Caspase-activated ROCK-1 allows erythroblast terminal maturation independently of cytokine-induced Rho signaling

A-S Gabet, S Coulon, A Fricot, J Vandekerckhove, Y Chang, J-A Ribeil, L Lordier, Y Zermati, V Asnafi, Z Belaid, N Debili, W Vainchenker, B Varet, O Hermine and G Courtois

Cell Death & Differentiation 18, 678-689 (April 2011) | doi:10.1038/cdd.2010.140

Stem cell factor (SCF) and erythropoietin are strictly required for preventing apoptosis and stimulating proliferation, allowing the differentiation of erythroid precursors from colony-forming unit-E to the polychromatophilic stage. In contrast, terminal maturation to generate reticulocytes occurs independently of cytokine signaling by a mechanism not fully understood. Terminal differentiation is characterized by a sequence of morphological changes including a progressive decrease in cell size, chromatin condensation in the nucleus and disappearance of organelles, which requires transient caspase activation. These events are followed by nucleus extrusion as a consequence of plasma membrane and cytoskeleton reorganization. Here, we show that in early step, SCF stimulates the Rho/ROCK pathway until the basophilic stage. Thereafter, ROCK-1 is activated independently of Rho signaling by caspase-3-mediated cleavage, allowing terminal maturation at least in part through phosphorylation of the light chain of myosin II. Therefore, in this differentiation system, final maturation occurs independently of SCF signaling through caspase-induced ROCK-1 kinase activation.

PTEN status switches cell fate between premature senescence and apoptosis in glioma exposed to ionizing radiation

J-J Lee, B C Kim, M-J Park, Y-S Lee, Y-N Kim, B L Lee and J-S Lee

Cell Death & Differentiation 18, 666-677 (April 2011) | doi:10.1038/cdd.2010.139

Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) has frequently been observed in human gliomas, conferring AKT activation and resistance to ionizing radiation (IR) and drug treatments. Recent reports have shown that PTEN loss or AKT activation induces premature senescence, but many details regarding this effect remain obscure. In this study, we tested whether the status of PTEN determined fate of the cell by examining PTEN-deficient U87, U251, and U373, and PTEN-proficient LN18 and LN428 glioma cells after exposure to IR. These cells exhibited different cellular responses, senescence or apoptosis, depending on the PTEN status. We further observed that PTEN-deficient U87 cells with high levels of both AKT activation and intracellular reactive oxygen species (ROS) underwent senescence, whereas PTEN-proficient LN18 cells entered apoptosis. ROS were indispensable for inducing senescence in PTEN-deficient cells, but not for apoptosis in PTEN-proficient cells. Furthermore, transfection with wild-type (wt) PTEN or AKT small interfering RNA induced a change from premature senescence to apoptosis and depletion of p53 or p21 prevented IR-induced premature senescence in U87 cells. Our data indicate that PTEN acts as a pivotal determinant of cell fate, regarding senescence and apoptosis in IR-exposed glioma cells. We conclude that premature senescence could have a compensatory role for apoptosis in the absence of the tumor suppressor PTEN through the AKT/ROS/p53/p21 signaling pathway.

cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production

N Vanlangenakker, T Vanden Berghe, P Bogaert, B Laukens, K Zobel, K Deshayes, D Vucic, S Fulda, P Vandenabeele and M J M Bertrand

Cell Death & Differentiation 18, 656-665 (April 2011) | doi:10.1038/cdd.2010.138

Three members of the IAP family (X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins-1/-2 (cIAP1 and cIAP2)) are potent suppressors of apoptosis. Recent studies have shown that cIAP1 and cIAP2, unlike XIAP, are not direct caspase inhibitors, but block apoptosis by functioning as E3 ligases for effector caspases and receptor-interacting protein 1 (RIP1). cIAP-mediated polyubiquitination of RIP1 allows it to bind to the pro-survival kinase transforming growth factor-β-activated kinase 1 (TAK1) which prevents it from activating caspase-8-dependent death, a process reverted by the de-ubiquitinase CYLD. RIP1 is also a regulator of necrosis, a caspase-independent type of cell death. Here, we show that cells depleted of the IAPs by treatment with the IAP antagonist BV6 are greatly sensitized to tumor necrosis factor (TNF)-induced necrosis, but not to necrotic death induced by anti-Fas, poly(I:C) oxidative stress. Specific targeting of the IAPs by RNAi revealed that repression of cIAP1 is responsible for the sensitization. Similarly, lowering TAK1 levels or inhibiting its kinase activity sensitized cells to TNF-induced necrosis, whereas repressing CYLD had the opposite effect. We show that this sensitization to death is accompanied by enhanced RIP1 kinase activity, increased recruitment of RIP1 to Fas-associated via death domain and RIP3 (which allows necrosome formation), and elevated RIP1 kinase-dependent accumulation of reactive oxygen species (ROS). In conclusion, our data indicate that cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent ROS production.

RASSF7 negatively regulates pro-apoptotic JNK signaling by inhibiting the activity of phosphorylated-MKK7

S Takahashi, A Ebihara, H Kajiho, K Kontani, H Nishina and T Katada

Cell Death & Differentiation 18, 645-655 (April 2011) | doi:10.1038/cdd.2010.137

Members of the Ras-association domain family (RASSF) of proteins influence apoptosis and cell cycling but little is known about the mechanisms. Here, we show that RASSF7 interacts with N-Ras and mitogen-activated protein kinase kinase 7 (MKK7) to negatively regulate c-Jun N-terminal kinase (JNK) signaling. Stress-induced JNK activation and apoptosis were markedly enhanced in cells depleted of RASSF7 or N-Ras by RNAi knockdown. An interaction with RASSF7 promoted the phosphorylated state of MKK7 but inhibited this kinase's ability to activate JNK. RASSF7 required its RA domain for both interaction with GTP-bound N-Ras and the anti-apoptotic response to stress stimuli. Following prolonged stress, however, RASSF7's anti-apoptotic effect was eliminated because of degradation of RASSF7 protein via the ubiquitin–proteasome pathway. Our results indicate that RASSF7 acts in concert with N-Ras to constitute a stress-sensitive temporary mechanism of apoptotic regulation. With initial stress, RASSF7/N-Ras promotes cell survival by inhibiting the MKK7/JNK pathway. However, with prolonged stress, RASSF7 protein undergoes degradation that allows cell death signaling to proceed. Our findings may account for the association of elevated RASSF7 with tumorigenesis.

A novel microtubule-modulating noscapinoid triggers apoptosis by inducing spindle multipolarity via centrosome amplification and declustering

P Karna, P C G Rida, V Pannu, K K Gupta, W B Dalton,H Joshi, V W Yang, J Zhou and R Aneja

Cell Death & Differentiation 18, 632-644 (April 2011) | doi:10.1038/cdd.2010.133

We have previously shown that a non-toxic noscapinoid, EM011 binds tubulin without altering its monomer/polymer ratio. EM011 is more active than the parent molecule, noscapine, in inducing G2/M arrest, inhibiting cellular proliferation and tumor growth in various human xenograft models. However, the mechanisms of mitotic-block and subsequent cell death have remained elusive. Here, we show that EM011-induced attenuation of microtubule dynamics was associated with impaired association of microtubule plus-end tracking proteins, such as EB1 and CLIP-170. EM011 treatment then led to the formation of multipolar spindles containing ‘real’ centrioles indicating drug-induced centrosome amplification and persistent centrosome declustering. Centrosome amplification was accompanied by an upregulation of Aurora A and Plk4 protein levels, as well as a surge in the kinase activity of Aurora A, suggesting a deregulation of the centrosome duplication cycle. Cell-cycle phase-specific experiments showed that the ‘cytotoxicity-window’ of the drug encompasses the late S-G2 period. Drug-treatment, excluding S-phase, not only resulted in lower sub-G1 population but also attenuated centrosome amplification and spindle multipolarity, suggesting that drug-induced centrosome amplification is essential for maximal cell death. Subsequent to a robust mitotic arrest, EM011-treated cells displayed diverse cellular fates suggesting a high degree of intraline variation. Some ‘apoptosis-evasive’ cells underwent aberrant cytokinesis to generate rampant aneuploidy that perhaps contributed to drug-induced cell death. These data indicate that spindle multipolarity induction by means of centrosome amplification has an exciting chemotherapeutic potential that merits further investigation.

