Adeno-associated virus perfusion enhanced expression: A commercially scalable, high titer, high quality producer cell line process.

With safety and efficacy demonstrated over hundreds of clinical trials in the last 30 years, along with at least six recent global marketing authorizations achieved since 2017, recombinant adeno-associated viruses (rAAVs) have been established as the leading therapeutic gene transfer vector for rare, monogenic diseases. Significant advances in manufacturing technology have been made in the last few decades to address challenges with GMP production of rAAV products, although yield, cost, scalability, and quality remain a challenge. With transient transfection processes established as a manufacturing platform for multiple commercial AAV products, there remains significant yield, cost, robustness, and scalability constraints that need to be resolved to enable a reliable supply of rAAV products for global patient access. The development of stable producer cell lines for rAAV products has enabled scalability and, in some cases, improvements in productivity. Herein we describe a novel AAV perfusion-enhanced expression (APEX) process, resulting in higher maximum cell densities in the production bioreactor with a 3- to 6-fold increase in volumetric productivity. This process has been successfully demonstrated across multiple serotypes in large scale cell culture with titers approaching 1 × 1012 GC/mL. The APEX production platform marks a significant leap forward in the efficient and effective manufacturing of rAAV vector products.

Unplanned Return to Hospital After Same Day Oral Cavity Resection: A Dual Institution Study.

OBJECTIVES: Primary objective: describe rates of 30-days unplanned readmission following outpatient resection of oral cavity cancer. Secondary objective: evaluate for patient and treatment factors associated with readmission. METHODS: Retrospective, dual-institution cohort study of 2 tertiary care referral centers involving adult patients undergoing resection of oral cavity cancer with plans for same-day discharge. Consecutive sample of 77 patients included. Primary outcome was unplanned readmission to emergency room or inpatient stay in the 30 days following surgery. Comparison testing was used between return and non-return groups. RESULTS: Among 77 patients treated with outpatient surgery for oral cavity cancer, 19 (25%) returned to the hospital within 30 days. Among the reasons for return, 16 (80%) were directly related to surgery, and 4 (20%) were related to perioperative medical complications not directly related to a surgical site. Among the 25 patients also undergoing sentinel lymph node biopsy with their oral cavity resection, none returned to the hospital for neck-related complications. While most patients could be safely observed and discharged after return to the hospital, 8 patients (10%) required inpatient readmission. No significant differences between return and non-return groups were identified, although there was a trend toward shorter driving distance from hospital for the return group (47.6 miles vs. 69.5 miles, P = 0.097). CONCLUSION: Unplanned return to the hospital following outpatient oral cavity resection is prevalent and primarily driven by postoperative primary resection site concerns. Among patients selected for same day discharge, no definite population at highest risk of unplanned return was identified.

Improved adeno-associated virus empty and full capsid separation using weak partitioning multi-column AEX chromatography.

Recombinant adeno-associated virus (rAAV) empty and full capsid separation has been a topic of interest in the rAAV gene therapy community for many years and the anion exchange chromatography (AEX) step has undergone various process optimizations to improve rAAV empty capsid separation, including AEX stationary phase, mobile phase, and process parameters. Here, we present a new AEX method that employs both weak partitioning chromatography (WPC) and multi-column chromatography (MCC) to achieve improved full rAAV percentage in the AEX pool. The WPC technology allows empty rAAV to be displaced by full rAAV during loading, while the MCC technology enables parallel column processing which further increases AEX step productivity. Our results show that, compared to baseline AEX batch chromatography, the AEX-WPC-MCC method demonstrated improvements in both AEX pool full rAAV percentage (∼ 20% increase) and rAAV genome recovery (∼ 20% increase). As a result, the productivity (full capsid generated per liter of AEX column per hour of processing time) of the AEX step increased by ∼34-fold from the baseline AEX batch run to the AEX-WPC-MCC run. It is foreseeable that this AEX-WPC-MCC method could find applications in large-scale rAAV manufacturing processes to improve AEX yield and reduce the cost of goods of rAAV manufacturing.

Tuning mobile phase properties to improve empty and full particle separation in adeno-associated virus productions by anion exchange chromatography.

