Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes

The increasing demand for a sustainable larviculture has promoted research regarding environmental parameters, diseases and nutrition, intersecting at the mucosal surface of the gastrointestinal tract of fish larvae. The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling. This study aimed at optimizing an LCM protocol for intestinal tissue of sea bass larvae. Furthermore, a 3'/5' integrity assay was developed for LCM samples of fish tissue, comprising low RNA concentrations. Furthermore, reliable reference genes for performing qPCR in larval sea bass gene expression studies were identified, as data normalization is critical in gene expression experiments using RT-qPCR. We demonstrate that a careful optimization of the LCM procedure allows recovery of high quality mRNA from defined cell populations in complex intestinal tissues. According to the geNorm and Normfinder algorithms, ef1a, rpl13a, rps18 and faua were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass across a range of experimental settings. The methodology developed here, offers a rapid and valuable approach to characterize cells/tissues in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof

T Cells Recognizing a Peptide Contaminant Undetectable by Mass Spectrometry

Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted β-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214 epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP206-214 using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP206-214. If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results

Falsification of biotechnology drugs: current dangers and/or future disasters?

Falsified medical products have become a global threat since they were first mentioned to the general public at the conference of experts on the rational use of drugs organized by the world health organization (WHO) in 1985. Today, official estimates of the annual death toll due to falsified medical products range between two hundred thousand and one million. Although the extent of this global problem is the most significant in the developing world, an increasing number of reports have demonstrated the presence of a substantial (black) market for falsified medical products in the developed world. In recent years, also biotechnology drugs (synthetic peptide drugs and protein drugs) have been reported to be prone for falsifications. Next to the traditional doping related substances and image-enhancing polypeptides (e.g., human growth hormone, melanotan II) also essential medicines such as insulin, oxytocin and monoclonal antibodies have been falsified. The danger regarding the use of these falsified polypeptide drugs lies in the fact that end-users have no guarantee of the safety and efficacy of these preparations. Multiple reports have namely described the presence of the wrong active pharmaceutical ingredient (API), the wrong dosage or the absence of the API. Additionally, adverse health effects have been reported in the past due to toxic contaminations and product or process related impurities. Moreover, also unauthorized polypeptides or polypeptides which failed clinical trials or are still subject of clinical or pre-clinical assessments have been found in seizures of regulatory agencies. It stands to reason that regulatory agencies and analytical laboratories handling falsified biotechnology drugs have stepped up efforts to counter these grievous practices. The analysis of these falsified polypeptides and putative impurities is however not always straightforward. Often (bio)analytical laboratories have to resort to a combination of electrophoretic techniques, immunological assays and mass spectrometry based approaches to merely identify the content of seized samples. In addition, the difference in size (peptide vs proteins vs monoclonal antibodies), complexity (e.g., isoforms, glycosylations) and different synthesis techniques (chemical synthesis, recombinant expression, native protein isolation) result in a wide range of putative health risks. This review therefore aims to provide a brief overview of the genuine biotherapeutics present on the market and their quality prerequisites. Next, we describe the identification strategy utilised by our lab to identify the API in falsified biotherapeutics, followed by a discussion of the putative hazards due to impurities and contaminations that were found or could be encountered in falsified biotherapeutics. Finally, we terminate with an educational prediction of what may happen in the future and possible ways to counteract putative future&nbsp;disasters.</p