Modulation of CD4+ T-cell activation by CD95 co-stimulation

M Paulsen, S Valentin, B Mathew, S Adam-Klages, U Bertsch, I Lavrik, P H Krammer, D Kabelitz and O Janssen

Cell Death & Differentiation 18, 619-631 (April 2011) | doi:10.1038/cdd.2010.134

CD95 is a dual-function receptor that exerts pro- or antiapoptotic effects depending on the cellular context, the state of activation, the signal threshold and the mode of ligation. In this study, we report that CD95 engagement modulates TCR/CD3-driven signaling pathways in resting T lymphocytes in a dose-dependent manner. While high doses of immobilized CD95 agonists silence T cells, lower concentrations augment activation and proliferation. We analyzed the co-stimulatory capacity of CD95 in detail in resting human CD4⁺ T cells, and demonstrate that low-dose ligand-induced co-internalization of CD95 and TCR/CD3 complexes enables non-apoptotic caspase activation, the prolonged activation of MAP kinases, the upregulation of antiapoptotic proteins associated with apoptosis resistance, and the activation of transcription factors and cell-cycle regulators for the induction of proliferation and cytokine production. We propose that the levels of CD95L on antigen-presenting cells (APCs), neighboring T cells or epithelial cells regulate inhibitory or co-stimulatory CD95 signaling, which in turn is crucial for fine-tuning of primary T-cell activation.

Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer

G Mudduluru, P Ceppi, R Kumarswamy, G V Scagliotti,M Papotti and H Allgayer

Oncogene , (14 February 2011) | doi:10.1038/onc.2011.13

Axl is a receptor that induces proliferation, migration and invasion in cancer. In this study, we show that specific microRNAs (miRNAs) target the 3′-UTR of Axl. Luciferase-reporter assays with wild-type and deleted miR-34 and miR-199a/b seed sequences of Axl 3′-UTR confirmed the specificity of targeting. An inverse correlation between Axl protein and miR-34a expression in a panel of non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and breast cancer (BRC) cell lines was observed, while miR-199a/b expression was completely suppressed. Pre-miR transfection inhibited in vitro migration and invasion and, in vivo, reduced the number of distant lung- or liver-metastases in a chorion-allantoic-membrane (CAM) assay. Moreover, methylation-specific PCR on bisulfite-converted DNA obtained from the cell lines showed that the miR-34a promoter methylation status was inversely correlated with its expression, and that miR-199a/b promoter regions were methylated in all cells tested. In a panel of NSCLC tissues (n=44), miR-34a and miR-199a/b were found to be downregulated and significantly co-expressed. A lower expression of all three miRs was significantly associated with squamous histotypes, and, in a preliminary series, NSCLC patients with miR-34a upregulation showed a positive association towards a longer survival. These results indicate that Axl receptor expression can be regulated by miR-34a and miR-199a/b, which are suppressed by promoter methylation in solid cancer cells.

Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death

Y Tang, Y Chen, H Jiang and D Nie

Cell Death & Differentiation 18, 602-618 (April 2011) | doi:10.1038/cdd.2010.117

Short-chain fatty acids (SCFAs) are the major by-products of bacterial fermentation of undigested dietary fibers in the large intestine. SCFAs, mostly propionate and butyrate, inhibit proliferation and induce apoptosis in colon cancer cells, but clinical trials had mixed results regarding the anti-tumor activities of SCFAs. Herein we demonstrate that propionate and butyrate induced autophagy in human colon cancer cells to dampen apoptosis whereas inhibition of autophagy potentiated SCFA induced apoptosis. Colon cancer cells, after propionate treatment, exhibited extensive characteristics of autophagic proteolysis: increased LC3-I to LC3-II conversion, acidic vesicular organelle development, and reduced p62/SQSTM1 expression. Propionate-induced autophagy was associated with decreased mTOR activity and enhanced AMP kinase activity. The elevated AMPKαphosphorylation was associated with cellular ATP depletion and overproduction of reactive oxygen species due to mitochondrial dysfunction involving the induction of MPT and loss of Δψ. In this context, mitochondria biogenesis was initiated to recover cellular energy homeostasis. Importantly, when autophagy was prevented either pharmacologically (3-MA or chloroquine) or genetically (knockdown of ATG5 or ATG7), the colon cancer cells became sensitized toward propionate-induced apoptosis through activation of caspase-7 and caspase-3. The observations indicate that propionate-triggered autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death, whereas application of an autophagy inhibitor (Chloroquine) is expected to enhance the therapeutic efficacy of SCFAs in inducing colon tumor cell apoptosis.

Study Helps Explain how Pathogenic E. coli Bacterium Causes Illness

WHAT:
Scientists at the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, have shown how the O157:H7 strain of Escherichia coli causes infection and thrives by manipulating the host immune response. The bacterium secretes a protein called NleH1 that directs the host immune enzyme IKK-beta to alter specific immune responses. This process not only helps the bacterium evade elimination by the immune system, it also works to prolong the survival of the infected host, enabling the bacterium to persist and ultimately spread to unaffected individuals. This finely balanced mechanism, observed in both laboratory and animal models, could be relevant to other pathogens involved in foodborne diseases.
While most E. coli strains help check the growth of harmful bacteria in the guts of animals and humans, a few E. coli strains, such as O157:H7, can cause severe diarrhea, abdominal cramps and, in rare cases, death. Human cases of E. coli O157:H7 have been linked to consumption of raw, undercooked, or spoiled meat.

NIAID researchers plan to use the new information to further study how the host immune system mounts a response to E. coliO157:H7 when infection begins and how the bacterium selectively blocks these defenses. Several foodborne pathogens, includingShigella and Salmonella, use a similar secretion system to disrupt host immune responses and infect gut cells.

ARTICLE:
F Wan, et al. IKK-beta phosphorylation regulates RPS3 nuclear translocation and NF-kappa B function during infection withEscherichia coli strain O157:H7. Nature Immunology. DOI 10.1038/ni.2007

WHO:
Michael Lenardo, M.D., Chief of the Molecular Development Section of the NIAID Laboratory of Immunology, is available to comment on this article.

