lipoxygenase system in ovulation. The aim of this study was to further characterize the effects of lipoxygenase inhibition and the interactions of the lipoxygenase and cyclooxygenase systems in the rat ovary during ovulation. The lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), was administered in vivo and in the isolated perfused rat ovary to determine its effect on ovulation rate. The in vivo study confirmed the inhibitory effect of NDGA, and in the perfusion experiments, NDGA caused a dose-dependent reduction in the ovulation rate. To further define the interaction between the lipoxygenase and cyclooxygenase systems, a second set of perfusions was performed with NDGA (10 microM) and the combination of NDGA (10 microM) plus a nonselective cyclooxygenase inhibitor, indomethacin (10 microM). NDGA significantly reduced the number of ovulations compared to that in controls. The ovulation rate for the combination of NDGA+indomethacin was also significantly lower than in controls but not different from that in the NDGA-treated group. Steroidogenesis was decreased only in the NDGA+indomethacin perfusions. Ovarian tissue PGE2 and PGF2alpha levels in the NDGA-treated ovaries were significantly suppressed compared to those in controls. Almost a complete block of PGE2 and PGF2alpha was seen in the NDGA+indomethacin group. LTB4 levels in the 10-h-perfused ovarian tissues were significantly decreased by NDGA compared to those in control tissues. Furthermore, LTB4 (3 microg added twice) completely reversed the inhibitory effect of 0.1 microM NDGA on ovulation rate and partially reversed the effect of 10 microM NDGA in the perfusion model. These results demonstrate that the products of the lipoxygenase pathway, especially LTB4, are important in the process of ovulation in this cyclically ovulating species. The interconnected lipoxygenase and cyclooxygenase pathways may optimize ovulation and facilitate steroidogenesis.

This study characterizes the efficacy and toxicity of: (a) free Adriamycin and N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-Adriamycin conjugate (P-A); (b) free and HPMA copolymer-meso-chlorin e6 monoethylene diamine disodium salt (Mce6) conjugate (P-C) and light-induced photodynamic therapy; and (c) combinations of the HPMA copolymer conjugates (P-A and P-C) in the destruction of human epithelial ovarian carcinoma heterotransplanted in the nude mouse (OVCAR-3). Eight-week-old female nu/nu mice were injected in both flanks with 0.04-0.05 cm3 OVCAR-3 solid tumor dispersed in media. When bilateral tumors reached a minimum volume of 0.18 cm3 (one axis, 2.0-mm minimum) and demonstrated consistent growth, the experiments were initiated. Drugs were given i.v. unless otherwise noted. Tumor-bearing mice were allocated to the following protocols: (a) Adriamycin at 1 mg/kg, P-A at 30 mg/kg (2.2 mg/kg Adriamycin equivalent), and controls (n = 6 each); (b) Mce6 and light (2 h after administration: 650 nm light for 15 min to deliver 220 J/cm2) at 1.25, 2.5, 5, and 10 mg/kg (n = 6 each), 2.5 mg/kg i.p. (n = 4), and controls (n = 6); (c) P-C at 12.5, 25, and 75 mg/kg (1.5, 2.9, and 8.7 mg/kg Mce6 equivalent, respectively with light (18 h after administration; 650 nm light for 15 min to deliver 220 J/cm2), P-C at 25 mg/kg (2.9 mg/kg Mce6 equivalent) with no light administration, and controls (n = 7 each); and (d) a combination of P-A (30 mg/kg, 2.2 mg/kg adriamycin equivalent) and P-C (12.5 and 75 mg/kg, 1.5 mg/kg and 8.7 mg/kg Mce6 equivalent, respectively) with and without light (n = 7 each; 18 h after administration; 650 nm light for 15 min to deliver 220 J/cm2) and controls (n = 12). Tumor volumes and animals weights were assessed for significant differences from the treated and controls groups by Student's t test. Adriamycin (1 mg/kg) and P-A (30 mg/kg. 2.2 mg/kg Adriamycin equivalent) caused less than a 10% weight loss, and treated tumor volumes (day 10-32) were significantly less than those of controls (all P0.045). Mce6 (2.5-10 mg/kg i.v.), caused tumor regression in 80% of tumors and a shock syndrome in 17-83%. i.p. dosing (2.5 mg/kg) was uniformly fatal. Mce6 at 1.25 mg/kg did not show reproducible efficacy. P-C with light (25 and 75 mg/kg, 2.9 and 8.7 mg/kg Mce6 equivalent, respectively) demonstrated significant tumor destruction (P0.003) but not complete ablation. The combinations of P-A (30 mg/kg, 2.2 mg/kg Adriamycin equivalent) plus P-C (12.5 and 75 mg/kg; 1.5 mg/kg and 8.7 mg/kg of Mce6 equivalent, respectively) with light resulted in tumor volumes that were significantly less than control tumor volumes and the tumor volumes of mice receiving either P-A (30 mg/kg, 2.2 mg/kg Adriamycin equivalent) or P-C with light (12.5 or 75 mg/kg. 1.5 or 8.7 mg/kg Mce6 equivalent, respectively) alone (all P0.02). P-C (75 mg/kg, 8.7 mg/kg Mce6 equivalent) added to P-A (30 mg/kg, 2.2 mg/kg Adriamycin equivalent) resulted in complete tumor ablation. Free Mce6 demonstrates a narrow margin of safety, which is extended by incorporation into HPMA copolymers. P-A demonstrates safety and efficacy in vivo. The combined chemotherapy and photodynamic therapy of P-A (30 mg/kg, 2.2 mg/kg Adriamycin equivalent) with P-C and light (12.5 and 75 mg/kg 1.5 and 8.7 mg/kg Mce6 equivalent, respectively) was nontoxic and allowed us to attain a significant improvement in tumor cures than those obtained by P-A or P-C with light alone.

