Chinese hamster ovary cells can produce galactose-α-1,3-galactose antigens on proteins

Chinese hamster ovary (CHO) cells are widely used for the manufacture of biotherapeutics, in part because of their ability to produce proteins with desirable properties, including 'human-like' glycosylation profiles. For biotherapeutics production, control of glycosylation is critical because it has a profound effect on protein function, including half-life and efficacy. Additionally, specific glycan structures may adversely affect their safety profile. For example, the terminal galactose-α-1,3-galactose (α-Gal) antigen can react with circulating anti α-Gal antibodies present in most individuals. It is now understood that murine cell lines, such as SP2 or NSO, typical manufacturing cell lines for biotherapeutics, contain the necessary biosynthetic machinery to produce proteins containing α-Gal epitopes. Furthermore, the majority of adverse clinical events associated with an induced IgE-mediated anaphylaxis response in patients treated with the commercial antibody Erbitux (cetuximab) manufactured in a murine myeloma cell line have been attributed to the presence of the α-Gal moiety. Even so, it is generally accepted that CHO cells lack the biosynthetic machinery to synthesize glycoproteins with α-Gal antigens. Contrary to this assumption, we report here the identification of the CHO ortholog of N-acetyllactosaminide 3-α-galactosyltransferase-1, which is responsible for the synthesis of the α-Gal epitope. We find that the enzyme product of this CHO gene is active and that glycosylated protein products produced in CHO contain the signature α-Gal antigen because of the action of this enzyme. Furthermore, characterizing the commercial therapeutic protein abatacept (Orencia) manufactured in CHO cell lines, we also identified the presence of α-Gal. Finally, we find that the presence of the α-Gal epitope likely arises during clonal selection because different subclonal populations from the same parental cell line differ in their expression of this gene. Although the specific levels of α-Gal required to trigger anaphylaxis reactions are not known and are likely product specific, the fact that humans contain high levels of circulating anti-α-Gal antibodies suggests that minimizing (or at least controlling) the levels of these epitopes during biotherapeutics development may be beneficial to patients. Furthermore, the approaches described here to monitor α-Gal levels may prove useful in industry for the surveillance and control of α-Gal levels during protein manufacture.National Center for Research Resources (U.S.) (Grant P41 RR018501-01

Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.

Blood pressure is a heritable trait influenced by several biological pathways and responsive to environmental stimuli. Over one billion people worldwide have hypertension (≥140 mm Hg systolic blood pressure or  ≥90 mm Hg diastolic blood pressure). Even small increments in blood pressure are associated with an increased risk of cardiovascular events. This genome-wide association study of systolic and diastolic blood pressure, which used a multi-stage design in 200,000 individuals of European descent, identified sixteen novel loci: six of these loci contain genes previously known or suspected to regulate blood pressure (GUCY1A3-GUCY1B3, NPR3-C5orf23, ADM, FURIN-FES, GOSR2, GNAS-EDN3); the other ten provide new clues to blood pressure physiology. A genetic risk score based on 29 genome-wide significant variants was associated with hypertension, left ventricular wall thickness, stroke and coronary artery disease, but not kidney disease or kidney function. We also observed associations with blood pressure in East Asian, South Asian and African ancestry individuals. Our findings provide new insights into the genetics and biology of blood pressure, and suggest potential novel therapeutic pathways for cardiovascular disease prevention

