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The acute hepatic porphyrias (AHPs) are inborn errors of heme biosynthesis, which include three autosomal dominant porphyrias, Acute Intermittent Porphyria (AIP), Hereditary Coproporphyria (HCP), and Variegate Porphyria (VP), and the ultra-rare autosomal recessive porphyria, δ-Aminolevulinic Acid Dehydratase Deficiency Porphyria (ADP). AIP, HCP, VP, and ADP each results from loss-of-function (LOF) mutations in their disease-causing genes: hydroxymethylbilane synthase (HMBS); coproporphyrinogen oxidase (CPOX); protoporphyrinogen oxidase (PPOX), and δ-aminolevulinic acid dehydratase (ALAD), respectively. During the 11-year period from January 1, 2007 through December 31, 2017, the Mount Sinai Porphyrias Diagnostic Laboratory diagnosed 315 unrelated AIP individuals with HMBS mutations, including 46 previously unreported mutations, 29 unrelated HCP individuals with CPOX mutations, including 11 previously unreported mutations, and 54 unrelated VP individuals with PPOX mutations, including 20 previously unreported mutations. Overall, of the 1692 unrelated individuals referred for AHP molecular diagnostic testing, 398 (23.5%) had an AHP mutation. Of the 650 family members of mutation-positive individuals tested for an autosomal dominant AHP, 304 (46.8%) had their respective family mutation. These data expand the molecular genetic heterogeneity of the AHPs and document the usefulness of molecular testing to confirm the positive biochemical findings in symptomatic patients and identify at-risk asymptomatic family members.

MicroRNAs (miRNAs) are class of small RNA molecules with major impact on gene regulation. We analyzed the potential of miRNAs secreted from pre-implantation embryos into the embryonic culture media as biomarkers to predict successful pregnancy. Using microarray analysis, we profiled the miRNome of the 56 spent culture media (SCM) after embryos transfer and found a total of 621 miRNAs in the SCM. On average, we detected 163 miRNAs in SCM of samples with failed pregnancies, but only 149 SCM miRNAs of embryos leading to pregnancies. MiR-634 predicted an embryo transfer leading to a positive pregnancy with an accuracy of 71% and a sensitivity of 85%. Among the 621 miRNAs, 102 (16.4%) showed a differential expression between positive and negative outcome of pregnancy with miR-29c-3p as the most significantly differentially expressed miRNA. The number of extracellular vehicles was lower in SCM with positive outcomes (3.8 × 109/mL EVs), as compared to a negative outcome (7.35 × 109/mL EVs) possibly explaining the reduced number of miRNAs in the SCM associated with failed pregnancies. The analysis of the miRNome in the SCM of couples undergoing fertility treatment lays the ground towards development of biomarkers to predict successful pregnancy and towards understanding the role of embryonic miRNAs found in the SCM.

Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content.

Organic semiconductor distributed feedback (DFB) lasers are of interest as external or chip-integrated excitation sources in the visible spectral range for miniaturized Raman-on-chip biomolecular detection systems. However, the inherently limited excitation power of such lasers as well as oftentimes low analyte concentrations requires efficient Raman detection schemes. We present an approach using surface-enhanced Raman scattering (SERS) substrates, which has the potential to significantly improve the sensitivity of on-chip Raman detection systems. Instead of lithographically fabricated Au/Ag-coated periodic nanostructures on Si/SiO2 wafers, which can provide large SERS enhancements but are expensive and time-consuming to fabricate, we use low-cost and large-area SERS substrates made via laser-assisted nanoreplication. These substrates comprise gold-coated cyclic olefin copolymer (COC) nanopillar arrays, which show an estimated SERS enhancement factor of up to ∼ 10(7). The effect of the nanopillar diameter (60-260 nm) and interpillar spacing (10-190 nm) on the local electromagnetic field enhancement is studied by finite-difference-time-domain (FDTD) modeling. The favorable SERS detection capability of this setup is verified by using rhodamine 6G and adenosine as analytes and an organic semiconductor DFB laser with an emission wavelength of 631.4 nm as the external fiber-coupled excitation source.

Cholesteryl ester storage disease (CESD) and Wolman disease are autosomal recessive later-onset and severe infantile disorders, respectively, which result from the deficient activity of lysosomal acid lipase (LAL). LAL is encoded by LIPA (10q23.31) and the most common mutation associated with CESD is an exon 8 splice junction mutation (c.894G>A; E8SJM), which expresses only ∼3%-5% of normally spliced LAL. However, the frequency of c.894G>A is unknown in most populations. To estimate the prevalence of CESD in different populations, the frequencies of the c.894G>A mutation were determined in 10,000 LIPA alleles from healthy African-American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish individuals from the greater New York metropolitan area and 6,578 LIPA alleles from African-American, Caucasian, and Hispanic subjects enrolled in the Dallas Heart Study. The combined c.894G>A allele frequencies from the two cohorts ranged from 0.0005 (Asian) to 0.0017 (Caucasian and Hispanic), which translated to carrier frequencies of 1 in 1,000 to ∼1 in 300, respectively. No African-American heterozygotes were detected. Additionally, by surveying the available literature, c.894G>A was estimated to account for 60% (95% confidence interval [CI]: 51%-69%) of reported mutations among multiethnic CESD patients. Using this estimate, the predicted prevalence of CESD in the Caucasian and Hispanic populations is ∼0.8 per 100,000 (∼1 in 130,000; 95% CI: ∼1 in 90,000 to 1 in 170,000). Conclusion: These data indicate that CESD may be underdiagnosed in the general Caucasian and Hispanic populations, which is important since clinical trials of enzyme replacement therapy for LAL deficiency are currently being developed. Moreover, future studies on CESD prevalence in African and Asian populations may require full-gene LIPA sequencing to determine heterozygote frequencies, since c.894G>A is not common in these racial groups. (HEPATOLOGY 2013;53:958–965)

X-linked protoporphyria (XLP) (MIM 300752) is a recently recognized erythropoietic porphyria due to gain-of-function mutations in the erythroid-specific aminolevulinate synthase gene (ALAS2). Previously, two exon 11 small deletions, c.1699_1670ΔAT (ΔAT) and c.1706_1709ΔAGTG (ΔAGTG), that prematurely truncated or elongated the ALAS2 polypeptide, were reported to increase enzymatic activity 20- to 40-fold, causing the erythroid accumulation of protoporphyrins, cutaneous photosensitivity and liver disease. The mutant ΔAT and ΔAGTG ALAS2 enzymes, two novel mutations, c.1734ΔG (ΔG) and c.1642C>T (p.Q548X), and an engineered deletion c.1670-1671TC>GA p.F557X were expressed, and their purified enzymes were characterized. Wild-type and ΔAGTG enzymes exhibited similar amounts of 54- and 52-kDa polypeptides on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), whereas the ΔAT and p.F557X had only 52-kDa polypeptides. Compared to the purified wild-type enzyme, ΔAT, ΔAGTG and Q548X enzymes had increased specific activities that were only 1.8-, 3.1- and 1.6-fold, respectively. Interestingly, binding studies demonstrated that the increased activity Q548X enzyme did not bind to succinyl-CoA synthetase. The elongated ΔG enzyme had wild-type specific activity, kinetics and thermostability; twice the wild-type purification yield (56 versus 25%); and was primarily a 54-kDa form, suggesting greater stability in vivo. On the basis of studies of mutant enzymes, the maximal gain-of function region spanned 57 amino acids between 533 and 580. Thus, these ALAS2 gain-of-function mutations increased the specific activity (ΔAT, ΔAGTG and p.Q548X) or stability (ΔG) of the enzyme, thereby leading to the increased erythroid protoporphyrin accumulation causing XLP.