Molecular characterization of apoptosis induced by CARF silencing in human cancer cells

C T Cheung, R Singh, A R Yoon, M K Hasan, T Yaguchi, S C Kaul, C O Yun and R Wadhwa

Cell Death & Differentiation 18, 589-601 (April 2011) | doi:10.1038/cdd.2010.129

Collaborator of ARF (CARF) was cloned as an ARF-interacting protein and shown to regulate the p53–p21^WAF1–HDM2 pathway, which is central to tumor suppression via senescence and apoptosis. We had previously reported that CARF inhibition in cancer cells led to polyploidy and caspase-dependent apoptosis, however, the mechanisms governing this phenomenon remained unknown. Thus, we examined various cell death and survival pathways including the mitochondrial stress, ataxia telangiectasia mutated (ATM)–ATR, Ras–MAP kinase and retinoblastoma cascades. We found that CARF is a pleiotropic regulator with widespread effects; its suppression affected all investigated pathways. Most remarkably, it protected the cells against genotoxicity; CARF knockdown elicited DNA damage response as evidenced by increased levels of phosphorylated ATM and γH2AX, leading to induction of mitotic arrest and eventual apoptosis. We also show that the CARF-silencing-induced apoptosis in vitro translates to in vivo. In a human tumor xenograft mouse model, treatment of developing tumors with short hairpin RNA (shRNA) against CARF via an adenovirus carrier induced complete suppression of tumor growth, suggesting that CARF shRNA is a strong candidate for an anticancer reagent. We demonstrate that CARF has a vital role in genome preservation and tumor suppression and CARF siRNA is an effective novel cancer therapeutic agent.

Profiling early lung immune responses in the mouse model of tuberculosis.

PLoS One. 2011 Jan 13;6(1):e16161.

Abstract

Tuberculosis (TB) is caused by the intracellular bacteria Mycobacterium tuberculosis, and kills more than 1.5 million people every year worldwide. Immunity to TB is associated with the accumulation of IFNγ-producing T helper cell type 1 (Th1) in the lungs, activation of M.tuberculosis-infected macrophages and control of bacterial growth. However, very little is known regarding the early immune responses that mediate accumulation of activated Th1 cells in the M.tuberculosis-infected lungs. To define the induction of early immune mediators in the M.tuberculosis-infected lung, we performed mRNA profiling studies and characterized immune cells in M.tuberculosis-infected lungs at early stages of infection in the mouse model. Our data show that induction of mRNAs involved in the recognition of pathogens, expression of inflammatory cytokines, activation of APCs and generation of Th1 responses occurs between day 15 and day 21 post infection. The induction of these mRNAs coincides with cellular accumulation of Th1 cells and activation of myeloid cells in M.tuberculosis-infected lungs. Strikingly, we show the induction of mRNAs associated with Gr1+ cells, namely neutrophils and inflammatory monocytes, takes place on day 12 and coincides with cellular accumulation of Gr1+ cells in M.tuberculosis-infected lungs. Interestingly, in vivo depletion of Gr1+ neutrophils between days 10-15 results in decreased accumulation of Th1 cells on day 21 in M.tuberculosis-infected lungs without impacting overall protective outcomes. These data suggest that the recruitment of Gr1+ neutrophils is an early event that leads to production of chemokines that regulate the accumulation of Th1 cells in the M.tuberculosis-infected lungs.

Reliability of the MODS assay decentralisation process in three health regions in Peru

Int J Tuberc Lung Dis. 2011 Feb;15(2):217-22, i.

OBJECTIVE: to deliver rapid isoniazid (INH) and rifampicin (RMP) drug susceptibility testing (DST) close to the patient, we designed a decentralisation process for the microscopic observation drug susceptibility (MODS) assay in Peru and evaluated its reliability.

METHODS: after 2 weeks of training, laboratory staff processed ≥ 120 consecutive sputum samples each in three regional laboratories. Samples were processed in parallel with MODS testing at an expert laboratory. Blinded paired results were independently analysed by the Instituto Nacional de Salud (INS) according to pre-determined criteria: concordance for culture, DST against INH and RMP and diagnosis of multidrug-resistant tuberculosis (MDR-TB) ≥ 95%, McNemar's P > 0.05, kappa index (κ) ≥ 0.75 and contamination 1-4%. Sensitivity and specificity for MDR-TB were calculated.

RESULTS: the accreditation process for Callao (126 samples, 79.4% smear-positive), Lima Sur (n = 130, 84%) and Arequipa (n = 126, 80%) took respectively 94, 97 and 173 days. Pre-determined criteria in all regional laboratories were above expected values. The sensitivity and specificity for detecting MDR-TB in regional laboratories were >95%, except for sensitivity in Lima Sur, which was 91.7%. Contamination was 1.0-2.3%. Mean delay to positive MODS results was 9.9-12.9 days.

CONCLUSION: technology transfer of MODS was reliable, effective and fast, enabling the INS to accredit regional laboratories swiftly.

PMID: 21219684 [PubMed - in process]

Performance of Serum C-Reactive Protein as a Screening Test for Smear-Negative Tuberculosis in an Ambulatory High HIV Prevalence Population

PLoS One. 2011 Jan 10; Volume 6, Number 1: e15248 Wilson, D., Badri, M., Maartens, G.

Delayed diagnosis has contributed to the high mortality of sputum smear-negative TB (SNTB) in high HIV prevalence countries. New diagnostic strategies for SNTB are urgently needed. C-reactive protein (CRP) is a non-specific inflammatory protein that is usually elevated in patients with TB, but its role in the diagnosis of TB is uncertain. To determine the diagnostic utility of CRP the researchers prospectively evaluated the performance of CRP as a screening test for SNTB in symptomatic ambulatory TB suspects followed up for 8 weeks in KwaZulu-Natal, South Africa. Confirmed TB was defined as positive culture or acid-fast bacilli with granulomata on histology, and possible TB as documented response to antitubercular therapy. The CRP quotient was defined as a multiple of the upper limit of normal of the serum CRP result. Three hundred and sixty four participants fulfilled entry criteria: 135 (37%) with confirmed TB, 114 (39%) with possible TB, and 115 (24%) without TB. The median CRP quotient was 15.4 (IQR 7.2; 23.3) in the confirmed TB group, 5.8 (IQR 1.4; 16.0) in the group with possible TB, and 0.7 (IQR 0.2; 2.2) in the group without TB (p<0.0001). The CRP quotient above the upper limit of normal had sensitivity 0.98 (95% CI 0.94; 0.99), specificity 0.59 (95% CI 0.50; 0.68), positive predictive value 0.74 (95% CI 0.67; 0.80), negative predictive value 0.96 (95% CI 0.88; 0.99), and diagnostic odds ratio 63.7 (95% CI 19.1; 212.0) in the confirmed TB group compared with the group without TB. Higher CRP quotients improved specificity at the expense of sensitivity. In high HIV prevalence settings, a normal CRP could be a useful test in combination with clinical evaluation to rule out TB in ambulatory patients. Point-of-care CRP should be further evaluated in primary care clinics

AstraZeneca Joins Fight against Tuberculosis

Guardian.co.uk, www.guardian.co.uk, March 2, 2011, by Julia Kollewe

AstraZeneca pharmaceutical company has joined its French rival, Sanofi-aventis, and the Universities of Cambridge and Lausanne in the More Medicines for Tuberculosis (MM4TB) consortium to develop new drugs for successful and shorter TB treatment. The consortium also includes the universities of Pavia, Italy, and Uppsala, Sweden, in addition to 19 other research groups from 13 countries. The consortium is led by TB expert Professor Stewart Cole of the École Polytechnique Fédérale de Lausanne, and will be funded by a €16million (US $22,215, 369) European Union grant. AstraZeneca will share its compounds and expertise, and research will be conducted at London hospitals and the John Innes Center, England, as well as in Russia, India, and South Africa. The aim is to have 10 to 20 compounds in the pipeline to develop two to three successful TB drugs.

