Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens.

BACKGROUND: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens. RESULTS: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%. CONCLUSION: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.

The performance of human papillomavirus high-risk DNA testing in the screening and diagnostic settings.

OBJECTIVE: We sought to evaluate the performance of the human papillomavirus high-risk DNA test in patients 30 years and older. MATERIALS AND METHODS: Screening (n=835) and diagnosis (n=518) groups were defined based on prior Papanicolaou smear results as part of a clinical trial for cervical cancer detection. We compared the Hybrid Capture II (HCII) test result with the worst histologic report. We used cervical intraepithelial neoplasia (CIN) 2/3 or worse as the reference of disease. We calculated sensitivities, specificities, positive and negative likelihood ratios (LR+ and LR-), receiver operating characteristic (ROC) curves, and areas under the ROC curves for the HCII test. We also considered alternative strategies, including Papanicolaou smear, a combination of Papanicolaou smear and the HCII test, a sequence of Papanicolaou smear followed by the HCII test, and a sequence of the HCII test followed by Papanicolaou smear. RESULTS: For the screening group, the sensitivity was 0.69 and the specificity was 0.93; the area under the ROC curve was 0.81. The LR+ and LR- were 10.24 and 0.34, respectively. For the diagnosis group, the sensitivity was 0.88 and the specificity was 0.78; the area under the ROC curve was 0.83. The LR+ and LR- were 4.06 and 0.14, respectively. Sequential testing showed little or no improvement over the combination testing. CONCLUSIONS: The HCII test in the screening group had a greater LR+ for the detection of CIN 2/3 or worse. HCII testing may be an additional screening tool for cervical cancer in women 30 years and older.

The processing of human pro-tumor necrosis factor

Human pro-TNF-$\alpha$ is a 26 kd type II transmembrane protein, and it is the precursor of 17 kd mature TNF. Pro-TNF release mature from its extracellular domain by proteolytic cleavage between resideu Ava ($-$1) and Val (+1). Both forms of TNF are biologically active and the native form of mature TNF is a bell-shaped trimer. The structure of pro-TNF was studied both in intact cell system and in an in vitro translation system by chemical crosslinking. We found that human pro-TNF protein exist as a trimer in intact cells (LPS-induced THP-1 cells and TNF cDNA transfected COS-3 cells) and this trimeric structure is assembled intracellularly, possibly in the ER. By analysis several deletion mutants, we observed a correlation between expression of pro-TNF cytotoxicity in a juxtacrine fashion and detection of the trimer, suggesting the trimeric structure is very important for its biologic activity. With a series of deletion mutants in the linking domain, we found that the small deletion did not block the cleavage and large deletion did regardless of the presence or absence of the native cleavage site, suggesting that the length of the residues between the plasma membrane and the base of the trimer determines the rate of the cleavage, possibly by blocking the accessibility of the cleavage enzyme to its action site. Our data also suggest that the native cleavage site is not sufficient for the release of mature TNF and alternative cleavage site(s) exists. ^

Functional and physical associations of beta1 integrins in the activation of human T lymphocytes