Tap-1 and Tap-2 genes code for a heterodimeric peptide transporter required for the normal maturation and surface expression of class I molecules. Polymorphic variants of these MHC encoded genes occur in rats and humans. After failing to amplify a 3' polymerase chain reaction (PCR) product from thymic and splenic cDNA of the nonobese nondiabetic (NON) strain, we considered it possible that Tap-1 polymorphism was present, since cDNA from CBA/J, C57BL/6, BALB/c, and NOD (nonobese diabetic) mice all yielded Tap-1 3' products. Overlapping PCR fragments spanning the highly conserved ATP-binding cassette (ABC) were generated for purposes of restriction endonuclease analysis, studies of IFN-gamma regulation, and sequencing. To avoid amplifying other members of the transporter family, we used a gel-purified 1670-bp Tap-1 PCR "long product" as template for nested PCR. Sequencing revealed three polymorphic alleles. The most divergent was for the NON strain and involved two non-conserved amino acid substitutions (Arg--Cys397 and Leu--Arg491) and three silent mutations. NON mice show an abnormal pattern of class I (Kb) expression and a sizeable reduction in the percentage of CD8+ cells in the blood and thymus. In F2 segregants, the low CD8 phenotype mapped to the MHC. Tap-1 genes of NON and C57BL/6 mice were equally sensitive to up-regulation by IFN-gamma. We conclude that the mouse Tap-1 transporter gene, like the Tap-2 of the rat and the Tap-1 and Tap-2 of the human, is polymorphic. The extensive variation and specific codon changes of Tap-1 in the NON mouse raise the possibility that this gene is the MHC locus responsible for altering the intrathymic development of CD8+ T cells.

Concanavalin A (Con A) administered in vivo inhibited the development of insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic mice. By experimental day 310 only one of 10 Con A-treated animals had developed IDDM compared to six of 11 cohort controls receiving buffered saline and 65% of colony controls. Treatment consisted of an initial 70-day course of Con A at a dose of 10 micrograms/g b. wt., i.p. in sterile saline every other day. After two deaths occurred in the Con A group, treatment was stopped at day 70. The remaining animals were given two 1-week booster doses beginning on experimental days 118 and 189. The single animal in the Con A group that eventually developed IDDM at day 293 had not received an injection for 107 days. Histological examination revealed peri-islet lymphocytosis but no islet infiltration in the Con A group. Surviving control animals showed both peri-islet and islet infiltrates. One-week Con A treatment significantly suppressed in vitro responses of spleen cells to Con A and allogeneic lymphocytes. The treatment increased the frequency of "blast-sized" cells in vivo and decreased the CD4+/CD8+ ratio among resting splenocytes. It is concluded that polyclonal T cell activation by Con A provides protection against autoimmune diabetes in nonobese diabetic mice concomitant with phenotypic and morphologic lymphocyte changes.