New Antibody Therapy to Tackle Tuberculosis

Wellcome Trust, www.wellcome.ac.uk, February 28, 2011

A team of researchers has developed an antibody-based therapy to prevent TB in mice. The team produced a human monoclonal antibody of the IgA type that can recognize the TB bacteria. The antibody works by attaching to the bacterial cells and triggering immune processes that prevent bacterial growth. When this antibody is combined with interferon, a modulator of the immune system, it offers protection against TB. The researchers are from the University of Dundee, Scotland; King’s College, London; and St. George’s Hospital Medical School, University of London. Each research team contributed a different type of expertise to the experiment: Professor Juraj Ivanyi of King’s College is an international expert in TB research, Dr. Jenny Woof of Dundee has experience in human IgA antibodies, and Dr. Rajko Reljic of St. George’s has expertise and special facilities for experimental models of infection. The study was funded jointly by the Wellcome Trust and the Dunhill Medical Trust and is published in the Journal of Immunology 2011;186:3113-3119.

Integrated mass spectrometry–based analysis of plasma glycoproteins and their glycan modifications

We present a protocol for the identification of glycosylated proteins in plasma followed by elucidation of their individual glycan compositions. The study of glycoproteins by mass spectrometry is usually based on cleavage of glycans followed by separate analysis of glycans and deglycosylated proteins, which limits the ability to derive glycan compositions for individual glycoproteins. The methodology described here consists of 2D HPLC fractionation of intact proteins and liquid chromatography–multistage tandem mass spectrometry (LC-MS/MSⁿ) analysis of digested protein fractions. Protein samples are separated by 1D anion-exchange chromatography (AEX) with an eight-step salt elution. Protein fractions from each of the eight AEX elution steps are transferred onto the 2D reversed-phase column to further separate proteins. A digital ion trap mass spectrometer with a wide mass range is then used for LC-MS/MSⁿ analysis of intact glycopeptides from the 2D HPLC fractions. Both peptide and oligosaccharide compositions are revealed by analysis of the ion fragmentation patterns of glycopeptides with an intact glycopeptide analysis pipeline.

Nature Protocols 6,253–269(2011)doi:10.1038/nprot.2010.176

Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos

Dynamic reprogramming of the genome takes place during the gamete-to-embryo transition. This transition defines a period of continuous and global change but has been difficult to study because of extremely limited material and varying degrees of chromatin compaction. Improved methods of detecting chromatin and gene expression changes in the germ line and in the preimplantation embryo would greatly enhance the understanding of this crucial developmental transition. Here we describe a protocol developed and used by us that improves the sensitivity of existing fluorescence in situhybridization (FISH) methods; the protocol described here has enabled us to visualize single-copy DNA targets and corresponding nascent RNA transcripts in preimplantation embryos and during spermatogenesis. Major improvements over alternative methods involve fixation and permeabilization steps. Chromatin epitopes can be visualized simultaneously by combining FISH with immunofluorescence; multicopy and repetitive element expression can also be reliably measured. This procedure (sample preparation and staining) requires 1–1.5 d to complete and will facilitate detailed examination of spatial relationships between chromatin epitopes, DNA and RNA during the dynamic transition from gamete to embryo.

 

Nature Protocols 6, 270–284 (2011) doi:10.1038/nprot.2010.195

Comprehensive cluster analysis with Transitivity Clustering

Transitivity Clustering is a method for the partitioning of biological data into groups of similar objects, such as genes, for instance. It provides integrated access to various functions addressing each step of a typical cluster analysis. To facilitate this, Transitivity Clustering is accessible online and offers three user-friendly interfaces: a powerful stand-alone version, a web interface, and a collection of Cytoscape plug-ins. In this paper, we describe three major workflows: (i) protein (super)family detection with Cytoscape, (ii) protein homology detection with incomplete gold standards and (iii) clustering of gene expression data. This protocol guides the user through the most important features of Transitivity Clustering and takes ~1 h to complete.

Nature Protocols 6, 285–295 (2011) doi:10.1038/nprot.2010.197

Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells

In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin⁻CD34⁺CD43⁺CD45⁺ multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5–19 d of culture with cytokines, depending on the cell type.

 

Nature Protocols 6, 296–313 (2011) doi:10.1038/nprot.2010.184

A practical guide to the synthesis of dinitroindolinyl-caged neurotransmitters

This protocol describes a method for efficient chemical synthesis of dinitroindolinyl derivatives of glutamate and γ-aminobutyric acid. These caged neurotransmitters are currently the most chemically and photochemically efficient probes for two-photon photolysis in living brain slices. The protocol only requires basic organic synthesis equipment, and no silica gel column chromatography or NMR spectroscopy is needed at any stage. HPLC is used to purify the caged transmitters at the end of the syntheses. Thus, the synthesis of dinitroindolinyl-caged neurotransmitters is within the scope of a modestly equipped chemistry laboratory.

Nature Protocols 6, 314–326 (2011) doi:10.1038/nprot.2010.193

A practical guide to the synthesis and use of membrane-permeant acetoxymethyl esters of caged inositol polyphosphates

This protocol describes a method for efficient chemical synthesis of an analog of inositol-1,4,5-trisphosphate (IP₃) hexakis acetoxymethyl ester having anortho-nitroveratryl photochemical caging group on the 6-hydroxyl position. The six esters render the probe membrane permeant, such that it can be loaded into intact living cells in vitro or in vivo. Inside cells, the caged IP₃ is inert until activated by two-photon excitation at 720 nm. The photoliberated signaling molecule can mobilize release of Ca²⁺ from intracellular stores on the endoplasmic reticulum. When co-loaded with the fluorescent Ca²⁺ indicator rhod-2, one laser can be used for stimulating and monitoring intracellular Ca²⁺signaling with single-cell resolution. This protocol has chemistry and biology sections; the former describes the organic synthesis of the caged IP₃, which requires 12 d, and the latter an application to a day-long study of astrocyte-regulated neuronal function in living brain slices acutely isolated from rats. As Ca²⁺ is the single most important intracellular second messenger and the IP₃-Ca²⁺ signaling cascade is used by many cells to produce increases in Ca²⁺ concentration, this method should be widely applicable for the study of a variety of physiological processes in intact biological systems.

Nature Protocols 6, 327–337 (2011) doi:10.1038/nprot.2010.194

Conditioned place preference behavior in zebrafish

This protocol describes conditioned place preference (CPP) in zebrafish following a single exposure to a substance. In the CPP paradigm, animals show a preference for an environment that has previously been associated with a substance (drug), thus indicating the positive-reinforcing qualities of that substance. The test tank consists of two visually distinct compartments separated by a central alley. The protocol involves three steps: the determination of initial preference, one conditioning session and the determination of final preference. This procedure is carried out in ~2 d; other reported CPP protocols take up to 2 weeks. An increase in preference for the drug-associated compartment is observed after a single exposure. Establishment of this high-throughput protocol in zebrafish makes it possible to investigate the molecular and cellular basis of choice behavior, reward and associative learning. The protocol is also a tool for testing psychoactive compounds in the context of a vertebrate brain.

Nature Protocols 6, 338–345 (2011) doi:10.1038/nprot.2010.201

Feeder-dependent and feeder-independent iPS cell derivation from human and mouse adipose stem cells

Adipose tissue is an abundantly available source of proliferative and multipotent mesenchymal stem cells with promising potential for regenerative therapeutics. We previously demonstrated that both human and mouse adipose-derived stem cells (ASCs) can be reprogrammed into induced pluripotent stem cells (iPSCs) with efficiencies higher than those that have been reported for other cell types. The ASC-derived iPSCs can be generated in a feeder-independent manner, representing a unique model to study reprogramming and an important step toward establishing a safe, clinical grade of cells for therapeutic use. In this study, we provide a detailed protocol for isolation, preparation and transformation of ASCs from fat tissue into mouse iPSCs in feeder-free conditions and human iPSCs using feeder-dependent or feeder/xenobiotic-free processes. This protocol also describes how ASCs can be used as feeder cells for maintenance of other pluripotent stem cells. ASC derivation is rapid and can be completed in <1 week, with mouse and human iPS reprogramming times averaging 1.5 and 2.5 weeks, respectively.

