Results from a 2020 field experiment encouraging voting by mail.

The ability to cast a mail ballot can safeguard the franchise. However, because there are often additional procedural protections to ensure that a ballot cast in person counts, voting by mail can also jeopardize people's ability to cast a recorded vote. An experiment carried out during the COVID-19 pandemic illustrates both forces. Philadelphia officials randomly sent 46,960 Philadelphia registrants postcards encouraging them to apply to vote by mail in the lead-up to the June 2020 primary election. While the intervention increased the likelihood a registrant cast a mail ballot by 0.4 percentage points (P = 0.017)-or 3%-many of these additional mail ballots counted only because a last-minute policy intervention allowed most mail ballots postmarked by Election Day to count.

Impervious to Elite Influence: Americans' ACA Attitudes, 2009-2020.

The Affordable Care Act (ACA) was a central issue dividing Republicans and Democrats for the decade following its 2010 enactment. Given that, it offers key lessons about policymaking and public opinion during a highly polarized political period. Here, I draw out some of those lessons from my 2023 book Stable Condition: Elites' Limited Influence on Health Care Attitudes, detailing how polarization shaped both the elite- and mass-level politics of the ACA. At the elite level, polarization and nationalization within the federal and state governments laid the groundwork for a highly complex law that was a patchwork of policies experienced very differently by different Americans. At the mass level, polarization and nationalization contributed to a remarkable level of stability in public opinion, so much so that even direct beneficiaries of the law did not typically become markedly more positive toward it. Elite efforts at opinion leadership, through policymaking and messaging, were largely unsuccessful.

Local demographic changes and US presidential voting, 2012 to 2016.

Immigration and demographic change have become highly salient in American politics, partly because of the 2016 campaign of Donald Trump. Previous research indicates that local influxes of immigrants or unfamiliar ethnic groups can generate threatened responses, but has either focused on nonelectoral outcomes or analyzed elections in large geographic units, such as counties. Here, we examine whether demographic changes at low levels of aggregation were associated with vote shifts toward an anti-immigration presidential candidate between 2012 and 2016. To do so, we compile a precinct-level dataset of election results and demographic measures for almost 32,000 precincts in the states of Florida, Georgia, Michigan, Nevada, Ohio, Pennsylvania, and Washington. We employ regression analyses varying model specifications and measures of demographic change. Our estimates uncover little evidence that influxes of Hispanics or noncitizen immigrants benefited Trump relative to past Republicans, instead consistently showing that such changes were associated with shifts to Trump's opponent.

Distinct Contributions of CD18 Integrins for Binding and Phagocytic Internalization of Pseudomonas aeruginosa.

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.

Motility-Independent Formation of Antibiotic-Tolerant Pseudomonas aeruginosa Aggregates.

Pseudomonas aeruginosa is a bacterial pathogen that causes severe chronic infections in immunocompromised individuals. This bacterium is highly adaptable to its environments, which frequently select for traits that promote bacterial persistence. A clinically significant temporal adaptation is the formation of surface- or cell-adhered bacterial biofilms that are associated with increased resistance to immune and antibiotic clearance. Extensive research has shown that bacterial flagellar motility promotes formation of such biofilms, whereupon the bacteria subsequently become nonmotile. However, recent evidence shows that antibiotic-tolerant nonattached bacterial aggregates, distinct from surface-adhered biofilms, can form, and these have been reported in the context of lung infections, otitis media, nonhealing wounds, and soft tissue fillers. It is unclear whether the same bacterial traits are required for aggregate formation as for biofilm formation. In this report, using isogenic mutants, we demonstrate that P. aeruginosa aggregates in liquid cultures are spontaneously formed independent of bacterial flagellar motility and independent of an exogenous scaffold. This contrasts with the role of the flagellum to initiate surface-adhered biofilms. Similarly to surface-attached biofilms, these aggregates exhibit increased antibiotic tolerance compared to planktonic cultures. These findings provide key insights into the requirements for aggregate formation that contrast with those for biofilm formation and that may have relevance for the persistence and dissemination of nonmotile bacteria found within chronic clinical infections.IMPORTANCE In this work, we have investigated the role of bacterial motility with regard to antibiotic-tolerant bacterial aggregate formation. Previous work has convincingly demonstrated that P. aeruginosa flagellar motility promotes the formation of surface-adhered biofilms in many systems. In contrast, aggregate formation by P. aeruginosa was observed for nonmotile but not for motile cells in the presence of an exogenous scaffold. Here, we demonstrate that both wild-type P. aeruginosa and mutants that genetically lack motility spontaneously form antibiotic-tolerant aggregates in the absence of an exogenously added scaffold. Additionally, we also demonstrate that wild-type (WT) and nonmotile P. aeruginosa bacteria can coaggregate, shedding light on potential physiological interactions and heterogeneity of aggregates.