Tetraspanins comprise a large family of integral membrane proteins involved in the regulation of cell adhesion, migration and fusion. In humans it consists of 33 members divided in four subfamilies. Here, we examined the spatial and temporal gene expression of four related tetraspanins during the embryonic development of Xenopus laevis by quantitative real-time PCR and in situ hybridization: Tspan3 (encoded by the gene Tm4sf8 gene) Tspan4 (encoded by the gene Tm4sf7), Tspan5 (encoded by the gene Tm4sf9) and Tspan7 (encoded by the gene Tm4sf2). These genes appeared first in the vertebrates during the evolution and are conserved across different species. In humans, they were associated with several diseases such as sclerosis, mental retardation and cancer; however their physiological role remained unclear. This work provides a comprehensive comparative analysis of the expression of these tetraspanins during the development of X. laevis. The more closely related tetraspanins Tspan3, Tspan4 and Tspan7 exhibited very similar spatial expression patterns, albeit differing in their temporal occurrence. The corresponding transcripts were found in the dorsal animal ectoderm at blastula stage. At early tailbud stages (stage 26) the genes were expressed in the migrating cranial neural crest located in the somites, developing eye, brain, and in otic vesicles. In contrast, Tspan5 appeared first at later stages of development and was detected prominently in the notochord. These data support close relatedness of Tspan3, Tspan4 and Tspan7. The expression of these tetraspanins in the cells with a high migratory potential, e.g. neural crest cells, suggests their role in the regulation of migration processes, characteristic for tetraspanin family members, during development. Similarity of the expression profiles might indicate at least partial functional redundancy, which is in concordance with earlier findings of tissue-limited or absent phenotypes in the knock-down studies of tetraspanins family members performed.

Malignant transformation is associated with enhancement of cell plasticity, which allows cancer cells to survive under different conditions by adapting to their microenvironment during growth and metastatic spread. Much effort has been devoted to understanding the molecular mechanisms of these processes. Although the importance of the extracellular matrix and of surface properties in these mechanisms is evident, the direct impact of distinct physical and chemical surfaces characteristics on cell fate remains unclear. Here, we have addressed this question using HT1080 fibrosarcoma cells as a model. To examine the relationship between surface topography, chemistry, and cell behavior, hydrophobic poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA-EDMA) and hydrophilic poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) surfaces with three different topographies (microporous, nanoporous, and nonporous) were generated. These surfaces were then modified by photoinitiated grafting of three different methacrylate monomers to create surface chemistry gradients of either negatively (AMPS) or positively (META) charged or zwitterionic (MDSA) functionalities. Our results show that AMPS promotes cell spreading, but that META abolishes cell growth. META and MDSA grafted on microporous BMA-EDMA produced superhydrophilic surfaces with high globularity and elasticity, which modified the cell phenotype by inhibiting cell spreading, followed by loss of mesenchymal characteristics and a reduction in protein levels of the mesenchymal markers N-cadherin, beta-catenin, p120 catenin, and also of the adaptor proteins vinculin and paxillin that are associated with adhesion and cancer cell invasion. The effect was strengthened along the gradient, suggesting that the density of the functional groups plays a role in this process. On the nanoporous surface, only MDSA grafting resulted in a significant increase in cell number, a reduction in N-cadherin expression, increased beta-catenin and p120 catenin levels, as well as the appearance of the epithelial marker E-cadherin. This indicates that the cancer cells have a high plasticity that is triggered by the collaborative effect of physical and chemical surface properties.

In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia.

collaboration au projet H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD); International audience; Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and.Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

In recent years there has been tremendous interest in both the basic biology and applications of extracellular vesicles (EVs) in translational cancer research. This includes a better understanding of their biogenesis and mechanisms of selective cargo packaging, their precise roles in horizontal communication, and their application as non-invasive biomarkers. The rapid advances in next-generation omics technologies are the driving forces for these discoveries. In this review, the authors focus on recent results of EV research in ovarian cancer. A deeper understanding of ovarian cancer-derived EVs, the types of cargo molecules and their biological roles in cancer growth, metastases and drug resistance, could have significant impact on the discovery of novel biomarkers and innovative therapeutics. Insights into the role of EVs in immune regulation could lead to novel approaches built on EV-based immunotherapy.

Cell populations often display heterogeneous behavior, including cell-to-cell variations in morphology, adhesion and spreading. However, better understanding the significance of such cell variations for the function of the population as a whole requires quantitative single-cell assays. To investigate adhesion variability in a CHO cell population in detail, we measured integrin-mediated adhesion to laminin and collagen, two ubiquitous ECM components, by AFM-based single-cell force spectroscopy (SCFS). CHO cells generally adhered more strongly to laminin than collagen but population adhesion force distributions to both ECM components were broad and partially overlapped. To determine the levels of laminin and collagen binding in individual cells directly, we alternatingly measured single cells on adjacent microstripes of collagen and laminin arrayed on the same adhesion substrate. In repeated measurements (≥60) individual cells showed a stable and ECM type-specific adhesion response. All tested cells bound laminin more strongly, but the scale of laminin over collagen binding varied between cells. Together, this demonstrates that adhesion levels to different ECM components are tightly yet differently set in each cell of the population. Adhesion variability to laminin was non-genetic and cell cycle-independent but scaled with the range of α6 integrin expression on the cell surface. Adhesive cell-to-cell variations due to varying receptor expression levels thus appear to be an inherent feature of cell populations and should to be considered when fully characterizing population adhesion. In this approach, SCFS performed on multifunctional adhesion substrates can provide quantitative single-cell information not obtainable from population-averaging measurements on homogeneous adhesion substrates.

The hc2 human papillomavirus DNA test (HC2) is effective when screening women for cervical dysplasia, and it might be effective in the screening for anal dysplasia. Differences between the anal and the cervical canals could affect the test performance. This prospective study (n = 292) measured the HC2 signal and in agreement with a histologic endpoint of high- grade dysplasia for anal specimens collected in various ways. Sensitivities were 91%, 85%, and 62% for specimens collected in a sample transport medium and a liquid-based cytology medium processed by Gyn or Non-Gyn protocol, respectively. HC2 sensitivity and specificity to predict high-grade anal dysplasia were similar for brush or swab specimen collections, but HC2 signal was 6 times higher with the brush. Specificity and sensi- tivity were similar whether the sample was collected first or after a cytology sample for brush or swab, but swab specimens at the second collection had an HC2 signal (mean) 48% lower than that of the first collection, and the swab cellularity was lower. The presence of maximum stool decreased the HC2 signal in anal swab specimens. Consensus polymerase chain reaction (PCR) confirmed that the 13 human papillomavirus probe types in HC2 were optimal for performance. HC2 could potentially be further investigated for use in screening anal dysplasia. A larger prospective study is indicated.

Human papillomavirus (HPV) DNA screening benefits cervical cancer diagnosis, but a few HPV infections result in cancer. Assays that predict cancer are desirable. A potential biomarker is the ratio of HPV E6–7 over E2 transcripts, which may increase during early cancer progression. Modified hybrid-capture technology detected, in separate wells, HPV E6–7 or E2 mRNA of HPV 16 or HPV 18 in samples. The limit of detection was approximately 1000 copies of in vitro transcribed RNA with linear dynamic range approximately four logs. No cross-reactivity between HPV 16 and HPV 18 mRNAs was detected. RNA of SiHa cells was stable in clinical specimen pools for 67 days, as determined by RT-PCR. The ratio of HPV E6–7:E2 mRNAs was relatively high for cancer cells lines, SiHa, Caski and HeLa cells preserved in clinical specimen pools. A broad distribution of HPV 16 E6–7:E2 mRNA ratio was detected in a small set of clinical specimens with various histological diagnoses. Some specimen ratios were so high for cancer cell lines, but the significance of the results needs to be determined. This method may help determine the pattern of gene expression in HPV-related disease or in other systems.

