Identification of imaging biomarkers for the assessment of tumour response to different treatments in a preclinical glioma model.

PURPOSE: Hypoxia-inducible factor 1α (HIF-1α) activity is one of the major players in hypoxia-mediated glioma progression and resistance to therapies, and therefore the focus of this study was the evaluation of HIF-1α modulation in relation to tumour response with the purpose of identifying imaging biomarkers able to document tumour response to treatment in a murine glioma model. METHODS: U251-HRE-mCherry cells expressing Luciferase under the control of a hypoxia responsive element (HRE) and mCherry under the control of a constitutive promoter were used to assess HIF-1α activity and cell survival after treatment, both in vitro and in vivo, by optical, MRI and positron emission tomography imaging. RESULTS: This cell model can be used to monitor HIF-1α activity after treatment with different drugs modulating transduction pathways involved in its regulation. After temozolomide (TMZ) treatment, HIF-1α activity is early reduced, preceding cell cytotoxicity. Optical imaging allowed monitoring of this process in vivo, and carbonic anhydrase IX (CAIX) expression was identified as a translatable non-invasive biomarker with potential clinical significance. A preliminary in vitro evaluation showed that reduction of HIF-1α activity after TMZ treatment was comparable to the effect of an Hsp90 inhibitor, opening the way for further elucidation of its mechanism of action. CONCLUSION: The results of this study suggest that the U251-HRE-mCherry cell model can be used for the monitoring of HIF-1α activity through luciferase and CAIX expression. These cells can become a useful tool for the assessment and improvement of new targeted tracers for potential theranostic procedures.

Validation of an engineered cell model for in vitro and in vivo HIF-1α evaluation by different imaging modalities.

PURPOSE: The aim of this study was to characterize a cell-based model for the molecular study of hypoxia-inducible factor (HIF)-1α activity, in the context of hypoxia, by means of different imaging techniques. PROCEDURES: Engineered U251-HRE glioma cells were used to analyze the molecular mechanisms underlying HIF-1α activity in vitro in relation to luciferase expression. The same cells were orthotopically implanted in mice to evaluate tumor progression and hypoxia induction by bioluminescence imaging, fluorescence imaging, positron emission tomography (PET), and magnetic resonance imaging (MRI). RESULTS: In vitro analyses highlighted the relationship between HIF-1α and luciferase activity in hypoxic conditions and after pharmacological treatments in U251-HRE cells. Through in vivo studies, it was possible to assess hypoxia establishment in relation to tumor growth by optical imaging, PET and MRI. CONCLUSIONS: The findings of this study indicate that the U251-HRE orthotopic murine model can be used to reliably evaluate processes modulating HIF-1α activity, using both molecular and preclinical non-invasive imaging techniques.

Therapeutic antibody targeting of Notch1 in T-acute lymphoblastic leukemia xenografts.

T-acute lymphoblastic leukemia (T-ALL) is characterized by several genetic alterations and poor prognosis in about 20-25% of patients. Notably, about 60% of T-ALL shows increased Notch1 activity, due to activating NOTCH1 mutations or alterations in the FBW7 gene, which confer to the cell a strong growth advantage. Therapeutic targeting of Notch signaling could be clinically relevant, especially for chemotherapy refractory patients. This study investigated the therapeutic efficacy of a novel anti-Notch1 monoclonal antibody by taking advantage of a collection of pediatric T-ALL engrafted systemically in NOD/SCID mice and genetically characterized with respect to NOTCH1/FBW7 mutations. Anti-Notch1 treatment greatly delayed engraftment of T-ALL cells bearing Notch1 mutations, including samples derived from poor responders or relapsed patients. Notably, the therapeutic efficacy of anti-Notch1 therapy was significantly enhanced in combination with dexamethasone. Anti-Notch1 treatment increased T-ALL cell apoptosis, decreased proliferation and caused strong inhibitory effects on Notch-target genes expression along with complex modulations of gene expression profiles involving cell metabolism. Serial transplantation experiments suggested that anti-Notch1 therapy could compromise leukemia-initiating cell functions. These results show therapeutic efficacy of Notch1 blockade for T-ALL, highlight the potential of combination with dexamethasone and identify surrogate biomarkers of the therapeutic response.

A Decision Support System for the assisted diagnosis of brain tumors: a feasibility study for ¹8F-FDG PET preclinical studies.

