Characterization of antibody-dependent cellular phagocytosis in patients infected with hepatitis C virus with different clinical outcomes.

Early neutralizing antibodies against hepatitis C virus (HCV) and CD8 + T cell effector responses can lead to viral clearance. However, these functions alone are not sufficient to protect patients against HCV infection, thus undefined additional antiviral immune mechanisms are required. In recent years, Fc-receptor-dependent antibody effector functions, particularly, antibody-dependent cellular phagocytosis (ADCP) were shown to offer immune protection against several RNA viruses. However, its development and clinical role in patients with HCV infection remain unknown. In this study, we found that patients with chronic GT1a or GT3a HCV infection had significantly higher concentrations of anti-envelope 2 (E2) antibodies, predominantly IgG1 subclass, than patients that cleared the viruses while the latter had antibodies with higher affinities. 97% of the patients with HCV had measurable ADCP of whom patients with chronic disease showed significantly higher ADCP than those who naturally cleared the virus. Epitope mapping studies showed that patients with antibodies that target antigenic domains on the HCV E2 protein that are known to associate with neutralization function are also strongly associated with ADCP, suggesting antibodies with overlapping/dual functions. Correlation studies showed that ADCP significantly correlated with plasma anti-E2 antibody levels and neutralization function regardless of clinical outcome and genotype of infecting virus, while a significant correlation between ADCP and affinity was only evident in patients that cleared the virus. These results suggest ADCP was mostly driven by antibody titer in patients with chronic disease while maintained in clearers due to the quality (affinity) of their anti-E2 antibodies despite having lower antibody titers.

Cationic Polysaccharides Bind to the Endothelial Cell Surface Extracellular Matrix Involving Heparan Sulfate.

Cationic polysaccharides have been extensively studied for drug delivery via the bloodstream, yet few have progressed to clinical use. Endothelial cells lining the blood vessel wall are coated in an anionic extracellular matrix called the glycocalyx. However, we do not fully comprehend the charged polysaccharide interactions with the glycocalyx. We reveal that the cationic polysaccharide poly(acetyl, arginyl) glucosamine (PAAG) exhibits the highest association with the endothelial glycocalyx, followed by dextran (neutral) and hyaluronan (anionic). Furthermore, we demonstrate that PAAG binds heparan sulfate (HS) within the glycocalyx, leading to intracellular accumulation. Using an in vitro glycocalyx model, we demonstrate a charge-based extent of association of polysaccharides with HS. Mechanistically, we observe that PAAG binding to HS occurs via a condensation reaction and functionally protects HS from degradation. Together, this study reveals the interplay between polysaccharide charge properties and interactions with the endothelial cell glycocalyx toward improved delivery system design and application.

Longitudinal Characterization of Phagocytic and Neutralization Functions of Anti-Spike Antibodies in Plasma of Patients after Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Phagocytic responses by effector cells to opsonized viruses have been recognized to play a key role in antiviral immunity. Limited data on coronavirus disease 2019 suggest that the role of Ab-dependent and -independent phagocytosis may contribute to the observed immunological and inflammatory responses; however, their development, duration, and role remain to be fully elucidated. In this study of 62 acute and convalescent patients, we found that patients with acute coronavirus disease 2019 can mount a phagocytic response to autologous plasma-opsonized Spike protein-coated microbeads as early as 10 d after symptom onset, while heat inactivation of this plasma caused 77-95% abrogation of the phagocytic response and preblocking of Fc receptors showed variable 18-60% inhibition. In convalescent patients, phagocytic response significantly correlated with anti-Spike IgG titers and older patients, while patients with severe disease had significantly higher phagocytosis and neutralization functions compared with patients with asymptomatic, mild, or moderate disease. A longitudinal subset of the convalescent patients over 12 mo showed an increase in plasma Ab affinity toward Spike Ag and preservation of phagocytic and neutralization functions, despite a decline in the anti-Spike IgG titers by >90%. Our data suggest that early phagocytosis is primarily driven by heat-liable components of the plasma, such as activated complements, while anti-Spike IgG titers account for the majority of observed phagocytosis at convalescence. Longitudinally, a significant increase in the affinity of the anti-Spike Abs was observed that correlated with the maintenance of both the phagocytic and neutralization functions, suggesting an improvement in the quality of the Abs.