Phosphatidylinositol-(3,4,5)-Trisphosphate Induces Phagocytosis of Nonmotile Pseudomonas aeruginosa.

Pathogenic bacteria that establish chronic infections in immunocompromised patients frequently undergo adaptation or selection for traits that are advantageous for their growth and survival. Clinical isolates of Pseudomonas aeruginosa, a Gram-negative, opportunistic bacterial pathogen, exhibit a temporal transition from a motile to a nonmotile phenotype through loss of flagellar motility during the course of chronic infection. This progressive loss of motility is associated with increased resistance to both antibiotic and immune clearance. We have previously shown that loss of bacterial motility enables P. aeruginosa to evade phagocytic clearance both in vitro and in vivo and fails to activate the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent phagocytic pathway. Therefore, we tested the hypothesis that clearance of phagocytosis-resistant bacteria could be induced by exogenously pretreating innate immune cells with the Akt-activating molecule phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). Here, we demonstrate that PIP3 induces the uptake of nonmotile P. aeruginosa by primary human neutrophils >25-fold, and this effect is phenocopied with the use of murine phagocytes. However, surprisingly, mechanistic studies revealed that the induction of phagocytosis by PIP3 occurs because polyphosphoinositides promote bacterial binding by the phagocytes rather than bypassing the requirement for PI3K. Moreover, this induction was selective since the uptake of other nonmotile Gram-negative, but not Gram-positive, bacteria can also be induced by PIP3 Since there is currently no treatment that effectively eradicates chronic P. aeruginosa infections, these findings provide novel insights into a potential methodology by which to induce clearance of nonmotile pathogenic bacteria and into the endogenous determinants of phagocytic recognition of P. aeruginosa.

A practical approach for O-linked mannose removal: the use of recombinant lysosomal mannosidase.

The methylotrophic yeast Pichia pastoris is an attractive expression system due to its ability to secrete large amounts of recombinant protein, with the potential for glycosylation. Advances in glycoengineering of P. pastoris have successfully demonstrated the humanization of both the N- and O-linked glycosylation pathways in this organism. However, in certain cases, the presence of O-linked glycans on a therapeutic protein may not be desirable. Recently, we have reported the in vitro utility of jack bean α-1,2/3/6-mannosidase to remove O-linked mannose from intact undenatured glycoproteins produced in glycoengineered P. pastoris. However, one caveat of this strategy is that jack bean mannosidase has yet to be cloned and as such is only available as crude cellular extracts. This raises several concerns for using this reagent to treat large preparations of therapeutic proteins generated in P. pastoris. Therefore, we postulated that lysosomal mannosidases which have been cloned and demonstrated to have similar activities to jack bean mannosidase on N-linked glycans would also process O-linked glycans in a similar fashion. To this end, we screened a panel of recombinant lysosomal mannosidases from different organisms and identified several which cannot only reduce extended O-linked mannose chains but which can also hydrolyze the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, not only do we show for the first time the utility of lysosomal mannosidase for O-linked mannose processing, but since this is a recombinant enzyme, it has several benefits over the use of crude jack bean mannosidase extracts.

Elimination of diaminopeptidase activity in Pichia pastoris for therapeutic protein production.

Yeast are important production platforms for the generation of recombinant proteins. Nonetheless, their use has been restricted in the production of therapeutic proteins due to differences in their glycosylation profile with that of higher eukaryotes. The yeast strain Pichia pastoris is an industrially important organism. Recent advances in the glycoengineering of this strain offer the potential to produce therapeutic glycoproteins with sialylated human-like N- and O-linked glycans. However, like higher eukaryotes, yeast also express numerous proteases, many of which are either localized to the secretory pathway or pass through it en route to their final destination. As a consequence, nondesirable proteolysis of some recombinant proteins may occur, with the specific cleavage being dependent on the class of protease involved. Dipeptidyl aminopeptidases (DPP) are a class of proteolytic enzymes which remove a two-amino acid peptide from the N-terminus of a protein. In P. pastoris, two such enzymes have been identified, Ste13p and Dap2p. In the current report, we demonstrate that while the knockout of STE13 alone may protect certain proteins from N-terminal clipping, other proteins may require the double knockout of both STE13 and DAP2. As such, this understanding of DPP activity enhances the utility of the P. pastoris expression system, thus facilitating the production of recombinant therapeutic proteins with their intact native sequences.

Computational modeling of revision total hip arthroplasty involving acetabular defects: A systematic review.

Revision total hip arthroplasty (rTHA) involving acetabular defects is a complex procedure associated with lower rates of success than primary THA. Computational modeling has played a key role in surgical planning and prediction of postoperative outcomes following primary THA, but modeling applications in rTHA for acetabular defects remain poorly understood. This study aimed to systematically review the use of computational modeling in acetabular defect classification, implant selection and placement, implant design, and postoperative joint functional performance evaluation following rTHA involving acetabular defects. The databases of Web of Science, Scopus, Medline, Embase, Global Health and Central were searched. Fifty-three relevant articles met the inclusion criteria, and their quality were evaluated using a modified Downs and Black evaluation criteria framework. Manual image segmentation from computed tomography scans, which is time consuming, remains the primary method used to generate 3D models of hip bone; however, statistical shape models, once developed, can be used to estimate pre-defect anatomy rapidly. Finite element modeling, which has been used to estimate bone stresses and strains, and implant micromotion postoperatively, has played a key role in custom and off-the-shelf implant design, mitigation of stress shielding, and prediction of bone remodeling and implant stability. However, model validation is challenging and requires rigorous evaluation and comparison with respect to mid- to long-term clinical outcomes. Development of fast, accurate methods to model acetabular defects, including statistical shape models and artificial neural networks, may ultimately improve uptake of and expand applications in modeling and simulation of rTHA for the research setting and clinic.

Production of sialylated O-linked glycans in Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N-linked glycans but up to now no one has addressed engineering the O-linked glycosylation pathway. Typically, O-linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with ß- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O-linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O-linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein-O-linked-mannose ß-1,2-N-acetylglucosaminyltransferase 1, resulted in the capping of the single O-linked mannose residues with N-acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O-linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N-linked glycosylated biotherapeutics to include molecules possessing O-linked glycans.

Effectiveness of Behaviorally Informed Letters on Health Insurance Marketplace Enrollment: A Randomized Clinical Trial.

Importance: Every year during the open enrollment period, hundreds of thousands of individuals across the Affordable Care Act marketplaces begin the enrollment process but fail to complete it, thereby resulting in coverage gaps or going uninsured. Objective: To investigate if low-cost ($0.55 per person) letters can increase health insurance enrollment. Design Setting and Participants: This intent-to-treat randomized clinical trial was conducted during the final 2 weeks of the 2015 open enrollment period among the 37 states on the HealthCare.gov platform. The trial targeted 744 510 individuals who started the enrollment process but had yet to complete it. Data were analyzed from January through August 2021. Interventions: Study participants were randomized to either a no-letter control group or to 1 of 8 letter variants that drew on evidence from the behavioral sciences about what motivates individuals to take action. Main Outcomes and Measures: The primary outcome was the health insurance enrollment rate at the end of the open enrollment period. Results: Of the 744 510 individuals (mean [SD] age, 41.9 [19.6] years; 53.9% women), 136 122 (18.3%) were in the control group and 608 388 (81.7%) were in the treatment group. Most lived in Medicaid nonexpansion states (72.7%), and a plurality were between 30 and 50 years old (41.0%). For race and ethnicity, 3.0% self-identified as Asian, 14.0% as Black, 5.1% as Hispanic, 39.8% as non-Hispanic White, and 38.2% as other or unknown. By the end of the open enrollment period, 4.0% of the control group enrolled in health insurance coverage. Comparatively, the enrollment rate in the pooled treatment group was 4.3%, which demonstrated an increase of 0.3 percentage points (95% CI, 0.2-0.4 percentage points; P<.001), yielding 1753 marginal enrollments. Letters that used action language caused larger enrollment effects, particularly among Black individuals (increase of 1.6 percentage points; 95% CI, 0.6-2.7 percentage points; P = .003) and Hispanic individuals (increase of 1.5 percentage points; 95% CI, 0.0-3.0 percentage points; P = .046) in Medicaid expansion states. Conclusions and Relevance: This randomized clinical trial shows that letters designed with best practices from the behavioral sciences literature were a low-cost way to increase health insurance enrollment in the Affordable Care Act marketplaces. More research is needed to understand what messages are most effective amid the recently passed American Rescue Plan. Trial Registration: ClinicalTrials.gov Identifier: NCT05010395.

