The results of this study demonstrate that IGF-1 and TGF-beta1 can act in combination to regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis.

Abstract. Chronic myelogenous leukemia (CML) has a typical progressive course with transition from a chronic phase to a terminal blast crisis phase. The mechanisms that lead to disease progression remain to be elucidated. To understand the role of aberrant methylation in the progression of CML, DNA methylation patterns in 16 patients with CML blast crisis were analyzed. Methylation status was analyzed by methylationspecific PCR (MSP) for 13 genes, including cell cycle regulating genes, DNA repair genes, apoptosis-related genes, a differentiation-associated gene and a cytokine signaling gene. The frequency of samples with methylation in each of the following genes were: p15, 18%; MGMT, 12%; RARβ, 12%; p16, 6%; DAPK, 6% and FHIT, 6%. In total, four (25%) cases showed methylation of at least one gene. None of the 16 cases showed hypermethylation of the hMLH1 or hMSH2 genes. These results suggest that hypermethylation of cell cycle control genes, but not DNA mismatch repair genes, play a significant role in the progression of CML. IntroductionChronic myelogenous leukemia (CML) has a typical progressive course with transition from the chronic phase to the terminal blast crisis phase. The mechanisms that lead to disease progression have yet to be elucidated. Cytogenetic and genetic changes occur in the majority of patients during disease progression. Approximately 70-80% of patients with CML blast crisis show additional chromosomal changes involving chromosomes 7, 8, 17, 19, 21 and 22, sometimes with duplication of the Ph chromosome (1). Genetic changes occurring in the progression to blast crisis include mutation of the p53 (20-30%), amplification of the c-myc (20%), deletion of the p16 (15%) and mutation of the Ras (6%) gene (2).DNA methylation at CpG sites in promoter regions is a frequent, acquired epigenetic event involved in the pathogenesis of various types of human malignancies. Methylation in the promoter region is capable of causing gene silencing, which may provide an alternative pathway to gene inactivation, in addition to deletions or mutations. The ABL1, calcitonin, ER and HIC1 genes were found to be frequently methylated in CML (3). Moreover, methylation of the ABL1 gene is associated with the progression of CML (4). These methylation phenotypes in CML provided a rationale for using demethylating agents such as 5-azacytidine and decitabine in a clinical setting, and preliminary clinical results were reported (3,5). To determine the role of aberrant methylation in the progression of CML, we analyzed DNA methylation patterns in CML blast crisis. Materials and methodsBone marrow cells were obtained from 16 patients who developed blast crisis during the follow-up of CML. Genomic DNA was extracted from low density mononuclear cells after the bone marrow cells were centrifuged in the presence of TRIzol reagent (Life Technologies Inc., Rockville, MD, USA). Control DNA was extracted from the peripheral blood of 10 healthy individuals. Methylation-specific PCR (MSP) was performed as previously described (6,7). B...

Organ culture studies involving whole explants of periosteum have been useful for studying chondrogenesis, but to date the standard culture model for these explants has required the addition of fetal bovine serum to the media. Numerous investigators have succeeded in culturing chondrocytes and embryonic cells in serum-free conditions but there have been no studies focused on achieving a defined, serum-free media for culturing periosteal explants. The purpose of the present investigation was to determine if whole periosteal explants can be grown and produce cartilage in serum-free conditions, and to define the minimum media supplements that would be conducive to chondrogenesis. 321 periosteal explants were obtained from the medial proximal tibiae of 31 two month-old NZ white rabbits and cultured using a published agarose suspension organ culture model and DMEM for six weeks. The explants were cultured with and without fetal bovine serum or bovine serum albumin and exposed to transforming growth factor beta alone, a combination of growth factors we call ChondroMix (10 ng/ml transforming growth factor beta, 50 ng/ml basic fibroblast growth factor, and 5 pg/ml growth hormone), and/or ITS+ (2.08 pg/ml each of insulin, transferrin, and selenious acid, plus 1.78 pg/ml linoleic acid and 0.42 mg/ml BSA). Maximal chondrogenic stimulation in this study was observed with the combination of ChondroMix and ITS+. However, the minimal requirement to match or exceed the level of chondrogenic stimulation seen in the standard model (TGF-1 in 100/0 FBS) was achieved simply by the addition of 2.0 pg/ml insulin in 0.10/0 BSA-containing medium (p < 0.05). Therefore, based on our results, it would be reasonable to assume that insulin is the component in ITS+ responsible for the observed increase in total cartilage growth. Lower concentrations of insulin were not effective, suggesting that the observed effect of insulin requires activation of the IGF-1 receptor.

