The cellular interaction of 5-fluorouracil and cisplatin in a human colon carcinoma cell line

The combination of 5-fluorouracil (5-FU) and cisplatin (CDDP) has been shown to have synergistic cytotoxicity in human tumours, but the biochemical mechanism for this interaction remains unclear. Therefore, the aim of this study was to investigate the interaction of 5-FU and CDDP in a human colon carcinoma cell line, NCI H548. A 24 h exposure to 5-FU resulted in a 5-FU IC50 value of 24.2 +/- 4.5 microM, Dm 22.6 microM; exposure to CDDP for 2 h resulted in a IC50 value of 20.8 +/- 8.0 microM, Dm 21.9 microM. When cells were exposed simultaneously to 5-FU for 24 h and CDDP for the initial 2 h, or when cells were treated with CDDP for 2 h followed by various concentrations of 5-FU for 24 h, no greater than additive cytotoxicity was observed. In contrast, when cells were treated with 5-FU for 24 h prior to CDDP for 2 h, a greater than additive interaction was noted (5-FU IC50 1.2 +/- 0.6 microM, Dm 1.3 microM, CI 0.45). Thymidine 10 microM partially reversed the growth inhibitory effects of the 5-FU/ CDDP combination. Using both immunological and biochemical assays, no notable differences in the total amount of TS enzyme or the fraction of bound TS enzyme could be detected in the absence or presence of CDDP. No notable differences could be detected in intracellular reduced folate pools, FdUMP or FUTP pools, or 5-FU incorporation into RNA or DNA with the addition of CDDP to 5-FU. DNA fragmentation studies revealed that the combination of 5-FU followed by CDDP demonstrated a greater degree of single-stranded DNA fragments in parental (P = 0.024) and newly synthesised DNA (P = 0.025) compared with the administration of CDDP prior to 5-FU or either drug alone. The increase in smaller DNA fragment size was reversed with the addition of thymidine (10 microM). These findings suggest that the interaction of 5-FU and CDDP is associated with a greater degree of fragmentation of both nascent and parental DNA.     

In vivo model of adeno-associated virus vector persistence and rescue

Gene therapy vectors based on human DNA viruses could be mobilized or rescued from individuals who are subsequently infected with the corresponding wild-type (wt) helper viruses. This phenomenon has been effectively modeled in vitro with both adenovirus (Ad) and adeno-associated virus (AAV) vectors but has not previously been studied in vivo. In the current study, we have developed an in vivo model to study the interactions of a recombinant AAV vector (AAV-CFTR) with wt AAV type 2 (AAV2) and a host range mutant Ad (Ad2HR405) for which monkey cells are permissive (D.E.Brough, S.A.Rice, S.Sell, and D.F.Klessig, J. Virol. 55:206-212, 1985). AAV-CFTR was administered to the respiratory epithelium of the nose or lung of rhesus macaques. Primary cells were harvested from the infusion site at time points up to 3 months after vector administration to confirm vector DNA persistence. Vector DNA was present in episomal form and could be rescued in vitro only by addition of wt AAV2 and Ad. In in vivo rescue studies, vector was administered before or after wt-AAV2 and Ad2HR405 infection, and the shedding of AAV-CFTR was examined. Ad2HR405 and wt-AAV2 infections were established in the nose with concomitant administration. wt-AAV2 replication occurred in the lung when virus was administered directly at a high titer to the lower respiratory tract. AAV-CFTR vector rescue was also observed in the latter setting. Although these studies were performed with small numbers of animals within each group, it appears that AAV-CFTR DNA persists in the primate respiratory tract and that this model may be useful for studies of recombinant AAV vector rescue.     

Management of renal trauma at a rural, level I trauma center

Eotaxin is a potent and selective CC chemokine for eosinophils and basophils. We established several monoclonal antibodies (Mabs) allowing the neutralization and measurement of human eotaxin. Using the Mabs as probes, we demonstrated that normal eosinophils contained intracellular granule-associated eotaxin. Quantification of cell-associated eotaxin in different leukocyte subsets revealed that it was principally expressed in eosinophils. Finally, we showed that normal eosinophils released eotaxin upon stimulation with either of two secretagogues, C5a or ionomycin. These findings raise the possibility that eosinophil-derived eotaxin contributes to the local accumulation of eosinophils at the site of inflammation.     

Characterization of a Schistosoma mansoni gene encoding a homologue of the Y-box binding protein

Hematopoietic stem cells (HSCs) support blood cells throughout life by utilizing their self-renewing and multilineage differentiating capabilities. Hematopoietic growth factors mediate their effects on stem cells by the tyrosine phosphorylation of proteins. Regulation of tyrosine phosphorylation is partially mediated by protein tyrosine phosphatases (PTPases). A possible mechanism by which hematopoietic stem cells maintain their self-renewing capacity and undifferentiated state is by controlling the balanced and opposing actions of protein tyrosine kinases (PTKs), receptors for growth factors, and PTPases. We have characterized the expression of PTPases in 5-fluorouracil (5-FU)-treated murine bone marrow cells, which represent a very primitive population of progenitors enriched for reconstituting stem cells, by using a consensus polymerase chain reaction (PCR) method. Several PTPases were expressed abundantly in the 5-FU-treated bone marrow stem cells. A novel PTP, termed protein tyrosine phosphatase receptor omicron (PTPRO), which is related to the homotypically adhering kappa, mu and PCP-2 receptor-type tyrosine phosphatases, was identified and characterized. We have cloned the murine and full-length human PTPRO cDNAs which share 89% homology, indicating that PTPRO is highly conserved between these species. The human PTPRO cDNA clone encodes a polypeptide of 1439 amino acids (aa) and has a calculated molecular mass of approximately 162 kDa. PTPRO consists of an extracellular segment containing a MAM domain, an immunoglobulin (Ig) domain, four fibronectin-type III (FN-III) repeats, a transmembrane segment, and two tandem intracellular PTP domains. The human PTPRO gene was assigned to human chromosome 1p35-pter using Southern blot analyses of genomic DNAs from rodent/human somatic hybrid cell lines containing human chromosome 1 or the p35-pter region of the chromosome. The mouse Ptpro gene was mapped to chromosome 4, closely linked to D4Mit16 and Elp1 (elliptocytosis-1), by using genomic DNAs from a (C57BL/6J x Mus spretus)F1 x Mus spretus backcross. In fetal tissues, PTPRO expression was observed in the brain and lung, whereas lower levels were observed in the kidney. In adult tissues, PTPRO was less restricted and was observed in the lung, heart, skeletal muscle, prostate, testis, and in various areas of the brain, indicating that PTPRO expression is developmentally regulated. Expression of PTPRO was also observed in human CD34+ bone marrow cells and 5-FU-treated murine primitive stem cells. These results suggest a potential role for PTPRO in stem cell adhesion and in mediating homophilic cell-cell interactions in other cell types.     

Angiolymphoid hyperplasia with eosinophilia of the parapharyngeal space

Angiolymphoid hyperplasia with eosinophilia (ALHE) is an uncommon benign condition characterized by cutaneous nodules with a predeliction for the head and neck region. Extracutaneous involvement is rare. We report a 44-year-old woman who had a large submucosal ALHE tumour in the parapharyngeal space. Our patient is of interest because of the unusual, and as far as we are aware from the literature, unique site and presentation of her lesion.