NLRP12 inhibits PRRSV-2 replication by promoting GP2a degradation via MARCH8.

NLRP12, a member of the NLR family, has been shown to exert a vital function in orchestrating immune responses. Here, using the immunosuppressive porcine reproductive and respiratory syndrome virus (PRRSV) as a model, the role of NLRP12 in virus infection was deciphered. We demonstrated that overexpression of NLRP12 significantly restrained PRRSV replication, while NLRP12 silencing resulted in increased viral titer. Mechanistically, NLRP12 interacts with glycoprotein 2a (GP2a) through its LRR domain and recruits the membrane-associated RING-CH E3 ubiquitin ligase 8 (MARCH8) via the PYD domain. NLRP12 facilitates the lysine-48 (K48)-linked polyubiquitination of GP2a at K128 and induces its lysosome degradation via the MARCH8-NDP52 (nuclear dot protein 52 kDa) pathway. To counteract this, PRRSV Nsp2 effectively prevented the polyubiquitination of GP2a induced by NLRP12 by its deubiquitinating activity. Meanwhile, the overexpression of Nsp4 decreased the mRNA of endogenous NLRP12 and cleaved NLRP12 in a 3C-like protease activity-dependent manner, which collaboratively counteracts the antiviral function of NLRP12. Collectively, this study revealed the mechanisms of the NLRP12-MARCH8-NDP52 axis in the host defense against PRRSV, which might be harnessed for the development of anti-PRRSV therapies.

Team Science Approaches to Unravel Monogenic Parkinson's Disease on a Global Scale.

BACKGROUND: Until recently, about three-quarters of all monogenic Parkinson's disease (PD) studies were performed in European/White ancestry, thereby severely limiting our insights into genotype-phenotype relationships at a global scale. OBJECTIVE: To identify the multi-ancestry spectrum of monogenic PD. METHODS: The first systematic approach to embrace monogenic PD worldwide, The Michael J. Fox Foundation Global Monogenic PD Project, contacted authors of publications reporting individuals carrying pathogenic variants in known PD-causing genes. In contrast, the Global Parkinson's Genetics Program's Monogenic Network took a different approach by targeting PD centers underrepresented or not yet represented in the medical literature. RESULTS: In this article, we describe combining both efforts in a merger project resulting in a global monogenic PD cohort with the buildup of a sustainable infrastructure to identify the multi-ancestry spectrum of monogenic PD and enable studies of factors modifying penetrance and expressivity of monogenic PD. CONCLUSIONS: This effort demonstrates the value of future research based on team science approaches to generate comprehensive and globally relevant results. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Engineering and characterizing porcine reproductive and respiratory syndrome virus with separated and tagged genes encoding the minor glycoproteins.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen affecting pigs and belongs to the enveloped plus-stranded RNA virus family Arteriviridae. A unique feature of Arteriviruses is that the genes encoding the structural proteins overlap at their 3` and 5` ends. This impedes mutagenesis opportunities and precludes the binding of short peptides for antibody detection, as this would alter the amino acids encoded by the overlapping gene. In this study, we aimed to generate infectious PRRSV variants with separated genes encoding the minor glycoproteins Gp2, Gp3, and Gp4, accompanied by appended tags for detection. All recombinant genomes facilitate the release of infectious virus particles into the supernatant of transfected 293 T cells, as evidenced by immunofluorescence of infected MARC-145 cells using anti-nucleocapsid antibodies. Furthermore, expression of Gp2-Myc and Gp3-HA was confirmed through immunofluorescence and western blot analysis with tag-specific antibodies. However, after two passages of Gp2-Myc and Gp3-HA viruses, the appended tags were completely removed as indicated by sequencing the viral genome. Recombinant viruses with separated Gp2 and Gp3 genes remained stable for at least nine passages, while those with Gp3 and Gp4 genes separated reverted to wild type after only four passages. Notably, this virus exhibited significantly reduced titers in growth assays. Furthermore, we introduced a tag to the C-terminus of Gp4. The Gp4-HA virus was consistently stable for at least 10 passages, and the HA-tag was detectable by western blotting and immunofluorescence.