Metastasis, the life-threatening aspect of cancer, is a systemic disease process. Considerable progress has been made in recent years regarding how tumor cells circulating in the blood and lymphatic systems interact with and extravasate into secondary sites, and what determines whether these disseminated tumors cells survive, remain dormant or go on to form macrometastases. New insights into the routes that tumor cells take once leaving the primary tumor have emerged. Novel concepts regarding early seeding of metastases coupled to parallel progression, self-seeding of primary tumors by circulating tumor cells and the induction of premetastatic niches in distant organs by primary tumors have come to the fore. The perceived role of the lymphatic system in determining patterns of metastasis formation in distant organs has been reassessed. Together these new insights have the potential to offer new therapeutic options. In particular, the regulation of tumor cell dormancy emerges as a key event in metastasis formation, and therapeutic control of dormancy holds the promise of rendering cancer a chronic rather than life-threatening disease.

Hereditary coproporphyria (HCP) is an autosomal dominant acute hepatic porphyria due to the half-normal activity of the heme biosynthetic enzyme, coproporphyrinogen oxidase (CPOX). The enzyme catalyzes the step-wise oxidative decarboxylation of the heme precursor, coproporphyrinogen III, to protoporphyrinogen IX via a tricarboxylic intermediate, harderoporphyrinogen. In autosomal dominant HCP, the deficient enzymatic activity results primarily in the accumulation of coproporphyrin III. To date, only a few homozygous HCP patients have been described, most having Harderoporphyria, a rare variant due to specific CPOX mutations that alter enzyme residues D400-K404, most patients described to date having at least one K404E allele. Here, we describe a Turkish male infant, the product of a consanguineous union, who presented with the Harderoporphyria phenotype including neonatal hyperbilirubinemia, hemolytic anemia, hepatosplenomegaly, and skin lesions when exposed to UV light. He was homoallelic for the CPOX missense mutation, c.980A > G (p.H327R), and had massively increased urinary uroporphyrins I and III (9,250 and 2,910 mu M, respectively) and coproporphyrins I and III (895 and 19,400 mu M, respectively). The patient expired at 5 months of age from an apparent acute neurologic porphyric attack. Structural studies predicted that p.H327R interacts with residue W399 in the CPOX active site, thereby accounting for the Harderoporphyria phenotype.

Intracellular signaling governed by serine/threonine kinases comprises the molecular interface between cell surface receptors and the nuclear transcriptional machinery. The protein kinase C (PKC) family members are involved in the control of many signaling processes directing cell proliferation, motility, and survival. Here, we examined a role of different PKC isoenzymes in protein phosphatase 2A (PP2A) and HRSL3 tumor suppressor–dependent cell death induction in the ovarian carcinoma cell line OVCAR-3. Phosphorylation and activity of PKC isoenzymes were measured in response to PP2A or phosphoinositide 3-kinase inhibition or HRSL3 overexpression. These experiments indicated a regulation of PKCθ, ϵ, ζ, and ι through PP2A and/or HRSL3, but not of PKCα and β. Using isoform-specific peptide inhibitors and overexpression approaches, we verified a contribution to PP2A- and HRLS3-dependent apoptosis only for PKCζ, suggesting a proapoptotic function of this kinase. We observed a significant proportion of human ovarian carcinomas expressing high levels of PKCζ, which correlated with poor prognosis. Primary ovarian carcinoma cells isolated from patients also responded to okadaic acid treatment with increased phosphorylation of PKCζ and apoptosis induction. Thus, our data indicate a contribution of PKCζ in survival control in ovarian carcinoma cells and suggest that upregulation or activation of tyrosine kinase receptors in this tumor might impinge onto apoptosis control through the negative regulation of the atypical PKCζ. Mol Cancer Res; 8(6); 919–34. ©2010 AACR.

Background It has been shown that women positive for HPV 16 and HPV 18 have an increased risk of high-grade cervical intraepithelial neoplasia (CIN) compared with women positive for other high-risk (HR) HPV types. In addition, HPV 18 and HPV 45 have been closely linked to aggressive and difficult to detect adenocarcinomas. Objectives To develop a test based on the Hybrid Capture® technology capable of specifically detecting the most important carcinogenic HPV types; 16, 18, and 45. Study design The assay is based on Hybrid Capture technology utilizing a mixture of short type-specific oligoribonucleotides to detect HPV types 16, 18, or 45. The assay utilizes no target amplification and shares workflow and critical reagents with the Digene HC2 HPV screening assay. Studies to evaluate specificity, performance of the test in comparison to HC2, and capability to detect a single genotype in the presence of multiple infections are described. Specificity was evaluated analytically using a panel of HR- and LR-HPV types to illustrate cross-reactivity. Performance in comparison to the HC2 test was evaluated by testing aliquots of the same prepared samples by the genotyping test and HC2. Ability to detect a single genotype during multiple infections was modeled by detecting HPV 16 plasmid in the presence of HPV 6 or HPV 31 at high copy numbers. Results The proposed genotyping assay specifically detects HPV 16, 18, and 45 with an analytical sensitivity of 5,000 copies per assay. The assay is highly specific and does not detect other tested high-risk or low-risk types at 10 8 copies per reaction. Utility of the genotyping test was demonstrated using clinical samples collected in Digene Specimen Transport Medium (STM) and results were confirmed by PCR. Conclusions The target-amplification free assay provides a genotyping method for highly specific detection of HPV 16, 18, and 45 without the complexity of PCR technology.

A novel DNA detection assay comprising Hybrid Capture® sample preparation, GP5+/6+ PCR with modifications and Luminex® 100 detection was developed and applied to genotyping of human papillomavirus (HPV) in cervical samples. Target-specific sample preparation was performed using magnetic beads conjugated with Hybrid Capture® (HC) antibody. DNA–RNA hybrids were formed between DNA target and RNA probes and captured on HC-beads. DNA on magnetic beads was amplified without elution using consensus GP5+/6+ PCR and then genotyped on Luminex® beads using hybridization probes for the 17 high-risk HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, 82 and an internal control. This new sequence-specific Hybrid Capture® sample preparation is fast, efficient and allows direct HPV genotyping by PCR. Compared to traditional non-sequence-specific sample preparation methods, HC sample preparation demonstrated slightly better detection of multiple HPV infections. The clinical utility of this method was demonstrated on cervical samples positive for HR HPV by the Hybrid Capture® 2 (hc2) screening assay.

Analyzing the growth of fibrosarcoma lines derived from IL-1alpha-, IL-1beta- , or IL-1alphabeta-knockout (-/-) mice in the immunocompetent host revealed that tumor-derived IL-1alpha and IL-1beta exert strong and opposing effects on immune response induction, which prohibited the evaluation of a potential impact on tumorigenicity. Therefore, in vivo growth of IL-1-deficient tumor lines was evaluated in nu/nu mice and was compared with in vitro growth characteristics. All IL-1-deficient fibrosarcoma lines grow in immunocompromised mice. However, IL-1alpha(-/-)beta-competent (comp) lines grow more aggressively, efficiently induce angiogenesis, and recruit inflammatory cells. Despite stronger tumorigenicity of IL-1beta(comp) lines, IL-1alpha strengthens anchorage-independent growth, but both IL-1alpha and IL-1beta support drug resistance. Corresponding to the aggressive growth, IL-1beta(comp) cells display increased matrix adhesion, motility, and cable formation on matrigel, likely supported by elevated alpha(v)/beta3 and matrix metalloroteinase expression. Recruitment of myeloid cells requires IL-1beta but is regulated by IL-1alpha, because inflammatory chemokine and cytokine expression is stronger in IL-1alpha(-/-)beta(comp) than in IL-1(wt) lines. This regulatory effect of tumor-derived IL-1alpha is restricted to the tumor environment and does not affect systemic inflammatory response induction by tumor-derived IL-1beta. Both sarcoma cell-derived IL-1alpha and IL-1beta promote tumor growth. However, IL-1alpha exerts regulatory activity on the tumor cell-matrix cross-talk, and only IL-1beta initiates systemic inflammation.

