Evaluation of molecular changes of distal organs after small bowel transplantation.

The ischemia and reperfusion of a jejunal graft during transplantation triggers the stress of endoplasmic reticulum thus inducing the synthesis of pro-inflammatory cytokines. Spreading of these signals stimulate immunological reactions in distal tissues, i.e. lung, liver and spleen. The aim of this study was to detect the molecular changes in liver and spleen induced by transplanted jejunal graft with one or six hours of reperfusion (group Tx1 and Tx6). Analysis of gene expression changes of inflammatory mediators (TNF-alpha, IL-10) and specific chaperones (Gadd153, Grp78) derived from endoplasmic reticulum (ER) was done and compared to control group. The qRT-PCR method was used for amplification of the specific genes. The levels of corresponding proteins were detected by Western blot with immunodetection. Protein TNF-alpha was in liver tissue significantly overexpressed in the experimental group Tx1 by 48 % (p<0.001). In the group Tx6 we found decreased levels of the same protein to the level of controls. However, the protein concentrations of TNF-alpha in spleen showed increased levels in group Tx1 by 31 % (p<0.001) but even higher levels in the group Tx6 by 115 % (p<0.001) in comparing to controls. Our data demonstrated that the spleen is more sensitive to post-transplantation inflammation than liver, with consequent stress of ER potentially inducing apoptosis and failure of basic functions of lymphoid tissue.

Sodium and potassium clearance rhythmicity in diabetic rats with insulin treatment.

The effect of insulin treatment on the daily distribution of the urinary volume and urinary sodium and potassium excreted, as well as their clearance rhythms in rats with streptozotocin (STZ)-induced diabetes was investigated. Normal(C), uncontrolled (D) and controlled insulin diabetic rats (DI), were studied during a light-dark (12 h:12 h) cycle and given food and water ad libitum. The DI rats showed a significant reduction in the urinary sodium and potassium excreted during 24 h with respect to the D rats, though these values were significantly higher than the C ones. A loss of the normal circadian rhythmicity of diuresis and both sodium and potassium clearance was observed in the D rats, together with higher values of M (MESOR) than in the C rats. These rhythms could be reestablished with continuous insulin infusion, their orthophases occurring near the C ones. However the M values of sodium and potassium clearance in DI rats are greater than C, showing higher values than this group during the rest phase. These results in DI rats may suggest that the constant rate infusion of insulin can be responsible for the high values of clearance of both ions at the rest phase and so for the incomplete renal rhythms restoration.

Circadian optimisation of irinotecan and oxaliplatin efficacy in mice with Glasgow osteosarcoma.

The relevance of circadian rhythms in irinotecan and oxaliplatin tolerability was investigated with regard to antitumour activity. Mice bearing Glasgow osteosarcoma (GOS) received single agent irinotecan (50 or 60 mg kg(-1) per day) or oxaliplatin (4 or 5.25 mg kg(-1) per day) at one of six dosing times expressed in hours after light onset (3, 7, 11, 15, 19 or 23 hours after light onset). Irinotecan (50 mg kg(-1) per day) and oxaliplatin (4 or 5.25 mg kg(-1) per day) were given 1 min apart at 7 or 15 hours after light onset, or at their respective times of best tolerability (7 hours after light onset for irinotecan and 15 hours after light onset for oxaliplatin) or worst tolerability (15 hours after light onset for irinotecan and 7 hours after light onset for oxaliplatin). Tumour growth rate was nearly halved and per cent increase in estimated life span (% ILS) was - doubled in the mice receiving irinotecan at 7 hours after light onset as compared to 15 hours after light onset (P<0.05). Results of similar magnitude were obtained with oxaliplatin for both endpoints, yet with 7 hours after light onset corresponding to least efficacy and 15 hours after light onset to best efficacy (P<0.05). Irinotecan addition to oxaliplatin proved therapeutic benefit only if the schedule consisted of irinotecan administration at 7 hours after light onset and oxaliplatin delivery at 15 hours after light onset, i.e. when both drugs were given near their respective "best" circadian times. These would correspond to the middle of the night for irinotecan and the middle of the day for oxaliplatin in humans.

Experimental chronotherapy of mouse mammary adenocarcinoma MA13/C with docetaxel and doxorubicin as single agents and in combination.