The Significance of the 98th Amino Acid in GP2a for Porcine Reproductive and Respiratory Syndrome Virus Adaptation in Marc-145 Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the pig industry. Marc-145 cells are widely used for PRRSV isolation, vaccine production, and investigations into virus biological characteristics. Despite their significance in PRRSV research, Marc-145 cells struggle to isolate specific strains of the North American virus genotype (PRRSV-2). The involvement of viral GP2a, GP2b, and GP3 in this phenomenon has been noted. However, the vital amino acids have not yet been identified. In this study, we increased the number of blind passages and successfully isolated two strains that were previously difficult to isolate with Marc-145 cells. Both strains carried an amino acid substitution in GP2a, specifically phenylalanine to leucine at the 98th amino acid position. Through a phylogenetic and epidemiologic analysis of 32 strains, those that were not amenable to isolation widely exhibited this mutation. Then, by using the PRRSV reverse genetics system, IFA, and Western blotting, we identified the mutation that could affect the tropism of PRRSV-2 for Marc-145 cells. Furthermore, an animal experiment was conducted. Through comparisons of clinical signs, mortality rates, and viral load in the organs and sera, we found that mutation did not affect the pathogenicity of PRRSV-2. In conclusion, our study firmly establishes the 98th amino acid in GP2a as a key determinant of PRRSV-2 tropism for Marc-145 cells.

Glycoprotein 2 as a gut gate keeper for mucosal equilibrium between inflammation and immunity.

Glycoprotein 2 (GP2) is a widely distributed protein in the digestive tract, contributing to mucosal barrier maintenance, immune homeostasis, and antigen-specific immune response, while also being linked to inflammatory bowel disease (IBD) pathogenesis. This review sheds light on the extensive distribution of GP2 within the gastrointestinal tract and its intricate interplay with the immune system. Furthermore, the significance of GP2 autoantibodies in diagnosing and categorizing IBD is underscored, alongside the promising therapeutic avenues for modulating GP2 to regulate immunity and maintain mucosal balance.

Adverse Drug Effect Profiles of Gp2b/3a Inhibitors: A Comparative Review of the Last Two Decades.

ST-Elevation Myocardial Infarction and non-ST Elevation Myocardial Infarction belong to the acute coronary syndrome group of diseases. These conditions are characterized by the complete or partial blockage of one or several coronary arteries, resulting in myocardial injury or necrosis. Various medications are used in their treatment, with the most recent addition being Glycoprotein IIb/IIIa inhibitors. They work by hindering the activity of glycoprotein IIb/IIIa receptors, which, in turn, prevents the clumping of platelets. Some of the GpIIb/IIIa inhibitors available in this category include abciximab, tirofiban, eptifibatide, roxifiban, and orbofiban. With this comprehensive literature review, we aimed to explore the potential adverse effects of these medications and compare the three in terms of their side effects profile. We searched through PubMed and Google Scholar and pinpointed 13 articles aligned with our inclusion criteria: six articles utilized eptifibatide, four were related to abciximab, and three used tirofiban. In 85% of the cases, a severe drop in platelet count, reaching as low as 1000/µL, was reported. Additionally, several other side effects were noted: one case documented multiple bruising spots appearing around the patient's body, two cases reported diffuse alveolar hemorrhage, and one case described a cardiac tamponade resulting from hemorrhagic pericarditis. Our study highlights the crucial significance of keeping a watchful eye on and comprehending the potential drawbacks linked to these medications in cardiovascular treatment. The necessity of researching these medications and their side effects is also evident, as this will significantly enhance the quality of treatment provided.

Single-cell profiling of GP2-enriched pancreatic progenitors to simultaneously create acinar, ductal, and endocrine organoids.