Nature Protocols 6, 346–358 (2011) doi:10.1038/nprot.2010.199

A differential proteome screening system for post-translational modification–dependent transcription factor interactions

Post-translational modifications (PTMs) of transcription factors alter interactions with co-regulators and epigenetic modifiers. For example, members of the C/EBP transcription factor family are extensively methylated on arginine and lysine residues in short, conserved, modular domains, implying modification-dependent cofactor docking. Here we describe array peptide screening (APS), a systematic and differential approach to detect PTM-dependent interactions in the human proteome using chemically synthesized, biotinylated peptides coupled to fluorophore-labeled streptavidin. Peptides with and without a modified residue are applied in parallel to bacterial expression libraries in an arrayed format. Interactions are detected and quantified by laser scanning to reveal proteins that differentially bind to nonmodified or modified peptides. We have previously used this method to investigate the effect of arginine methylation of C/EBPβ peptides. The method enables determination of PTM-dependent transcription factor interactions, quantification of relative binding affinities and rapid protein classification, all independently of the transactivation potential of peptides or cellular abundance of interactors. The protocol provides a cost-effective alternative to mass spectrometric approaches and takes 3–4 d to complete.

Nature Protocols 6, 359–364 (2011) doi:10.1038/nprot.2011.303

Analysis of protein-ligand interactions by fluorescence polarization

Quantification of the associations between biomolecules is required both to predict and understand the interactions that underpin all biological activity. Fluorescence polarization (FP) provides a nondisruptive means of measuring the association of a fluorescent ligand with a larger molecule. We describe an FP assay in which binding of fluorescein-labeled inositol 1,4,5-trisphosphate (IP₃) to N-terminal fragments of IP₃ receptors can be characterized at different temperatures and in competition with other ligands. The assay allows the standard Gibbs free energy (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) changes of ligand binding to be determined. The method is applicable to any purified ligand-binding site for which an appropriate fluorescent ligand is available. FP can be used to measure low-affinity interactions in real time without the use of radioactive materials, it is nondestructive and, with appropriate care, it can resolve ΔH° and ΔS°. The first part of the protocol, protein preparation, may take several weeks, whereas the FP measurements, once they have been optimized, would normally take 1–6 h.

Nature Protocols 6, 365–387 (2011) doi:10.1038/nprot.2011.305

Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage

Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix.

The protocol comprises (i) cartilage culture and optional cartilage extraction, (ii) a quick and simple colorimetric assay for quantitating aggrecan and (iii) neoepitope western blotting to identify specific aggrecan fragments partitioning to the medium or cartilage compartments. The crucial difference between the methods for mice and larger animals is that the proportion of aggrecan in a given sample is normalized to total aggrecan rather than to tissue wet weight. This necessary break from tradition arises because tiny volumes of liquid clinging to mouse cartilage can increase the apparent tissue wet weight, causing unacceptable errors. The protocol has broad application for the in vitro analysis of transgenic mice, particularly those with mutations that affect cartilage remodeling, arthritic disease and skeletal development. The protocol is robust, reliable and takes 7–11 d to complete.

Nature Protocols 6, 388–404 (2011) doi:10.1038/nprot.2010.179 Published online 03 March 2011

Dynamic clamp with StdpC software

Dynamic clamp is a powerful method that allows the introduction of artificial electrical components into target cells to simulate ionic conductances and synaptic inputs. This method is based on a fast cycle of measuring the membrane potential of a cell, calculating the current of a desired simulated component using an appropriate model and injecting this current into the cell. Here we present a dynamic clamp protocol using free, fully integrated, open-source software (StdpC, for spike timing-dependent plasticity clamp). Use of this protocol does not require specialist hardware, costly commercial software, experience in real-time operating systems or a strong programming background. The software enables the configuration and operation of a wide range of complex and fully automated dynamic clamp experiments through an intuitive and powerful interface with a minimal initial lead time of a few hours. After initial configuration, experimental results can be generated within minutes of establishing cell recording.

Nature Protocols 6, 405–417(2011) doi:10.1038/nprot.2010.200 Published online 03 March 2011

Revisiting the T-cell receptor alpha/delta locus and possible associations with multiple sclerosis

A role for T cells in the pathogenesis of multiple sclerosis (MS) is well supported, evidenced by myriad immunological studies, as well as the unequivocal genetic influence of the major histocompatibility complex (MHC). Despite many attempts, no convincing genetic associations have been made between T-cell receptor (TCR) gene loci and MS. However, these studies may not be definitive because of small sample sizes and under-representative marker coverage of the chromosomal regions being investigated. To explore potential roles between the TCR alpha locus and MS, we have genotyped a large family-based cohort, including 1360 affected individuals and 1659 of their unaffected first-degree relatives, at 40 single-nucleotide polymorphism (SNP) markers within the TCR alpha/delta locus. This represents the largest TCR alpha-MS study to date. From this screen, we identified three potential loci of interest in TCR alpha variable and constant gene regions using the transmission disequilibrium test. Although SNPs implicating each of these regions of interest will require genotyping in independent replication cohorts, these findings suggest a role for TCR gene polymorphisms in MS susceptibility. In the context of these findings we review the evidence.

Genes and Immunity (2011) 12, 59–66; doi:10.1038/gene.2010.65; published online 27 January 2011

A genome-wide admixture scan for ancestry-linked genes predisposing to sarcoidosis in African-Americans

Genome-wide linkage and association studies have uncovered variants associated with sarcoidosis, a multiorgan granulomatous inflammatory disease. African ancestry may influence disease pathogenesis, as African-Americans are more commonly affected by sarcoidosis. Therefore, we conducted the first sarcoidosis genome-wide ancestry scan using a map of 1384 highly ancestry-informative single-nucleotide polymorphisms genotyped on 1357 sarcoidosis cases and 703 unaffected controls self-identified as African-American. The most significant ancestry association was at marker rs11966463 on chromosome 6p22.3 (ancestry association risk ratio (aRR)=1.90; P=0.0002). When we restricted the analysis to biopsy-confirmed cases, the aRR for this marker increased to 2.01;P=0.00007. Among the eight other markers that demonstrated suggestive ancestry associations with sarcoidosis were rs1462906 on chromosome 8p12, which had the most significant association with European ancestry (aRR=0.65; P=0.002), and markers on chromosomes 5p13 (aRR=1.46;P=0.005) and 5q31 (aRR=0.67; P=0.005), which correspond to regions we previously identified through sib-pair linkage analyses. Overall, the most significant ancestry association for Scadding stage IV cases was to marker rs7919137 on chromosome 10p11.22 (aRR=0.27; P=2 × 10⁻⁵), a region not associated with disease susceptibility. In summary, through admixture mapping of sarcoidosis we have confirmed previous genetic linkages and identified several novel putative candidate loci for sarcoidosis.