Adhesion involves interactions between cells or cells with extracellular matrix components and is a fundamental process for all multicellular organisms as well as many pathogenic microbes. Integrins are heterodimeric transmembrane proteins that function as adhesion molecules and transduce signals between the extracellular environment and the intracellular cytoskeletal machinery. β1 integrin subfamily is highly expressed on T lymphocytes and mediates cell spreading, adhesion and coactivation. T lymphocytes have an important role in the regulation and homeostasis of the immune system therefore, the goals of this study were to first to investigate β1 integrin interaction with fibronectin binding protein A (FnbpA), a surface protein expressed on gram-negative bacteria Staphylococcus aureus. Second, characterize the association and function of a non-integrin surface protein, CD98, with β1 integrins on T lymphocytes. ^ FnbpA binds to fibronectin (FN), also a ligand for α5β1 and α4β1 integrins on T lymphocytes. Since both bacterial proteins FnbpA and T cell integrins utilize FN, it was of interest to determine the effects FnbpA on T cell activation. Results demonstrated that recombinant FnbpA (rFnbpA) coimmobilized with OKT3 mediated T cell coactivation in a soluble FN-dependent manner. Integrin α5β1 was identified as the main integrin utilized by Staphylococcus aureus FnbpA from studies using soluble antibodies to inhibit T cell proliferation and parallel plate flow chamber assays. The mechanism of rFnbpA-mediated coactivation was one that used soluble FN as a bridge between rFnbpA and integrin α5β1 on the T lymphocyte. ^ Since integrins are utilized by T lymphocytes and bacterial proteins, it was of interest to identify proteins involved in integrin regulation. Anti-CD98 mAb 80A10 was identified and characterized from a screen to identify surface proteins involved in integrin signaling and functions. CD98 is a non-integrin protein that was sensitive to integrin inhibition in human T lymphocyte aggregation and activation, thus suggested that CD98 shared a common signaling pathway with integrins. These results led to the question of whether CD98 physically associates with β1 integrins. Fluorescence microscopy and biochemical analysis determined that CD98 is specifically associated with β1 integrin on human T lymphocytes and may be part of a larger multimolecular signaling complex. ^

The molecular cloning and characterization of human heparanase cDNA and the immunochemical localization of heparanase in metastatic melanomas

Heparanase, an endo-$\beta$-D-glucuronidase, has been associated with melanoma metastasis. Polyclonal antibodies directed against the murine N-terminal heparanase peptide detected a M$\sb{\rm r}\sim 97,000$ protein upon SDS-polyacrylamide gel electrophoresis of mouse melanoma and human melanoma cell lysates. In an indirect immunocytochemical study, metastatic human A375-SM and mouse B16-BL6 melanoma cells were stained with the anti-heparanase antibodies. Heparanase antigen was localized in the cytoplasm of permeabilized melanoma cells as well as at the cell surface of unpermeabilized cells. Immunohistochemical staining of frozen sections from syngeneic mouse organs containing micrometastases of B16-BL6 melanoma demonstrated heparanase localized in metastatic melanoma cells, but not in adjacent normal tissues. Similar studies using frozen sections of malignant melanomas resected from patients indicated that heparanase is localized in invading melanoma cells, but not in adjacent connective tissues.^ Monoclonal antibodies directed against murine heparanase were developed and characterized. Monoclonal antibody 10E5, an IgM, precipitated and inhibitated the enzymatic activity of heparanase. A 2.6 kb cDNA was isolated from a human melanoma $\lambda$gt11 cDNA library using the monoclonal antibody 10E5. Heparan sulfate cleavage activity was detected in the lysogen lysates from E. Coli Y1089 infected with the $\lambda$gt11 cDNA and this activity was inhibited in the presence of 10-fold excess of heparin, a potent inhibitor of heparanase. The nucleotide sequence of the cDNA was determined and insignificant homology was found with the gene sequences currently known. The cDNA hybridized to a 3.2-3.4 kb mRNA in human A375 melanoma, WI-38 fibroblast, and THP-1 leukemia cells using Northern blots.^ Heparanase expression was examined using Western and Northern blots. In comparison to human A375-P melanoma cells, the quantity of 97,000 protein recognized by the polyclonal anti-heparanase antibodies doubled in the metastatic variant A375-SM cells and the quantity of 3.2-3.4 kb mRNA doubled in A375MetMix, a metastatic variant similar to A375-SM cells. In B16 murine melanoma cell, the intensity of the 97,000 protein increased more than 2 times comparing with B16-F1 cells. The extent in the increase of the protein and the mRNA levels is comparable to the change of heparanase activity observed in those cells.^ In summary, the studies suggest that (a) the N-terminus of the heparanase molecule in mouse and human is antigenically related; (b) heparanase antigens are localized at the cell surface and in the cytoplasm of metastatic human and mouse melanoma cells; (c) heparanase antigens are localized in invasive and metastatic murine and human melanomas in vivo, but not in adjacent normal tissues; (d) heparanase molecule appeared to be differentially expressed at the transcriptional as well as at the translational level; and (e) the size of human heparanase mRNA is 3.2-3.4 kilobase. ^