A Low-Cost, High-Fidelity Simulator for Transabdominal Chorionic Villus Sampling.

INTRODUCTION: Chorionic villus sampling (CVS) remains essential for first-trimester genetic diagnosis, yet clinical volume may be insufficient to train new clinicians in the technique. Available simulation models are expensive, require animal parts or specialized resins, and cannot be stored for repeated use. METHODS: We present a model for trans-abdominal CVS (TA-CVS) which is constructed from readily available materials costing less than $10 and can be refrigerated and re-used to train maternal-fetal medicine fellows in CVS. RESULTS: All three attending physicians performing TA-CVS at our institution described the model as an accurate visual and tactile simulation, prompting its integration into our fellowship curriculum. To date, two senior fellows have achieved competency on the simulator and begun to perform clinical CVS under supervision, one of whom is an author on this paper. Both fellows and attendings indicated that the simulator provided a valuable tool for repeated practice prior to clinical CVS. Simulators are now maintained on the unit and have been re-used for 3 months and dozens of simulated procedures each without any apparent qualitative degradation in performance. DISCUSSION/CONCLUSION: We describe a low-cost easily constructed, durable, high-fidelity simulator for TA-CVS.

Predicting primary cesarean delivery in pregnancies complicated by gestational diabetes mellitus.

BACKGROUND: Prediction models have shown promise in helping clinicians and patients engage in shared decision-making by providing quantitative estimates of individual risk of important clinical outcomes. Gestational diabetes mellitus is a common complication of pregnancy, which places patients at higher risk of primary CD. Suspected fetal macrosomia diagnosed on prenatal ultrasound is a well-known risk factor for primary CD in patients with gestational diabetes mellitus, but tools incorporating multiple risk factors to provide more accurate CD risk are lacking. Such tools could help facilitate shared decision-making and risk reduction by identifying patients with both high and low chances of intrapartum primary CD. OBJECTIVE: This study aimed to develop and internally validate a multivariable model to estimate the risk of intrapartum primary CD in pregnancies complicated by gestational diabetes mellitus undergoing a trial of labor. STUDY DESIGN: This study identified a cohort of patients with gestational diabetes mellitus derived from a large, National Institutes of Health-funded medical record abstraction study who delivered singleton live-born infants at ≥34 weeks of gestation at a large tertiary care center between January 2002 and March 2013. The exclusion criteria included previous CD, contraindications to vaginal delivery, scheduled primary CD, and known fetal anomalies. Candidate predictors were clinical variables routinely available to a practitioner in the third trimester of pregnancy found to be associated with an increased risk of CD in gestational diabetes mellitus. Stepwise backward elimination was used to build the logistic regression model. The Hosmer-Lemeshow test was used to demonstrate goodness of fit. Model discrimination was evaluated via the concordance index and displayed as the area under the receiver operating characteristic curve. Internal model validation was performed with bootstrapping of the original dataset. Random resampling with replacement was performed for 1000 replications to assess predictive ability. An additional analysis was performed in which the population was stratified by parity to evaluate the model's predictive ability among nulliparous and multiparous individuals. RESULTS: Of the 3570 pregnancies meeting the study criteria, 987 (28%) had a primary CD. Of note, 8 variables were included in the final model, all significantly associated with CD. They included large for gestational age, polyhydramnios, older maternal age, early pregnancy body mass index, first hemoglobin A1C recorded in pregnancy, nulliparity, insulin treatment, and preeclampsia. Model calibration and discrimination were satisfactory with the Hosmer-Lemeshow test (P=.862) and an area under the receiver operating characteristic curve of 0.75 (95% confidence interval, 0.74-0.77). Internal validation demonstrated similar discriminatory ability. Stratification by parity demonstrated that the model worked well among both nulliparous and multiparous patients. CONCLUSION: Using information routinely available in the third trimester of pregnancy, a clinically pragmatic model can predict intrapartum primary CD risk with reasonable reliability in pregnancies complicated by gestational diabetes mellitus and may provide quantitative data to guide patients in understanding their individual primary CD risk based on preexisting and acquired risk factors.