Genes and Immunity (2011) 12, 67–77; doi:10.1038/gene.2010.56; published online 23 December 2010

Characterization of the macrophage transcriptome in glomerulonephritis-susceptible and -resistant rat strains

Crescentic glomerulonephritis (CRGN) is a major cause of rapidly progressive renal failure for which the underlying genetic basis is unknown. Wistar–Kyoto (WKY) rats show marked susceptibility to CRGN, whereas Lewis rats are resistant. Glomerular injury and crescent formation are macrophage dependent and mainly explained by seven quantitative trait loci (Crgn1–7). Here, we used microarray analysis in basal and lipopolysaccharide (LPS)-stimulated macrophages to identify genes that reside on pathways predisposing WKY rats to CRGN. We detected 97 novel positional candidates for the uncharacterized Crgn3–7. We identified 10 additional secondary effector genes with profound differences in expression between the two strains (>5-fold change, <1% false discovery rate) for basal and LPS-stimulated macrophages. Moreover, we identified eight genes with differentially expressed alternatively spliced isoforms, by using an in-depth analysis at the probe level that allowed us to discard false positives owing to polymorphisms between the two rat strains. Pathway analysis identified several common linked pathways, enriched for differentially expressed genes, which affect macrophage activation. In summary, our results identify distinct macrophage transcriptome profiles between two rat strains that differ in susceptibility to glomerulonephritis, provide novel positional candidates for Crgn3–7 and define groups of genes that play a significant role in differential regulation of macrophage activity.

Genes and Immunity (2011) 12, 78–89; doi:10.1038/gene.2010.61; published online 23 December 2010

Complement factor H deficiency and endocapillary glomerulonephritis due to paternal isodisomy and a novel factor H mutation

Complement factor H (CFH) is a regulator of the alternative complement activation pathway. Mutations in the CFH gene are associated with atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis type II and C3 glomerulonephritis. Here, we report a 6-month-old CFH-deficient child presenting with endocapillary glomerulonephritis rather than membranoproliferative glomerulonephritis (MPGN) or C3 glomerulonephritis. Sequence analyses showed homozygosity for a novelCFH missense mutation (Pro139Ser) associated with severely decreased CFH plasma concentration (<6%) but normal mRNA splicing and expression. The father was heterozygous carrier of the mutation, but the mother was a non-carrier. Thus, a large deletion in the maternal CFH locus or uniparental isodisomy was suspected. Polymorphic markers across chromosome 1 showed homozygosity for the paternal allele in all markers and a lack of the maternal allele in six informative markers. This combined with a comparative genomic hybridization assay demonstrated paternal isodisomy. Uniparental isodisomy increases the risk of homozygous variations in other genes on the affected chromosome. Therefore, we analyzed other susceptibility genes on chromosome 1 and found no sequence variation in membrane cofactor protein, but homozygosity for the common deletion of CFH-related proteins 1 and 3, which may contribute to the early onset of disease.

Genes and Immunity (2011) 12, 90–99; doi:10.1038/gene.2010.63; published online 27 January 2011

Association of EBF1, FAM167A(C8orf13)-BLK andTNFSF4 gene variants with primary Sjögren's syndrome

We performed a candidate gene association study in 540 patients with primary Sjögren's Syndrome (SS) from Sweden (n=344) and Norway (n=196) and 532 controls (n=319 Swedish, n=213 Norwegian). A total of 1139 single-nucleotide polymorphisms (SNPs) in 84 genes were analyzed. In the meta-analysis of the Swedish and Norwegian cohorts, we found high signals for association between primary SS and SNPs in three gene loci, not previously associated with primary SS. These are the early B-cell factor 1 (EBF1) gene, P=9.9 × 10⁻⁵, OR 1.68, the family with sequence similarity 167 member A–B-lymphoid tyrosine kinase (FAM167A–BLK) locus, P=4.7 × 10⁻⁴, OR 1.37 and the tumor necrosis factor superfamily (TNFSF4=Ox40L) gene, P=7.4 × 10⁻⁴, OR 1.34. We also confirmed the association between primary SS and the IRF5/TNPO3 locus and the STAT4 gene. We found no association between the SNPs in these five genes and the presence of anti-SSA/anti-SSB antibodies. EBF1, BLK and TNFSF4 are all involved in B-cell differentiation and activation, and we conclude that polymorphisms in several susceptibility genes in the immune system contribute to the pathogenesis of primary SS.

Genes and Immunity (2011) 12, 100–109; doi:10.1038/gene.2010.44; published online 23 September 2010

Replication of top markers of a genome-wide association study in multiple sclerosis in Spain

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with presumed autoimmune origin, triggered by genetic and environmental risk factors. A recent genome-wide association study conducted on MS identified new biallelic markers outside the HLA (human leucocyte antigen) region involved in disease susceptibility: rs1109670(DDEF2); rs1458175 (PDZRN4); rs1529316 and rs2049306 (CSMD1);rs16914086 (TBC1D2); rs1755289 (SH3GL2); rs1841770 (ZIC1); rs651477(EN1); rs7607490 (TRIB2); rs397020 (C20orf46); rs908821 (SLC25A36);rs7672826 (MGC45800) and rs9523762 (GPC5). We aimed at replicating these top association signals in a Spanish cohort of 2863 MS patients and 2930 sex- and age-matched controls. Only rs9523762 mapping in the GPC5gene was significantly associated (G allele, P=1.6 × 10⁻⁵; odds ratio (95%confidence interval)=1.23 (1.12–1.36)), supporting a role for this proteoglycan in MS predisposition. The independent replication of association signals to validate data generated by genome-wide association scans is a first step in the effort to improve patient care.

Genes and Immunity (2011) 12, 110–115; doi:10.1038/gene.2010.52; published online 14 October 2010

An autoimmune-associated variant in PTPN2 reveals an impairment of IL-2R signaling in CD4+ T cells

The IL-2/IL-2R signaling pathway has an important role in autoimmunity. Several genes identified in genome-wide association (GWA) studies encode proteins in the IL-2/IL-2R signaling cascade that are associated with autoimmune diseases. One of these, PTPN2, encodes a protein tyrosine phosphatase that is highly expressed in T cells and regulates cytokine signaling. An intronic risk allele in PTPN2, rs1893217(C), correlated with decreased IL-2R signaling in CD4⁺ T cells as measured by phosphorylation of STAT5 (phosphorylated STAT5 (pSTAT5)). We modeled an additive single nucleotide polymorphism (SNP) genotype, in which each copy of the risk allele conferred a decrease in IL-2R signaling (P=4.4 × 10⁻⁸). Decreased pSTAT5 impacted IL-2Rβ chain signaling resulting in reduced FOXP3 expression in activated cells. This phenotype was not due to overt differences in expression of the IL-2R, molecules in the IL-2R signaling cascade or defects in STAT5. However, the rs1893217(C) risk variant did correlate with decreased PTPN2 expression in CD4⁺CD45RO T cells (P=0.0002). Thus, the PTPN2rs1893217(C) risk allele associated with reduced pSTAT5 in response to IL-2 and reduced PTPN2 expression. Together, these data suggest that decreased expression of PTPN2 may indirectly modulate IL-2 responsiveness. These findings, identified through genotype/phenotype relationships, may lead to identification of novel mechanisms underlying dysregulation of cytokine signaling in autoimmunity.