There is evidence that cell-associated IL-1 alpha supports immune response induction. Here we explored the impact of malignant cell-derived IL-1 on immunogenicity, immune response induction and tumor-induced immunosuppression using 3-methylcholanthrene-induced fibrosarcoma lines derived from wild-type (wt), IL-1 alpha-, IL-1 beta- or IL-1a beta-knockout (IL-1 alpha(-/-), IL-1 beta(-/-), IL-1 alphabeta(-/-)) C57BL6 mice. The wt, IL-1 alpha(-/-), IL-1 beta(-/-) and IL-1 alphabeta(-/-) fibrosarcoma lines express MHC class I molecules at a high level. The lines do not differ in their susceptibility toward NK cells, macrophages, and allogeneic CTL, or in their capacity as stimulators of an allogeneic response. However, IL-1 beta(-/-) tumors rarely grow in the syngeneic host, which is the consequence of a strong T helper and CTL response induction by IL-1 alpha-competent, IL-1 beta(-/-) tumors. On the other hand, IL-1 beta-competent, IL-1 alpha(-/-) tumors strongly assist CD11b(+)Gr-1(+) myeloid-derived suppressor cell and regulatory T cell expansion, which both suppress with high efficacy activated T helper cell proliferation and CTL lysis. In IL-1 alphabeta(-/-) tumors, the absence of IL-1 alpha becomes decisive, i.e. despite reduced suppressor cell recruitment, tumor growth was unimpaired due to inefficient immune response induction. Thus, sarcoma cell-derived IL-1 alpha and IL-1 beta do not act in concert. Induction of a strong immune response by IL-1 alpha demands therapeutic exploitation, which may become more efficient if systemic induction of immunosuppression by IL-1 beta can also be circumvented.

Low-frequency blood pressure oscillations (Mayer waves) are discussed as a marker for sympathetic modulation of vascular tone. However, the factors that determine the frequency response of the vasculature to sympathetic stimuli are not fully understood. Possible mechanisms include functions related to α1-adrenergic receptors (α1-AR) and postreceptor processes involved in the vascular contractile response. The purpose of the present study was to examine the hypothesis that expression levels of α1-AR and their subtype distribution determine velocity and magnitude of α1-AR-mediated vascular smooth muscle cell (VSMC) contraction. α1A-, α1B-, and α1D-AR subtypes were transfected into VSMCs from rat aorta and characterized immunocytochemically via confocal microscopy. Functional studies in isolated cells were performed using video microscopy. The α1-AR agonist phenylephrine produced dose-dependent contractions of VSMCs. All transfected groups were more sensitive to phenylephrine compared with controls. Maximal contraction velocity almost doubled in transfected cells. However, no differences in observed parameters were found between the three transfected groups. Contractile properties in response to membrane depolarization with KCl were similar in all groups. In conclusion, α1-AR density determines velocity and sensitivity of α1-AR-mediated contraction in VSMCs. α1-AR subtype distribution does not appear to influence vasoconstriction to sympathetic stimuli.

HRSL3 (also known as H-REV107-1) belongs to a class II tumor suppressor gene family and is downregulated in several human tumors including ovarian carcinomas. To unravel the mechanism of HRSL3 tumor suppressor action, we performed a yeast two-hybrid screen and identified the alpha-isoform of the regulatory subunit A of protein phosphatase 2A (PR65alpha) as a new interaction partner of HRSL3. Interaction between HRSL3 and PR65alpha was confirmed in vitro and by co-immunoprecipitation in mammalian cells. We demonstrate that HRSL3 binds to the endogenous PR65alpha, thereby partially sequestering the catalytic subunit PR36 from the PR65 protein complex, and inhibiting PP2A catalytic activity. Furthermore, binding of HRSL3 to PR65 induces apoptosis in ovarian carcinoma cells in a caspase-dependent manner. Using several mutant HRSL3 constructs, we identified the N-terminal proline-rich region within the HRSL3 protein as the domain that is relevant for both binding of PR65alpha and induction of programmed cell death. This suggests that the negative impact of HRSL3 onto PP2A activity is important for the HRSL3 pro-apoptotic function and indicates a role of PP2A in survival of human ovarian carcinomas. The analysis of distinct PP2A target molecules revealed PKCzeta as being involved in HRSL3 action. These data implicate HRSL3 as a signaling regulatory molecule, which is functionally involved in the oncogenic network mediating growth and survival of ovarian cancer cells.

The authors wish to thank Dr R Simpson and Dr D Taylor for critical reading of the manuscript and acknowledge the Horizon 2020 European Cooperation in Science and Technology programme and its support of our European Network on Microvesicles and Exosomes in Health & Disease (ME-HaD; BM1202 www.cost.eu/COST_Actions/bmbs/Actions/BM1202). In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

H-REV107-1, a known member of the class II tumor suppressor gene family, is involved in the regulation of differentiation and survival. We analyzed H-REV107-1 in non-small cell lung carcinomas, in normal lung, and in immortalized and tumor-derived cell lines. Sixty-eight percent of lung tumors revealed positive H-REV107-1-specific staining. Furthermore, survival analysis demonstrated a significant association of cytoplasmic H-REV107-1 with decreased patient survival. This suggested that H-REV107-1, known as a tumor suppressor, plays a different role in non-small cell lung carcinomas. Knock-down of H-REV107-1 expression in lung carcinoma cells inhibited anchorage-dependent and anchorage-independent growth whereas overexpression of H-REV107-1 induced tumor cell proliferation. Consistent with results of the survival analysis, cytoplasmic localization of the protein was essential for this growth-inducing function. Analysis of signaling pathways potentially involved in this process demonstrated that overexpression of H-REV107-1 stimulated RAS-GTPase activity, ERK1,2 phosphorylation, and caveolin-1 expression in the cell lines analyzed. These results indicate that H-REV107-1 is deficient in its function as a tumor suppressor in non-small cell lung carcinomas and is required for proliferation and anchorage-independent growth in cells expressing high levels of the protein, thus contributing to tumor progression in a subset of non-small cell lung carcinomas.

H-rev107-1 is a growth inhibitory RAS target gene capable of suppressing anchorage independent growth in vitro and in vivo. Using a tumour tissue array with 241 matched tumour and normal tissue cDNA pools, we found down-regulation of H-REV107-1 in 7 out of 14 ovary-derived cDNAs. RT-PCR analysis and immunohistochemical investigation confirmed expression of H-REV107-1 in normal ovarian epithelial cells but down-regulation in high grade ovarian carcinomas. H-REV107-1 is also strongly expressed in immortalized rat and human ovarian epithelial cells in vitro, but suppressed in transformed cells by two different mechanisms. KRAS-transformed rat ovarian cells and PA1 teratocarcinoma cells, reversibly repress H-REV107-1 via MAP/ERK signaling. In contrast, treatment of A27/80 and OVCAR-3 epithelial ovarian cancer cells with IFNgamma stimulated H-REV107-1 expression. In NIH3T3 cells harbouring an estrogen-inducible IRF-1, H-rev107-1 is directly induced after activation of IRF-1, indicating that H-rev107-1 is a target of IRF-1. Stimulation of H-REV107-1 expression was also observed in ovarian epithelial cells suggesting that IRF-1 is involved in H-REV107-1 regulation in human ovarian epithelium. In the IFNgamma-sensitive cell line A27/80, H-REV107-1 suppresses colony formation. A27/80 and OVCAR-3 cells overexpressing H-REV107-1 protein underwent apoptosis. These results demonstrate down-regulation of the class II tumour suppressor H-REV107-1 in human ovarian carcinomas and suggest an involvement of H-REV107-1 in interferon-dependent cell death.