The AccuFlow sensor: a novel digital health tool to assess intrapartum blood loss at cesarean delivery.

OBJECTIVES: During obstetric hemorrhage, peripheral vasoconstriction maintains heart rate and blood pressure until compensatory mechanisms are overwhelmed and patients deteriorate rapidly. Real-time perfusion measurements could quantify vasoconstriction, improving early recognition of hemorrhage and facilitating early intervention to reduce morbidity and mortality. The AccuFlow device makes rapid, non-invasive, quantitative measurements of perfusion, but has not been studied for hemorrhage detection or used in surgical settings. This study evaluated feasibility, tolerability, and preliminary efficacy of the AccuFlow for assessment of blood loss at cesarean delivery (CD). METHODS: In this pilot study, sensors were applied to the wrist, forearm, bicep, and chest wall of 25 patients undergoing scheduled CD. Postoperatively, sensors were removed and patients rated the AccuFlow and the standard anesthesia monitoring equipment on a validated comfort rating scale for wearable computers (CRS). Blood loss was estimated by the surgical team (EBL) and calculated from change in hematocrit, weight, and height (CBL). CRS scores were compared via Wilcoxon signed ranks tests. Coefficients of correlation between sensor readings and CBL, and between EBL and CBL, were compared using Fisher's R-to-z transformation. RESULTS: There were no safety events; no participants requested device removal. CRS ratings of the AccuFlow and the standard monitoring equipment were similar (7.2 vs. 8.8, p=0.25). Change in wrist perfusion from delivery to dressing placement was more strongly correlated with CBL than was EBL (R=-0.48 vs. R=0.087, p=0.03). CONCLUSIONS: The AccuFlow sensor is well-tolerated and shows promise in detecting intrapartum hemorrhage, though larger studies are needed.

The Impact of Maternal Obesity on Neonatal Outcomes of Pregnancies Complicated by Fetal Growth Restriction.

OBJECTIVE: This study aimed to characterize the relationship between maternal obesity, fetal abdominal size, and neonatal morbidity in pregnancies complicated by fetal growth restriction (FGR). STUDY DESIGN: Pregnancies complicated by FGR, which resulted in delivery of a live, singleton, nonanomalous infant at a single center between 2002 and 2013 were identified in a large, National Institutes of Health-funded database of detailed pregnancy and delivery information extracted by trained research nurses. Pregnancies complicated by diabetes were excluded. Fetal biometry measurements from third trimester ultrasounds performed at the same institution were extracted from another institutional database. Pregnancies were divided into cohorts based on fetal abdominal circumference (AC) gestational age percentile (<10th centile, 10-29th centile, 30-49th centile, and ≥50th centile) at the ultrasound closes to the date of delivery. Obesity was defined by prepregnancy body mass index >30 kg/m2. The primary outcome was a composite of neonatal morbidity (CM) including 5-minute Apgar < 7, arterial cord pH <7.0, sepsis, respiratory support, chest compressions, phototherapy, exchange transfusion, hypoglycemia requiring treatment, or neonatal death. Outcomes were compared between women with versus without prepregnancy obesity overall and then stratified by AC cohort. RESULTS: A total of 379 pregnancies met criteria; CM occurred in 136 (36%). Overall, there was no difference in CM between infants born to women with versus without obesity (risk ratio (RR): 1.11, 95% confidence interval: 0.79-1.56). When stratified according to AC at ultrasound closest to delivery, there was higher prevalence of CM occurring among women with prepregnancy obesity than those without prepregnancy obesity when the fetal AC was >50th or 30 to 49th centile However, these differences did not reach statistical significance. CONCLUSION: Our study identified no significant difference in risk of CM among growth-restricted infants of obese versus nonobese mothers, including among infants with very small AC. More research is needed to further examine the potential relationships postulated here. KEY POINTS: · No significant differences in neonatal outcomes of FGR pregnancies in obese versus nonobese patients.. · No significant differences in AC percentile distribution in FGR pregnancies in obese versus nonobese.. · Pregnancies complicated by obesity had a higher need for cardiac support but not chest compressions..