Genes and Immunity (2011) 12, 116–125; doi:10.1038/gene.2010.54; published online 23 December 2010

Transcriptional repression and DNA looping associated with a novel regulatory element in the final exon of the lymphotoxin-beta gene

Transcriptional regulation has a critical role in the coordinate and context-specific expression of a cluster of genes encoding members of the tumour necrosis factor (TNF) superfamily found at chromosome 6p21, comprising TNF, LTA (encoding lymphotoxin-alpha) and LTB (encoding lymphotoxin-beta). This is important, as dysregulated expression of these genes is implicated in susceptibility to many autoimmune, inflammatory and infectious diseases. We describe here a novel regulatory element in the fourth exon of LTB, which is highly conserved, localises to the only CpG island in the locus, and is associated with a DNaseI hypersensitive site and specific histone modifications. We find evidence of binding by Yin Yang 1 (YY1), cyclic AMP response element (CRE)-binding protein (CREB) and CCCTC-binding factor (CTCF) to this region in Jurkat T cells, which is associated with transcriptional repression on reporter gene analysis. Chromatin conformation capture experiments show evidence of DNA looping, involving interaction of this element with the LTB promoter, LTA promoter and TNF 30
untranslated region (UTR). Small interfering RNA (siRNA) experiments demonstrate a functional role for YY1 and CREB in LTB expression. Our findings provide evidence of additional complexity in the transcriptional regulation of LTB with implications for coordinate expression of genes in this important genomic locus.

Genes and Immunity (2011) 12, 126–135; doi:10.1038/gene.2010.62; published online 20 January 2011

Effect of amino acid substitutions in the human IFN-γR2 on IFN-γ responsiveness

Patients with interferon-γ receptor (IFN-γR) null mutations have severe infections with poorly pathogenic Mycobacteria. The IFN-γR complex involves two IFN-γR1 and two IFN-γR2 chains, in which several amino acid substitutions, some linked to disease and some apparently naturally occurring, have been described. We developed a model system to study functional effects of genetic variations in IFN-γR2. We retrovirally transduced wild-type IFN-γR2 and IFN-γR2 carrying presently known amino acid substitutions in various human cell lines, and next determined the IFN-γR2 expression pattern as well as IFN-γ responsiveness. We determined that the T58R, Q64R, E147K and K182E variants of IFN-γR2 are fully functional, although the Q64R variant may be expressed higher on the cell membrane. The R114C, T168N and G227R variants were identified in patients that had disseminated infections with non-tuberculousMycobacteria. Of these genetic variants, T168N was confirmed to be completely non-functional, whereas the novel variant G227R, and the previously reported R114C, were partial functional. The impaired IFN-γresponsiveness of R114C and G227R is mainly due to reduced receptor function, although expression on the cell membrane is reduced as well. We conclude that the T58R, Q64R, E147K and K182E variants are polymorphisms, whereas the R114C, T168N and G227R constitute mutations associated with disease.

Genes and Immunity (2011) 12, 136–144; doi:10.1038/gene.2010.74; published online 20 January 2011

Members 6B and 14 of the TNF receptor superfamily in multiple sclerosis predisposition

TNFRSF6B and TNFRSF14 genes were recently associated with Crohn's disease and rheumatoid arthritis. TNFRSF14 is known as herpes virus entry mediator (HVEM), and herpes viruses have been involved in the aetiology of multiple sclerosis (MS). MS patients present human herpes virus 6 (HHV6) in active plaques and increased antibody responses to HHV6. We aimed to ascertain the role of these genes in MS susceptibility and to investigate the relationship of the gene encoding the widely expressed HVEM receptor with the active replication of HHV6 found in some MS patients. Genotyping of 1370 Spanish MS patients and 1715 ethnically matched controls was performed. HHV6A DNA levels (surrogate of active viral replication) were analysed in serum of MS patients during a 2-year follow-up. Both polymorphisms were associated with MS predisposition, with stronger effect in patients with HHV6 active replication—TNFRSF6B-rs4809330^*A: P=0.028, OR=1.13; TNFRSF14-rs6684865^*A: overall P=0.0008, OR=1.2; and HHV6-positive patients vs controls: P=0.017, OR=1.69.

Genes and Immunity (2011) 12, 145–148; doi:10.1038/gene.2010.42; published online 21 October 2010

Let-7/miR-98 regulate Fas and Fas-mediated apoptosis

Fas is ubiquitously expressed on a variety of cells and triggers apoptosis, which have critical roles in the immune system. MicroRNAs (miRNAs) have been recently identified as regulators that modulate target gene expression and are involved in diverse biological processes, such as cell proliferation and apoptosis. This study was undertaken to investigate the contribution of miRNA in the regulation of Fas expression and Fas-mediated apoptosis. Bioinformatics analysis indicated that Fas was a potential target of let-7/miR-98 family. Indeed ectopic expression of let-7/miR-98 reduced, whereas knockdown of endogenous let-7/miR-98 increased the expression of Fas at both mRNA and protein levels. Let-7/miR-98 was verified to target Fas 3′ untranslated region directly by site-directed gene mutagenesis and reporter gene assay. More importantly, introduction of let-7/miR-98 could decrease the sensitivity to Fas-induced apoptosis. Furthermore, let-7/miR-98 expression was reduced in activation-induced cell death process, accompanied by increased expression of Fas. In conclusion, our study first demonstrated that let-7/miR-98 regulated Fas expression and the sensitivity of Fas-mediated apoptosis.

Genes and Immunity (2011) 12, 149–154; doi:10.1038/gene.2010.53; published online 13 January 2011

Prostate's PTEN prognosis

U.S. and European academics have separately identified new biochemical signatures that could improve prostate cancer diagnosis and monitoring.^{1, 2} ProteoMediX AG holds rights to the European data and plans to develop a blood-based test that will complement PSA testing to increase diagnostic accuracy. Metamark Genetics Inc. has licensed the American team's results and plans to develop an objective histological test that will complement Gleason scoring to improve prognostic prediction.

The standard screening procedure to detect prostate cancer includes a blood test that measures levels of prostate-specific antigen (KLK3; PSA).³ Although PSA testing can detect prostate cancer with high sensitivity, the test has poor specificity. The reason for the high rate of false positives is that unrelated factors including prostate inflammation can increase PSA levels.

If serum PSA concentration exceeds a certain threshold, a patient may elect to undergo prostate biopsy, in which a tissue sample is extracted and examined microscopically for cellular abnormalities. A trained pathologist then determines the presence of cancerous cells and assigns a Gleason score that ranges from 2–10, with the upper number representing the worst prognosis.

Gleason scores are used to determine how aggressively to treat the cancer, with options that range from watchful waiting to radical prostatectomy. The score is a good way to measure progression but can vary depending on the pathologist who examines the sample.

To improve decision making, the academics at the Swiss Federal Institute of Technology Zurich (ETHZ) and theDana-Farber Cancer Institute both used a mouse model of cancer induced by deleting Pten (Mmac1; Tep1) specifically in the prostate.

PTEN is the second most frequently mutated tumor suppressor gene behind p53, and genetic alterations of at least one PTEN allele have been reported in 70%–80% of prostate cancers.⁴

“We didn't think that searching in a sea of diverse patients with different stages of cancer was the most rational strategy to identify new cancer markers,” said Wilhelm Krek, a professor of cell biology at ETHZ who led one of the groups. “This mouse model is a highly reproducible, robust and controlled system that maximized our chance of identifying relevant prostate-specific biomarkers.”

In collaboration with Ruedi Aebersold, professor and head of the Institute of Molecular Systems Biology at ETHZ, the group used mass spectrometry to identify and quantify glycosylated proteins in prostate tissue and serum collected from wild-type or Pten mutant mice. The authors limited their analysis to glycosylated proteins, which can be selected for during sample preparation, to maximize the approach's sensitivity.

After identifying 126 serum proteins that were expressed differently in Pten-deleted mouse prostate tissue compared with control tissue, the researchers set out to validate their findings in the clinic. They collected serum from 77 prostate cancer patients and 66 controls with benign prostatic hyperplasia (BPH) and performed targeted mass spectrometry to measure the levels of candidate biomarkers in each patient group.