Partisanship, Messaging, and the COVID-19 Vaccine: Evidence From Survey Experiments.

PURPOSE: To investigate partisanship in COVID-19 attitudes, and assess partisan or scientific messaging effects on COVID-19 vaccination intentions. DESIGN: Two-wave survey with two-arm randomized experiment. SETTING: Recruited Pennsylvania residents online. SAMPLE: 2037 (May 2020) and 1577 (October 2020) Pennsylvania residents, aged 18-94 years. INTERVENTION: Respondents saw messaging that presented either President Trump or scientists endorsing the vaccine, then reported their vaccination intentions. MEASURES: Likert scale items measuring COVID-19 attitudes (May), including mask wearing and vaccination intentions (May and October). ANALYSIS: Partisan differences in attitudes were analyzed by chi-square; differences in support for mask wearing and vaccination intentions were also analyzed by Mann-Whitney U. The messaging experiment was analyzed by chi-square, Mann-Whitney U, and survey-weighted multivariate logistic regression. RESULTS: Significant partisan differences were found in all attitudes. The partisan split in support for mask wearing increased from May to October, whereas the split in vaccination intentions decreased. Compared to partisan messaging, scientific messaging increased overall odds of intending to vaccinate by 32% in May (adjusted odds ratio [AOR]=1.32, 95% confidence interval [CI] = 1.06-1.65), and increased odds among Democrats by 142% in October (AOR = 2.42, CI = 1.29-4.55). Scientific messaging had no significant effect on independents or Republicans. CONCLUSION: Partisan COVID-19 attitudes were widespread and persistent. Partisan endorsement of the vaccine positively influenced those with congruent beliefs, while scientific messaging produced consistent effects across political affiliation.

Elimination of ß-mannose glycan structures in Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, such as those containing ß-mannose (Man) linkages, can elicit an immune response or bind to Man receptors, thus reducing their efficacy. Recent studies have confirmed that P. pastoris has four genes from the ß-mannosyl transferase (BMT) family and that Bmt2p is responsible for the majority of ß-Man linkages on glycans. While expressing recombinant human erythropoietin (rhEPO) in a developmental glycoengineered strain devoid of BMT2 gene expression, cross-reactivity was observed with an antibody raised against host cell antigens. Treatment of the rhEPO with protein N-glycosidase F eliminated cross-reactivity, indicating that the antigen was associated with the glycan. Thorough analysis of the glycan profile of rhEPO demonstrated the presence of low amounts of α-1,2-mannosidase resistant high-Man glycoforms. In an attempt to eliminate the α-mannosidase resistant glycoforms, we used a systemic approach to genetically knock-out the remaining members of the BMT family culminating in a quadruple bmt2,4,1,3 knock-out strain. Data presented here conclude that the additive elimination of Bmt2p, Bmt3p and Bmt1p activities are required for total abolition of ß-Man-associated glycans and their related antigenicity. Taken together, the elimination of ß-Man containing glycoforms represents an important step forward for the Pichia production platform as a suitable system for the production of therapeutic glycoproteins.

Not by turnout alone: Measuring the sources of electoral change, 2012 to 2016.