Rationale: Pancreatic lineage specification follows the formation of tripotent pancreatic progenitors (PPs). Current protocols rebuilding PPs in vitro have an endocrine lineage bias and are mostly based on PDX1/NKX6-1 coexpression neglecting other markers decisive for PP heterogeneity and lineage potential. However, true tripotent PPs are of utmost interest to study also exocrine disorders such as pancreatic cancer and to simultaneously generate all three pancreatic lineages from the same ancestor. Methods: Here, we performed a comprehensive compound testing to advance the generation of multipotent progenitors, which were further characterized for their trilineage potential in vitro and in vivo. The heterogeneity and cell-cell communication across the PP subpopulations were analyzed via single-cell transcriptomics. Results: We introduce a novel PP differentiation platform based on a comprehensive compound screening with an advanced design of experiments computing tool to reduce impurities and to increase Glycoprotein-2 expression and subsequent trilineage potential. Superior PP tripotency was proven in vitro by the generation of acinar, endocrine, and ductal cells as well as in vivo upon orthotopic transplantation revealing all three lineages at fetal maturation level. GP2 expression levels at PP stage ascribed varying pancreatic lineage potential. Intermediate and high GP2 levels were superior in generating endocrine and duct-like organoids (PDLO). FACS-based purification of the GP2high PPs allowed the generation of pancreatic acinar-like organoids (PALO) with proper morphology and expression of digestive enzymes. scRNA-seq confirmed multipotent identity, positioned the GP2/PDX1/NKX6-1high population next to human fetal tip and trunk progenitors and identified novel ligand-receptor (LR) interactions in distinct PP subpopulations. LR validation experiments licensed midkine and VEGF signaling to increase markers labelling the single cell clusters with high GP2 expression. Conclusion: In this study, we guide human pluripotent stem cells into multipotent pancreatic progenitors. This common precursor population, which has the ability to mature into acinar, ductal and functional ß-cells, serves as a basis for studying developmental processes and deciphering early cancer formation in a cell type-specific context. Using single-cell RNA sequencing and subsequent validation studies, we were able to dissect PP heterogeneity and specific cell-cell communication signals.

Unique Structure and Distinctive Properties of the Ancient and Ubiquitous Gamma-Type Envelope Glycoprotein.

After the onset of the AIDS pandemic, HIV-1 (genus Lentivirus) became the predominant model for studying retrovirus Env glycoproteins and their role in entry. However, HIV Env is an inadequate model for understanding entry of viruses in the Alpharetrovirus, Gammaretrovirus and Deltaretrovirus genera. For example, oncogenic model system viruses such as Rous sarcoma virus (RSV, Alpharetrovirus), murine leukemia virus (MLV, Gammaretrovirus) and human T-cell leukemia viruses (HTLV-I and HTLV-II, Deltaretrovirus) encode Envs that are structurally and functionally distinct from HIV Env. We refer to these as Gamma-type Envs. Gamma-type Envs are probably the most widespread retroviral Envs in nature. They are found in exogenous and endogenous retroviruses representing a broad spectrum of vertebrate hosts including amphibians, birds, reptiles, mammals and fish. In endogenous form, gamma-type Envs have been evolutionarily coopted numerous times, most notably as placental syncytins (e.g., human SYNC1 and SYNC2). Remarkably, gamma-type Envs are also found outside of the Retroviridae. Gp2 proteins of filoviruses (e.g., Ebolavirus) and snake arenaviruses in the genus Reptarenavirus are gamma-type Env homologs, products of ancient recombination events involving viruses of different Baltimore classes. Distinctive hallmarks of gamma-type Envs include a labile disulfide bond linking the surface and transmembrane subunits, a multi-stage attachment and fusion mechanism, a highly conserved (but poorly understood) "immunosuppressive domain", and activation by the viral protease during virion maturation. Here, we synthesize work from diverse retrovirus model systems to illustrate these distinctive properties and to highlight avenues for further exploration of gamma-type Env structure and function.

Human glycoprotein-2 expressed in Brunner glands - A putative autoimmune target and link between Crohn's and coeliac disease.