Involvement of human chromosome 17 in the control of the metastatic phenotype in melanoma

Fusion of nonmetastatic murine melanoma K1735 C19H cells with metastatic human melanoma A375 C15N cells resulted in a hybrid (termed H7) which was highly metastatic in a nude mouse model. The H7 hybrid retained chromosome 17 as the sole intact human chromosome in the cell. A lung bioassay showed that the K1735 C19H cells were present in the lungs of nude mice with s.c. tumors, yet at 6-weeks after tumor resection, no cells remained in the lung and therefore did not form lung metastases. Examination of various phenotypic properties such as in vivo and in vitro growth demonstrated that phenotypically the H7 hybrid was most like the K1735 C19H cell line except for its metastatic ability. In contrast the H7 hybrid cells containing single or multiple copies of human chromosome 17 with a point mutation at codon 249 (arg-gly) of the p53 gene, readily formed lung metastases. A plasmid containing the human p53 from the H7 hybrid and four other contructs with mutations at codon 143 (val-arg), 175 (arg-his), 249 (arg-ser) and 273 (arg-his) were transfected into K1735 C19H cells. K1735 C19H cells expressing human p53 genes with mutations at codons 249, both the arg-ser mutation and the mutation from the H7 hybrid and 273 produced significantly more lung metastases.^ In vitro assays demonstrated that responses to various cytotoxic and DNA damaging agents varied with the presence of mutant p53 and with the type of agent used. When cultured in mouse lung-conditioned medium, the K1735 C19H cell line was growth-inhibited, while cells containing a mutant human p53 (either on the whole chromosome 17, as in the H7 hybrid cells or from a stably transfected construct) were growth stimulated. Western blot analysis of lung-conditioned media derived from either 6-month or 15-month old mice has detected high levels of soluble Fas ligand in the medium from older animals. Comparison of the levels of Fas receptor on the K1735 C19H cell line and the H7 hybrid were almost identical, but 50% of the K1735 C19H cells were killed in the presence of anti-Fas antibody as opposed to 7% of the H7 hybrid cells. The growth-inhibitory effects of the lung-conditioned medium on the K1735 C19H cells were abrogated by coculture with Fas-Fc, which competes with the Fas ligand for receptor binding. Growth-inhibition of the K1735 C19H was 54% when cultured in 60 $\mu$g/0.2 ml lung-conditioned medium and a control Fc, with only 9% inhibition in 60 $\mu$g/0.2 ml lung-conditioned medium and Fas-Fc. Growth of the H7 cells and K1735 C19H cells transfected with various mutant human p53 genes were unchanged by the presence of either the control Fc or the Fas-Fc. This indicates that the presence of human chromosome 17, and mutant p53 in part protects the cells from Fas:Fas ligand induced apoptosis, and allows the growth of lung metastases. ^

Federally regulated institutional review boards: The aspects of a "model" IRB---Is the current system in need of a tune up or complete overhaul?