Changes in partisan outcomes between consecutive elections must come from changes in the composition of the electorate or changes in the vote choices of consistent voters. How much composition versus conversion drives electoral change has critical implications for the policy mandates of election victories and campaigning and governing strategies. Here, we analyze electoral change between the 2012 and 2016 U.S. presidential elections using administrative data. We merge precinct-level election returns, the smallest geography at which vote counts are available, with individual-level turnout records from 37 million registered voters in six key states. We find that both factors were substantively meaningful drivers of electoral change, but the balance varied by state. We estimate that pro-Republican Party (GOP) conversion among two-election voters was particularly important in states including Ohio, Michigan, and Pennsylvania where the pro-GOP swings were largest. Our results suggest conversion remains a crucial component of electoral change.

Cisplatin increases immune activity of monocytes and cytotoxic T-cells in a murine model of epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is an immunologically active malignancy, but thus far immune therapy has had limited success in clinical trials. One barrier to implementation of efficacious immune therapies is a lack of knowledge of the effect of chemotherapy on the monocyte-derived component of the immune infiltrate within the tumor. We utilized the ID8 murine EOC model to investigate alterations within tumor ascites that occur following administration of platinum chemotherapy. Cisplatin treatment resulted in a significant increase in monocytes within the ascites of tumor bearing mice. We identified that CD11b+ cells from the ascites of mice that have been treated with cisplatin elicits an increase in IFN-É£ expression from CD8+ T-cells compared to CD11b+ cells from a mouse treated with vehicle control (604.0 pg/mL v. 4328.0 pg/mL; p < .0001). Splenocytes derived from tumor bearing mice released increase levels of IFN-É£ after treatment with cisplatin when incubated with dendritic cells (DCs) and tumor antigen (62.0 v. 92.1 pg/mL; p = .03). Cisplatin induced an increase in T-cell and monocyte/macrophage activation markers (CD62L and CD301). Levels of IL-10, IL-6, and VEGF in the cell free ascites of mice treated with cisplatin decreased (p > .05). These results indicate that treatment with cisplatin leads to an increase of anti-tumor activity within the ascites related to alterations in the ascites monocytes. Further investigation of these findings in humans is necessary to identify how these cells behave in different patient subgroups and if there is a role for monocyte directed therapy in conjunction with T-cell directed therapy and/or chemotherapy.

Sex Hormones and Aging Modulate Interferon Lambda 1 Production and Signaling by Human Uterine Epithelial Cells and Fibroblasts.

Estradiol (E2) and progesterone (P) have potent effects on immune function in the human uterine endometrium which is essential for creating an environment conducive for successful reproduction. Type III/lambda (λ) interferons (IFN) are implicated in immune defense of the placenta against viral pathogens, which occurs against the backdrop of high E2 and P levels. However, the effect of E2 and P in modulating the expression and function of IFNλ1 in the non-pregnant human uterine endometrium is unknown. We generated purified in vitro cultures of human uterine epithelial cells and stromal fibroblast cells recovered from hysterectomy specimens. Poly (I:C), a viral dsRNA mimic, potently increased secretion of IFNλ1 by both epithelial cells and fibroblasts. The secretion of IFNλ1 by epithelial cells significantly increased with increasing age following poly (I:C) stimulation. Stimulation of either cell type with E2 (5x10-8M) or P (1x10-7M) had no effect on expression or secretion of IFNλ1 either alone or in the presence of poly (I:C). E2 suppressed the IFNλ1-induced upregulation of the antiviral IFN-stimulated genes (ISGs) MxA, OAS2 and ISG15 in epithelial cells, but not fibroblasts. Estrogen receptor alpha (ERα) blockade using Raloxifene indicated that E2 mediated its inhibitory effects on ISG expression via ERα. In contrast to E2, P potentiated the upregulation of ISG15 in response to IFNλ1 but had no effect on MxA and OAS2 in epithelial cells. Our results demonstrate that the effects of E2 and P on IFNλ1-induced ISGs are cell-type specific. E2-mediated suppression, and selective P-mediated stimulation, of IFNλ1-induced ISG expression in uterine epithelial cells suggest that the effects of IFNλ1 varies with menstrual cycle stage, pregnancy, and menopausal status. The suppressive effect of E2 could be a potential mechanism by which ascending pathogens from the lower reproductive tract can infect the pregnant and non-pregnant endometrium.