In the past decade, recombinant adeno-associated virus (rAAV) has gained increased attention as a prominent gene therapy technology to treat monogenetic diseases. One of the challenges in rAAV production is the enrichment of full rAAV particles containing the gene of interest (GOI) payload. By adjusting the mobile phase properties of anion-exchange chromatography (AEX), it was demonstrated that empty and full separation of rAAV was improved in monolith based preparative AEX chromatography. When compared to the baseline method using NaCl, the use of tetraethylammonium acetate (TEA-Ac) in the AEX mobile phase resulted in enhanced resolution from 0.75 to 1.23 between "Empty" and "Full" peaks by salt linear gradient elution, as well as increased the percentage of full rAAV particles from 20% to 36% and genome recovery from 59% to 62%. Furthermore, a dual wash plus step elution AEX method was developed. Wherein, the first wash step harnesses TEA-Ac to separate empty and full capsids, which is followed by a second wash step that ensures no TEA-Ac salt is carried over into AEX eluate. The resulting optimized AEX purification method has the potential to be adapted for manufacturing and purification processes involving various rAAV production platforms that experience empty and full rAAV separation challenges.

Guideline-compliant adjuvant chemotherapy for resected high risk oral cavity cancer: Homefield advantage.

PURPOSE: Factors that are associated with failure to receive guideline-compliant adjuvant chemotherapy after resection of high-risk oral cavity cancer are understudied. Here, we performed a retrospective cohort study of surgically treated patients with oral cavity squamous cell carcinoma to determine rates of guideline-compliant adjuvant chemotherapy and to examine patient factors associated with receiving guideline-compliant chemotherapy. STUDY DESIGN: Retrospective cohort. SETTING: Two tertiary care referral centers. METHODS: Patients with resected high-risk oral cavity squamous cell carcinoma and known adjuvant therapy details were included. Extranodal extension or positive margins were considered high-risk features for which adjuvant chemoradiation was indicated. Patient factors were examined to determine associations with receiving on-guidelines treatment. Univariable and multivariable logistic regression were used to determine significance of associations. RESULTS: 75 patients were included. 36 (48 %) patients received guideline-compliant cisplatin. In total, 39 (52 %) patients did not receive guideline-compliant chemotherapy. On multivariable analysis, meeting with a university medical oncologist was significantly associated with the receipt of guideline-compliant cisplatin (OR 6.38, 95 % CI 2.26-20.0, p < 0.001). CONCLUSION: Adherence to on-guidelines treatment can be difficult to achieve in patients with advanced stage head and neck cancer. Meeting with university medical oncology is associated with an increased chance of receiving guideline-compliant chemotherapy.

Synchrotron radiation circular dichroism spectroscopy reveals that gold and silver nanoparticles modify the secondary structure of a lung surfactant protein B analogue.

Inhaled nanoparticles (NPs) depositing in the alveolar region of the lung interact initially with a surfactant layer and in vitro studies have demonstrated that NPs can adversely affect the biophysical function of model pulmonary surfactants (PS), of which surfactant protein B (SP-B) is a key component. Other studies have demonstrated the potential for NPs to modify the structure and function of proteins. It was therefore hypothesised that NPs may affect the biophysical function of PS by modifying the structure of SP-B. Synchrotron radiation circular dichroism (SRCD) spectroscopy was used to explore the effect of various concentrations of gold nanoparticles (AuNPs) (5, 10, 20 nm), silver nanoparticles (AgNPs) (10 nm) and silver citrate on the secondary structure of surfactant protein B analogue, SP-B1-25, in a TFE/PB dispersion. For Au and Ag NPs the SRCD spectra indicated a concentration dependent reduction in the α-helical structure of SP-B1-25 (5 nm AuNP ≈ 10 nm AgNP ≫ 10 nm AuNP > 20 nm AuNP). For AuNPs the effect was greater for the 5 nm size, which was not fully explained by consideration of surface area. The impact of the 10 nm AgNPs was greater than that of the 10 nm AuNPs and the effect of AgNPs was greater than that of silver citrate at equivalent Ag mass concentrations. For 10 nm AuNPs, SRCD spectra for dispersions in, the more physiologically relevant, DPPC showed a similar concentration dependent pattern. The results demonstrate the potential for inhaled NPs to modify SP-B1-25 structure and thus potentially adversely impact the physiological function of the lung, however, further studies are necessary to confirm this.

Utility of SNOMED CT in automated expansion of clinical terms in discharge summaries: Testing issues of coverage.