The information about the range and distribution of Human papillomavirus load in clinical specimens is important for the design of accurate clinical tests. The amount of Human papillomavirus in cervical specimens was estimated using the digene HC2 HPV DNA Test® (QIAGEN). This semi-quantitative assay is based on linear signal amplification with an analytical limit-of-detection of approximately 2500 virus copies per assay and 3–4 log dynamic range. The dynamic range of the assay was extended by a serial dilution strategy. Two large sets of positive specimens (n = 501 and 569) were analyzed and 9–11% of specimens was estimated to contain more than 7 × 107 copies of virus. The viral load was also assessed for an assortment of specimens with known cytology diagnoses (n = 9435) and histological diagnoses (n = 2056). The percentage of specimens with more than 7 × 107 copies of virus was estimated to be 0.89 for normal cells, 4.2 for atypical cells (unknown significance), 14.31 for cells of low-grade lesions and 22.24 for cells of high-grade lesions. The viral load increased with disease severity, but its broad distribution may not support its use as a disease biomarker. This information is important for assay design and automation, where cross-reactivity and sample-to-sample contamination must be addressed rigorously.

The knowledge gained from comprehensive profiling projects that aim to define the complex genomic alterations present within cancers will undoubtedly improve our ability to detect and treat those diseases, but the influence of these resources on our understanding of basic cancer biology is still to be demonstrated. Extracellular vesicles have gained considerable attention in past years, both as mediators of intercellular signalling and as potential sources for the discovery of novel cancer biomarkers. In general, research on extracellular vesicles investigates either the basic mechanism of vesicle formation and cargo incorporation, or the isolation of vesicles from available body fluids for biomarker discovery. A deeper understanding of the cargo molecules present in extracellular vesicles obtained from patients with urogenital cancers, through high-throughput proteomics or genomics approaches, will aid in the identification of novel diagnostic and prognostic biomarkers, and can potentially lead to the discovery of new therapeutic targets.

Setleis syndrome is characterized by bitemporal scar-like lesions and other characteristic facial features. It results from recessive mutations that truncate critical functional domains in the basic helix-loop-helix (bHLH) transcription factor, TWIST2, which regulates expression of genes for facial development. To date, only four nonsense or small deletion mutations have been reported. In the current report, the clinical findings in a consanguineous Turkish family were characterized. Three affected siblings had the characteristic features of Setleis syndrome. Homozygosity for the first TWIST2 missense mutation, c.326T>C (p.Leu109Pro), was identified in the patients. In silico analyses predicted that the secondary structure of the mutant protein was sustained, but the empirical force field energy increased to an unfavorable level with the proline substitution (p.Leu109Pro). On a crystallographically generated dimer, p.Leu109 lies near the dimer interface, and the proline substitution is predicted to hinder dimer formation. Therefore, p.Leu109Pro-TWIST2 alters the three dimensional structure and is unable to dimerize, thereby hindering the binding of TWIST2 to its target genes involved in facial development.

Extracellular membrane vesicles derived from the endosomal compartments and released by the fusion of the multivesicular bodies with the cell membrane are referred as exosomes (Exo) [Van Niel et al., J Biochem 140:13-21, 2006]. They function as mediators of intercellular communication and are employed by the organism in the regulation of systemic and local processes. Meantime, Exo are recognized as an indispensable entity of physiological fluids [Caby et al., Int Immunol 17:879-887, 2005; Lasser et al., J Transl Med 9:9, 2011; Lasser et al., Am J Rhinol Allergy 25:89-93, 2011]. Exo and other types of extracellular vesicles, e.g., exosome-like vesicles [van Niel et al., Gastroenterology 121:337-349, 2001] and microvesicles (MV) [Daveloose et al., Thromb Res 22:195-201, 1981], contain multiple functional molecules including lipids [Vidal et al., J Cell Physiol 140:455-462, 1989]; proteins [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; mRNA [Valadi et al., Nat Cell Biol 9:654-659, 2007]; DNA [Waldenstrom et al., PLoS One 7:e34653, 2012]; noncoding RNA, e.g., miRNA [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; and retrotransposon elements [Balaj et al., Nat Commun 2:180, 2011]. Assessment of the biological functions of Exo showed that they deliver specifically their cargo from the donor to recipient cells. Albeit the molecular mechanisms of this process are not fully understood, approaches for the application of Exo and MV as a tool for a cell-specific delivery of signalling molecules were successfully tested in in vitro and in vivo models [Maguire et al., Mol Ther 20:960-971, 2012]. Ovarian cancer cells release Exo, which bind stroma cells as well as donor cancer cells [Escrevente et al., BMC Cancer 11:108, 2011]. Here we describe an experimental approach for the assessment of Exo interaction and uptake by target cells. Methods for the isolation and purification of Exo from cell culture supernatants are included. To allow visualization of vesicle uptake, labelling of Exo with different fluorescent dyes, such as CFSE, PKH, DHPE, and DiOC18, is presented. Finally, we explain qualitative and quantitative analysis of Exo uptake by immunofluorescence and flow cytometry, respectively.

Focal facial dermal dysplasia (FFDD) Type IV is a rare syndrome characterized by facial lesions resembling aplasia cutis in a preauricular distribution along the line of fusion of the maxillary and mandibular prominences. To identify the causative gene(s), exome sequencing was performed in a family with two affected siblings. Assuming autosomal recessive inheritance, two novel sequence variants were identified in both siblings in CYP26C1-a duplication of seven base pairs, which was maternally inherited, c.844_851dupCCATGCA, predicting p.Glu284fsX128 and a missense mutation, c.1433G>A, predicting p.Arg478His, that was paternally inherited. The duplication predicted a frameshift mutation that led to a premature stop codon and premature chain termination, whereas the missense mutation was not functional based on its in vitro expression in mammalian cells. The FFDD skin lesions arise along the sites of fusion of the maxillary and mandibular prominences early in facial development, and Cyp26c1 was expressed exactly along the fusion line for these facial prominences in the first branchial arch in mice. Sequencing of four additional, unrelated Type IV FFDD patients and eight Type II or III TWIST2-negative FFDD patients revealed that three of the Type IV patients were homozygous for the duplication, whereas none of the Type II or III patients had CYP26C1 mutations. The seven base pairs duplication was present in 0.3% of healthy controls and 0.3% of patients with other birth defects. These findings suggest that the phenotypic manifestations of FFDD Type IV can be non-penetrant or underascertained. Thus, FFDD Type IV results from the loss of function mutations in CYP26C1.

Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are inborn errors of heme biosynthesis with the same phenotype but resulting from autosomal recessive loss-of-function mutations in the ferrochelatase (FECH) gene and gain-of-function mutations in the X-linked erythroid-specific 5-aminolevulinate synthase (ALAS2) gene, respectively. The EPP phenotype is characterized by acute, painful, cutaneous photosensitivity and elevated erythrocyte protoporphyrin levels. We report the FECH and ALAS2 mutations in 155 unrelated North American patients with the EPP phenotype. FECH sequencing and dosage analyses identified 140 patients with EPP: 134 with one loss-of-function allele and the common IVS3-48T>C low expression allele, three with two loss-of-function mutations and three with one loss-of-function mutation and two low expression alleles. There were 48 previously reported and 23 novel FECH mutations. The remaining 15 probands had ALAS2 gain-of-function mutations causing XLP: 13 with the previously reported deletion, c.1706_1709delAGTG, and two with novel mutations, c.1734delG and c.1642C>T(p.Q548X). Notably, XLP represented ~10% of EPP phenotype patients in North America, two to five times more than in Western Europe. XLP males had twofold higher erythrocyte protoporphyrin levels than EPP patients, predisposing to more severe photosensitivity and liver disease. Identification of XLP patients permits accurate diagnosis and counseling of at-risk relatives and asymptomatic heterozygotes.

Extracellular membrane vesicles derived from the endosomal compartments and released by the fusion of the multivesicular bodies with the cell membrane are referred as exosomes (Exo) [Van Niel et al., J Biochem 140:13-21, 2006]. They function as mediators of intercellular communication and are employed by the organism in the regulation of systemic and local processes. Meantime, Exo are recognized as an indispensable entity of physiological fluids [Caby et al., Int Immunol 17:879-887, 2005; Lasser et al., J Transl Med 9:9, 2011; Lasser et al., Am J Rhinol Allergy 25:89-93, 2011]. Exo and other types of extracellular vesicles, e.g., exosome-like vesicles [van Niel et al., Gastroenterology 121:337-349, 2001] and microvesicles (MV) [Daveloose et al., Thromb Res 22:195-201, 1981], contain multiple functional molecules including lipids [Vidal et al., J Cell Physiol 140:455-462, 1989]; proteins [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; mRNA [Valadi et al., Nat Cell Biol 9:654-659, 2007]; DNA [Waldenstrom et al., PLoS One 7:e34653, 2012]; noncoding RNA, e.g., miRNA [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; and retrotransposon elements [Balaj et al., Nat Commun 2:180, 2011]. Assessment of the biological functions of Exo showed that they deliver specifically their cargo from the donor to recipient cells. Albeit the molecular mechanisms of this process are not fully understood, approaches for the application of Exo and MV as a tool for a cell-specific delivery of signalling molecules were successfully tested in in vitro and in vivo models [Maguire et al., Mol Ther 20:960-971, 2012]. Ovarian cancer cells release Exo, which bind stroma cells as well as donor cancer cells [Escrevente et al., BMC Cancer 11:108, 2011]. Here we describe an experimental approach for the assessment of Exo interaction and uptake by target cells. Methods for the isolation and purification of Exo from cell culture supernatants are included. To allow visualization of vesicle uptake, labelling of Exo with different fluorescent dyes, such as CFSE, PKH, DHPE, and DiOC18, is presented. Finally, we explain qualitative and quantitative analysis of Exo uptake by immunofluorescence and flow cytometry, respectively.

Extracellular micro- and nano-scale membrane vesicles produced by different cells are recognized as an essential entity of physiological fluids in a variety of organisms and function as mediators of intercellular communication employed for the regulation of multiple systemic and local processes. In the last decade, an exponential amount of experimental work was dedicated to exploring the biogenesis and secretion mechanisms, physiological and pathological functions and potential applications of the extracellular vesicles (EVs). Noteworthy is the large heterogeneity of in vitro and in vivo models applied, technical approaches developed in these studies and the diversity of designations assigned to different or similar types of EVs. Hence, there is a clear necessity for a uniform nomenclature and standardization of methods to isolate and characterize these vesicles. In April 2012, the first meeting of the International Society for Extracellular Vesicles (ISEV) took place bringing together this exponentially grown scientific community. The University of Gothenburg (Krefting Research Centre) together with the Interim Board of the Society created in September 2011 (Jan Lotvall, Clotilde Thery, Xandra Breakefield, Marca Wauben, Yong Song Gho, Lawrence Rajendran, Graca Raposo, Douglas Taylor, Margareta Sjostrand and Esbjorn Telemo) organized this fantastic event that counted 488 registered and contributing participants. This meeting report provides a retrospective summary of the broad spectrum of ISEV-2012 sessions. Again, we emphasize novel findings, discussions and decisions met by the community during the meeting. 2012 Elisa Araldi et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited

Tumour initiation and progression are driven by constitutively activated oncogenes mediating deregulation of the balance between cell deathand survival pathways. Among the most relevant signalling cascades activated in the majority of tumour types, the RAS/mitogen-activated protein kinase (Ras/MAPK), the phosphatidyl inositol-3kinase/protein kinase B (PI3K/PKB) and the protein kinases C (PKC) signalling cascades were postulated (Weinstein, 1987; Nicosia et al., 2003; Roberts and Der, 2007; McCubrey et al., 2007; Breitkreutz et al., 2007). These cascades define individual characteristics of particular tumours and consequently their individual responsiveness to cancer therapy. In this chapter, we will address the characteristics of the apoptotic signalling pathways in ovarian carcinomas. Particular attention will be given to the HRS family of tumour suppressor genes encoding proteins with phospholipase activity and suppressed in the majority of ovarian malignancies. We will describe signalling cascades down regulating two well-characterized members of this family H-REV107-1/HRLS3/PLA2G16 and TIG3/RARRES/RIG1 in tumour cells. Furthermore, potential therapeutic consequences of the re-expression of these genes defined as a class II tumour suppressors will be discussed.

We have developed a new research assay that combines sequence-specific sample preparation and isothermal amplification for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae infections. The assay targets both the omp gene and the cryptic plasmid of C. trachomatis and the multicopy opa gene of N. gonorrhoeae , which are amplified and detected in a single reaction. We evaluated the ability of the assay to detect C. trachomatis and N. gonorrhoeae infections in first-catch urine, swab, and liquid-based cytology samples. Total agreement between the new assay and APTIMA Combo 2 varied between 95.3% and 100%, depending on the sample type and target detected. Total agreement between the new assay and BD ProbeTec varied between 96.7% and 100%, depending on the sample type and target detected. The assay has a simple work flow, and endpoint results can be achieved in 3 h, including sample preparation. The assay described here was evaluated for research use and was compared to commercially available assays.

Background The focal facial dermal dysplasias (FFDDs) are a group of inherited disorders of facial development, characterised by bitemporal or preauricular scar-like defects, the former resembling ‘forceps marks’. Recently, different homozygous TWIST2 nonsense mutations were reported in unrelated Setleis syndrome (FFDD Type III) patients from consanguineous families, consistent with autosomal recessive inheritance. Mexican-Nahua sibs with facial and ophthalmologic features of FFDD type III were evaluated. Methods Genomic DNAs were isolated for sequencing of the TWIST2 gene. The clinical features and inheritance of all previously reported FFDD patients were reviewed. Results The affected sibs were homozygous for a novel TWIST2 frameshift mutation, c.168delC (p.S57AfsX45). Notably, both parents and two heterozygous sibs had distichiasis and partial absence of lower eyelashes. The FFDD subtypes were reclassified: the ‘Brauer-Setleis’ phenotype (autosomal dominant with variable expressivity) as FFDD type II; and patients with preauricular lesions as a new subtype, FFDD type IV. Conclusions FFDD type III heterozygotes with TWIST2 mutations may have syndromic manifestations. Review of previous FFDD patients resulted in reclassification of the subtypes.