Glycoprotein 2 (GP2) is an autoantigen in Crohn's (CD) and coeliac disease (CeD). We assessed GP2-isoform (GP21-4)-expression in intestinal biopsies of paediatric patients with CD, CeD, ulcerative colitis (UC), and healthy children (HC). Transcription of GP21-4 was elevated in proximal small intestine in CeD and CD patients (only GP22/4) compared to jejunum (CeD/CD) and large bowel (CD). CeD patients demonstrated higher duodenal GP22/4-mRNA levels compared to HC/UC patients whereas CD patients showed higher GP24-mRNA levels compared to UC patients. Duodenal synthesis of only small GP2 isoforms (GP23/4) was demonstrated in epithelial cells in patients/HC and in Brunner glands (also large isoforms) with a more frequent apical location in CD/CeD patients. All four GP2 isoforms interacted with gliadin and phosphopeptidomannan. Gliadin digestion improved binding to GP2 isoforms. GP21-4 binding to CeD/CD-related antigens, elevated duodenal GP21-4-mRNA transcription, and GP2-protein secretion in Brunner glands of CeD/CD patients suggest an autoimmune CeD/CD link.

A Single Amino Acid Substitution in Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 2 Significantly Impairs Its Infectivity in Macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted tropism for macrophages and CD163 is a key receptor for infection. In this study, the PRRSV strain NCV1 was passaged on MARC-145 cells for 95 passages, and two plaque-clones (C1 and C2) were randomly selected for further analysis. The C1 virus nearly lost the ability to infect porcine alveolar macrophages (PAMs), as well as porcine kidney cells expressing porcine CD163 (PK15-pCD163), while the C2 virus replicates well in these two cell types. Pretreatment of MARC-145 cells with an anti-CD163 antibody nearly blocked C1 virus infection, indicating that the virus still required CD163 to infect cells. The C1 virus carried four unique amino acid substitutions: three in the nonstructural proteins and a K160I in GP2. The introduction of an I160K substitution in GP2 of the C1 virus restored its infectivity in PAMs and PK15-pCD163 cells, while the introduction of a K160I substitution in GP2 of the low-passaged, virulent PRRSV strain NCV13 significantly impaired its infectivity. Importantly, pigs inoculated with the rNCV13-K160I mutant exhibited lower viremia levels and lung lesions than those infected with the parental rNCV13. These results demonstrated that the K160 residue in GP2 is one of the key determinants of PRRSV tropism.

Diagnostic and prognostic role of pancreatic secretory granule membrane major glycoprotein 2 (GP2) immunohistochemistry: A TMA study on 27,681 tumors.

Pancreatic secretory granule membrane major glycoprotein 2 (GP2) is a membrane component of zymogen granules which is abundantly secreted by pancreatic acinar cells. Because RNA based analyses suggest a strict limitation of GP2 expression to the pancreas in normal tissues, and a strong preference to pancreatic cancer among tumors, GP2 expression analysis might have diagnostic utility. To better understand the role of GP2 protein expression, GP2 was successfully analyzed in 27,965 tumor samples from 132 different tumor types and subtypes as well as 8 samples each of 76 different normal tissue types by immunohistochemistry in a tissue microarray format (TMA). GP2 immunostaining was seen in 14 of 16 (87.5 %) acinar cell carcinomas, 6 of 507 (1.2 %) ductal adenocarcinomas, and 3 of 99 neuroendocrine neoplasms of the pancreas (3.0 %). GP2 was also found in 23 extra-pancreatic tumor entities including several types of neuroendocrine neoplasms (14.3-58.8 %), prostatic adenocarcinomas (8.2-18.8 %), various other adenocarcinomas (0.1-7.7 %), and several categories of benign and malignant salivary gland tumors (2.3-3.1 %). A strong GP2 positivity was only seen in 6 tumor categories including 50 % of 16 pancreatic acinus cell carcinomas, 11.8 % of 17 neuroendocrine tumors of the lung, 1.3 % of 80 primary Gleason 4 + 4 % and 0.6 % of 181 recurrent prostate cancers, as well as 0.8 % of 133 adenocarcinomas of the lung. In a cohort of 14,747 prostate cancers with follow up data, GP2 immunostaining was strongly linked to advanced pT stage, high Gleason grade, lymph node metastasis, and recurrence free survival (p < 0.0001 each). The prognostic impact of GP2 positivity was independent of established parameters in TMPRSS2:ERG fusion-negative cancers (p < 0.0001). In summary, our data show that GP2 is preferentially expressed in acinar cell carcinomas of the pancreas but the glycoprotein can - rarely - also be expressed in a variety of other tumor entities.