We studied the tracking of 51chromium-labeled (resting) and Con A-induced (activated) tritiated thymidine-labeled syngeneic lymphocytes to adrenal, blood, brain, heart, liver, lymph nodes, pancreas, spleen, testis/ovary, thymus and thyroid in prediabetic, nonobese diabetic (NOD) mice and in age- and sex-matched C57BL/6 mice. 51Chromium-labeled cells showed no significant difference between strains in tracking to any tissue except lymph nodes (decreased in NOD vs. C57, P less than .05). Con A incubation resulted in no difference between strains in lymphocyte tracking to lymph nodes, but NOD mice had increased pancreatic tracking with Con A-incubated cells compared to C57 mice (P less than .05). Female NOD mice had reduced thymic tracking (P = .001) compared to C57 controls. Positive selection experiments showed the Con A-responsive cell to be a T cell. Both Lyt2+ (CD8+) and L3T4+ (CD4+) enriched T cell populations showed a labeling response to Con A. After 48 h of Con A incubation, the L3T4+/Lyt2+ ratio increased in splenocytes from NOD mice (P less than .05), whereas it decreased in C57 controls (P less than .01). Over the course of 6 days in culture, NOD splenocytes exposed to Con A characteristically exhibited a delayed expansion of the Lyt2+ population. We conclude that Con A incubation of NOD splenocytes results in T cells that, when reinfused, avoid the thymus and track preferentially to the pancreas. Con A immunomodulation as potential treatment for autoimmune disease warrants further study in this murine model.

The effects of 3 weeks of daily subcutaneous injections of a serotonin agonist (fluoxetine, 20 mg/kg) or agent having antiglucocorticoid properties (RU-486, 20 mg/kg) on food intake and body growth were examined in C57BL/6 ob/ob and lean mice. Fluoxetine injections during ad libitum feeding led to depressed food intake and body weight in both obese and lean mice over the initial 10 days of treatment with full recovery of both intake and weight by the end of the study. Fluoxetine treatment of mice restricted to 3.2 g of laboratory diet per day caused small but persistent depressions of body weight in both phenotypes. RU-486 did not affect food intake weight or weight gain in either phenotype or feeding condition and neither drug affected carcass composition. Insulin levels were also unaffected by treatment but final corticosterone concentrations were consistently higher in fluoxetine-treated mice and lower in RU-486-treated mice. Although transient benefits on feeding and weight gain were observed with fluoxetine administration, the present results show that neither fluoxetine nor RU-486 administered parenterally provides significant long-term improvement in the metabolic, growth or behavioral disturbances that characterize ob/ob mice.

Human ovarian epithelial carcinoma (BG-1) was heterotransplanted in female nude athymic mice and growth was evaluated in intact, surgically castrated, and gonadotropin-releasing hormone (GnRH) agonist (Lupron-SR)-treated mice. Tumor volume was represented as percent of tumor volume on day 0 and measured every other day from the administration of drug and/or the attainment of a minimum tumor volume of 0.5 cm3 on each side of the animal. Tumors in surgically castrated mice had significantly accelerated growth compared with control tumors grown in intact mice (1810 +/- 247.2 versus 1253.6 +/- 44.6%, respectively; P less than .05). Treatment with GnRH agonist significantly reduced tumor growth in intact mice at days 16, 18, and 20 compared with normal control tumors and placebo-treated tumors in intact mice (P less than .02). Gonadotropin levels in pooled serum of mice were reduced from normal levels with GnRH agonist treatment (control: mLH 16.9 +/- 2.2 ng/mL, mFSH 6.6 +/- 0.3 ng/mL; GnRH agonist-treated: mLH 9.3 +/- 1.8 ng/mL, mFSH 4.9 +/- 0.7 ng/mL; surgically castrated: mLH 32.2 +/- 2.7 ng/mL, mFSH 19.4 +/- 0.5 ng/mL). This human tumor model appears responsive to gonadotropins as evidenced by the ability of a GnRH agonist to inhibit growth of BG-1. These results suggest that GnRH agonist therapy may be a useful adjuvant in the treatment of human ovarian epithelial carcinoma.

