95 MAGE-A1 protein expression pattern in > 5,000 tumor and healthy tissue samples: Validation of MAGE-A1 as an ideal target for TCR-based cell therapy

BackgroundCancer testis antigens (CTAs) are considered attractive targets for T cell receptor (TCR)-based cellular therapies as their expression in healthy adults is considered restricted to the immune-privileged testis. However, low-level expression of some CTAs in healthy tissue has been observed, resulting in significant on-target/off-cancer toxicity. Melanoma associated antigen 1 (MAGE-A1) is a member of the MAGE-A CTA family, whose members are known to influence cellular signaling pathways through their E3 ubiquitin ligase-binding MAGE homology domain. MAGE-A proteins are frequently expressed in different cancer types, have been linked to oncogenic activity and their expression has been associated with poor prognosis.1 Literature data suggest that in healthy tissues MAGE-A1 is detected in testis, only, with one exception suggesting MAGE-A1 RNA expression in cerebellum and cerebrum.2 Therefore, to evaluate MAGE-A1 as a potential target for cellular immunotherapies, an in-depth analysis of MAGE-A1 expression in > 70 different healthy tissue types and > 5,000 cancer biopsies was conducted, aiming to assess if MAGE-A1 represents a valid and safe target.MethodsA MAGE-A1 antibody with high specificity (TK-AbMA1P) was identified and characterized for immunohistochemistry. A large panel of > 70 different healthy tissue types and > 5,000 tumor biopsies was explored and scored for MAGE-A1 expression by tissue microarray. Identified cancer entities with relevant MAGE-A1 expression were further investigated to assess spatial intratumoral MAGE-A1 expression distribution and expression consistency between primary tumor and lymph node/distant metastases.ResultsCharacterization of TK-AbMA1P demonstrated fully paralog-selective staining for MAGE-A1. Analysis of MAGE-A1 expression in over 70 different healthy tissues confirmed strictly selective expression of MAGE-A1 in testis. An extended analysis of various CNS tissues including cerebellum and cerebrum did not reveal any expression in CNS. The analysis of > 5,000 tumor biopsies showed significant MAGE-A1 expression in distinct subgroups of multiple major tumor types with high unmet medical need. Substantial expression was detected for example in non-small-cell lung cancer, various breast cancer subtypes, gastrointestinal and urogenital cancers, among others. Extended analysis of the MAGE-A1 positive tumors demonstrated highly homogenous and consistent spatial intratumoral distribution of MAGE-A1 expression as well as between primary tumor and metastases.ConclusionsThis analysis confirms that MAGE-A1 is a highly selectively expressed CTA and demonstrates relevant expression in various indications with high unmet medical need, suggesting that MAGE-A1 is an ideal target for highly potent TCR-based adoptive cell therapy.ReferencesWeon JL, Potts PR. The MAGE protein family and cancer. Curr Opin Cell Biol 2015;37:1–8.Morgan RA, Chinnasamy N, Abate-Daga D, Gros A, Robbins PF, Zheng Z, Dudley ME, Feldman SA, Yang JC, Sherry RM, Phan GQ, Hughes MS, Kammula US, Miller AD, Hessman CJ, Stewart AA, Restifo NP, Quezado MM, Alimchandani M, Rosenberg AZ, Nath A, Wang T, Bielekova B, Wuest SC, Akula N, McMahon FJ, Wilde S, Mosetter B, Schendel DJ, Laurencot CM, Rosenberg SA. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy. J Immunother 2013;36(2):133–51.Ethics ApprovalThis study was approved by the Ethics Commission of the Ärztekammer Hamburg; approval number WF-049/09. Participants gave informed consent before taking part.

Minocycline black bone disease in arthroplasty: a systematic review

Background Minocycline black bone disease is a rare finding that can cause concern when unexpectedly encountered during routine arthroplasty. Prolonged minocycline use can cause selective staining of subchondral bone, whilst peri-articular soft tissue and cartilage appear uninvolved. Methods A systematic review according to PRISMA guidelines was performed to identify all reported cases in the literature. Results Including the patient we present, eleven cases of minocycline black bone disease encountered during arthroplasty have been reported in the literature. All cases have had an excellent outcome, with no complications reported to date. Conclusions Minocycline black bone disease can be a concerning intra-operative finding when unexpectedly encountered during routine arthroplasty, but should not affect the operative plan. Surgeons should exclude alternative causes of bone discolouration when the history is unclear.