Identification of cell-surface glycans that mediate motility-dependent binding and internalization of Pseudomonas aeruginosa by phagocytes.

Phagocytic cells are critical to host defense against Pseudomonas aeruginosa, a Gram-negative bacterium that is an opportunistic pathogen. Accordingly, susceptible individuals frequently have impaired innate immune responses, including those with cystic fibrosis or neutropenia. Previous studies identified that the downregulation, or loss, of bacterial flagellar motility enables bacteria to evade interactions with phagocytic cells that result in phagocytic uptake of the bacteria. However, the mechanistic bases for motility-dependent interactions between P. aeruginosa and host cell surfaces that lead to phagocytic uptake of the bacteria are poorly understood. A recent insight is that exogenous addition of a negatively charged phospholipid, phosphatidylinositol-(3,4,5)-triphosphate (PIP3), promotes the engagement of non-motile strains of P. aeruginosa with phagocytes leading to uptake of the bacteria. Thus, we hypothesized that the engagement of P. aeruginosa by phagocytic cells is mediated by motility-dependent interactions with cell-surface polyanions. Here we report that endogenous polyanionic N-linked glycans and heparan sulfate mediate bacterial binding of P. aeruginosa by human monocytic cells. These specific interactions resulted in P. aeruginosa phagocytosis, bacterial type 3 secretion system (T3SS)-mediated cellular intoxication and the IL-1ß response of host innate immune cells. Importantly, the bacterial interactions with the glycans were motility-dependent and could be recapitulated with purified, immobilized glycans. Therefore, this work describes novel interactions of P. aeruginosa with specific phagocyte cell-surface glycans that modulate relevant host innate immune responses to the bacteria, including phagocytosis, inflammation and cytotoxicity.

Binding of DC-SIGN to glycoproteins expressed in glycoengineered Pichia pastoris.

Previous studies have shown that glycoproteins expressed in wild-type Pichia pastoris bind to Dendritic cell-SIGN (DC-Specific Intercellular adhesion molecule-3 Grabbing Nonintegrin), a mannose-binding receptor found on dendritic cells in peripheral tissues which is involved in antigen presentation and the initiation of an immune response. However, the binding of DC-SIGN to glycoproteins purified from P. pastoris strains engineered to express humanized N- and O-linked glycans has not been tested to date. In this study, the binding of glycoproteins with specific high-mannose or human N- and O-linked glycan structures to DC-SIGN was tested. Proteins with humanized N-glycans including Man5 structures and O-glycans (up to as many as 24) with single mannose chain length showed DC-SIGN binding that was comparable to that measured for a CHO-produced IgG1 which lacks O-linked mannose. Glycoproteins with wild-type N-glycans and mannotriose and higher O-glycans bound to DC-SIGN in a manner that was strongly inhibited by either the use of enzymatic N-deglycosylation or sodium meta-periodate oxidation. Mannan purified from humanized P. pastoris also showed lower ability to inhibit DC-SIGN binding to glycoproteins with wild type fungal glycosylation than mannan purified from wild type strains. This study shows that humanized P. pastoris can produce glycoproteins that do not bind to DC-SIGN.

Optimization of erythropoietin production with controlled glycosylation-PEGylated erythropoietin produced in glycoengineered Pichia pastoris.

Pichia pastoris is a methylotropic yeast that has gained great importance as an organism for protein expression in recent years. Here, we report the expression of recombinant human erythropoietin (rhEPO) in glycoengineered P. pastoris. We show that glycosylation fidelity is maintained in fermentation volumes spanning six orders of magnitude and that the protein can be purified to high homogeneity. In order to increase the half-life of rhEPO, the purified protein was coupled to polyethylene glycol (PEG) and then compared to the currently marketed erythropoiesis stimulating agent, Aranesp(®) (darbepoetin). In in vitro cell proliferation assays the PEGylated protein was slightly, and the non-PEGylated protein was significantly more active than comparator. Pharmacodynamics as well as pharmacokinetic activity of PEGylated rhEPO in animals was comparable to that of Aranesp(®). Taken together, our results show that glycoengineered P. pastoris is a suitable production host for rhEPO, yielding an active biologic that is comparable to those produced in current mammalian host systems.