Hybrid Capture 2 (HC2) Human Papillomavirus (HPV) DNA Test® is FDA approved and is a proven aid in detecting HPV infections of the cervix and as an aid in diagnosing, with cytology, cervical disease. A prospective feasibility study was conducted to determine if HC2 testing has utility when screening for high-grade anal dysplasia (AIN2+). We enrolled 298 patients (45% HIV+) who had AIN2+ screening with cytology, histology and HC2 testing for two specimens: a swab into liquid-based cytology medium and either a swab or a brush collection in specimen transport medium (STM). High-resolution anoscopy was performed on all patients with biopsy of AIN2+ suspicious lesions. Cytology was benign (42%), atypical squamous cells of undetermined significance (30%), low-grade squamous intraepithelial lesion (18%), high-grade squamous intraepithelial lesion (1%), ASCUS possibly high-grade dysplasia (1.7%) and nondiagnostic (7%) and 36% had AIN2+ histology. Sensitivity and specificity for predicting AIN2+ histology for any abnormal cytology were 77 and 52%, whereas HC2 sensitivity and specificity were 91 and 40% (p = 0.005 for sensitivity), respectively. There was no significant difference in HC2 sensitivity or specificity between brush and swab or STM and residual cells from cytology. AIN2+ was found in 20% of patients with benign cytology. Only nine AIN2+ specimens were HC2−. This prospective study indicates that HC2 may be useful when screening for anal dysplasia; however, a larger study is recommended.

Thermophilic helicase dependent amplification (tHDA), which employs helicase to unwind double-stranded DNA at constant temperature, is a relatively new isothermal nucleic acid amplification technology. In this study, the development and optimization of a 4-plex tHDA assay for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are described. tHDA is combined with sequence-specific sample preparation on magnetic beads and homogeneous endpoint fluorescence detection using dual-labeled probes. This 4-plex tHDA assay was applied to the detection of 2 genes on CT and a multicopy gene on NG in the presence of an internal control. The assay showed high analytical sensitivity and specificity of simultaneous CT/NG detection and is compatible with a wide variety of sample types and media. The isothermal reaction conditions and homogeneous endpoint detection utilized in this assay are well suited for laboratory automation and high-throughput screening applications as well as for point-of-care testing.

Fabry disease, an X-linked lysosomal storage disorder, results from the deficient activity of α-galactosidase A (α-Gal A). In affected males, the clinical diagnosis is confirmed by the markedly decreased α-Gal A activity. However, in female heterozygotes, the α-Gal A activity can range from low to normal due to random X-chromosomal inactivation, and diagnostic confirmation requires identification of the family's α-Gal A gene mutation. In a young female who had occasional acroparesthesias, corneal opacities, and 15 to 50% of the lower limit of normal leukocyte α-Gal A activity, α-Gal A sequencing in two expert laboratories did not identify a confirmatory mutation, presenting a diagnostic dilemma. A renal biopsy proved diagnostic and renewed efforts to detect an α-Gal A mutation. Subsequent gene dosage analyses identified a large α-Gal A deletion confirming her heterozygosity, and she was started on enzyme replacement therapy. Thus, gene dosage analyses can detect large deletions (>50bp) in suspect heterozygotes for X-linked and autosomal dominant diseases that are "sequencing cryptic," resolving molecular diagnostic dilemmas.

BackgroundThe incidence of cancer in patients with neurological diseases, who have been treated with LiCl, is below average. LiCl is a well-established inhibitor of Glycogen synthase kinase-3, a kinase that controls several cellular processes, among which is the degradation of the tumour suppressor protein p53. We therefore wondered whether LiCl induces p53-dependent cell death in cancer cell lines and experimental tumours.ResultsHere we show that LiCl induces apoptosis of tumour cells bothin vitroandin vivo. Cell death was accompanied by cleavage of PARP and Caspases-3, -8 and -10. LiCl-induced cell death was not dependent on p53, but was augmented by its presence. Treatment of tumour cells with LiCl strongly increased TNF-α and FasL expression. Inhibition of TNF-α induction using siRNA or inhibition of FasL binding to its receptor by the Nok-1 antibody potently reduced LiCl-dependent cleavage of Caspase-3 and increased cell survival. Treatment of xenografted rats with LiCl strongly reduced tumour growth.ConclusionsInduction of cell death by LiCl supports the notion that GSK-3 may represent a promising target for cancer therapy. LiCl-induced cell death is largely independent of p53 and mediated by the release of TNF-α and FasL.Key words: LiCl, TNF-α, FasL, apoptosis, GSK-3, FasL

Infection with a high-risk carcinogenic type of human papillomavirus (HPV) is necessary for the development of cervical cancer. The digene HC2 HPV Test (HC2) is an important screening tool but lacks genotyping capability. To address this issue, we developed an assay for the rapid genotyping of HPV in cervical specimens. The three steps of this assay include Hybrid Capture target enrichment, whole-genome amplification, and Luminex XMAP detection. The assay includes the simultaneous detection of two genomic regions from each of 17 high-risk and two low-risk HPV types most associated with disease. The assay performance was tested on HPV plasmids as well as clinical specimens. An analytical limit of detection of 100 copies or less was demonstrated for linear, circular, and integrated HPV DNA. This finding is at least 1 log lower than the HC2 assay limit of detection. There was no cross-reactivity among the HPV types up to 1,000,000 copies. There was also no substantial assay interference from substances in cervical specimens. Although the clinical performance of the assay was not formally tested, the assay had good agreement (Cohen's kappa equal to 0.72) with both a PCR-based HPV genotyping assay (n = 131) and the HC2 assay (n = 502) using representative cervical specimens. This assay may be easy to automate and could be applied for the detection of other targets in future studies.

For most Mendelian disorders, targeted genome sequencing is an effective method to detect causative mutations. However, sequencing PCR-amplified exonic regions and their intronic boundaries can miss large deletions or duplications and mutations that lead to PCR failures in autosomal dominant disorders and in heterozygote detection for X-linked diseases. Here, a method is described for detecting large (>50 bp) deletions/duplications in the X-linked α-galactosidase A (GLA) gene, which cause Fabry disease. Briefly, multiplex PCR mixtures were designed to amplify each GLA exon and an unrelated internal control exon to normalize GLA exonic amplicon peak heights. For each normalized GLA amplicon, the normal control female to male peak-height ratios were 1.8 to 2.2 (expected 2.0), whereas the expected ratios for deletions or duplications would be ∼1.0 or 3.0, respectively. Using this method, three novel deletions, c.369+3_547+954del4096insT, c.194+2049_369+773del2619insCG, and c.207_369+651del814ins231, were detected in unrelated women with signs and/or symptoms suggestive of Fabry disease, but no "sequencing-detectable" mutations. The deletions were confirmed by sequencing their respective GLA RT-PCR products. This method can identify gene rearrangements that may be cryptic to genomic DNA sequencing and can be readily adapted to other X-linked or autosomal dominant genes.

