Nonmyeloablative stem cell therapy enhances microcirculation and tissue regeneration in murine inflammatory bowel disease.

BACKGROUND AND AIMS: Reduced microcirculation has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Stem cells or endothelial progenitor cells are thought to contribute to tissue regeneration through neoangiogenesis or vasculogenesis in ischemia- or inflammatory-related diseases. We therefore hypothesized that adult stem cells facilitate epithelial repair in IBD. METHODS: Moderate-severe colitis in mice was induced by dextran sulfate sodium (DSS) and 2.0 x 10(6) immortalized CD34(-) stem cells infused twice via the tail vein during an observation period of 35 days in a nonmyeloablative setting. RESULTS: Here, we demonstrate that adult stem cells home to the damaged digestive tract in the large intestine and facilitate mucosal repair in moderate-severe colitis. Nonmyeloablative stem cell therapy resulted in increased survival in severe colitis (P < .0001). Moreover, clinical activity and histologic evaluation of the colitis severity score were reduced significantly in moderate (P = .0003 or P = .03) and severe (P < .0001 or P < .03) colitis after 35 days, in addition to the DSS-induced shortening of colon length (P = .002 and P < .0002). Genetically marked stem cells were detected predominantly in the submucosa of the damaged colon epithelium. Epithelial repair in experimental IBD was mediated either by induction of improved vasculogenesis or by the differentiation of the transplanted stem cells into endothelial cells, as demonstrated by the promotion of Tie2 activity in the infused cells at the site of the damaged mucosa. CONCLUSIONS: Our findings indicate that systemically administered adult stem cells respond to an adequate tissue lesion in murine IBD by enhancing microcirculation, resulting in accelerated tissue repair.

Validation and application of a high-performance liquid chromatographic-based assay for determination of the inosine 5'-monophosphate dehydrogenase activity in erythrocytes.

Thiopurine drug monitoring has become an important issue in treating children with acute lymphoblastic leukaemia (ALL). In this population, a genetic polymorphism causes wide differences in the activity of thiopurine S-methyletransferase (TPMT)--the rate-limiting enzyme of the thiopurine degradation metabolism--leading to the necessity of drug dose adjustments. It is not yet known if similar differences exist in the inosine 5'-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205), the rate-limiting enzyme of the thiopurine synthesis. To test this, we established and validated a high-performance liquid chromatographic (HPLC)-based assay to determine the IMPDH enzyme activity in erythrocytes. The remarkable features of this assay are its simple erythrocyte separation/haemolysis and assay conditions and a distinct segregation of xanthosine 5'-monophosphate (XMP) from the clear supernatant after precipitation. The probes were processed without a time-consuming extraction and heating procedure and the assay demonstrated a good intra- and interday stability as well as a recovery rate of approximately 100%. The IMPDH enzyme activity was measured in erythrocytes of 75 children with diagnosis of ALL before starting antileukaemic therapy and their activity compared to those of 35 healthy adult controls. The measured enzyme activity was wide ranging in both groups. The individual enzyme activity differences observed in children with ALL might led to differences in the thionucleotide levels in those undergoing the standard thiopurine dose regimen.

Histochemical assessment of early myocardial infarction using 2,3,5-triphenyltetrazolium chloride in blood-perfused porcine hearts.

INTRODUCTION: Although the chemical mechanism of the triphenyltetrazolium (TTC) reaction, for macroscopic detection of myocardial infarction, has been described previously, literature reports on correct tissue preparation and the use of this technique in intact large animals are lacking. METHODS: We investigated the special requirements for TTC staining in blood-perfused porcine hearts, validated the various handling steps and provided detailed information for precise and easy use of this histochemical method. The left anterior descending coronary artery was occluded for 45 min followed by 6 h of reperfusion in an open chest preparation using anesthetised domestic pigs. The hearts were excised and the organ-handling steps and TTC-staining procedure validated. RESULTS: The protocol includes (i) intracoronary saline perfusion, (ii) pressure-controlled determination of the non-ischemic region by Evans blue dye, (iii) a freeze-thaw cycle, (iv) a triphenyltetrazolium incubation period, and (v) a bleach cycle with 4% paraformaldehyde. The TTC-staining results were confirmed by histology of transitional regions of the infarct area, area-at-risk and non-risk-region. DISCUSSION: If some special features associated with blood-perfused porcine hearts are considered carefully, reliable results for subsequent infarct size calculations can be obtained and large potential errors excluded.