The ETHZ team also used computational modeling to find the sets of proteins that were most predictive of PTENstatus, prostate cancer diagnosis or Gleason score. The approach identified a four-protein signature that could improve the accuracy of prostate cancer diagnosis when combined with PSA measurement.

Separately, each test distinguished prostate cancer from BPH with sensitivity and specificity of about 80% and 60%, respectively. Combined, sensitivity and specificity increased to 85% and 79%, respectively.

Results were published in the Proceedings of the National Academy of Sciences.

In a separate study, the Dana-Farber researchers sought to identify new prognostic markers for prostate cancer. Their first step was generating a double-knockout mouse with exacerbated disease in which both Pten and mothers against decapentaplegic homolog 4 (Madh4; Smad4; Dpc4) were deleted in the prostate. SMAD4 is a DNA-binding transcriptional regulator thought to play a role in prostate cancer progression.

Whereas Pten-deleted mice developed localized lesions in the prostate that were only minimally metastatic, the double knockouts developed aggressive metastatic disease. Subsequent analyses showed that the aggressive disease partly stemmed from upregulation of cyclin D1 (Ccnd1; Bcl1) and osteopontin (Opn; Spp1), a pair of targets downstream from Smad4.

Those two targets increased tumor growth and metastasis when expressed in prostate tumor cells that were implanted into mice.

The prognostic value of all four proteins was validated by immunohistochemical staining of tissue samples from 405 tumor specimens from men diagnosed with prostate cancer who underwent radical prostatectomy. Staining for the four proteins predicted clinical outcome significantly better than Gleason scoring, and the combination of the two measures further increased prognostic accuracy compared with either alone.

Results were published in Nature.

Study leader Ronald DePinho, professor of medicine and genetics at Harvard Medical School and director of the Belfer Institute for Applied Cancer Science at Dana-Farber, told SciBX the goal was to “identify genes that were functional determinants of cancer progression because we thought they would be the most accurate predictors of prognosis.”

“These two papers propose the means to fulfill major unmet needs in prostate cancer diagnosis and prognosis,” said Pier Paolo Pandolfi, professor of medicine and pathology at Harvard Medical School and director of research at the Cancer Center at Beth Israel Deaconess Medical Center who has pioneered the development of Pten-deficient mouse models.

Pandolfi noted that even prostate cancers without PTEN mutation frequently have decreased expression of PTEN, suggesting that these gene signatures could be applicable to many patients regardless of PTEN mutational status.

Pandolfi did say that large, multicenter prospective trials are needed to validate the findings.

“One advantage of prostate cancer is that there is a great lag between diagnosis and eventual outcome, so it provides an opportunity to follow patients' progress and collect samples over time, making it easier to validate and compare tests with respect to other, more aggressive cancers,” he said.

 

ProteoMediX was founded in 2010 as a spinoff of ETHZ to develop a test based on the candidate glycoprotein signatures identified in the PNAS publication. The company is developing ELISA-based assays for prostate cancer based on the findings and hopes to begin pivotal clinical trials in two to three years.

“Our big advantage is we already know the signature we want to look for, and we now need to develop the assays,” CEO and cofounder Ralph Schiess told SciBX. “There is a huge demand for improved tests for prostate cancer diagnosis, and while mass spectrometry is a powerful discovery tool, it would not have the throughput necessary to handle that demand.”

“While the company is developing the commercial aspect, at ETH Zurich we will continue to use mass spectrometry to examine this signature in larger patient cohorts, to look for new and improved signatures and to test whether we can predict PTEN mutation status in other cancer types,” said Krek, also a cofounder of ProteoMediX.

Metamark was founded in 2007 to develop prognostic tests for cancer based on the work of DePinho and Lynda Chin, a professor of dermatology at Harvard Medical School and scientific director of the Belfer Institute who also was an author of the Nature paper.

CSO Peter Blume-Jensen told SciBX that the company “is developing tests that use quantitative immunofluorescence to accurately measure protein levels in tissue samples, and we intend to automate the assays as much as possible. Gleason scoring is somewhat subjective and can vary from doctor to doctor, and we want to try to remove that element of uncertainly in favor of an evidence-based, quantitative test.”

He said the Nature paper “lays a strong biochemical foundation to build a prognostic test that can complement or replace Gleason scoring, to help determine the risk of prostate cancer progression and recurrence before treatment decisions are made.”

The company plans to finalize its choice of markers by the end of 2011 and to launch a validation study in 2Q12.

“The company goes beyond looking at correlative prognostic biomarkers—it is trying to look at functionally significant drivers of cancer aggression,” said Chin. “If you look at validated diagnostic and prognostic biomarkers in clinical use, every single one is functionally important. They are not just correlative; they also play a role in disease.”

Cain, C. SciBX 4(9); doi:10.1038/scibx.2011.241
Published online March 3 2011

Promising New Gene Therapy for HIV Immunity

Scientists are working to genetically engineer the immune cells of HIV patients so they become HIV-resistant, according to interim results presented Wednesday at the 18th Conference on Retroviruses and Opportunistic Infections in Boston.
Dr. Carl June, a gene-therapy expert at the University of Pennsylvania, and colleagues collected CD4 T cells from HIV patients' own blood for the procedure. The team used "zinc fingers" to snip out a single gene in the collected T cells so that they lacked CCR5, a receptor HIV needs to enter the cell. The engineered cells were then injected back into their donor.
The first of nine patients in the study received a single infusion in July 2009. In all nine, the engineered cells remain HIV-free and have multiplied to an average 6 percent of the patients' T cells. No serious adverse events were reported, though all experienced temporary symptoms such as headache, chills or fever.
"It's a big accomplishment because this is the first successful attempt at genetic editing," said June. "It gives us an essential tool."
"This is elegant work, scientifically very sound, and an important 'proof of concept,'" said Dr. Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases. NIAID funded research that was foundational to the study. California-based Sangamo BioSciences, which developed the zinc fingers used like molecular scissors to delete or insert specific genes, is funding human testing of the T cells.
"It's a step," said co-investigator Pablo Tebas. "It's a step in a long process." Plans for a clinical trial of the therapy are underway.

Half of Men Have HPV Infections: Study

New research suggests that half of adult males may be infected with human papillomavirus, certain strains of which cause most cases of genital warts and cervical cancer. The STD also is linked to anal, penile, head, and neck cancers. And while women, especially as they age, appear better able to clear HPV infection, men seem to lack this capability, said study leader Anna Giuliano.
"The biology seems to be very similar to women," said Giuliano, of the H. Lee Moffitt Cancer Center and Research Institute in Tampa, Fla. "What is different is men seem to have high prevalence of genital HPV infections throughout their lifespans."
The research team examined HPV infection rates among 1,159 men in the United States, Brazil, and Mexico. The men, who were recruited from the general population, universities, and organized health care systems, all were HIV-negative with no history of cancer. They were assessed every six months, with median follow-up of 27.5 months.
"We found that there is a high proportion of men who have genital HPV infections," Giuliano said. "At enrollment, it was 50 percent." The rate of new HPV acquisition among the men was similar to that noted among women. About 6 percent of men per year became infected with HPV type 16, which causes cervical cancer in women and other cancers in men.
Although US doctors are permitted to vaccinate both young females and males against HPV, the shots are part of routine recommendations for young females only.
These latest results "must surely strengthen the argument for vaccination of men, both for their own protection, and that of their partners," said a statement by Dr. Anne Szarewski of London's Wolfson Institute of Preventive Medicine.
The full study, "Incidence and Clearance of Genital Human Papillomavirus Infection in Men (HIM): A Cohort Study," was published in the Lancet (2011;doi:10.1016/S0140-6736(10)62342-2).