Analysis of tissues following mesenchymal stromal cell therapy in humans indicates limited long-term engraftment and no ectopic tissue formation.

Mesenchymal stromal cells (MSCs) are explored as a novel treatment for a variety of medical conditions. Their fate after infusion is unclear, and long-term safety regarding malignant transformation and ectopic tissue formation has not been addressed in patients. We examined autopsy material from 18 patients who had received human leukocyte antigen (HLA)-mismatched MSCs, and 108 tissue samples from 15 patients were examined by PCR. No signs of ectopic tissue formation or malignant tumors of MSC-donor origin were found on macroscopic or histological examination. MSC donor DNA was detected in one or several tissues including lungs, lymph nodes, and intestine in eight patients at levels from 1/100 to <1/1,000. Detection of MSC donor DNA was negatively correlated with time from infusion to sample collection, as DNA was detected from nine of 13 MSC infusions given within 50 days before sampling but from only two of eight infusions given earlier. There was no correlation between MSC engraftment and treatment response. We conclude that MSCs appear to mediate their function through a "hit and run" mechanism. The lack of sustained engraftment limits the long-term risks of MSC therapy.

Docetaxel induces apoptosis in hormone refractory prostate carcinomas during multiple treatment cycles.

Caspase-cleaved proteins are released from disintegrated apoptotic cells and can be detected in the circulation. We here addressed whether caspase-cleaved cytokeratin 18 (CK18-Asp396) can be used as a serum biomarker for assessment of the clinical efficiency of chemotherapy in hormone-refractory prostate cancer (HRPC). A total of 82 patients with HRPC were evaluated during 751 treatment cycles, either with estramustine (EMP)/vinorelbine or with EMP/docetaxel. The levels of CK18-Asp396 and of total CK18 were measured in patient serum before and during therapy by ELISA. Docetaxel induced significant increases in serum CK18-Asp396 (P<0.0001) and total CK18 (P<0.0002), suggesting induction of apoptosis. Similarly, vinorelbine induced increases in both CK18-Asp396 and CK18 (P<0.001 and 0.011). In contrast, EMP induced increases in total serum CK18 (P<0.0001), but not in CK18-Asp396 (P=0.13). The amplitudes of docetaxel-induced increases were associated with baseline prostate-specific antigen (PSA) and CK18 serum levels in these patients, consistent with tumoral origin of caspase-cleaved fragments. Docetaxel induced significant increases in CK18-Asp396 during second-, third- and fourth-line therapy and induced increased levels of CK18-Asp396 during treatment cycles 1-8. In contrast, vinorelbine induced significant increases only during cycles 1-3. In a subgroup of 32 patients that received EMP/vinorelbine in second line followed by EMP/docetaxel in third line, docetaxel induced stronger increases than vinorelbine (P=0.008). These results show that the CK18-Asp396 serum marker can be used to assess tumour apoptosis in vivo and suggest that the clinical efficiency of docetaxel in HRPC is due to induction of apoptosis during multiple treatment cycles.

A novel assay for discovery and characterization of pro-apoptotic drugs and for monitoring apoptosis in patient sera.

We have developed an apoptosis assay based on measurement of a neoepitope of cytokeratin-18 (CK18-Asp396) exposed after caspase-cleavage and detected by the monoclonal antibody M30. The total amount of caspase-cleaved CK18 which has accumulated in cells and tissue culture media during apoptosis is measured by ELISA. The sensitivity is sufficient for use in the 96-well format to allow high-through-put screening of drug libraries. We here describe strategies allowing classification of pro-apoptotic compounds according to their profiles of induction of apoptosis in the presence of pharmacological inhibitors. The time course of induction of CK18 cleavage can furthermore be used to distinguish structurally similar compounds. We propose that compounds that induce rapid CK18 cleavage have mechanisms of actions distinct from conventional genotoxic and microtubuli-targeting agents, and we present one example of an agent that induces almost immediate mitochondrial depolarization and cytochrome c release. Finally, CK18-Asp396 cleavage products are released from cells in tissue culture, and presumably from tumor cells in vivo. These products can be measured in sera from cancer patients. We present evidence suggesting that it will be possible to use the M30-ELISA assay for measuring chemotherapy-induced apoptosis in patient sera, opening possibilities for monitoring therapy.

Skin blood flow in the human hand in relation to applied pressure.

Our aim was to study the relationship between external pressure and skin blood flow (BF) in the area of the palm (P) of the human hand and the relationship between BF and pressure pain thresholds. A special probe was designed for measuring simultaneously the skin BF using a laser Doppler flowmeter and the pressure using an algometer. The normal BF in the distal phalanx of the index finger and in the middle of the thenar musculature was higher than in the middle phalanx of the middle finger and the middle of P. A pressure of 15-33 kPa was required to reduce BF by 50% and 30-52 kPa to reduce by 85% except in P where the pressure had to be much higher; for 50% of the subjects the reduction in BF did not reach 85% even at 100 kPa. After the removal of the pressure the BF was 170%-230% of the normal readings. The BF did not correlate with the pressure pain thresholds. These results indicate that continuously applied pressure in the hand should not be above 50 kPa to avoid tissue damage due to the cessation of BF in the skin.

The modern endoscopic approach to ureterocele.