Dominant stop-loss <em>HNRNPA1</em> variants in juvenile-onset myopathy

\ua9 2024 The Author(s). Muscle &amp; Nerve published by Wiley Periodicals LLC. Introduction/Aims: Heterogeneous nuclear ribonucleoprotein A1 is involved in nucleic acid homeostatic functions. The encoding gene HNRNPA1 has been associated with several neuromuscular disorders including an amyotrophic lateral sclerosis-like phenotype, distal hereditary motor neuropathy, multisystem proteinopathy, and various myopathies. We report two unrelated individuals with monoallelic stop loss variants affecting the same codon of HNRNPA1. Methods: Two individuals with unsolved juvenile-onset myopathy were enrolled under approved institutional protocols. Phenotype data were collected and genetic analyses were performed, including whole-exome sequencing (WES). Results: The two probands (MNOT002-01 and K1440-01) showed a similar onset of slowly progressive extremity and facial weakness in early adolescence. K1440-01 presented with facial weakness, winged scapula, elevated serum creatine kinase (CK) levels, and mild neck weakness. MNOT002-01 also exhibited elevated CK levels along with facial weakness, cardiomyopathy, respiratory dysfunction, pectus excavatum, a mildly rigid spine, and loss of ambulation. On quadriceps muscle biopsy, K1440-01 displayed rounded myofibers, mild variation in fiber diameter, and type 2 fiber hypertrophy, while MNOT002-01 displayed rimmed vacuoles. Monoallelic stop-loss variants in HNRNPA1 were identified for both probands: c.1119A&gt;C p.*373Tyrext*6 (K1440-01) and c.1118A&gt;C p.*373Serext*6 (MNOT002-01) affect the same codon and are both predicted to lead to the addition of six amino acids before termination at an alternative stop codon. Discussion: Both stop-loss variants in our probands are likely pathogenic. Our findings contribute to the disease characterization of pathogenic variants in HNRNPA1. This gene should be screened in clinical diagnostic testing of unsolved cases of sporadic or dominant juvenile-onset myopathy

Staphylococcus aureus Surface Protein SdrE Binds Complement Regulator Factor H as an Immune Evasion Tactic

Similar to other highly successful invasive bacterial pathogens, Staphylococcus aureus recruits the complement regulatory protein factor H (fH) to its surface to inhibit the alternative pathway of complement. Here, we report the identification of the surface-associated protein SdrE as a fH-binding protein using purified fH overlay of S. aureus fractionated cell wall proteins and fH cross-linking to S. aureus followed by mass spectrometry. Studies using recombinant SdrE revealed that rSdrE bound significant fH whether from serum or as a purified form, in both a time- and dose-dependent manner. Furthermore, rSdrE-bound fH exhibited cofactor functionality for factor I (fI)-mediated cleavage of C3b to iC3b which correlated positively with increasing amounts of fH. Expression of SdrE on the surface of the surrogate bacterium Lactococcus lactis enhanced recruitment of fH which resulted in increased iC3b generation. Moreover, surface expression of SdrE led to a reduction in C3-fragment deposition, less C5a generation, and reduced killing by polymorphonuclear cells. Thus, we report the first identification of a S. aureus protein associated with the staphylococcal surface that binds factor H as an immune evasion mechanism

Harmful and beneficial aspects of Parthenium hysterophorus: an update

Parthenium hysterophorus is a noxious weed in America, Asia, Africa and Australia. This weed is considered to be a cause of allergic respiratory problems, contact dermatitis, mutagenicity in human and livestock. Crop production is drastically reduced owing to its allelopathy. Also aggressive dominance of this weed threatens biodiversity. Eradication of P. hysterophorus by burning, chemical herbicides, eucalyptus oil and biological control by leaf-feeding beetle, stem-galling moth, stem-boring weevil and fungi have been carried out with variable degrees of success. Recently many innovative uses of this hitherto notorious plant have been discovered. Parthenium hysterophorus confers many health benefits, viz remedy for skin inflammation, rheumatic pain, diarrhoea, urinary tract infections, dysentery, malaria and neuralgia. Its prospect as nano-medicine is being carried out with some preliminary success so far. Removal of heavy metals and dye from the environment, eradication of aquatic weeds, use as substrate for commercial enzyme production, additives in cattle manure for biogas production, as biopesticide, as green manure and compost are to name a few of some other potentials. The active compounds responsible for hazardous properties have been summarized. The aim of this review article is to explore the problem P. hysterophorus poses as a weed, the effective control measures that can be implemented as well as to unravel the latent beneficial prospects of this weed

