Bone marrow mesenchymal stem cell transplantation in acute brain trauma. Improvement of brain glucose metabolism in a rat model.

AIM: This study was performed to evaluate the effects of intravenously transplanted rat bone-marrow derived mesenchymal stem cells (rBMSCs) in an acute brain trauma model using serial 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) in rat models. ANIMALS, METHODS: Trauma models were made using a controlled cortical impact injury device. The stem cell treatment group was treated with intravenous injections of BMSCs, and models without stem cell therapy comprised the control group. Serial 18F-FDG PET images were obtained 1, 7, 14, 21, and 28 days after trauma. The difference in 18F-FDG uptake between day 1 and each time point after trauma was analyzed with SPM2 (uncorrected p < 0.005). RESULTS: The stem cell treatment group demonstrated significantly higher 18F-FDG uptake in the right parietal region at 14 days after trauma than at 1 day after trauma. An increase in glucose metabolism in the right parietal cortex appeared on days 21 and 28 after trauma in the group without stem cell treatment. The 18F-FDG uptake in the brain was improved over a broader area, including the right parietal and right primary somatosensory cortex, on days 21 and 28 after trauma in the stem cell treatment group compared with the group without stem cell treatment. CONCLUSION: BMSC therapy in trauma models led to improved glucose metabolism. This result might support the therapeutic effect of stem cells in brain trauma.

A simple and efficient in vitro method for metabolism studies of radiotracers.

In vitro metabolism of acetylcholinesterase inhibitors containing 3-[(18)F]fluoromethylbenzyl- ([(18)F]1) and 4-[(18)F]fluorobenzyl-piperidine moieties ([(18)F]2) was studied and compared with the in vivo metabolism. Defluorination of the [(18)F]1 mainly occurred to generate [(18)F]fluoride ion both in vitro and in vivo. In contrast, the [(18)F]2 was converted into an unknown polar metabolite in both metabolism methods and another metabolite, 4-[(18)F]fluorobenzoic acid in vitro. These results demonstrated that the in vitro method can be used to predict the in vivo metabolism of both radiotracers.

Detection of rifampin-resistant Mycobacterium tuberculosis in sputa by nested PCR-linked single-strand conformation polymorphism and DNA sequencing.

Either PCR-mediated single strand conformation polymorphism (SSCP) analysis or DNA sequencing of rpoB DNA (157 bp) can be used as a rapid screening method for the detection of mutations related to the rifampin resistance of Mycobacterium tuberculosis. However, due to the nonspecific amplification of rpoB DNA from nontuberculous mycobacteria these methods cannot be directly applied to clinical specimens such as sputa. We developed a nested PCR method that can specifically amplify the rpoB DNA of M. tuberculosis on the basis of rpoB DNA sequences of 44 mycobacteria. Nested PCR-linked SSCP analysis and the DNA sequencing method were applied directly in order to detect M. tuberculosis and determine its rifampin susceptibility in 56 sputa. The results obtained by nested PCR-SSCP and DNA sequencing were concordant with those of conventional drug susceptibility testing and DNA sequencing performed with culture isolates.

Differentiation of mycobacterial species by PCR-restriction analysis of DNA (342 base pairs) of the RNA polymerase gene (rpoB).

PCR amplification-restriction analysis (PRA) of rpoB DNA (342 bp), which comprises the Rif(r) region, was used for the differential identification of 49 mycobacteria. The DNA had been used previously for the identification of mycobacterial species by comparative sequence analysis (B. J. Kim et al., J. Clin. Microbiol. 37:1714-1720, 1999). Digestion with four restriction enzymes (HaeIII, HindII, MvaI, and AccII), which were selected on the basis of rpoB DNA sequences, generated distinctive PRA patterns that allowed not only the reference strains but also the clinical isolates of mycobacteria to be distinguished. Both rapidly and slowly growing mycobacteria were distinctly differentiated by HaeIII digestion of the amplified rpoB DNA. By HindII digestion the Mycobacterium tuberculosis complex was distinguished from the other mycobacteria. Furthermore, six subspecies of Mycobacterium kansasii (subspecies I to VI) as well as the closely related Mycobacterium gastri, and other closely related species, were distinguished by simultaneous digestion of MvaI and AccII. According to the rpoB PRA scheme, 240 strains of clinical isolates could be identified. It was also possible to detect and identify M. tuberculosis directly from sputa and bronchoalveolar lavage specimens. These results suggest that PRA of rpoB DNA is a simple and feasible method not only for the differentiation of culture isolates but also for the rapid detection and identification of pathogenic mycobacteria in primary clinical specimens.

Antiviral effects of 28-deacetylsendanin on herpes simplex virus-1 replication.

The compound purified from the fruit of Melia azedarach exerted an antiviral effect on herpes simplex virus-1 (HSV-1) in Vero cells. It was identified as 28-deacetylsendanin (28-DAS). The 50% inhibitory concentration (IC50) of 28-DAS was 1.46 microg/ml without cytotoxicity at 400 microg/ml on Vero cells. Electron microscopy showed that low electron-dense cores of newly synthesized nucleocapsids remained in swollen nuclei and no extracellular virus particles were observed at 15 h p.i. Consistent with this result, it was confirmed by a plaque assay that few infectious progeny viruses were released from the 28-DAS-treated virus-infected cells at 24 h p.i. Intracellular viruses in 28-DAS-treated virus-infected cells were 23% of untreated and infected cells. The synthesis of thymidine kinase (TK) was reduced by 28-DAS at early stage. In conclusion, 28-DAS inhibited the replication of HSV-1, reduced the synthesis of HSV-1 TK, and led to the formation of defective nucleocapsids.

Over-expression of urokinase receptor in human epidermoid-carcinoma cell line (HEp3) increases tumorigenicity on chorio-allantoic membrane and in severe-combined-immunodeficient mice.

Using chorio-allantoic membranes (CAMs) of chick embryos and severe-combined-immunodeficient (SCID) mice, we investigated the effects of urokinase-type plasminogen-activator receptor (u-PAR) over-expression on the process of invasion and tumorigenicity. By the transfection of u-PAR cDNA, 3 u-PAR-over-expressing clones expressing 1.6- to 4.6-fold more u-PAR mRNA than parent cells were obtained from a human epidermoid-carcinoma cell line, HEp3, that expresses urokinase-type plasminogen activator (u-PA) and u-PAR. All the u-PAR-over-expressing clones showed greater invasiveness (13 to 29%) than that of parent HEp3 cells on CAMs. Immunohistochemistry revealed densely stained u-PAR-positive cells near the margin of the tumor, where a u-PAR-over-expressing clone, designated SM-3, was invading thickened fibrous tissue on CAMs. Three u-PAR-overexpressing clones formed larger tumors (>40 mm3) than did parent HEp3 cells on CAMs. Moreover, when the u-PAR-overexpressing clone (SM-3) was injected s.c. into the back of the SCID mice it produced a larger tumor volume than the control (HEp3) and down-regulated (AS-2) clones and significantly shortened the survival of SCID mice. These results demonstrate that increased u-PAR expression is an important factor in determining the malignant phenotype that makes cancer cells more invasive and tumorigenic.

Alanine metabolism and gluconeogenesis in the rat.

The metabolism of alanine and several other gluconegoneic substrates was studied in anesthtized fed and fasted rats, i.e., rats with low and high rates of gluconeogenesis. Glutamine was released by the hindquarter (muscle) in both groups, whereas lactate, pyruvate, and alanine were taken up in fed rats and were released during starvation. Despite this, blood levels of alanine, lactate, and pyruvate were diminished in fasting rats, suggesting increased extraction by liver. Treatment of fasted rats for 24 h with phloridzin caused glycosuria and secondarily led to hypoglycemia and an intensification of the chargesobserved with fasting, i.e., hyperketonemia, hyperglucagonemia, and increased gluconeogenesis (assessed by urea N excretion). Blood alanine was decreased, even though the release of alanine from muscle was increased. Pretreatment with triamcinolone and administration of exogenous alanine both attenuated the hypoglycemia and ketosis, It is concluded that 1) in states of heightened gluconeogenesis, alanine release from muscle may not keep pace with extraction by liver and blood alanine decreases; 2) the release of alanine, lactate, and pyruvate from muscle parallel each other suggesting common control factors; and 3) in the red state muscle is an important site of lactate disposition.

Serum insulin and growth hormone response patterns in monozygotic twin siblings of patients with juvenile-onset diabetes.

To detect abnormalities in the secretion of insulin and growth hormone in monozygotic twin siblings of patients with juvenile-onset diabetes, their responses during oral, cortisone-primed oral, and intravenous, glucose tolerance tests and intravenous tolbutamide tests were compared to those of matched controls. The twins had higher mean serum insulin levels during all tests, but differences reached statistical significance (P less than 0.02) only in the cortisone-primed test. Growth hormone levels were higher in the twins (P less than 0.04) in the intravenous tolbutamide tolerance test. The frequency of abnormal oral glucose tolerance tests among controls, diabetic monozygotic twins and the offspring of two diabetic parents was also compared. Twins and controls had nearly the same frequency of normal tests; however, the diabetic offspring had a significantly higher (P less than 0.001) prevalence of abnormal tests. These data suggest that magnitudes of environmental and genetic factors operating in monozygotic "pre-diabetic" children of diabetic parents.