Expression of the Heterotrimeric GP2/GP3/GP4 Spike of an Arterivirus in Mammalian Cells.

Equine arteritis virus (EAV), an enveloped positive-strand RNA virus, is an important pathogen of horses and the prototype member of the Arteiviridae family. Unlike many other enveloped viruses, which possess homotrimeric spikes, the spike responsible for cellular tropism in Arteriviruses is a heterotrimer composed of 3 glycoproteins: GP2, GP3, and GP4. Together with the hydrophobic protein E they are the minor components of virus particles. We describe the expression of all 3 minor glycoproteins, each equipped with a different tag, from a multi-cassette system in mammalian BHK-21 cells. Coprecipitation studies suggest that a rather small faction of GP2, GP3, and GP4 form dimeric or trimeric complexes. GP2, GP3, and GP4 co-localize with each other and also, albeit weaker, with the E-protein. The co-localization of GP3-HA and GP2-myc was tested with markers for ER, ERGIC, and cis-Golgi. The co-localization of GP3-HA was the same regardless of whether it was expressed alone or as a complex, whereas the transport of GP2-myc to cis-Golgi was higher when this protein was expressed as a complex. The glycosylation pattern was also independent of whether the proteins were expressed alone or together. The recombinant spike might be a tool for basic research but might also be used as a subunit vaccine for horses.

Adhesion of Enteropathogenic, Enterotoxigenic, and Commensal Escherichia coli to the Major Zymogen Granule Membrane Glycoprotein 2.

Pathogenic bacteria, such as enteropathogenic Escherichia coli (EPEC) and enterotoxigenic E. coli (ETEC), cause diarrhea in mammals. In particular, E. coli colonizes and infects the gastrointestinal tract via type 1 fimbriae (T1F). Here, the major zymogen granule membrane glycoprotein 2 (GP2) acts as a host cell receptor. GP2 is also secreted by the pancreas and various mucous glands, interacting with luminal type 1 fimbriae-positive E. coli. It is unknown whether GP2 isoforms demonstrate specific E. coli pathotype binding. In this study, we investigated interactions of human, porcine, and bovine EPEC and ETEC, as well as commensal E. coli isolates with human, porcine, and bovine GP2. We first defined pathotype- and host-associated FimH variants. Second, we could prove that GP2 isoforms bound to FimH variants to various degrees. However, the GP2-FimH interactions did not seem to be influenced by the host specificity of E. coli. In contrast, soluble GP2 affected ETEC infection and phagocytosis rates of macrophages. Preincubation of the ETEC pathotype with GP2 reduced the infection of cell lines. Furthermore, preincubation of E. coli with GP2 improved the phagocytosis rate of macrophages. Our findings suggest that GP2 plays a role in the defense against E. coli infection and in the corresponding host immune response. IMPORTANCE Infection by pathogenic bacteria, such as certain Escherichia coli pathotypes, results in diarrhea in mammals. Pathogens, including zoonotic agents, can infect different hosts or show host specificity. There are Escherichia coli strains which are frequently transmitted between humans and animals, whereas other Escherichia coli strains tend to colonize only one host. This host specificity is still not fully understood. We show that glycoprotein 2 is a selective receptor for particular Escherichia coli strains or variants of the adhesin FimH but not a selector for a species-specific Escherichia coli group. We demonstrate that GP2 is involved in the regulation of colonization and infection and thus represents a molecule of interest for the prevention or treatment of disease.

Glycoprotein 2 in health and disease: lifting the veil.