Intrauterine Fetal Demise Associated with Vascular Malperfusion and Multiple Uterine Leiomyomata: A Report of Two Cases.

BACKGROUND: Uterine leiomyomata have been loosely associated with intrauterine fetal demise (IUFD), largely attributed to fetal growth restriction from cavitary distortion. We present two cases of IUFD in patients with non-distorting leiomyomata and pathologic placental findings of maternal vascular malperfusion (MVM) and fetal vascular malperfusion (FVM). CASE REPORT: Case 1 details a 28w3d IUFD associated with large leiomyomata (largest 11.9 × 7.6 × 9.7 cm), post-partum deep vein thrombosis, and severe pre-eclampsia histologic features. Case 2 details a 25w2d IUFD associated with smaller leiomyomata (largest 3.1 × 3.0 × 3.3 cm). Both placentas demonstrated MVM, including parenchymal thrombi and accelerated villous maturity, and FVM, including avascular stem villi. DISCUSSION: As the placentas in both cases demonstrated findings consistent with altered placental perfusion, we posit that leiomyomata in these cases may have been associated with both maternal and fetal vascular malperfusion, ultimately contributing to fetal demise.

Macrophages bind LDL using heparan sulfate and the perlecan protein core.

The retention of low-density lipoprotein (LDL) is a key process in the pathogenesis of atherosclerosis and largely mediated via smooth-muscle cell-derived extracellular proteoglycans including the glycosaminoglycan chains. Macrophages can also internalize lipids via complexes with proteoglycans. However, the role of polarized macrophage-derived proteoglycans in binding LDL is unknown and important to advance our understanding of the pathogenesis of atherosclerosis. We therefore examined the identity of proteoglycans, including the pendent glycosaminoglycans, produced by polarized macrophages to gain insight into the molecular basis for LDL binding. Using the quartz crystal microbalance with dissipation monitoring technique, we established that classically activated macrophage (M1)- and alternatively activated macrophage (M2)-derived proteoglycans bind LDL via both the protein core and heparan sulfate (HS) in vitro. Among the proteoglycans secreted by macrophages, we found perlecan was the major protein core that bound LDL. In addition, we identified perlecan in the necrotic core as well as the fibrous cap of advanced human atherosclerotic lesions in the same regions as HS and colocalized with M2 macrophages, suggesting a functional role in lipid retention in vivo. These findings suggest that macrophages may contribute to LDL retention in the plaque by the production of proteoglycans; however, their contribution likely depends on both their phenotype within the plaque and the presence of enzymes, such as heparanase, that alter the secreted protein structure.

Incidence of Gestational Diabetes Diagnosed on the 100-G Glucose Tolerance Test in Pregnancies with a 50-G Glucose Challenge Test Result of 200 mg/dL or Greater.