The therapeutic index of docetaxel, doxorubicin and their combination may be improved by an adequate selection of the circadian time of administration. The present study constitutes a prerequisite to testing the clinical relevance of chronotherapy in human breast cancer. Three experiments were performed in C3H/HeN mice. Each treatment modality was administered i.v. once a week for 3 weeks at one of six circadian stages, during the light span, when the mice were resting: 3, 7, and 11 h after light onset (HALO), or during darkness, when the mice were active: 15, 19, and 23 HALO. The circadian time dependency of single agent tolerability was investigated in healthy mice using four dose levels for docetaxel (38.8, 23.3, 14, and 8.4 mg/kg/injection) and for doxorubicin (13.8, 8.3, 5 and 3 mg/kg/injection; experiment 1). The circadian time dependency of each single agent efficacy was studied in MA13/C-bearing mice, using two dose levels of docetaxel (38.8 or 23.3 mg/kg/injection) or doxorubicin (8.3 or 5 mg/kg/injection; experiment 2). The toxicity and the efficacy of the simultaneous docetaxel-doxorubicin combination were assessed as a function of dosing time in experiment 3. Two combinations were tested (A, 16.3 mg/kg/injection of docetaxel and 2.5 mg/kg/injection of doxorubicin; and B, 11.6 and 3.5 mg/kg/injection, respectively) at each of the above six circadian times. Mortality, body weight change, and tumor size were recorded for 60-70 days in each experiment. Single agent docetaxel or doxorubicin was significantly best tolerated near the middle of the rest span (7 HALO) and most toxic in the middle of the activity phase (19 HALO). Docetaxel or doxorubicin as a single drug were also most effective at 7 HALO, irrespective of dose. Treatment at 7 HALO produced highest rates of complete tumor inhibition (81% versus 11% at 3 HALO for docetaxel, p from chi2 <0.001, and 69% versus 44% at 11 HALO for doxorubicin, not significant) and highest day 60 survival rate (100% versus 28% at 3 HALO for docetaxel, p from chi2 <0.001 and 89% versus 69% at 15 HALO for doxorubicin, not significant). Docetaxel-doxorubicin combinations were most effective following dosing in the beginning of the rest span or short after the onset of the activity span, with regard to the rates of both complete tumor inhibitions (45% at 3 HALO versus 15% at 19 HALO) and day 70 survival rates (85% and 80% at 3 and 7 HALO respectively, versus 20% at 19 HALO). The efficacy of single agent docetaxel or doxorubicin and that of their combination varied largely as a function of circadian dosing time. Single agent docetaxel at 7 HALO was the best treatment option in this model with regard to both tolerability and efficacy. This timing may correspond to the middle of the night in cancer patients.

Variations and interrelation between vasopressin and plasma osmolality in diabetic rats with insulin treatment.

Although it is well known that plasma osmolality and plasma vasopressin (VP) levels in diabetes mellitus are higher than in non-diabetic conditions (and that these levels return to normality with insulin therapy), there are no existing studies which examine for insulin-dependent diabetes, either the persistence of daily rhythmic variations of VP or the relationship between this variation and daily osmotic oscillations. We have therefore examined nycthaemeral variations in both plasma osmolality and plasma VP in normal (C), uncontrolled (D) and controlled insulin-dependent streptozotocin diabetic rats (DI). The uncontrolled streptozotocin treated rats presented, a loss of VP rhythmicity, together with higher values of VP than in both normal and controlled diabetic rats. The VP rhythm, however, could be restored with insulin treatment. Furthermore, the temporal VP/osmolality ratio in uncontrolled diabetic rats is higher than in normal rats, although this ratio does not show the daily rhythmic pattern that is present in both normal and diabetic rats treated with insulin. This may indicate that the lack of rhythmicity in osmotic regulation is responsible for the absence of a circadian rhythm in VP. As a result, we conclude that in uncontrolled diabetic rats, the higher VP levels and the loss of VP circadian rhythmicity could be due to a higher sensitivity in the osmoregulatory system, together with an absence of circadian variation of this system. This circadian variation could be responsible for the plasma VP rhythmicity in both normal and controlled diabetic rats.

Circadian rhythms of food and water intake and urine excretion in diabetic rats.

The light/dark (L/D) rhythms of food and water intake and urine output were studied in normal and diabetic rats for 7 consecutive days at 4-h intervals. The control rats showed the highest values of these parameters during the dark phase (83.68% food, 68.71% water, and 67.44% urine). The diabetic rats also maintained this nocturnal predominance, although the values were less marked (59.55% food, 55% water, and 56% urine). A circadian rhythm of food (phi = 3.31 h) and water (phi = 3.54 h) intake as well as of the volume of urine excreted (phi = 1.10) was detected in the control animals. The diabetic rats, inspite of presenting polyphagia, maintain the circadian rhythm of food intake, whereas a loss of the normal circadian variation of drinking intake was observed as well as the absence of circadian rhythm in urinary excretion. It was concluded that in streptozotocin-diabetic rats we have observed a loss of the normal patterns of water intake and urinary excretion, perhaps masked by the polydipsia and polyuria, whereas the circadian rhythm of food intake remains.

Circadian rhythms of plasma corticosterone at different times after induction of diabetes. Responses to corticoadrenal stimulation in light and dark phases.

In this study we have tried to determine the effects of streptozotocin-induced (50 mg/kg) diabetes (15 and 30 day duration) on circadian rhythms of plasma corticosterone concentrations and on the responsiveness of the adrenal glands to exogenously administered ACTH at the time of maximum and minimum levels of plasma corticosterone. Rats were kept under controlled lighting 12h light/12h dark (12L/12D) and fed ad libitum. The corticosteroid circadian pattern in control (C) rats is characterized as one in which peak corticosterone concentrations occur at the beginning of the dark phase (activity period), with a decrease over the remainder of the 24h period. Circadian rhythmicity of plasma corticosterone concentration was absent in the diabetic rats 15 days after induction (D15 rats), with higher mean levels than the C. However, in the diabetic rats 30 days after induction (D30 rats) there is a recovery of this rhythm with similar acrophase and amplitude to the C rats. One hour after stimulation by ACTH (5 IU/kg) at the time of maximum and minimum levels of plasma corticosterone, the C rats showed similar plasma corticosterone levels. In the D15 rats, levels of corticosterone in the light phase one hour after ACTH administration were higher than in the dark phase; being lower than C in this phase. The loss of capacity to respond during the dark phase may be due to adrenal blunting in this phase with high levels of plasma corticosterone. In D30 rats, there is a more noticeable loss of capacity for adrenal response in the light than in the dark phase, with values lower than C and D15 rats in both phases. These findings suggest that the duration of diabetes has a significant role in both plasma corticosterone rhythms and adrenal sensitivity to ACTH administration.