In 2020, we discovered glycoprotein 2 (GP2) variants associated with pancreatic cancer susceptibility in a genome-wide association study involving the Japanese population. Individuals carrying a missense coding variant (rs78193826) in the GP2 gene resulting in a p.V432M substitution had an approximately 1.5-fold higher risk of developing pancreatic cancer than those without this variant. GP2 is expressed on the inner surface of zymogen granules in pancreatic acinar cells, which are responsible for the sorting, storage and secretion of digestive enzymes. Upon neuronal, hormonal, or other stimulation, GP2 is cleaved from the membrane of zymogen granules and then secreted into the pancreatic duct and intestinal lumen. While the functions of GP2 remain poorly understood, emerging evidence suggests that it plays an antibacterial role in the gastrointestinal tract after being secreted from pancreatic acinar cells. Impaired GP2 functions may facilitate the adhesion of bacteria to the intestinal mucosa. In this review article, we summarize the role of GP2 in health and disease, emphasizing its functions in the gastrointestinal tract, as well as genetic variations in the GP2 gene and their associations with disease susceptibility. We hope that its robust genetic associations with pancreatic cancer, coupled with its emerging role in gastrointestinal mucosal immunity, will spur renewed research interest in GP2, which has been understudied over the past 30 years compared with its paralog uromodulin (UMOD).

Common Variants in NUS1 and GP2 Genes Contributed to the Risk of Gestational Diabetes Mellitus.

Background: Recently, NUS1 and GP2 genes were reported to be associated with the risk of type 2 diabetes (T2D) in a Japanese population. Given the sharing of pathogenic contribution from genetic factors between T2D and gestational diabetes mellitus (GDM), we conducted the study to systematically examine the relationship of NUS1 and GP2 genes with the susceptibility to GDM in Chinese Han population. Methods: A total of 4,250 subjects comprised of 1,282 patients with GDM and 2,968 controls were recruited, and 20 tag single nucleotide polymorphisms (SNPs) (10 from NUS1 and 10 from GP2) were selected for genotyping. Association analyses were conducted for GDM and its related biomedical indexes including fasting glucose and HbA1c levels. Results: Two SNPs, rs80196932 from NUS1 (P=2.93×10-5) and rs117267808 from GP2 (P=5.68×10-5), were identified to be significantly associated with the risk of GDM. Additionally, SNP rs80196932 was significantly associated with HbA1c level in both patients with GDM (P=0.0009) and controls (P=0.0003), while SNP rs117267808 was significantly associated with fasting glucose level in both patients with GDM (P=0.0008) and controls (P=0.0007). Serum levels of protein NUS1 and GP2 were measured for the study subjects, and significant differences were identified among groups with different genotypes of SNP rs80196932 and rs117267808, respectively. Conclusions: Our findings indicate that NUS1 and GP2 genes contribute to the risk of GDM, which would help to offer the potential to improve our understanding of the etiology of GDM and, in turn, could facilitate the development of novel medicines and treatments for GDM.

Tunable expression rate control of a growth-decoupled T7 expression system by L-arabinose only.

BACKGROUND: Precise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell. In this article we show that genes under control of a normally Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible PT7-lacO promoter can be induced solely with L-arabinose in a newly constructed Escherichia coli expression host BL21-AI, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production. RESULTS: Here, we show that BL21-AI is able to precisely regulate protein production rates on a cellular level in an L-arabinose concentration-dependent manner and simultaneously allows for reallocation of metabolic resources due to L-arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2. We have successfully characterized the system under relevant fed-batch like conditions in microscale cultivation (800 µL) and generated data proofing a relevant increase in specific yields for 6 different Escherichia coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting. CONCLUSIONS: In all cases tested, BL21-AI outperformed the parental strain BL21-AI, operated in growth-associated production mode. Specific MP-GFP fusion proteins yields have been improved up to 2.7-fold. Therefore, this approach allows for fine tuning of MP production or expression of multi-enzyme pathways where e.g. particular stoichiometries have to be met to optimize product flux.

Antibodies Against Glycoprotein 2 Are Specific Biomarkers for Pediatric Crohn's Disease.

