Control of acute myeloid leukemia by a trifunctional NKp46-CD16a-NK cell engager targeting CD123.

CD123, the alpha chain of the IL-3 receptor, is an attractive target for acute myeloid leukemia (AML) treatment. However, cytotoxic antibodies or T cell engagers targeting CD123 had insufficient efficacy or safety in clinical trials. We show that expression of CD64, the high-affinity receptor for human IgG, on AML blasts confers resistance to anti-CD123 antibody-dependent cell cytotoxicity (ADCC) in vitro. We engineer a trifunctional natural killer cell engager (NKCE) that targets CD123 on AML blasts and NKp46 and CD16a on NK cells (CD123-NKCE). CD123-NKCE has potent antitumor activity against primary AML blasts regardless of CD64 expression and induces NK cell activation and cytokine secretion only in the presence of AML cells. Its antitumor activity in a mouse CD123+ tumor model exceeds that of the benchmark ADCC-enhanced antibody. In nonhuman primates, it had prolonged pharmacodynamic effects, depleting CD123+ cells for more than 10 days with no signs of toxicity and very low inflammatory cytokine induction over a large dose range. These results support clinical development of CD123-NKCE.

Nonalcoholic hepatic steatosis in Zucker diabetic rats: spontaneous evolution and effects of metformin and fenofibrate.

No specific treatment for nonalcoholic hepatic fatty liver disease has been defined. We followed the spontaneous evolution of liver steatosis and tested the therapeutic usefulness of metformin and fenofibrate in a model of steatosis, the Zucker diabetic fatty (ZDF) rat. ZDF and control rats were studied at 7, 14, and 21 weeks. After initial study at 7 weeks, ZDF rats received no treatment, metformin or fenofibrate until studies at 14 or 21 weeks. ZDF rats were obese, hypertriglyceridemic, insulin resistant at 7 weeks, type 2 diabetic at 14, diabetic with insulin deficiency at 21. They had steatosis at 7 weeks with increased hepatic expression and activity of lipogenesis. Steatosis was unchanged at 14 and 21 weeks despite lower expression and activity of lipogenesis. Metformin and fenofibrate did not modify energy intake or expenditure or the evolution of diabetes. Both compounds decreased plasma triacylglycerol (TAG) concentrations. Hepatic TAG content was reduced by fenofibrate at 14 and 21 weeks but only at 21 weeks by metformin. Metformin had no significant effects on the expression in liver of genes of fatty acids metabolism. The beneficial effect of fenofibrate occurred despite increased expression of genes involved in the uptake and activation of fatty acids. Acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase I (CPTI) mRNA levels were increased by fenofibrate showing evidence of increased lipid oxidation. To conclude, metformin had only moderate effects on liver steatosis. The effects of fenofibrate was more marked but remained mild.

Diabetic cardiomyopathy: effects of fenofibrate and metformin in an experimental model--the Zucker diabetic rat.

BACKGROUND: Diabetic cardiomyopathy (DCM) contributes to cardiac failure in diabetic patients. It is characterized by excessive lipids accumulation, with increased triacylglycerol (TAG) stores, and fibrosis in left ventricle (LV). The mechanisms responsible are incompletely known and no specific treatment is presently defined. We evaluated the possible usefulness of two molecules promoting lipid oxidation, fenofibrate and metformin, in an experimental model of DCM, the Zucker diabetic rat (ZDF). METHODS: ZDF and controls (C) rats were studied at 7, 14 and 21 weeks. After an initial study at 7 weeks, ZDF rats received no treatment, metformin or fenofibrate until final studies (at 14 or 21 weeks). C rats received no treatment. Each study comprised measurements of metabolic parameters (plasma glucose, TAG, insulin levels) and sampling of heart for histology and measurements of TAG content and relevant mRNA concentration. RESULTS: ZDF rats were insulin-resistant at 7 weeks, type 2 diabetic at 14 weeks and diabetic with insulin deficiency at 21 weeks. Their plasma TAG levels were increased. ZDF rats had at 7 weeks an increased LV TAG content with some fibrosis. LV TAG content increased in untreated ZDF rats at 14 and 21 weeks and was always higher than in C. Fibrosis increased also moderately in untreated ZDF rats. Metformin and fenofibrate decreased plasma TAG concentrations. LV TAG content was decreased by metformin (14 and 21 weeks) and by fenofibrate (14 weeks). Fibrosis was reduced by fenofibrate only and was increased by metformin. Among the mRNA measured, fenofibrate increased Acyl-CoA Oxidase mRNA level, metformin decreased Acyl-CoA Synthase and increased AdipoR1 and pro-inflammatory mRNA levels. CONCLUSION: Fenofibrate had favourable actions on DCM. Metformin had beneficial effect on TAG content but not on fibrosis. PPARalpha agonists could be useful for the prevention and treatment of DCM.