Transforming growth factor-pl (TGF-PI) has been shown to stimulate chondrogenesis in periosteal explants cultured in agarose suspension. TGF-Ps exert their cellular effects through a heteromeric cell membrane receptor complex consisting of TGF-P type I and type I1 receptors. In this study, the spatial and temporal expressions of the type I receptor (TPR-I), type I1 receptor (TPR-11) and endogenous TGF-P1 in periosteal explants cultured in vitro were examined using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The temporal changes in the expression of the TPR-I and TPR-I1 mRNAs correlated with that of TGF-PI. Exogenous administration of TGF-PI upregulated the expression of both receptors and of the TGF-PI ligand in a biphasic pattern. The earlier peak of upregulation was observed at 7 days in culture. A later peak of upregulation was seen at 42 days, at which time cartilage formation reached a maximum. Imniunohistocheniical studies demonstrated co-localization of TPR-I and TPR-I1 simultaneously among the same cells expressing TGF-PI. TGF-P1 treatment increased the expression of TGF-PI, TPR-I and TPR-I1 in mesenchymal cells in the cambium layer at 7 days in culture. Small round chondrocytes showed widely distributed immunoreactivity of TGF-PI, TPR-I and TPR-I1 in the 42-day explants treated with TGF-PI . These observations support the hypothesis that TGF-P1 regulates the initiation and formation of cartilage during periosteal chondrogenesis.

Background: To improve the prognosis of patients with urachal cancer and establish an effective chemotherapeutic regimen for distant metastases. Methods: We conducted a retrospective study to evaluate the efficacy and safety of a modified combination of 5-fluorouracil, leucovorin and oxaliplatin (mFOLFOX6) therapy in patients with metastatic urachal cancer. Results: Five patients were treated with mFOLFOX6. Their median age was 65 years (range 41-80). The median follow-up time was 42 months (range 18-46). Two of the 5 patients (40%) showed an objective response: 1 achieved a clinically complete response and 1 a partial response. The grade 3/4 toxicity associated with this regimen was primarily neutropenia, but febrile neutropenia was not observed. Oxaliplatin treatment was discontinued because of a grade 2 allergic reaction in 1 patient. Grade 2 peripheral sensory neuropathy caused by oxaliplatin was observed in 2 patients, and the OPTIMOX (stop and go) approach had to be adopted. Conclusions: mFOLFOX6 appears to be effective for the treatment of metastatic urachal cancer.

Intravascular large B-cell lymphoma (IVLBCL) is a rare lymphoma characterized by the presence of large tumour cells within the blood vessels. It has been considered that IVLBCL is a highly malignant disease with poor prognosis. However, it has been shown that a therapeutic effect resembling that of conventional B-cell lymphomas may be obtained with the application of systemic chemotherapy at the early stage of this disease. Although involvement in the lung is often detected at autopsy, early diagnosis is quite difficult. In this report, we present a case of IVLBCL with pulmonary involvement where 18-fluorodeoxyglucose positron emission tomography (FDG-PET) was useful in the early diagnosis. Neither computed tomography (CT) nor 67 gallium scintigraphy could reveal the presence of disease in the lung. Histological evidence of IVLBCL was obtained by TBLB after FDG uptake in the lung was confirmed by FDG-PET. The patient exhibited a good response to the subsequent combination chemotherapy. We propose that FDG-PET is a powerful tool for the early diagnosis of IVLBCL with pulmonary involvement, if the possibility of this disease presents in the patient with respiratory symptoms without abnormal findings by CT and 67 gallium scintigraphy.

These results suggest that elastase and superoxide are involved in OA lung injury.

We investigated the involvement of IL-8 in the delayed vascular permeability (VP) in rabbit lipopolysaccharide (LPS)-pleurisy.