OBJECTIVE: This study aimed to assess the positive predictive value (PPV) of a 1-hour, 50-g glucose challenge test (GCT) result ≥200 mg/dL for the diagnosis of gestational diabetes mellitus (GDM) on a 3-hour, 100-g glucose tolerance test (GTT). STUDY DESIGN: Pregnancies between 2008 and 2016 with a GCT result ≥200 mg/dL were identified retrospectively. GCT and GTT dates and results, demographics, and working due date (EDD) were extracted. Gestational age at testing was calculated from test date and EDD. As some clinicians presumptively diagnose GDM in such cases, if a GTT result was not available, clinic notes were reviewed to determine whether a GTT was ordered. Positive predictive values (PPV) were calculated at GCT cut-offs at and beyond 200 mg/dL. Subgroups were compared including early GCT (<16 weeks) versus routine GCT (24-28 weeks), GTT result normal versus GTT diagnostic of GDM, and GTT ordered versus GTT not ordered. Rates of use of medication for glycemic control were assessed among these groups. RESULTS: Of 236 pregnant women with a GCT result ≥200 mg/dL, 115 (48%) GTT was ordered for 115 (49%), whereas 123 (52%) were managed as presumed GDM. Of 100 (87%) who completed the test, 81 (81%) were diagnosed with GDM with a median intertest interval of 14 days. No statistically significant differences were found between groups stratified by GTT result. Use of rates of metformin, glyburide, and insulin were similar between those diagnosed with GDM by GTT and those diagnosed with GDM by GCT alone. CONCLUSION: A GCT result of ≥200 mg/dL has a PPV of 81% for diagnosis of GDM by GTT in a contemporary U.S. population, with a median intertest interval of 14 days between GCT and GTT. However, those diagnosed by GCT alone were as likely as those diagnosed by GTT to require medication for glycemic control, including insulin, suggesting that requiring a GTT may result in underdiagnosis and delayed treatment of GDM. KEY POINTS: · A 50-g GCT result of 200 mg/dL or greater has a PPV of 81% for GDM on the 100 g GTT.. · Patients diagnosed with GDM by GCT alone were as likely to require insulin as those diagnosed by GTT.. · 81% of patients diagnosed with GDM on the GTT completed their GTT at least 1 week after the GCT, thus requiring GTT in this population may lead to unnecessary delays in care..

De Novo Engineering of Metal-Organic Framework-Printed In Vitro Diagnostic Devices for Specific Capture and Release of Tumor Cells.

Herein, a paper-based in vitro diagnostic device (IVD) is developed via inkjet printing of de novo engineered, boronic acid-rich metal-organic frameworks (BMOFs). The newly developed BMOFs simultaneously possess crystalline and amorphous structure, mesopore size, large surface area, and retain a high level of boronic acid integration. After printing the BMOFs on the filter paper, the BMOF-printed paper IVD shows a rapid response time (40 min) towards cancer cell capture and its maximum cell capture capacity reaches approximately (4.5 ±1.1) ×104 cells cm-2 . Furthermore, the BMOF-printed IVD shows nine times higher capture ability of cancer cells than non-cancerous cells, suggesting its excellent selectivity. Importantly, the pH-tunable affinity of BMOF to glucose enables its dual-responsive behavior without affecting cell viability. In addition, a desired cell pattern could be achieved by directly drawing BMOFs onto a silicon substrate, highlighting its capacity as a miniaturized device for tumor cell capture and analysis. This simple and label-free nanoplatform enables new opportunities for designing MOF-based smart devices for diverse biomedical applications such as a cost-effective IVD technologies for cancer diagnosis, genotyping, and prognosis.

Redox Active Cerium Oxide Nanoparticles: Current Status and Burning Issues.

Research on cerium oxide nanoparticles (nanoceria) has captivated the scientific community due to their unique physical and chemical properties, such as redox activity and oxygen buffering capacity, which made them available for many technical applications, including biomedical applications. The redox mimetic antioxidant properties of nanoceria have been effective in the treatment of many diseases caused by reactive oxygen species (ROS) and reactive nitrogen species. The mechanism of ROS scavenging activity of nanoceria is still elusive, and its redox activity is controversial due to mixed reports in the literature showing pro-oxidant and antioxidant activity. In light of its current research interest, it is critical to understand the behavior of nanoceria in the biological environment and provide answers to some of the critical and open issues. This review critically analyzes the status of research on the application of nanoceria to treat diseases caused by ROS. It reviews the proposed mechanism of action and shows the effect of surface coatings on its redox activity. It also discusses some of the crucial issues in deciphering the mechanism and redox activity of nanoceria and suggests areas of future research.

Hyaluronidase-4 is produced by mast cells and can cleave serglycin chondroitin sulfate chains into lower molecular weight forms.