Adiponectin receptors: expression in Zucker diabetic rats and effects of fenofibrate and metformin.

The insulin-sensitizing adipokine, adiponectin, acts through 2 receptors, AdipoR1 and AdipoR2. A decreased expression of these receptors could contribute to insulin resistance and diabetes. We determined if the expression of adiponectin receptors is decreased in an experimental model, the Zucker diabetic rat (ZDF), and if a peroxisome proliferator-activated receptor alpha agonist, fenofibrate, and metformin could increase these expressions. The ZDF and control (L) rats were studied at 7, 14, and 21 weeks. After initial study at 7 weeks, ZDF received no treatment (n = 10), metformin (n = 10), or fenofibrate (n = 10) until final studies at 14 or 21 weeks. The L rats received no treatment. AdipoR1 and R2 expressions were measured in liver, muscle, and white adipose tissue (WAT). As expected, ZDF rats were insulin resistant at 7 weeks, had type 2 diabetes mellitus at 14 weeks, and had diabetes with insulin deficiency at 21 weeks. Compared with L rats, AdipoRs messenger RNA was decreased only in the WAT (P < .05) of 7-week-old ZDF rats, but was unchanged in muscle and increased in liver. Metformin and fenofibrate decreased plasma triacylglycerols (P < .01) as expected. The only effect of fenofibrate on AdipoRs was a moderate increase (P < .01) of both receptors' messenger RNA in liver. Metformin increased AdipoR1 and R2 expression in muscle (P < .01) and AdipoR1 (P < .01) in WAT. These results do not support an important role for decreased AdipoRs expression in the development of insulin resistance and diabetes. Parts of the actions of fenofibrate and of metformin could be mediated by a stimulation of the expression of these receptors in liver and in insulin-sensitive, glucose-utilizing tissues (muscle, WAT), respectively.

Determination of protein replacement rates by deuterated water: validation of underlying assumptions.

H(2)O administration has recently been proposed as a simple and convenient method to measure protein synthesis rates. (2)H(2)O administration results in deuterium labeling of free amino acids such as alanine, and incorporation into proteins of labeled alanine can then be used to measure protein synthesis rates. We examined first whether during (2)H(2)O administration plasma free alanine enrichment is a correct estimate of the enrichment in the tissue amino acid pools used for protein synthesis. We found that, after (2)H(2)O administration, deuterium labeling in plasma free alanine equilibrated rapidly with body water, and stable enrichment values were obtained within 20 min. Importantly, oral administration of (2)H(2)O induced no difference of labeling between portal and peripheral circulation except for the initial 10 min after a loading dose. The kinetics of free alanine labeling were comparable in various tissues (liver, skeletal muscle, heart) and in plasma with identical plateau values. We show next that increased glycolytic rate or absorption of unlabeled amino acids from ingested meals do not modify alanine labeling. Calculated synthesis rates of mixed proteins were much higher (20- to 70-fold) in plasma and liver than in muscle and heart. Last, comparable replacement rates of apoB100-VLDL were obtained in humans by using the kinetics of incorporation into apoB100 of infused labeled leucine or of alanine labeled by (2)H(2)O administration. All of these results support (2)H(2)O as a safe, reliable, useful, and convenient tracer for studies of protein synthesis, including proteins with slow turnover rate.

