Multi-cistronic vector encoding optimized safety switch for adoptive therapy with T-cell receptor-modified T cells.

T-cell receptor (TCR) gene transfer is an attractive strategy to equip T cells with defined antigen-specific TCRs using short-term in vitro procedures to target both hematological malignancies and solid tumors. TCR gene transfer poses different safety issues that might warrant the inclusion of a suicide gene. High affinity TCRs may result in on-target toxicity, and off-target reactivity directed against healthy tissue can be observed due to mixed TCR dimers. Inclusion of a suicide gene as a safety switch may abrogate these unwanted toxicities. Human CD20 has been proposed as a nonimmunogenic suicide gene targeted by widely used clinical-grade anti-CD20 antibodies that can additionally function as a selection marker. However, transduction of T cells with a multi-cistronic vector encoding both TCR and CD20 resulted in poor coexpression. In this study, we demonstrated that codon optimization of TCR and CD20 resulted in profound coexpression of both the preferentially expressed antigen in melanoma (PRAME)-TCR and CD20, allowing selective as well as efficient elimination of these engineered T cells in vitro. These results demonstrate the great potential of codon optimized CD20 to be broadly used in clinical trials as a safety switch.

Low-dose acarbose does not delay digestion of starch but reduces its bioavailability.

AIMS: Slowly digestible starch is associated with beneficial health effects. The glucose-lowering drug acarbose has the potential to retard starch digestion since it inhibits alpha-amylase and alpha-glucosidases. We tested the hypothesis that a low dose of acarbose delays the rate of digestion of rapidly digestible starch without reducing its bioavailability and thereby increasing resistant starch flux into the colon. METHODS: In a crossover study, seven healthy males ingested corn pasta (50.3 g dry weight), naturally enriched with (13)C, with and without 12.5 mg acarbose. Plasma glucose and insulin concentrations, and (13)CO(2) and hydrogen excretion in breath were monitored for 6 h after ingestion of the test meals. Using a primed continuous infusion of D-[6,6-(2)H(2)] glucose, the rate of appearance of starch-derived glucose was estimated, reflecting intestinal glucose absorption. RESULTS: Areas under the 2-h postprandial curves of plasma glucose and insulin concentrations were significantly decreased by acarbose (-58.1 +/- 8.2% and -72.7 +/- 7.4%, respectively). Acarbose reduced the overall 6-h appearance of exogenous glucose (bioavailability) by 22 +/- 7% (mean +/-se) and the 6-h cumulative (13)CO(2) excretion by 30 +/- 6%. CONCLUSIONS: These data show that in healthy volunteers a low dose of 12.5 mg acarbose decreases the appearance of starch-derived glucose substantially. Reduced bioavailability seems to contribute to this decrease to a greater extent than delay of digestion. This implies that the treatment effect of acarbose could in part be ascribed to the metabolic effects of colonic starch fermentation.

Influence of a subsequent meal on the oro-cecal transit time of a solid test meal.

BACKGROUND: Oro-cecal transit time (OCTT) is determined for clinical diagnostics of intestinal complaints and research purposes. Ingestion of a subsequent meal during the test period shortens the OCTT of a liquid test meal (glucose solution), as previously reported. This study was conducted to determine whether the same phenomenon occurs after ingestion of a solid test meal. MATERIALS AND METHODS: The OCTT of a pancake was measured with the lactose-[(13)C]-ureide breath test on two occasions in 28 volunteers. All the volunteers took the same subsequent meal once at 4 h and at 6 h after ingestion of the pancake. RESULTS: In 16 of the 56 tests no increase in breath-(13)CO(2) was observed. No statistically significant difference was found between the OCTTs of the test meal after ingestion of the subsequent meal at 4 h or 6 h (367; 311-405 min and 290; 370-405 min, median quartiles, respectively) (P = 0.14, n = 18). Only a subgroup (n = 4) with a short OCTT in the test with the 4-h subsequent meal (278; 259-296 min) tended to have a longer OCTT in the test with the 6-h subsequent meal (390; 379-401 min; P = 0.059). CONCLUSION: The effect of the ingestion of a subsequent meal on the transit time of a test meal is shown to be dependent on the physical form and/or caloric content of the test meal.

[Two sisters with progressive familial intrahepatic cholestasis]. / Twee zusjes met progressieve familiaire intrahepatische cholestase.

A few months after birth two sisters aged 5 and 9 years had developed cholestasis and abnormal liver functions with symptoms including itching and jaundice. The younger sister also developed rickets and clotting disorders. On clinical, biochemical and immunohistopathological grounds the diagnosis of 'progressive familial intrahepatic cholestasis (PFIC) type 2' was made. Medical treatment was not succesfull in reducing symptoms. An ileocolonic bypass in the younger child was not effective. Subsequently, both patients underwent partial external biliary diversion (PEBD). Except for a period of intermittent itching in the younger child, both patients remained free of symptoms 2 years after PEBD. In cases where an early diagnosis is made, PEBD could delay or even prevent the necessity of liver transplantation.

[Nutrition and health--carbohydrates, the risks to health from the postprandial glycaemic response and the possibilities for its manipulation]. / Voeding en gezondheid--koolhydraten, het gezondheidsrisico van de postprandiale glykemische respons en de mogelijkheden voor beïnvloeding.