Thirty C57BI mice were randomized into two groups. Group 1 served as controls while Group 2 was given 10% V/V ethanol with the drinking water. Whole blood- associated acetaldehyde (WBAA) was measured on capillary blood samples using a fluorigenic high performance chromatographic assay. WBAA peaked at Day 2. A stable mean plateau of 263 +/- 71 SD with a range of 160-400 nmoles/g hemoglobin WBAA was found in the group consuming ethanol compared with 122 +/- 17 SD and a range of 88-150 nmoles/g hemoglobin for controls (p less than 0.001). When ethanol was discontinued, levels of WBAA declined and became similar to those of controls by 9 days following cessation of ethanol. The quantitative difference between ethanol-consuming and control animals and also the rapid rise of whole blood-associated acetaldehyde and the relatively slow decline following cessation of ethanol intake indicate that such a test might be a useful monitor of drinking behavior.

Subcutaneous methadone administration (2.5 mg/kg/day) to female mice beginning 2 weeks prior to conception and continuing through weaning resulted in a significant increase in the number of circulating platelets (1650 × 103 vs 1150 × 103/mm3), an increase in marrow megakaryocytes per high power field (12.22 vs 9.59) and an increase in the percentage of young megakaryocytes (34.5 vs 28.4) in the offspring but not in the mothers. The effect was maximal 14 days after birth and diminished by 28 days. Injection of 5 mg/kg/day resulted in similar findings but the changes persisted for 35 days. These findings are consistent with those noted in the infants of human polydrug abusers and suggest that methadone plays a role in similar abnormalities in humans.

Ocular lesions secondary to sodium cyanate administration in the beagle are described. Fifteen dogs were given sodium cyanate at doses of 30 to 170 mg/kg/day, 5 days per week. Fourteen developed cataracts which were generally posterior and subcapsular. Five animals developed corneal lesions. Control beagles and cyanate-treated rats and monkeys did not develop lesions. The one drug-treated beagle without ocular lesions received a maximum dose of 30 mg/kg/day of sodium cyanate for 19 months and achieved a mean carbamylatin of 0.39 residue of cyanate per mol of hemoglobin. Animals developing cataracts received a maximum dose of 119+/-23 mg/kg/day for 28+/-10 months and achieved a mean carbamylation of 0.69+/-0.13 residue/mol. Animals developing corneal lesions received a maximum dose of 113+/-22 mg/kg/day for 36+/-7 months and achieved a mean carbamylation of 0.78+/-0.09 residue/mol. Younger animals appeared more susceptible to the cataractogenic effects of the drug than older ones. Lens intracellular sodium was increased by 100% in the cataractous lenses while potassium was significantly decreased in the lenses with the greatest opacity. Extracellular space was increased in the cataractous lenses by 61%. Reduced glutathione levels were decreased by 34% and lactate leakage into the medium was increased by 12%. Rubidium-86 uptake was increased by 26% in the cataractous lenses, probably reflecting an inadequate compensatory response to increased passive fluxes of cations resulting from a structural or enzymatic lesion.

The viscosity of cystic fibrosis fluids leads to clinical difficulties in adequate clearance of the respiratory and gastrointestinal tracts and in delivery of gases and nutrients to their respective sites. Various sulfhydryl agents are effective in reducing in vitro sputum viscosity. Present therapy for cystic fibrosis involves the aerosolization of N-acetylcysteine, presenting problems of toxicity and inadequate delivery to the small airways. We have been assessing various prodrugs which would liberate the free thiol in vivo and which would be orally absorbed. The free thiol of WR 2721, at a concentration of 1 mM, reduces sputum viscosity by 70% in 15 min. WR 2721 is hydrolyzed to the free thiol chemically at an acid pH and enzymatically by homogenates of the liver and gut. It is well absorbed (23%) after oral administration to rabbits. Clinical studies indicate that orally administered WR 2721 is without toxicity and reduces sputum viscosity in cystic fibrosis patients in 30 to 60 min after administration.