Contrasting circadian rhythms of blood pressure among inbred rat strains: recognition of dipper and non-dipper patterns.

OBJECTIVE: The non-dipper pattern, i.e. the lack of nocturnal blood pressure (BP) fall, carries a high risk of cardiovascular complications, both in hypertensive and normotensive subjects. Without genetic engineering, experimental demonstration of the non-dipper phenomenon is lacking. The purpose of this study was to assess the haemodynamic and behavioural daily parameters among various strains of rats - spontaneously hypertensive rats (SHR), Wistar-Kyoto (WKY) and Fischer 344 (F344) - in order to characterize their circadian patterns and to detect a non-dipper animal model. METHODS: Changes in BP, heart rate (HR), and spontaneous locomotor activity (SLA) were recorded continuously for 11 days using telemetry in freely moving 10-week-old male SHR, WKY and F344 rats, in standardized laboratory conditions. Variations in haemodynamic and behavioural parameters were assessed in terms of day/night differences and spectral power corresponding with the 24-h period. RESULTS: All rats exhibited clear circadian variations in HR and in SLA, in synchrony with the light cycle. Light/dark differences in BP were significantly lower in F344 compared with those of SHR and WKY. The smaller circadian changes in BP observed in F344 were also demonstrated using spectral analysis: the peak detected at 24-h was reduced in F344 compared with SHR and WKY. CONCLUSION: The inbred F344 strain lacks the typical circadian BP rhythm while oscillations of HR and SLA are maintained, suggesting different regulatory mechanisms. The F344 strain may represent a useful animal model for studying the effects of drugs aimed at restoring the dipper status.

Different vascular responsiveness to angiotensin II in two normotensive rat strains.

Rats of the Fischer 344 (F344) and Wistar Kyoto (WKY) strains are known to present differences in stimuli responses involving the renin-angiotensin system and in cardiovascular responses to an acoustic startle stimulus. Here we compared the vascular reactivity to angiotensin II (ANG II) of these normotensive, inbred rat strains. Blood pressure (BP) and heart rate (HR) were recorded in conscious rats, before and after a neurohumoral blockade obtained by successive administration of chlorisondamine, enalapril, a V1-vasopressinergic receptor antagonist (Manning compound) and atropine methyl nitrate. BP was restored by a constant infusion of noradrenaline. Boluses of ANG II ranging from 0.001 to 1280 ng/kg were injected randomly. Average dose-response curves were established. After neurohumoral blockade, the minimum mean BP (MBP) produced by hydralazine (3 mg/kg, i.v.) and the maximum MBP produced by noradrenaline (60 microg/mL and 800 microL/min, i.v.) were used to reflect arterial wall structure. The maximal systolic blood pressure (SBP) and pulse pressure (PP) responses to ANG II were higher in F344 compared with WKY (+86 +/- 3 mmHg vs. +71 +/- 3 mmHg, P < 0.01 for SBP, +31 +/- 2 mmHg vs. +18 +/- 1 mmHg, P < 0.001 for PP). After the ANG II type 1 (AT1) receptor blocker valsartan, ANG II had no significant effect on BP. F344 and WKY exhibited the same maximum MBP in response to noradrenaline. However, MBP level following hydralazine was higher in F344 (F344: 48 +/- 2 mmHg vs. WKY: 37 +/- 3 mmHg, P < 0.01). The amplification in F344 of the vasoconstrictive response to ANG II mediated by AT1 receptors is compatible with a high number of AT1 receptors in this strain. In F344, the exaggerated systolic and PP responses to ANG II and the higher MBP level after hydralazine most likely reflect a structural modification of the arterial wall such as hypertrophic remodelling in F344.