Carbohydrates are the most important source of food-derived energy. The metabolic effects of different types of carbohydrate can vary considerably, partially due to differences in glucose and insulin response. Several studies indicate that postprandial hyperglycaemia is associated with an increased risk of cardiovascular disease in patients with diabetes mellitus. For these patients, and possibly also for individuals with impaired glucose tolerance as well as for the healthy population at large, it may be of benefit to prevent postprandial hyperglycaemia. In order to manipulate the postprandial glycaemic response, an understanding of the underlying mechanisms of this response is crucial. The postprandial blood glucose level is influenced by a number of factors, such as the amount and type of ingested carbohydrates, gastric emptying rate and digestion and secretion of gastrointestinal and other hormones. Different approaches can be chosen to prevent postprandial hyperglycaemia, including changes in the diet and the use of drugs that delay gastric emptying or digestion of carbohydrates. The administration of gastrointestinal hormones or manipulation of the secretion of these hormones, are also possibilities. Investigating the regulation of the postprandial blood-glucose concentration and its possible manipulation could result in new approaches to preventing postprandial hyperglycaemia.

Oro-cecal transit time: influence of a subsequent meal.

BACKGROUND: Small intestinal and oro-cecal transit time (OCTT) is determined for clinical diagnostics and research purposes. Experimental protocols used vary with respect to the inclusion of a subsequent meal during the test period. This study was conducted to elucidate whether the ingestion of a subsequent meal during the test period influences the OCTT of the test meal. MATERIALS AND METHODS: The OCTT of a liquid test meal, measured with the lactose-[(13)C]ureide breath test, was compared between four groups of healthy volunteers (n = 36) who consumed the subsequent meal at different time points. Also, the OCTT was determined twice in eight subjects; a subsequent meal was ingested after 180 min (test A) and after 360 min (test B). RESULTS: An apparently meal-related increase in median OCTT was observed. The OCTT of the eight volunteers measured in test A (210; 210-349 median; quartiles) was significantly shorter than that found in test B (345; 300-375 min, P = 0.016). As result of the ingestion of the subsequent meal at 180 min the OCTT was shortened by 90; 64-116 min in 7/8 subjects. CONCLUSION: These data indicate that the ingestion of a subsequent meal affects the OCTT of a liquid test meal. This phenomenon could be explained by the increased intestinal motility in response to a meal, and should be taken into account when designing protocols for measurements of the OCTT and in the interpretation of small intestinal absorption studies.

Chemical RNA labeling without 3' end bias using fluorescent cis-platin compounds.

Recently, fluorescent, monofunctional cis-platin derivatives have been developed to chemically label nucleic acids for use in fluorescent hybridization assays. Here we show by hybridizations to microarrays containing oligonucleotide probes for the 3' ends, middle parts, and 5' ends of mRNAs, that this labeling methodology bypasses the problem of the 3' end bias that is characteristic of the conventional enzymatic oligo(dT)-primed, reverse transcription labeling of mRNAs.

The 13C/2H-glucose test for determination of small intestinal lactase activity.

To diagnose hypolactasia, determination of lactase enzyme activity in small intestinal biopsy material is considered to be the golden standard. Because of its strongly invasive character and the sampling problems, alternative methods have been looked for. We analysed the 13C-glucose response in serum after consumption of 25 g of naturally enriched 13C-lactose. As an internal standard, 0.5 g of 2H-glucose was added and the 2H-glucose response in serum was measured simultaneously. The studies were performed in healthy volunteers with a background of genetically determined lactase nonpersistence (n = 12; low lactase activity) and lactase persistence (n = 27; high lactase activity). The results were compared with those of the lactose hydrogen breath test, the lactose 13CO2 breath test and the previously described 13C-lactose digestion test. After consumption of 13C-lactose and 2H-glucose, the mean ratio 13C-glucose/2H-glucose concentration in serum at 45-75 min was 0.26 +/- 0.09 in the low lactase activity group and 0.93 +/- 0.17 in the high lactase activity group (P < 0.01). Threshold of the ratio between digesters and maldigesters was calculated as 0.46. Accuracy of the new test was superior to all other tests. We conclude that the 13C/2H-glucose test has the potential of determining the small intestinal lactase activity in vivo and of estimating the amount of lactose which is digested in the small intestine.

Digestion of so-called resistant starch sources in the human small intestine.

BACKGROUND: Resistant starch sources, which are only partially digested in the small intestine, can be used to increase colonic availability of short-chain fatty acids. OBJECTIVE: To study the characteristics of the fermentation of resistant starch, the digestion of resistant starch in the small intestine has to be quantified. We compared the metabolic fates of highly digestible cornstarch (DCS), Hylon VII (type 2 resistant starch), and Novelose 330 (type 3 resistant starch), which are of corn origin and, therefore, naturally enriched in (13)C. DESIGN: After administration of 40 g starch or glucose to 7 healthy volunteers, glucose and exogenous glucose concentrations in serum and (13)CO(2) excretion in breath were analyzed for 6 h. (13)C abundance in carbon dioxide was analyzed by isotope ratio mass spectrometry (IRMS) and (13)C abundance in glucose by gas chromatography-combustion IRMS. RESULTS: By comparing the area under the curve (2 h) of exogenous glucose concentration in serum ((13)C glycemic index) after intake of starch or glucose, (13)C glycemic indexes for DCS, Hylon VII, and Novelose 330 were calculated to be 82 +/- 23%, 44 +/- 16%, and 43 +/- 15%, respectively. Comparison of 6-h cumulative percentage dose recovery in breath showed that 119 +/- 28% of DCS, 55 +/- 23% of Hylon VII, and 50 +/- 26% of Novelose 330 was digested in the small intestine. CONCLUSION: The exogenous glucose response in serum and the (13)CO(2) excretion in breath can be used to estimate small intestinal digestion of resistant starch, which amounts to approximately 50%.