Mast cells represent a heterogeneous cell population that is well-known for the production of heparin and the release of histamine upon activation. Serglycin is a proteoglycan that within mast cell α-granules is predominantly decorated with the glycosaminoglycans heparin or chondroitin sulfate (CS) and has a known role in granule homeostasis. Heparanase is a heparin-degrading enzyme, is present within the α-granules, and contributes to granule homeostasis, but an equivalent CS-degrading enzyme has not been reported previously. In this study, using several approaches, including epitope-specific antibodies, immunohistochemistry, and EM analyses, we demonstrate that human HMC-1 mast cells produce the CS-degrading enzymes hyaluronidase-1 (HYAL1) and HYAL4. We observed that treating the two model CS proteoglycans aggrecan and serglycin with HYAL1 and HYAL4 in vitro cleaves the CS chains into lower molecular weight forms with nonreducing end oligosaccharide structures similar to CS stub neoepitopes generated after digestion with the bacterial lyase chondroitinase ABC. We found that these structures are associated with both the CS linkage region and with structures more distal toward the nonreducing end of the CS chain. Furthermore, we noted that HYAL4 cleaves CS chains into lower molecular weight forms that range in length from tetra- to dodecasaccharides. These results provide first evidence that mast cells produce HYAL4 and that this enzyme may play a specific role in maintaining α-granule homeostasis in these cells by cleaving CS glycosaminoglycan chains attached to serglycin.

Engineering nanomedicines through boosting immunogenic cell death for improved cancer immunotherapy.

Current cancer immunotherapy has limited response rates in a large variety of solid tumors partly due to the low immunogenicity of the tumor cells and the immunosuppressive tumor microenvironment (ITM). A number of clinical cancer treatment modalities, including radiotherapy, chemotherapy, photothermal and photodynamic therapy, have been shown to elicit immunogenicity by inducing immunogenic cell death (ICD). However, ICD-based immunotherapy is restricted by the ITM limiting its efficacy in eliciting a long-term antitumor immune response, and by severe systemic toxicity. To address these challenges, nanomedicine-based drug delivery strategies have been exploited for improving cancer immunotherapy by boosting ICD of the tumor cells. Nanosized drug delivery systems are promising for increasing drug accumulation at the tumor site and codelivering ICD inducers and immune inhibitors to simultaneously elicit the immune response and relieve the ITM. This review highlights the recent advances in nanomedicine-based immunotherapy utilizing ICD-based approaches. A perspective on the clinical translation of nanomedicine-based cancer immunotherapy is also provided.

Platelet Factor 4 Binds to Vascular Proteoglycans and Controls Both Growth Factor Activities and Platelet Activation.

Platelet factor 4 (PF4) is produced by platelets with roles in both inflammation and wound healing. PF4 is stored in platelet α-granules bound to the glycosaminoglycan (GAG) chains of serglycin. This study revealed that platelet serglycin is decorated with chondroitin/dermatan sulfate and that PF4 binds to these GAG chains. Additionally, PF4 had a higher affinity for endothelial-derived perlecan heparan sulfate chains than serglycin GAG chains. The binding of PF4 to perlecan was found to inhibit both FGF2 signaling and platelet activation. This study revealed additional insight into the ways in which PF4 interacts with components of the vasculature to modulate cellular events.

Soluble LILRA3 promotes neurite outgrowth and synapses formation through a high-affinity interaction with Nogo 66.

Inhibitory proteins, particularly Nogo 66, a highly conserved 66-amino-acid loop of Nogo A (an isoform of RTN4), play key roles in limiting the intrinsic capacity of the central nervous system (CNS) to regenerate after injury. Ligation of surface Nogo receptors (NgRs) and/or leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse orthologue the paired immunoglobulin-like receptor B (PIRB) by Nogo 66 transduces inhibitory signals that potently inhibit neurite outgrowth. Here, we show that soluble leukocyte immunoglobulin-like receptor A3 (LILRA3) is a high-affinity receptor for Nogo 66, suggesting that LILRA3 might be a competitive antagonist to these cell surface inhibitory receptors. Consistent with this, LILRA3 significantly reversed Nogo-66-mediated inhibition of neurite outgrowth and promoted synapse formation in primary cortical neurons through regulation of the ERK/MEK pathway. LILRA3 represents a new antagonist to Nogo-66-mediated inhibition of neurite outgrowth in the CNS, a function distinct from its immune-regulatory role in leukocytes. This report is also the first to demonstrate that a member of LILR family normally not expressed in rodents exerts functions on mouse neurons through the highly homologous Nogo 66 ligand.