In an attempt to understand the variability of the hematologic response to oral sodium cyanate, iron metabolism was studied in a group of 39 patients with sickel cell disease. Eleven of the 39 patients were found to have no stainable iron in the marrow despite the fact that patients with sickle cell disease are generally considered to have hemosiderosis. The mean per cent saturation and total iron-binding capacity were in the low-normal range in sickle cell patients whether or not stainable iron was present in the bone marrow aspirate. Serum ferritin concentrations, on the other hand, were found to be high in both groups (greater than 500 mu g/liter) when compared to controls (60 mu g/liter). The high serum ferritin levels denoted significant total-body iron deposition which may be unavailable for normal metabolic processes. One patient with no stainable iron in the bone marrow aspirate did respond to iron therapy alone with an increase in hemoglobin concentration. Serum ceruloplasmin levels were also found to be high in sickle cell disease patients. The ability to respond to oral cyanate therapy was correlated with the amount of stainable iron in the bone marrow aspirate. These studies emphasize the necessity of a reevaluation of iron metabolism in the pathophysiology and treatment of sickle cell disease.

Earlier studies have shown that WR 2721 [H2N-(CH2)3-NH(CH2)2SPO3H2] is converted to its free thiol form, N-2-mercaptoethyl-1,3-diaminopropane (MDP), at the acidic pH of the stomach. MDP is a radioprotective compound and a mucolytic agent capable of decreasing sputum viscosity in the lungs of patients with cystic fibrosis. Conversion of WR 2721 and MDP to the corresponding sulfonic acid (MDP-SO3H) permits quantitative determination of these compounds in physiological fluids by use of an automatic amino acid analyzer. After oral administration of WR 2721 to human patients and rabbits it is converted to MDP and the free thiol form of the drug associates with plasma proteins by mixed disulfide linkage. The plasma proteins serve as a depot and reservoir of MDP for potential exchange at the tissues. When incubated with whole sputum or with purified mucin solutions in vitro, MDP decreased the viscosity of these solutions by reduction of the accessible disulfide bonds of the mucin molecule and was subsequently found in mixed disulfide association with the mucin molecule. The association of MDP with proteins via mixed disulfide linkage has important implications for the development of optimal dose regimens for administration of WR 2721 to patients.

Among female nonobese diabetic mice, ketotic insulin-dependent diabetes mellitus (IDDM) develops spontaneously in 80% between 12 and 26 weeks of age. This condition resembles human type I diabetes. IDDM developed in 0 of 11 (0%) mice prophylactically treated with 10 mg of cyclosporine A (CyA) per kg s.c. every 4th day from 8 to 26 weeks of age; 8 of 10 (80%) of sex- and age-matched controls developed IDDM; 2 of 8 (25%) followed up to 5 months beyond the time of drug administration developed IDDM. The distribution of specific radioactivity ([3H]CyA) was used to calculate the concentrations of CyA in serum, blood cells and various organs. Serum values of CyA produced by radioimmunoassay were higher than those estimated by the [3H]CyA method. Pancreata of CyA-treated mice were histologically normal. Pancreata of control mice showed lymphocytic infiltration of the islets of Langerhans. Neither hepatotoxicity nor nephrotoxicity assessed by biochemical and histological data was detectable in CyA-treated mice. The insulin secretory capacity of trypan blue viable functional pancreatic islets isolated post-treatment; was significantly lower in controls than in CyA-treated mice; islet content of insulin was not statistically different between controls and CyA-treated mice. We conclude that low nontoxic doses of CyA abrogate completely the development of diabetes in the female nonobese diabetic mouse and abolish lymphocytic infiltration of the islets of Langerhans against which there is autoimmunity. The effect of CyA persists well past the duration of therapy.