Exosomes are most important intercellular communicators and tetraspanins/tetraspanin-complexes have been suggested to play an important role in exosomal target cell selection. We have shown that only exosomes expressing a Tspan8–CD49d complex preferentially bind endothelial cells, which initiates angiogenesis. This finding was unexpected as in the exosome donor cell Tspan8 is associated with CD49c and the tetraspanins CD9 and CD151. In view of the discussed therapeutic power of exosomes as message/drug transporter, it became important to clarify the mechanisms accounting for the distinct Tspan8-web in the cell membrane versus exosomes. We therefore compared the route of Tspan8 and Tspan8-chimera internalization, where the N- and/or C-terminal regions were exchanged with the corresponding regions of CD9 or CD151. Activation-induced Tspan8-internalization proceeds more rapidly than CD9 internalization and is accompanied by disassembly of the Tspan8–CD9–CD151 membrane complex in resting cells. Tspan8-internalization relies on the association of the Tspan8 N-terminal region with intersectin-2, a multimodular complex involved in clathrin-coated pit internalization. Internalization and recovery of Tspan8 in early endosomes is further promoted by the recruitment of CD49d such that only in PMA-activated cells a Tspan8–INS2–CD49d–clathrin complex is recovered in cholesterol-depletion-resistant membrane microdomains. PMA-induced Tspan8-internalization promotes cell migration, but reduces matrix and cell adhesion. Thus, stimulation initiates tetraspanin-web rearrangements, which have strong functional consequences for the cell, exosome-delivery and exosome target selection. This knowledge will be essential for generating tailored therapeutic exosomes.

Tumor-derived exosomes containing the tetraspanin Tspan8 can efficiently induce angiogenesis in tumors and tumor-free tissues. However, little information exists on exosome–endothelial cell (EC) interactions or the proangiogenic role of tetraspanins, which are a constitutive component of exosomes. In this study, we used a rat adenocarcinoma model (AS-Tspan8) to explore the effects of exosomal Tspan8 on angiogenesis. Tspan8 contributed to a selective recruitment of proteins and mRNA into exosomes, including CD106 and CD49d, which were implicated in exosome-EC binding and EC internalization. We found that EC internalized Tspan8-CD49d complex–containing exosomes. Exosome uptake induced vascular endothelial growth factor (VEGF)–independent regulation of several angiogenesis-related genes, including von Willebrand factor, Tspan8, chemokines CXCL5 and MIF, chemokine receptor CCR1, and, together with VEGF, VEGF receptor 2. EC uptake of Tspan8-CD49d complex–containing exosomes was accompanied by enhanced EC proliferation, migration, sprouting, and maturation of EC progenitors. Unraveling these new pathways of exosome-initiated EC regulation could provide new options for therapeutic interference with tumor-induced angiogenesis. Cancer Res; 70(4); 1668–78

Background and Purpose— The cause of initial ischemic stroke in up to 30% of young patients remains unclear. Fabry disease, due to deficient α-galactosidase A (α-Gal A) activity, is a vascular endothelial glycosphingolipid storage disease typically presenting in childhood. With advancing age, patients develop renal, cardiac, and cerebrovascular disease and die prematurely. A European study suggested an increased prevalence of unrecognized Fabry disease in patients with cryptogenic stroke. We hypothesized that α-Gal A deficiency is a rare cause of initial early-onset ischemic stroke in men. Methods— The Stroke Prevention in Young Men Study enrolled >550 men (15 to 49 years) with first ischemic stroke in the Baltimore–Washington area in 2004 to 2007. Frozen plasma samples were assayed for α-Gal A activity, and DNA from patients with consistently low plasma α-Gal A activities were sequenced. Results— The study sample consisted of 558 men (42% African-American; median age 44 years). Stroke was cryptogenic in 154 men (40% African-American). In 10 patients with low plasma α-Gal A activities, DNA sequencing identified alterations in the α-Gal A gene in 2 patients. The polymorphism, D313Y, which results in low plasma enzyme activity, but near normal levels of cellular activity was seen in one European-American male. The Fabry disease-causing A143T mutation was seen in an African-American male with cryptogenic stroke (0.18% of all strokes: upper 95% CI=0.53%; 0.65% of cryptogenic strokes: upper 95% CI=1.92%). Conclusions— In this biracial population, unrecognized Fabry disease is a rare but treatable cause of initial ischemic stroke in young men.

Embryonic stem cells are immortal, can self renew, and differentiate into all cells of the body. The adult organism maintains adult stem cells in regenerative organs that can differentiate into all cells of the respective organ. Virchow's hypothesis that cancer may arise from embryonic-like cells has received strong support, as it was demonstrated that tumors contain few cells, known as cancer stem or cancer-initiating cells (CIC), that account for primary and metastatic tumor growth. CIC are mostly defined by expression of CIC-markers that are associated and correlated with the potential of CIC to grow in xenogeneic mice. CIC marker profiles have been elaborated for many tumors, with several markers as CD24, CD44, CD133, CD166, EpCAM, and some integrins, being expressed by tumors of different histological type. Their function in promoting CIC maintenance and activity is largely unknown. The fate of stem cells, determined by their position, is minutely regulated by few adjacent cells creating a niche. CIC also require a niche, mostly for settlement and growth in distant organs. This so called pre-metastatic niche is initiated by the primary tumor before metastasizing cell arrival. How do CIC prepare the pre-metastatic niche? Cancer cells secrete a matrix that serves a cross-talk with surrounding tissues. Additionally, cancer cells can abundantly deliver exosomes, which function as long-distance intercellular communicators. Studies on a rat pancreatic adenocarcinoma support our hypothesis that tumor-derived matrix and exosomes are the main actors in forming the pre-metastatic niche with CIC markers being engaged in matrix preparation and/or exosome delivery.

Background: Nucleic acid amplification technologies significantly improved the limit of detection (LOD) for diagnostic assays. The ability of these assays to amplify fewer than 10 target copies of DNA or RNA imposes new requirements on the preparation of clinical samples. We report a statistical method to determine how large of an aliquot is necessary to reproducibly provide a detectable number of cells. Methods: We determined the success probability (p) based on aliquot size and sample volume. The binomial distribution, based on p and the concentration of cells in sample, was used to calculate the probability of getting no target objects in an aliquot and to determine the minimum number of objects per aliquot necessary to generate a reproducible clinical assay. Results: The described method was applied to find a minimum aliquot volume required for a set LOD, false-negative rate (FNR), and %CV. For example, to keep FNR Conclusion: This probability model is a useful tool to predict the impact of aliquot volume on the LOD and reproducibility of clinical assays. Even for samples for which pathogens are homogeneously distributed, it is theoretically impossible to collect a single pathogen consistently if the concentration of pathogen is below a certain limit.

The glycosylphosphatidylinositol-anchored molecule C4.4A, which shares structural features with uPAR, is frequently expressed on carcinomas with upregulated expression during tumor progression. Moreover, rare expression on nontransformed epithelial cells is strongly increased during tissue remodeling, e.g., during wound healing. This strictly regulated expression prompted us to define transcriptional activation of the C4.4A gene. C4.4A transcription was analyzed in 2 syngenic rat tumor cell lines with low or high metastatic potential, respectively. Though genomic C4.4A DNA was present in both lines, C4.4A mRNA and transcription of a reporter construct containing the C4.4A promoter was only observed in the metastasizing subline. Deletions and point mutations in the C4.4A promoter-driven reporter construct revealed that activation of the TATA-less, GC-rich core promoter (-1 to -50 bp) does not suffice to initiate transcription that requires coactivation of a proximal response element (-71 to -88 bp) and can be further increased by more distal response elements (-89 to -133 bp). Mobility-shift and cotransfection studies showed that Sp3 binding enhances C4.4A transcription, whereas potential Sp1 binding sites were ineffective. C4.4A transcription essentially requires C/EBPbeta binding to a TRE/CCAAT composite element (-71 to -88 bp) as measured by ChIP assay. C4.4A transcription is strikingly enhanced by cotransfection with JunD or c-Jun, such that C4.4A is most strongly transcribed even in the C4.4A-negative tumor cell line after cotransfection with C/EBPbeta plus JunD or c-Jun. Thus, upregulation of C/EBPbeta during tumor progression and wound repair may well provide a sufficient trigger for transcription of the C4.4A gene.