Institutional Review Boards (IRBs) are the primary gatekeepers for the protection of ethical standards of federally regulated research on human subjects in this country. This paper focuses on what general, broad measures that may be instituted or enhanced to exemplify a "model IRB". This is done by examining the current regulatory standards of federally regulated IRBs, not private or commercial boards, and how many of those standards have been found either inadequate or not generally understood or followed. The analysis includes suggestions on how to bring about changes in order to make the IRB process more efficient, less subject to litigation, and create standardized educational protocols for members. The paper also considers how to include better oversight for multi-center research, increased centralization of IRBs, utilization of Data Safety Monitoring Boards when necessary, payment for research protocol review, voluntary accreditation, and the institution of evaluation/quality assurance programs. ^ This is a policy study utilizing secondary analysis of publicly available data. Therefore, the research for this paper focuses on scholarly medical/legal journals, web information from the Department of Health and Human Services, Federal Drug Administration, and the Office of the Inspector General, Accreditation Programs, law review articles, and current regulations applicable to the relevant portions of the paper. ^ Two issues are found to be consistently cited by the literature as major concerns. One is a need for basic, standardized educational requirements across all IRBs and its members, and secondly, much stricter and more informed management of continuing research. There is no federally regulated formal education system currently in place for IRB members, except for certain NIH-based trials. Also, IRBs are not keeping up with research once a study has begun, and although regulated to do so, it does not appear to be a great priority. This is the area most in danger of increased litigation. Other issues such as voluntary accreditation and outcomes evaluation are slowing gaining steam as the processes are becoming more available and more sought after, such as JCAHO accrediting of hospitals. ^ Adopting the principles discussed in this paper should promote better use of a local IRBs time, money, and expertise for protecting the vulnerable population in their care. Without further improvements to the system, there is concern that private and commercial IRBs will attempt to create a monopoly on much of the clinical research in the future as they are not as heavily regulated and can therefore offer companies quicker and more convenient reviews. IRBs need to consider the advantages of charging for their unique and important services as a cost of doing business. More importantly, there must be a minimum standard of education for all IRB members in the area of the ethical standards of human research and a greater emphasis placed on the follow-up of ongoing research as this is the most critical time for study participants and may soon lead to the largest area for litigation. Additionally, there should be a centralized IRB for multi-site trials or a study website with important information affecting the trial in real time. There needs to be development of standards and metrics to assess the performance of the IRBs for quality assurance and outcome evaluations. The boards should not be content to run the business of human subjects' research without determining how well that function is actually being carried out. It is important that federally regulated IRBs provide excellence in human research and promote those values most important to the public at large.^

The application of principal component analysis on human development indicators: Temporal approach from 1999 to 2010

Background and Objective. Ever since the human development index was published in 1990 by the United Nations Development Programme (UNDP), many researchers started searching and corporative studying for more effective methods to measure the human development. Published in 1999, Lai’s “Temporal analysis of human development indicators: principal component approach” provided a valuable statistical way on human developmental analysis. This study presented in the thesis is the extension of Lai’s 1999 research. ^ Methods. I used the weighted principal component method on the human development indicators to measure and analyze the progress of human development in about 180 countries around the world from the year 1999 to 2010. The association of the main principal component obtained from the study and the human development index reported by the UNDP was estimated by the Spearman’s rank correlation coefficient. The main principal component was then further applied to quantify the temporal changes of the human development of selected countries by the proposed Z-test. ^ Results. The weighted means of all three human development indicators, health, knowledge, and standard of living, were increased from 1999 to 2010. The weighted standard deviation for GDP per capita was also increased across years indicated the rising inequality of standard of living among countries. The ranking of low development countries by the main principal component (MPC) is very similar to that by the human development index (HDI). Considerable discrepancy between MPC and HDI ranking was found among high development countries with high GDP per capita shifted to higher ranks. The Spearman’s rank correlation coefficient between the main principal component and the human development index were all around 0.99. All the above results were very close to outcomes in Lai’s 1999 report. The Z test result on temporal analysis of main principal components from 1999 to 2010 on Qatar was statistically significant, but not on other selected countries, such as Brazil, Russia, India, China, and U.S.A.^ Conclusion. To synthesize the multi-dimensional measurement of human development into a single index, the weighted principal component method provides a good model by using the statistical tool on a comprehensive ranking and measurement. Since the weighted main principle component index is more objective because of using population of nations as weight, more effective when the analysis is across time and space, and more flexible when the countries reported to the system has been changed year after year. Thus, in conclusion, the index generated by using weighted main principle component has some advantage over the human development index created in UNDP reports.^