Decision support systems for the assisted medical diagnosis offer the main feature of giving assessments which are poorly affected from arbitrary clinical reasoning. Aim of this work was to assess the feasibility of a decision support system for the assisted diagnosis of brain cancer, such approach presenting potential for early diagnosis of tumors and for the classification of the degree of the disease progression. For this purpose, a supervised learning algorithm combined with a pattern recognition method was developed and cross-validated in ¹8F-FDG PET studies of a model of a brain tumour implantation.

Daily rhythms in blood and milk lead toxicokinetics following intravenous administration of lead acetate to dairy cows in summer.

The objective of this study was to investigate circadian variations of blood and milk lead toxicokinetics in dairy cows in summer. Twenty lactating Holstein animals were randomly assigned to four treatments corresponding to different hours after onset of light (HALO): 2, 8, 14, and 20. Cows received a single intravenous administration of 2.5 mg/kg lead as lead acetate. Blood and milk samples were taken and analyzed by atomic absorption spectrophotometry. For each toxicokinetic parameter, a one-way analysis of variance (ANOVA) was performed to outline the existence of daily variations. Significant blood differences as a function of HALO were found for the hybrid constant of distribution (alpha), hybrid constant of elimination (beta), elimination half-life (t(1/2)beta), area under the curve (AUC), volume of distribution at steady state (V(ss)) and clearance (Cl(B)) (p<0.05). Half-life of elimination presented two peaks at 2 and 14 HALO. Milk data showed significant differences for maximum concentration and AUC (p<0.05). The ratio AUC(milk)/AUC(blood) was utilized to estimate penetration of lead in milk. It differed significantly throughout the day (p<0.05). Milk data for the significant parameters could be fitted to circadian rhythms. No circadian rhythms were detected in blood parameters or in the ratio AUC(milk)/AUC(blood).

Diet and cooling interactions on physiological responses of grazing dairy cows, milk production and composition.

The objective of this trial was to evaluate the effects of diet and cooling in the holding pen before milking on rectal temperature, respiration rate and milk production and composition. Fifty-eight lactating Holstein cows were used in a factorial split-plot design, at Rafaela Experimental Station from 12 January to 3 March 2003. The treatments were combinations of two diets: control (CD) and balanced (BD) with two levels of cooling before milking: none (NSF) and a sprinkler and fans (SF). Forage:concentrate ratios for CD and BD were 81:19 and 68:32, respectively. Cows were milked twice daily. Milk production was recorded daily, and milk composition (fat, protein, lactose and urea) was analysed twice a week. The physiological data were recorded once a week, before the cattle entered the holding pen and after milking, in the afternoon. Average maximum weekly temperature humidity index was 75.4 and ranged from 61.4 to 83. There were highly significant effects of cooling on physiological responses. Milk production was affected by diet and cooling, with no interaction; the highest and lowest production of milk was 22.42 and 20.07 l/cow per day, for BD+SF and CD+NSF, respectively. Protein was affected by diet, and was higher for BD (3.17 vs. 3.08%). There were interaction effects on milk fat at the 8% level, the highest concentration being 3.65% for BD+NFS. It was concluded that under grazing conditions, cooling by sprinkler and fans before milking improves the comfort of dairy cows, and that the effects on milk production and composition are enhanced when diets are specially formulated for heat-stress periods.

Hydrogenated fish fat for grazing dairy cows in summer.

Thirty-two multiparous Holando argentino cows in mid lactation were randomly assigned to two treatments: control or HFF (hydrogenated fish fat) at Rafaela, 31 degrees 11' South, during summer 1997/1998, to evaluate the effect of using HFF as a supplement under grazing conditions. Animals in both treatments grazed an alfalfa pasture, and were confined from 1000 hours to 1700 hours daily in a shaded pen where water was provided ad libitum. During each milking, animals in the control group received 3.73 kg dry matter per cow each day (DM cow(-1) day(-1)) concentrate (15% crude protein; 8.69 MJ energy for location/kg DM). Cows in the HFF group received 3.25 kg DM cow(-1) day(-1) concentrate, plus 0.200 kg DM cow(-1) day(-1) HFF. Both diets presented similar energy, protein and neutral detergent fibre contents. The trial was performed during a strong "El Niño" event, which resulted in a total rainfall of 396.3 mm (80% higher than normal). The mean temperature was 23.7 (SD 3.2) degrees C and the mean temperature humidity index was 72.9 (SD 4.96). Production data were analysed using a completely randomised design with analysis of covariance. Supplementation with HFF produced an increase in daily milk production (26.4 (SD 2.46) l/cow compared to 23.9 (SD 2.68) l/cow for the controls; P<0.05). Milk fat production was higher for HFF (P<0.05): 941 (SD 96) g cow(-1) day(-1) as compared to controls, which yielded 846 (SD 95) g cow(-1) day(-1). Milk protein yields also differed significantly (P<0.05), the respective values for HFF and controls being 795 (SD 72) g cow(-1) day(-1) and 715 (SD 83) g cow(-1) day(-1). It was concluded that hydrogenated fish fat could be a good ingredient to sustain high yields and elevated maintenance requirements in a grazing system during hot conditions.

