We cloned and analyzed the integrated human papillomavirus type 16 (HPV-16) genomes that are present in the human cervical carcinoma cell lines SiHa and CaSki. The single HPV-16 genome in the SiHa line was cloned as a 10-kilobase (kb) Hindlll fragment. Integration of the HPV-16 genome occurred at bases 3132 and 3384 with disruption of the E2 and E4 open reading frames (ORFs). An additional 52-base-pair deletion of HPV-16 sequences fused the E2 and E4 ORFs. The 5' portion of the disrupted E2 ORF terminated immediately in the contiguous human right-flanking sequences. Heteroduplex analysis of this cloned integrated viral genome with the prototype HPV-16 DNA revealed no other deletions, insertions, or rearrangements. DNA sequence analysis of the El ORF, however, revealed the presence of an additional guanine at nucleotide 1138, resulting in the fusion of the Ela and Elb ORFs into a single El ORF. Sequence analysis of the human flanking sequences revealed one-half of an Alu sequence at the left junction and a sequence highly homologous to the human 0 repeat in the right-flanking region. Analysis of the three most abundant BamHI clones from the CaSki line showed that these consisted of (i) full-length, 7.9-kb HPV-16 DNA; (ii) a 6.5-kb genome resulting from a 1.4-kb deletion of the long control region; and (iii) a 10.5-kb clone generated by a 2.6-kb tandem repeat of the 3' early region. These HPV-16 genomes were arranged in the host chromosomes as head-to-tail, tandemly repeated arrays. Transcription analysis revealed expression of the HPV-16 genome in each of these two cervical carcinoma cell lines, albeit at significantly different levels. Preliminary mapping of the viral RNA with subgenomic strand-specific probes indicated that viral transcription appeared to be derived primarily from the E6 and E7 ORFs.

Neurofibromatosis-1 (NF1) is an autosomal dominant disorder with marked variability of expression. Analysis of the NF1 gene (NF1) has detected a variety of mutations without any clear correlation with phenotype. However, deletions which remove all of NF1 have been reported in a small number of patients who have minor facial abnormalities, mental retardation, learning disabilities, and early or excessive burden of cutaneous or plexiform neurofibromas. The purpose of this study was to determine whether these phenotypic traits are associated with whole gene deletions. Out of 406 of our NF1 patients, 70 patients had manifestations previously associated with gene deletions. Thirty-five of these patients from 26 families were available for study. By fluorescence in situ hybridization (FISH) analysis, 4 were found to have deletions of the entire gene, including 2 sporadic cases, 1 familial case, and 1 case where family history could not be verified. In addition, the mother of the familial case was found to be mosaic for the deletion. Our results suggest that although large NF1 deletions occur with relatively high frequency in patients with certain findings, the presence of a deletion cannot be predicted solely on the basis of clinical phenotype.

BackgroundFibromyalgia (FM) is a clinical syndrome characterized by chronic pain and allodynia. The diagnosis of FM has been one of exclusion as a test to confirm the diagnosis is lacking. Recent data highlight the role of the immune system in FM. Aberrant expressions of immune mediators, such as cytokines, have been linked to the pathogenesis and traits of FM. We therefore determined whether cytokine production by immune cells is altered in FM patients by comparing the cellular responses to mitogenic activators of stimulated blood mononuclear cells of a large number of patients with FM to those of healthy matched individuals.MethodsPlasma and peripheral blood mononuclear cells (PBMC) were collected from 110 patients with the clinical diagnosis of FM and 91 healthy donors. Parallel samples of PBMC were cultured overnight in medium alone or in the presence of mitogenic activators; PHA or PMA in combination with ionomycin. The cytokine concentrations of IFN-γ, IL-5, IL-6, IL-8, IL-10, MIP-1β , MCP-1, and MIP1-α in plasma as well as in cultured supernatants were determined using a multiplex immunoassay using bead array technology.ResultsCytokine levels of stimulated PBMC cultures of healthy control subjects were significantly increased as compared to matched non-stimulated PBMC cultures. In contrast, the concentrations of most cytokines were lower in stimulated samples from patients with FM compared to controls. The decreases of cytokine concentrations in patients samples ranged from 1.5-fold for MIP-1β to 10.2-fold for IL-6 in PHA challenges. In PMA challenges, we observed 1.8 to 4-fold decreases in the concentrations of cytokines in patient samples.ConclusionThe cytokine responses to mitogenic activators of PBMC isolated from patients with FM were significantly lower than those of healthy individuals, implying that cell-mediated immunity is impaired in FM patients. This novel cytokine assay reveals unique and valuable immunologic traits, which, when combined with clinical patterns, can offer a diagnostic methodology in FM.

Cellular cholesterol metabolism is regulated primarily through the coordinate expression of two proteins, the low density lipoprotein (LDL) receptor and 3-hydroxy-

IntroductionChronic idiopathic myelofibrosis (IM) is a hematological malignancy characterized by splenomegaly, a leukoerythroblastic blood picture, teardrop poikilocytosis (dacrocytosis), varying degrees of bone marrow (BM) fibrosis, and extramedullary hematopoiesis. [1][2][3][4] IM is thought to originate at the level of the multipotent hematopoietic stem cell (HSC). [1][2][3][4] The HSC defect results in a profound hyperplasia of morphologically abnormal megakaryocytes and clonal populations of monocytes, which have been shown to locally release fibrogenic growth factors, leading to BM fibrosis. [5][6][7] Rare CD34 ϩ HSC/hematopoietic progenitor cells (HPCs) circulate in the peripheral blood (PB) of healthy individuals. [8][9] Increased numbers of CD34 ϩ cells can be mobilized into the PB following the administration of a variety of cytokines and/or chemotherapeutic agents. [9][10][11] Barosi et al recently demonstrated that the PB of IM patients contained 360 times more CD34 ϩ cells than normal controls and 18 to 30 times more CD34 ϩ cells than patients with other Philadelphia chromosome-negative (Ph -) myeloproliferative disorders (MPDs). 12 In addition, the PB CD34 ϩ cell number was further shown to be related to disease progression and to serve as a biomarker for disease activity. 12 The number of assayable HPCs present in the PB of IM patients have also been shown to be increased. [13][14][15] Therefore, IM represents a unique situation in which the numbers of CD34 ϩ cells appearing in the PB are frequently markedly increased in the absence of extrinsic stimuli.Although the CD34 antigen is expressed by both the HSC and HPC, the functional potential of the CD34 ϩ cells in the PB of IM patients has not been well defined. In this report we have further phenotyped the CD34 ϩ cells of IM patients and examined the multilineage differentiation potential of these cells, as well as their ability to repopulate immunodeficient mice. In addition, we have demonstrated the utility of this in vivo model to analyze the cellular and molecular events that occur during the transition of IM to acute leukemia. Patients, materials, and methods Patients and healthy control subjectsAll human tissue samples were obtained after informed consent following the guidelines of the institutional review board of the University of Illinois College of Medicine. PB samples were obtained from: (1) healthy donors in steady-state hematopoiesis; (2) healthy donors mobilized with granulocyte colony-stimulating factor (G-CSF; Amgen, Thousand Oaks, CA) at 5 g/kg/d subcutaneously; and (3) patients with IM or polycythemia vera (PV) who met the World Health Organization (WHO) diagnostic criteria for IM and PV, [1][2][3][4]16 as well as patients with secondary myelofibrosis associated with pulmonary hypertension. 17 None of the patients were receiving cytotoxic agents at the time of study and none had evidence of transformation to acute leukemia. The specific clinical characteristics of the patients whose CD34 ϩ cells were examined for BM-repopulati...