IL-1β Promotes TGF-β1 and IL-2 Dependent Foxp3 Expression in Regulatory T Cells

Earlier, we have shown that GM-CSF-exposed CD8α− DCs that express low levels of pro-inflammatory cytokines IL-12 and IL-1β can induce Foxp3+ Tregs leading to suppression of autoimmunity. Here, we examined the differential effects of IL-12 and IL-1β on Foxp3 expression in T cells when activated in the presence and absence of DCs. Exogenous IL-12 abolished, but IL-1β enhanced, the ability of GM-CSF-exposed tolerogenic DCs to promote Foxp3 expression. Pre-exposure of DCs to IL-1β and IL-12 had only a modest effect on Foxp3− expressing T cells; however, T cells activated in the absence of DCs but in the presence of IL-1β or IL-12 showed highly significant increase and decrease in Foxp3+ T cell frequencies respectively suggesting direct effects of these cytokines on T cells and a role for IL-1β in promoting Foxp3 expression. Importantly, purified CD4+CD25+ cells showed a significantly higher ability to maintain Foxp3 expression when activated in the presence of IL-1β. Further analyses showed that the ability of IL-1β to maintain Foxp3 expression in CD25+ T cells was dependent on TGF-β1 and IL-2 expression in Foxp3+Tregs and CD25− effectors T cells respectively. Exposure of CD4+CD25+ T cells to IL-1β enhanced their ability to suppress effector T cell response in vitro and ongoing experimental autoimmune thyroidits in vivo. These results show that IL-1β can help enhance/maintain Tregs, which may play an important role in maintaining peripheral tolerance during inflammation to prevent and/or suppress autoimmunity

Genome-wide association meta-analysis of spontaneous coronary artery dissection identifies risk variants and genes related to artery integrity and tissue-mediated coagulation

Spontaneous coronary artery dissection (SCAD) is an understudied cause of myocardial infarction primarily affecting women. It is not known to what extent SCAD is genetically distinct from other cardiovascular diseases, including atherosclerotic coronary artery disease (CAD). Here we present a genome-wide association meta-analysis (1,917 cases and 9,292 controls) identifying 16 risk loci for SCAD. Integrative functional annotations prioritized genes that are likely to be regulated in vascular smooth muscle cells and artery fibroblasts and implicated in extracellular matrix biology. One locus containing the tissue factor gene F3, which is involved in blood coagulation cascade initiation, appears to be specific for SCAD risk. Several associated variants have diametrically opposite associations with CAD, suggesting that shared biological processes contribute to both diseases, but through different mechanisms. We also infer a causal role for high blood pressure in SCAD. Our findings provide novel pathophysiological insights involving arterial integrity and tissue-mediated coagulation in SCAD and set the stage for future specific therapeutics and preventions

Plasma miRNA as Biomarkers for Assessment of Total-Body Radiation Exposure Dosimetry

The risk of radiation exposure, due to accidental or malicious release of ionizing radiation, is a major public health concern. Biomarkers that can rapidly identify severely-irradiated individuals requiring prompt medical treatment in mass-casualty incidents are urgently needed. Stable blood or plasma-based biomarkers are attractive because of the ease for sample collection. We tested the hypothesis that plasma miRNA expression profiles can accurately reflect prior radiation exposure. We demonstrated using a murine model that plasma miRNA expression signatures could distinguish mice that received total body irradiation doses of 0.5 Gy, 2 Gy, and 10 Gy (at 6 h or 24 h post radiation) with accuracy, sensitivity, and specificity of above 90%. Taken together, these data demonstrate that plasma miRNA profiles can be highly predictive of different levels of radiation exposure. Thus, plasma-based biomarkers can be used to assess radiation exposure after mass-casualty incidents, and it may provide a valuable tool in developing and implementing effective countermeasures

Metabolomic Profiling Reveals a Role for Androgen in Activating Amino Acid Metabolism and Methylation in Prostate Cancer Cells

Prostate cancer is the second leading cause of cancer related death in American men. Development and progression of clinically localized prostate cancer is highly dependent on androgen signaling. Metastatic tumors are initially responsive to anti-androgen therapy, however become resistant to this regimen upon progression. Genomic and proteomic studies have implicated a role for androgen in regulating metabolic processes in prostate cancer. However, there have been no metabolomic profiling studies conducted thus far that have examined androgen-regulated biochemical processes in prostate cancer. Here, we have used unbiased metabolomic profiling coupled with enrichment-based bioprocess mapping to obtain insights into the biochemical alterations mediated by androgen in prostate cancer cell lines. Our findings indicate that androgen exposure results in elevation of amino acid metabolism and alteration of methylation potential in prostate cancer cells. Further, metabolic phenotyping studies confirm higher flux through pathways associated with amino acid metabolism in prostate cancer cells treated with androgen. These findings provide insight into the potential biochemical processes regulated by androgen signaling in prostate cancer. Clinically, if validated, these pathways could be exploited to develop therapeutic strategies that supplement current androgen ablative treatments while the observed androgen-regulated metabolic signatures could be employed as biomarkers that presage the development of castrate-resistant prostate cancer

Preventing Staphylococcus aureus Sepsis through the Inhibition of Its Agglutination in Blood

Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci - coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA) – were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans