Moving towards high density clinical signature studies with a human proteome catalogue developing multiplexing mass spectrometry assay panels

A perspective overview is given describing the current development of multiplex mass spectrometry assay technology platforms utilized for high throughput clinical sample analysis. The development of targeted therapies with novel personalized medicine drugs will require new tools for monitoring efficacy and outcome that will rely on both the quantification of disease progression related biomarkers as well as the measurement of disease specific pathway/signaling proteins

Biobank resources for future patient care: developments, principles and concepts

The aim of the overview is to give a perspective of global biobank development is given in a view of positioning biobanking as a key resource for healthcare to identify new potential markers that can be used in patient diagnosis and complement the targeted personalized drug treatment. The fast progression of biobanks around the world is becoming an important resource for society where the patient benefit is in the focus, with a high degree of personal integrity and ethical standard. Biobanks are providing patient benefits by large scale screening studies, generating large database repositories. It is envisioned by all participating stakeholders that the biobank initiatives will become the future gateway to discover new frontiers within life science and patient care. There is a great importance of biobank establishment globally, as biobanks has been identified as a key area for development in order to speed up the discovery and development of new drugs and protein biomarker diagnostics. One of the major objectives in Europe is to establish concerted actions, where biobank networks are being developed in order to combine and have the opportunity to share and build new science and understanding from complex disease biology. These networks are currently building bridges to facilitate the establishments of best practice and standardizations

Localization of tamoxifen in human breast cancer tumors by MALDI mass spectrometry imaging

Background: Tamoxifen is used in endocrine treatment of breast cancer to inhibit estrogen signaling. A set of strati‐ ed ER‐positive and ER‐negative tumor sections was subjected to manual deposition of tamoxifen solution in order to investigate its spatial distribution upon exposure to interaction within thin tissue sections. Methods: The localization of tamoxifen in tumor sections was assessed by matrix assisted laser deposition/ioniza‐ tion mass spectrometry imaging. The images of extracted ion maps were analyzed for comparison of signal intensity distributions. Results: The precursor ion of tamoxifen (m/z 372.233) displayed heterogeneous signal intensity distributions in his‐ tological compartments of tumor tissue sections. The levels of tamoxifen in tumor cells compared with stroma were higher in ER‐positive tissues, whereas ER‐negative tissue sections showed lower signal intensities in tumor cells. Conclusions: The experimental model was successfully applied on frozen tumor samples allowing for di erentiation between ER groups based on distribution of tamoxifen

Low Levels of Perforin Expression in CD8+ T Lymphocyte Granules in Lymphoid Tissue during Acute Human Immunodeficiency Virus Type 1 Infection

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) responses are detectable shortly after the acute phase of HIV infection, but they cannot control viral replication and prevent development of chronic immune suppression. This article describes a defect in the coexpression of perforin in granzyme A-positive CD8+ T cells in lymphoid tissue from patients with acute HIV infection and a reduction in the perforin-dependent nuclear translocation of granzyme A. Furthermore, intracellular levels of HIV DNA and RNA found in lymphoid tissue were higher (10-100 times) than those found in blood, and blood samples showed more-coordinated cellular perforin/granzyme A expression. This suggests that mechanisms inhibiting CTL-mediated cytotoxicity are operative in lymphoid tissue early in the course of HIV infectio

Integrated proteogenomic approach identifying a protein signature of COPD and a new splice variant of SORBS1

Translation of genomic alterations to protein changes in chronic obstructive pulmonary disease (COPD) is largely unexplored. Using integrated proteomic and RNA sequencing analysis of COPD and control lung tissues, we identified a protein signature in COPD characterised by extracellular matrix changes and a potential regulatory role for SUMO2. Furthermore, we identified 61 differentially expressed novel, non-reference, peptides in COPD compared with control lungs. This included two peptides encoding for a new splice variant of SORBS1, of which the transcript usage was higher in COPD compared with control lungs. These explorative findings and integrative proteogenomic approach open new avenues to further unravel the pathology of COPD

Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention

Expression of Tissue factor in Adenocarcinoma and Squamous Cell Carcinoma of the Uterine Cervix: Implications for immunotherapy with hI-con1, a factor VII-IgGFc chimeric protein targeting tissue factor

<p>Abstract</p> <p>Background</p> <p>Cervical cancer continues to be an important worldwide health problem for women. Up to 35% of patients who are diagnosed with and appropriately treated for cervical cancer will recur and treatment results are poor for recurrent disease. Given these sobering statistics, development of novel therapies for cervical cancer remains a high priority. We evaluated the expression of Tissue Factor (TF) in cervical cancer and the potential of hI-con1, an antibody-like-molecule targeted against TF, as a novel form of immunotherapy against multiple primary cervical carcinoma cell lines with squamous- and adenocarcinoma histology.</p> <p>Methods</p> <p>Because TF is a transmembrane receptor for coagulation factor VII/VIIa (fVII), in this study we evaluated the <it>in vitro </it>expression of TF in cervical carcinoma cell lines by immunohistochemistry (IHC), real time-PCR (qRT-PCR) and flow cytometry. Sensitivity to hI-con1-dependent cell-mediated-cytotoxicity (IDCC) was evaluated in 5-hrs-⁵¹chromium-release-assays against cervical cancer cell lines <it>in vitro</it>.</p> <p>Results</p> <p>Cytoplasmic and/or membrane TF expression was observed in 8 out of 8 (100%) of the tumor tissues tested by IHC and in 100% (11 out of 11) of the cervical carcinoma cell lines tested by real-time-PCR and flow cytometry but not in normal cervical keratinocytes (<it>p </it>= 0.0023 qRT-PCR; <it>p </it>= 0.0042 flow cytometry). All primary cervical cancer cell lines tested overexpressing TF, regardless of their histology, were highly sensitive to IDCC (mean killing ± SD, 56.2% ± 15.9%, range, 32.4%-76.9%, <it>p </it>< 0.001), while negligible cytotoxicity was seen in the absence of hI-con1 or in the presence of rituximab-control-antibody. Low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (<it>p </it>= 0.025) while human serum did not significantly decrease IDCC against cervical cancer cell lines (<it>p </it>= 0.597).</p> <p>Conclusions</p> <p>TF is highly expressed in squamous and adenocarcinoma of the uterine cervix. hI-con1 induces strong cytotoxicity against primary cervical cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of cervical cancer refractory to standard treatment modalities.</p

Both Conventional and Interferon Killer Dendritic Cells Have Antigen-Presenting Capacity during Influenza Virus Infection

Natural killer cells are innate effector cells known for their potential to produce interferon-γ and kill tumour and virus-infected cells. Recently, B220+CD11cintNK1.1+ NK cells were found to also have antigen-presenting capacity like dendritic cells (DC), hence their name interferon-producing killer DC (IKDC). Shortly after discovery, it has already been questioned if IKDC really represent a separate subset of NK cells or merely represent a state of activation. Despite similarities with DCs, in vivo evidence that they behave as bona fide APCs is lacking. Here, using a model of influenza infection, we found recruitment of both conventional B220− NK cells and IKDCs to the lung. To study antigen-presenting capacity of NK cell subsets and compare it to cDCs, all cell subsets were sorted from lungs of infected mice and co-cultured ex vivo with antigen specific T cells. Both IKDCs and conventional NK cells as well as cDCs presented virus-encoded antigen to CD8 T cells, whereas only cDCs presented to CD4 T cells. The absence of CD4 responses was predominantly due to a deficiency in MHCII processing, as preprocessed peptide antigen was presented equally well by cDCs and IKDCs. In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung. In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset

LPS unmasking of Shigella flexneri reveals preferential localisation of tagged outer membrane protease IcsP to septa and new poles

The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP(HA) was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP(HA) was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP(HA) was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP(HA) and IcsA showed that IcsP(HA) preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP(HA) in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP(HA) was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP(HA) detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.Elizabeth Ngoc Hoa Tran, Matthew Thomas Doyle, Renato Moron