Sixty years have now passed since that brief 18-month span of time that brought insulin from the realm of hypothesis to the reality of treatment for human diabetes. There were rumblings around the turn of the century that the "discovery" of insulin was inevitable, although there is no doubt that the tenacity, energy, and observations of Drs. Banting and Best and their colleagues in Toronto ushered in the "insulin era." The euphoria that followed has been well-described by a number of observers. Lives could be saved. In the ensuing 60 years, however, an increasing prevalence of morbidity concomitant with an increasing prevalence of morbidity been observed, as well as increasing insight into the contribution of hyperglycemia to diabetic "sequelae." C. H. Best himself did not let the miracle and wonder of insulin treatment obscure the necessity of viewing the hormone as a drug, and wrote in 1974: "We hope for and expect more physiological methods of giving insulin." The symposium on the following pages was designed to explore the optimal use of insulin as a pharmacologic agent. To place the present and future in perspective and also celebrate 60 years of insulin use, a retrospective look is included (Table 1). The 19th century generated the intellectual ferment that culminated in the insulin era. Claude Bernard isolated glycogen and contributed greatly to our knowledge of carbohydrate metabolism. Both he and Schiff explored the potential role of the pancreas by ligating and sclerosing the pancreatic ducts with oil or paraffin.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of rhodotorulic acid (RA) as an iron-chelating drug was suggested by experiments in hypertransfused rats in which urinary and fecal iron excretion were significantly enhanced in response to RA. The toxicity of the drug appears to be minimal at a parenteral dose less than 250 mg/kg. An increased excretion of zinc was the only notable side effect of the drug at the doses used. When administered i.v. to humans, RA was only 16% more effective than desferrioxamine (DF). Pharmacokinetic studies showed that RA persisted in the bloodstream of dogs 6 times longer than desferrioxamine after an intravenous injection. Accordingly RA was evaluated as a potential repository drug. While animal experiments were encouraging, human subjects experienced a painful local reaction to RA administered either i.m. or s.c. as a suspension in physiological saline. Accordingly it appears that RA is best looked at as a second line drug, unless a means can be found to obviate local inflammatory reactions.

The recent suggestion that the administration of aspirin might be useful in the treatment of sickle-cell anemia has been further studied and found to be without basis. After incubation with aspirin, sickle-cell erythrocytes are not inhibited from sickling after deoxygenation. In addition, although aspirin does transfer the acetyl group to hemoglobin both in vitro and in vivo , in our experiments the reaction does not result in any alteration in the oxygen equilibrium of either intact erythrocytes or hemoglobin in solution. Furthermore, patients maintained on long-term aspirin administration also showed no shift in the oxygen affinity of their blood.

The rat ovarian perfusion model with bursa removed and intact was used to further characterize the effects of interleukin-1 beta (IL-1 beta) and the natural IL-1 receptor anatagonist (IRAP) on ovulation, steroidogenesis, and prostaglandin production. Twenty-six- to 27-day-old female Sprague-Dawley rats were injected sc with 25 IU PMSG, and 48 h later, the right ovary was dissected (with bursa removed and intact for various experiments) and placed in the perfusion chamber. Ovaries were exposed to various doses of IL-1 beta alone, IL-1 beta with LH, and IL-1 beta with LH and isobutylmethylxanthine (IBMX). The natural IL-1 receptor antagonist was also added to the chambers with LH and IBMX. IL-1 beta at 0.8 (n = 3) and 8.0 (n = 4) nM did not induce LH-independent ovulation in PMSG-stimulated ovaries with bursa removed. In bursa-intact perfusions (n = 3), one ovulation was produced in each compared to control ovaries with bursa intact (n = 3) given an ovulatory trigger of LH alone [2.3 +/- 0.6 (+/- SD) ovulations; P < 0.02]. IL-1 beta enhanced, in a dose- and gonadotropin-dependent fashion, the production of prostaglandin E2 (PGE2) in PMSG-stimulated ovaries with bursa removed given an ovulatory trigger of LH and IBMX compared to that in controls. PGF2 alpha and 6-keto-PGF1 alpha were also modulated by IL-1 beta. Estradiol and progesterone production were not affected. The natural IRAP inhibited ovulation (7.8 +/- 3.9 ovulations vs. 12.4 +/- 1.5; P < 0.04) in PMSG-stimulated ovaries given LH and IBMX as the ovulatory trigger compared to that in controls. This inhibition of ovulation was not associated with reduced steroid or PG levels. IL-1 beta appears to play a potentially significant role in the process of ovulation. The functional importance of the bursa in this model is highlighted in this study. IL-1 beta modulates PG, but not steroid, production. IRAP inhibited ovulation without significantly affecting PG or steroid production.