Targeted Delivery and Redox Activity of Folic Acid-Functionalized Nanoceria in Tumor Cells.

Cerium oxide nanoparticles (nanoceria) are promising catalytic nanomaterials that are widely reported to modulate intracellular reactive oxygen species (ROS). In this study, nanoceria were synthesized by flame spray pyrolysis and functionalized with a cell-targeting ligand, folic acid (FA). The surface functionalization of nanoceria was stable, and FA enhanced the uptake of nanoceria via folate receptors. Internalized nanoceria and FA-nanoceria were localized predominantly in the cytoplasm. FA-nanoceria modulated intracellular ROS to a greater extent than the nanoceria in colon carcinoma cells, but induced ROS in ovarian cancer cells, likely due to their enhanced uptake. Together these data demonstrated that the functionalization of nanoceria with FA modulated their endocytosis and redox activity, and they may find application in the delivery of anticancer drugs in the future.

Structure-Activity Relationships of Bioengineered Heparin/Heparan Sulfates Produced in Different Bioreactors.

Heparin and heparan sulfate are structurally-related carbohydrates with therapeutic applications in anticoagulation, drug delivery, and regenerative medicine. This study explored the effect of different bioreactor conditions on the production of heparin/heparan sulfate chains via the recombinant expression of serglycin in mammalian cells. Tissue culture flasks and continuously-stirred tank reactors promoted the production of serglycin decorated with heparin/heparan sulfate, as well as chondroitin sulfate, while the serglycin secreted by cells in the tissue culture flasks produced more highly-sulfated heparin/heparan sulfate chains. The serglycin produced in tissue culture flasks was effective in binding and signaling fibroblast growth factor 2, indicating the utility of this molecule in drug delivery and regenerative medicine applications in addition to its well-known anticoagulant activity.

Bioengineered human heparin with anticoagulant activity.

Heparin is a carbohydrate anticoagulant used clinically to prevent thrombosis, however impurities can limit its efficacy. Here we report the biosynthesis of heparin-like heparan sulfate via the recombinant expression of human serglycin in human cells. The expressed serglycin was also decorated with chondroitin/dermatan sulfate chains and the relative abundance of these glycosaminoglycan chains changed under different concentrations of glucose in the culture medium. The recombinantly expressed serglycin produced with 25mM glucose present in the culture medium was found to possess anticoagulant activity one-seventh of that of porcine unfractionated heparin, demonstrating that bioengineered human heparin-like heparan sulfate may be a safe next-generation pharmaceutical heparin.

Curcumin-Loading-Dependent Stability of PEGMEMA-Based Micelles Affects Endocytosis and Exocytosis in Colon Carcinoma Cells.

Polymeric micelles were formed from poly(poly(ethylene glycol) methyl ether methacrylate)-block-poly(styrene) (P(PEGMEMA)-b-PS) block copolymer of two different chain lengths. The micelles formed were approximately 16 and 46 nm in diameter and used to encapsulate curcumin. Upon loading of the curcumin into the micelles, their size increased to approximately 34 and 80 nm in diameter, respectively, with a loading efficiency of 58%. The unloaded micelles were not cytotoxic to human colon carcinoma cells, whereas only the smaller loaded micelles were cytotoxic after 72 h of exposure. The micelles were rapidly internalized by the cells within minutes of exposure, with the loaded micelles internalized to a greater extent owing to their enhanced stability compared to that of the unloaded micelles. The larger micelles were more rapidly internalized and exocytosed than the smaller micelles, demonstrating the effect of micelle size and drug loading on drug delivery and cytotoxicity.