RETRACTED: Modified high-performance liquid chromatographic conditions for determination of inosine 5'-monophosphate dehydrogenase activity inerythrocytes.

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Calpain inhibition reduces infarct size and improves global hemodynamics and left ventricular contractility in a porcine myocardial ischemia/reperfusion model.

Calpains, a family of Ca2+-dependent cysteine proteases, are activated during myocardial ischemia and reperfusion. This study investigates the cardioprotective effects of calpain inhibition on infarct size and global hemodynamics in an ischemia/reperfusion model in pigs, using the calpain inhibitor A-705253. The left anterior descending coronary artery was occluded for 45 min and reperfused for 6 h. A bolus of 1.0 mg/kg A-705253 or distilled water was given intravenously 15 min prior to induction of ischemia and a constant plasma level of A-705253 was maintained by continuous infusion of 1.0 mg/kg A-705253 during reperfusion. Infarct size was assessed histochemically using triphenyltetrazolium chloride staining. Macromorphometric findings were verified by light microscopy on hematoxylin-eosin- and Tunel-stained serial sections. Global hemodynamics, including the first derivate of the left ventricular pressure (dP / dtmax), were measured continuously throughout the experiment. A-705253 reduced the infarct size by 35% compared to controls (P < 0.05). Hemodynamic alterations, including heart rate, aortic blood pressure, central venous pressure and left atrial pressure, were attenuated mainly during ischemia and the first 2 h during reperfusion by A-705253. Cardiac function improved, as determined by dP / dtmax, after 6 h of reperfusion (P < 0.003). Our results demonstrate that myocardial protection can be achieved by calpain inhibition, which decreases infarct size and improves left ventricular contractility and global hemodynamic function. Hence, the calpain-calpastatin system might play an important pathophysiological role in porcine myocardial ischemia and reperfusion damage and A-705253 could be a promising cardioprotective agent.

Interference free and simplyfied liquid chromatography-based determination of thiopurine S-methyltransferase activity in erythrocytes.

The determination of the thiopurine S-methyltransferase activity (TPMT; EC 2.1.1.67) has become an important issue during thiopurine therapy due to its known genetic polymorphism resulting in a wide range of TPMT activity. Therefore, the standard thiopurine drug regimen is associated with increased hematopoetic toxicity in patients with low or absent TPMT activity, whereas patients with high activity may be insufficiently treated. However, presently available methods are labour intensive and time consuming and tend towards too high or too low enzyme activity due to their methodological approach. The use of instable substrate solutions (6-MP or 6-TG), organic solvents like dimethyl sulfoxide and too high substrate and co-substrate saturation concentrations contribute to this phenomenon. We therefore, established an optimized and fast isocratic HPLC linked TPMT assay based on the enzymatic methylation of mercaptopurine or thioguanine in RBC lysates with S-adenosyl-l-methionine as methyl donor. Unspecific non-enzymatic methylation was not detectable. The recovery of 6-methyl-mercaptopurine was 97-102%, the intra- and interday variation between 1.0 and 5.0%, respectively. The assay dispenses with a time consuming extraction procedure with organic solvents, a heating step, and a gradient elution and is therefore, favourable for clinical routine application. The TPMT activity was measured in 62 untreated children with acute lymphoblastic leucemia at the time of diagnosis (activity = 34.0+/-10.6 nmol/g Hb/h, range: 11.5-55.4 nmol/g Hb/h) and in 12 adult healthy volunteers (62.8+/-7.7 nmol/g Hb/h, range: 48-82 nmol/g Hb/h) reflecting the wide measurable TPMT activity found in erythrocytes.