PURPOSE: During the last 20 years the surgical approach to ureterocele has evolved from major open surgery to minimally invasive endoscopic puncture. We believe that the endoscopic approach decreases the need for open surgical procedures. We identified specific factors that predict the need for repeat surgery. MATERIALS AND METHODS: We reviewed the charts of 60 new patients with ureterocele treated with primary endoscopic incision between 1991 and 1995. Followup ranged from 4 to 62 months (mean 20). Mode of presentation, ureterocele location, associated vesicoureteral reflux and association of the ureterocele with a duplex system were evaluated. Ureterocele wall thickness was assessed subjectively via radiographic and cystoscopic methods, and categorized as thin, intermediate and thick. RESULTS: All 9 patients with a single system ureterocele had an intravesical ureterocele. No patient had associated reflux nor did any require a secondary open procedure. In 3 cases new onset ipsilateral reflux into the ureterocele spontaneously resolved. Of the 51 patients with a duplex system and associated ureterocele 19 (37%) required a secondary open procedure. The ureterocele was intravesical and ectopic in 22 (43%) and 29 (57%) cases, respectively. Reflux was associated with the ureterocele in 27 patients (53%), and 12 (44%) required a secondary open procedure. A total of 11 patients underwent ureteral reimplantation of 15 refluxing renal units and only 2 renal units required ureteral tapering. Reflux is no longer present in 14 of the 15 renal units (93%). Patients with a thick walled ureterocele required repeat puncture more frequently than those with a nonthick ureterocele. CONCLUSIONS: With the use of modern endoscopic techniques children with intravesical and single system ureteroceles require secondary open surgery less frequently than those with ectopic and duplex system ureteroceles. The mode of presentation does not predict the need for a repeat open procedure. Thick walled ureteroceles require repeat endoscopic puncture more frequently than thin and intermediate walled ureteroceles.

Shelf-life of prepeeled potato cultivated, stored, and processed by various methods.

The effects of cultivation conditions, winter storage, peeling method, browning prevention chemicals replacing sodium bisulfite, and packing methods on the sensory, nutritional and microbiological quality of pre-peeled potato were examined. Two different cultivation lots of the potato variety Van Gogh were used. Cultivation and harvesting conditions and peeling method were the most important facts reducing the sensory quality, especially the appearance, of prepeeled and sliced potatoes. Cooking and baking of potatoes decreased the appearance defects detected in raw potatoes. The levels of vitamin C in packaged samples decreased during winter storage. Cooking for 10 min and keeping potatoes at 60 degrees C for 1 h after cooking also decreased the content of vitamin C. In potato samples immediately after treatments aerobic bacteria were present at levels of 400 to 2,950 CFU/g and lactic acid bacteria at levels of 8 to 16 CFU/g. The number of aerobic bacteria did not increase during storage, and the number of lactic acid bacteria increased at the most to 90 CFU/g. Peeling, washing and packaging methods, cultivation conditions, and winter storage did not have important effects on the number of microbes present.

Decreased nitrogen rates and irrigation effect on celery yield and internal quality.

The effect of decreased nitrogen rates (90, 60 and 30 kg N/ha) with and without irrigation on celery yield quantity and internal quality were studied in field experiments in 1993 and 1994. The decreased nitrogen rates reduced yields in both years. In the dry and warm conditions of 1994 nitrogen x irrigation interaction was clearly observed; irrigation increased yield more at higher nitrogen rates than at lower nitrogen rates. In the rainy conditions of 1993 irrigation did not effect the yield level. On the other hand, in both years, decreased nitrogen rates increased dry matter, vitamin C and dietary fiber contents, and nitrogen had a minor effect on total sugar content. In 1994 alpha- and beta carotene and thiamin were also analyzed but nitrogen had no effect on them. Irrigation increased dry matter and total sugar contents at the two lowest nitrogen rates in 1994, but decreased vitamin C contents in both years. There was also slight evidence that irrigation might decrease alpha- and beta-carotene and thiamin contents.

Soil forming and plant density effects on carrot yield and internal quality.

The effects of soil forming (SF) and plant density (PD) on the carrot yield, mean root weight and internal quality was studied in field experiments in 1993 and 1994. 'Fontana BZ' carrots were grown in flat land, a narrow ridge, a broad ridge, and a compacted broad ridge soil configurations with low (LD) and high (HD) target plant densities, four and seven hundred thousand carrots per hectar. The total and marketable yields were larger in flat land and narrow ridge than in the broad- and compacted broad ridges in 1993 and in 1994 at HD. The number of marketable carrots were highest with the flat land soil configurations in both years. A dry spring in 1993 favored flat land growing conditions; in ridges the fine sand dried quickly. SF did not influence the mean weight of a marketable carrot in 1993, but in 1994 the narrow ridge configuration resulted in heavier carrots than the flat land or broad ridge growing conditions. In the climatically more unfavorable year of 1993, SF and PD affected quality; dry matter was lower in flat land than in the ridges. At HD, the flat land soil configuration produced higher glucose and fructose than carrots grown in the narrow and broad ridges. Dietary fiber and vitamin C were higher in narrow ridge than in compacted broad ridge grown carrots. At LD the flat land and broad ridge produced highest and compacted broad ridge the lowest beta-carotene contents; alpha-carotene was higher at LD than at HD.

Effect of various commercially available enzymes in the liquid chromatographic determination with external standardization of thiamine and riboflavin in foods.

The efficacy of various commercially available enzymes in the determination of thiamine and riboflavin in foods was studied by liquid chromatography (LC) using external standards. Different enzymes, as well as the same enzyme produced by different manufacturers, very strongly affected the determination of both vitamins. The recoveries for different foods ranged from 85 to over 100% for thiamine and from 80 to 100% for riboflavin. The present LC method was accurate and precise when tested on a food and a feed reference material, and coefficients of variation were 5.5% for thiamine and 10% for riboflavin in a rye flour reference material tested for 8 months.