Chronobiological variations of indomethacin pharmacokinetic parameters in sheep.

A two-phase study to investigate the influence of administration time on pharmacokinetics of indomethacin in sheep was performed. In phase I, 12 animals were allocated to four groups, each corresponding to a different time: 08:00, 14:00, 20:00, 02:00 h. Sheep received an intravenous administration of 1 mg/kg indomethacin. In phase II, each group was administered indomethacin with a 12-h difference compared to Phase I. The trial was performed in autumn, and animals were subjected to a natural light:dark cycle of 10:14 h. Blood samples were taken and processed by high performance liquid chromatography (HPLC) with ultraviolet detection. For each pharmacokinetic parameter, an analysis of variance was performed to outline the existence of chronobiological variations. Concentration at zero time (C0), hybrid constant for distribution and its half life, hybrid constant for elimination and its half-life, volume of distribution (V(d)), area under the curve (AUC(infinity)) and clearance rate (Cl), presented chronobiological variations (P < 0.05) and were fitted to a cosine equation. The following parameters adjusted to circadian rhythms: C(0) (acrophase: 13.9788 h); AUC(infinity) (acrophase: 13.4377 h); V(d) (acrophase: 0.8245 h) and Cl (acrophase: 1.4965 h). It was concluded that pharmacokinetic parameters of intravenously injected indomethacin in sheep would behave in a different, though predictable, manner according to the animal's biological clock.

Chronotoxicology of florfenicol.

Sixty 3-month-old homozygote male mice were studied for circadian rhythmicity in the toxicity of florfenicol overdose. Animals were kept under a regimen of 12h light, 12h darkness (12:12 LD) with food and water available ad libitum. The LD50 (median lethal) dose was determined in a preliminary experiment and was administered to groups of 10 mice at six different clock times (hours) after light onset (HALO): 0, 4, 8, 12, 16, and 20 HALO. Cosinor analysis verified a statistically significant (P < .04) circadian rhythm in the toxic effect (mortality) of florfenicol. Mortality was greatest when the drug was injected 4h after the commencement of the activity span (16 HALO) and least when injected 4h after the start of the diurnal rest span (4 HALO). Mortality was 2.5 times greater when drug injection was given at 16 HALO than at 4 HALO.

Evaluation of different shades to improve dairy cattle well-being in Argentina.

Two tree shades (TS1 and TS2) and an artificial shade structure (AS) were evaluated using black globe temperatures (BGTs) to assess their effectiveness in reducing heat load. The artificial structure consisted of a black woven polypropylene cloth providing 80% shade, mounted on 2.5-m-high eucalyptus posts. The work was carried out at Rafaela Experimental Station, Argentina, during the summer (January and February) 1994. BGTs and floor temperatures were measured in concrete floor holding pens with and without artificial shade. The results showed no difference between TS1, TS2 and AS, their average BGTs being 30.2 (SD 0.58), 29.0 (SD 0.70) and 30.2 (SD 0.74) degrees C, respectively. BGTs under all three shades were significantly lower (P < 0.01) than the average BGT recorded outside: 35.5 (SD 1.12) degrees C. Average BGTs in holding pens were 32.4 (SD 1.38) and 39.9 (SD 1.91) degrees C for shaded and non-shaded areas (P < 0.01). The corresponding average floor temperatures were 27.8 (SD 0.68) degrees C and 47.7 (SD 2.13) degrees C (P < 0.01). To assess the effects of shade on animal well-being, afternoon rectal temperatures (RT) and respiratory rate (respirations per minute, RR) of lactating cows were recorded twice a week. Rectal temperatures were significantly higher for non-shaded cows (P < 0.01), mean RT being 40.1 (SD 0.65) degrees C vs 39.3 (SD 0.42) degrees C for the shaded animals. Corresponding RRs were 78.9 (SD 18.0) and 60.7 (SD 10.6) (P < 0.05). It was concluded that: (1) tree and artificial shades produced similar effects, (2) shading the holding pen with an 80% shading cloth was effective in reducing heat load and floor temperatures, and (3) access to shade in our pasture-based system improved animal well-being.