BACKGROUND: Serological markers can assist in accurate differentiation between Crohn's disease (CD) and ulcerative colitis (UC). One such marker is anti-glycoprotein 2 (anti-GP2) which was shown to be a specific marker for CD in adult patients. The aim of our study was to assess the utility of anti-GP2 and GP2 as biomarkers for pediatric CD, and determine whether they correlate with disease activity. METHODS: Serum samples were tested by ELISA for anti-GP2 isoform 4 IgG and IgA, and also for GP2. Results were correlated with demographic and clinical data. RESULTS: The cohort consisted of 53 pediatric patients with CD, 42 with UC, and 53 controls. Levels of anti-GP2 were significantly increased in pediatric patients with CD in comparison with patients with UC, and control subjects, with high positive predictive value for both IgG and IgA (97.9% and 82.6%, respectively). While specificity of anti-GP2 IgG and IgA was very high (98.7% and 90.0%, respectively), sensitivity was low (42.0% and 35.5% for IgG and IgA, respectively). In CD, anti-GP2 correlated with disease activity, and decreased in treatment-naïve patients following successful induction therapy. A higher IgA anti-GP2 was also demonstrated in patients with ileo-colonic involvement, and was associated with a younger age. Finally, positive GP2 level was identified in only 1/211 serum samples. CONCLUSIONS: A positive anti-GP2 level is highly associated with CD, while a negative result does not exclude CD. Additional studies are required to determine whether these markers can be used in pediatric patients with CD for risk stratification.

Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in E. coli.

The site-specific incorporation of noncanonical amino acids (ncAAs) into proteins by amber stop codon suppression has become a routine method in academic laboratories. This approach requires an amber suppressor tRNACUA to read the amber codon and an aminoacyl-tRNA synthetase to charge the tRNACUA with the ncAA. However, a major drawback is the low yield of the mutant protein in comparison to the wild type. This effect primarily results from the competition of release factor 1 with the charged suppressor tRNACUA for the amber codon at the A-site of the ribosome. A number of laboratories have attempted to improve the incorporation efficiency of ncAAs with moderate results. We aimed at increasing the efficiency to produce high yields of ncAA-functionalized proteins in a scalable setting for industrial application. To do this, we inserted an ncAA into the enhanced green fluorescent protein and an antibody mimetic molecule using an industrial E. coli strain, which produces recombinant proteins independent of cell growth. The controlled decoupling of recombinant protein production from cell growth considerably increased the incorporation of the ncAA, producing substantially higher protein yields versus the reference E. coli strain BL21(DE3). The target proteins were expressed at high levels, and the ncAA was efficiently incorporated with excellent fidelity while the protein function was preserved.

Over-Production of Therapeutic Growth Factors for Articular Cartilage Regeneration by Protein Production Platforms and Protein Packaging Cell Lines.

This review article focuses on the current state-of-the-art cellular and molecular biotechnology for the over-production of clinically relevant therapeutic and anabolic growth factors. We discuss how the currently available tools and emerging technologies can be used for the regenerative treatment of osteoarthritis (OA). Transfected protein packaging cell lines such as GP-293 cells may be used as "cellular factories" for large-scale production of therapeutic proteins and pro-anabolic growth factors, particularly in the context of cartilage regeneration. However, when irradiated with gamma or x-rays, these cells lose their capacity for replication, which makes them safe for use as a live cell component of intra-articular injections. This innovation is already here, in the form of TissueGene-C, a new biological drug that consists of normal allogeneic primary chondrocytes combined with transduced GP2-293 cells that overexpress the growth factor transforming growth factor ß1 (TGF-ß1). TissueGene-C has revolutionized the concept of cell therapy, allowing drug companies to develop live cells as biological drug delivery systems for direct intra-articular injection of growth factors whose half-lives are in the order of minutes. Therefore, in this paper, we discuss the potential for new innovations in regenerative medicine for degenerative diseases of synovial joints using mammalian protein production platforms, specifically protein packaging cell lines, for over-producing growth factors for cartilage tissue regeneration and give recent examples. Mammalian protein production platforms that incorporate protein packaging eukaryotic cell lines are superior to prokaryotic bacterial expression systems and are likely to have a significant impact on the development of new humanized biological growth factor therapies for treating focal cartilage defects and more generally for the treatment of degenerative joint diseases such as OA, especially when injected directly into the joint.