A Simple Technique for the Identification of Environmental DNA (eDNA ) in the Water Samples

The environmental DNA(eDNA) is the DNA that is shed by the organisms in their environment by different ways viz. , mucous, faeces, skin, eggs, sperms and also when these organisms die due to natural death or disease. The eDNA will persist for several days. Identification of eDNA is a useful method of determining the organisms present in an aquatic environment like amphibians, reptiles, fishes , insects and larval forms of some of these organisms. By analysing the e-DNA it is possible to monitor the species distribution in water bodies like lakes and ponds simply by collecting a sample of water. The technique can be applied for the survey of the water bodies on a large scale for the genomic, taxonomic as well as pollutional studies. The DNA isolation procedures that are available are laborious and time consuming. Therefore, during the present study, a simplified method was devised i. e. , isolation of eDNA with ethanol after which Feulgen stain was applied to identify and confirm it, as it is an easy method before proceeding to work with the isolated eDNA using other techniqnies for further studies. The Feulgen method is used for the selective staining and the localisation of the DNA in the tissues but is adopted during the present study for the water samples for quick identification of eDNA. The smear of eDNA stained with Feulgen showed dark pink or magenta colour under the microscope where it was concentrated but stained lightly when dispersed and fragmented as observed in the present study. Further studies of the isolated eDNA are in progress in our laboratory for quantifying and sequencing eDNA using latest techniques like next generation sequencing for the identification of fish species in the lakes.

A Fused Thiophene-S,S-Dioxide-Based Super-Photostable Fluorescent Marker for Lipid Droplets

Lipid droplets (LDs) are essential organelle in most eukaryotes, and tracking intracellular LDs dynamics using synthetic small molecules is crucial for biological studies. However, only a limited number of fluorescent markers that satisfy all requirements, such as the selective staining of LDs, high photostability, and sufficient biocompatibility, have been developed. Herein, we report a series of donor-p-acceptor dyes based on the thiophene-containing fused polycyclic scaffold [1]benzothieno[3,2-<i>b</i>][1]benzothiophene (BTBT), in which either or both thiophene rings are oxidized into thiophene-<i>S</i>,<i>S</i>-dioxide to form an electron-accepting building block. Among these dyes, LAQ1 satisfied all the aforementioned requirements, and allowed us capturing ultra-small LDs on the endoplasmic reticulum (ER) membrane by stimulation emission depletion (STED) microscopy with a super-resolution below the diffraction limit of light. Moreover, the extremely high photostability of LAQ1 enabled recording the lipolysis of LDs and the concomitant lipogenesis as well as long-term trajectory analysis of micro LDs at the single particle level in living cells.

A Fused Thiophene-S,S-Dioxide-Based Super-Photostable Fluorescent Marker for Lipid Droplets

Lipid droplets (LDs) are essential organelle in most eukaryotes, and tracking intracellular LDs dynamics using synthetic small molecules is crucial for biological studies. However, only a limited number of fluorescent markers that satisfy all requirements, such as the selective staining of LDs, high photostability, and sufficient biocompatibility, have been developed. Herein, we report a series of donor-p-acceptor dyes based on the thiophene-containing fused polycyclic scaffold [1]benzothieno[3,2-<i>b</i>][1]benzothiophene (BTBT), in which either or both thiophene rings are oxidized into thiophene-<i>S</i>,<i>S</i>-dioxide to form an electron-accepting building block. Among these dyes, LAQ1 satisfied all the aforementioned requirements, and allowed us capturing ultra-small LDs on the endoplasmic reticulum (ER) membrane by stimulation emission depletion (STED) microscopy with a super-resolution below the diffraction limit of light. Moreover, the extremely high photostability of LAQ1 enabled recording the lipolysis of LDs and the concomitant lipogenesis as well as long-term trajectory analysis of micro LDs at the single particle level in living cells.

Fluorescent small-molecule agonists as follicle-stimulating hormone receptor imaging tools

Discovery of a potent, small-molecule, fluorescent agonist of the follicle-stimulating hormone receptor (FSHR) for selective staining of FSHR-expressing cells.

Highly selective staining and quantification of intracellular lipid droplets with a compact push–pull fluorophore based on benzothiadiazole

A robust lipophilic dye, based on the structures of the benzothiadiazole heterocycle, was shown to be a potent fluorescent stain for the selective imaging of lipid droplets (LDs) within both live and fixed human cells.