OBJECTIVE: This study tests coverage of SNOMED CT as an expansion source in the process of automated expansion of clinical terms found in discharge summaries. Term expansion is commonly used as a technique in knowledge extraction, query formulation and semantic modelling among other applications. However, characteristics of the sources might affect credibility of outputs, and coverage is one of them. METHOD: We developed an automated method for testing coverage of more than one source at a time. We used several methods to clean our corpus of discharge summaries before we extracted text fragments as candidates for clinical concepts. We then used Unified Medical Language System (UMLS) sources and UMLS REST API to filter concepts from the pool of text fragments. Statistical measures like true positive rate and false negative rate were used to decide on the coverage of the source. We also tested the coverage of the individual SNOMED CT hierarchies using the same methods. RESULTS: Findings suggest that a combination of four terminologies tested (SNOMED CT, NCI, LNC and MSH) achieves over 90% of coverage for term expansion. We also found that the SNOMED CT hierarchies that hold clinically relevant concepts provided 60% of coverage. CONCLUSION: We believe that our findings and the method we developed will be of use to both scientists and practitioners working in the domain of knowledge extraction.

Hydrodynamic Mixing Tunes the Stiffness of Proteoglycan-Mimicking Physical Hydrogels.

Self-assembling hydrogels are promising materials for regenerative medicine and tissue engineering. However, designing hydrogels that replicate the 3-4 order of magnitude variation in soft tissue mechanics remains a major challenge. Here hybrid hydrogels are investigated formed from short self-assembling ß-fibril peptides, and the glycosaminoglycan chondroitin sulfate (CS), chosen to replicate physical aspects of proteoglycans, specifically natural aggrecan, which provides structural mechanics to soft tissues. Varying the peptide:CS compositional ratio (1:2, 1:10, or 1:20) can tune the mechanics of the gel by one to two orders of magnitude. In addition, it is demonstrated that at any fixed composition, the gel shear modulus can be tuned over approximately two orders of magnitude through varying the initial vortex mixing time. This tuneability arises due to changes in the mesoscale structure of the gel network (fibril width, length, and connectivity), giving rise to both shear-thickening and shear-thinning behavior. The resulting hydrogels range in shear elastic moduli from 0.14 to 220 kPa, mimicking the mechanical variability in a range of soft tissues. The high degree of discrete tuneability of composition and mechanics in these hydrogels makes them particularly promising for matching the chemical and mechanical requirements of different applications in tissue engineering and regenerative medicine.

Environmentally Relevant Iron Oxide Nanoparticles Produce Limited Acute Pulmonary Effects in Rats at Realistic Exposure Levels.

Iron is typically the dominant metal in the ultrafine fraction of airborne particulate matter. Various studies have investigated the toxicity of inhaled nano-sized iron oxide particles (FeOxNPs) but their results have been contradictory, with some indicating no or minor effects and others finding effects including oxidative stress and inflammation. Most studies, however, did not use materials reflecting the characteristics of FeOxNPs present in the environment. We, therefore, analysed the potential toxicity of FeOxNPs of different forms (Fe3O4, α-Fe2O3 and γ-Fe2O3) reflecting the characteristics of high iron content nano-sized particles sampled from the environment, both individually and in a mixture (FeOx-mix). A preliminary in vitro study indicated Fe3O4 and FeOx-mix were more cytotoxic than either form of Fe2O3 in human bronchial epithelial cells (BEAS-2B). Follow-up in vitro (0.003, 0.03, 0.3 µg/mL, 24 h) and in vivo (Sprague-Dawley rats, nose-only exposure, 50 µg/m3 and 500 µg/m3, 3 h/d × 3 d) studies therefore focused on these materials. Experiments in vitro explored responses at the molecular level via multi-omics analyses at concentrations below those at which significant cytotoxicity was evident to avoid detection of responses secondary to toxicity. Inhalation experiments used aerosol concentrations chosen to produce similar levels of particle deposition on the airway surface as were delivered in vitro. These were markedly higher than environmental concentrations. No clinical signs of toxicity were seen nor effects on BALF cell counts or LDH levels. There were also no significant changes in transcriptomic or metabolomic responses in lung or BEAS-2B cells to suggest adverse effects.

Equine pregnancy-specific glycoprotein CEACAM49 secreted by endometrial cup cells activates TGFB.

In early equine pregnancy, a highly invasive trophoblast cell subpopulation, the chorionic girdle cells, invade the endometrium and form endometrial cups (EC). These cells express classical MHC molecules, thereby stimulating a humoral and cellular immune response, resulting in a massive accumulation of maternal CD4+ and CD8+ T cells around the EC. Nevertheless, no immediate destruction of endometrial cups by maternal lymphoid cells occurs, presumably due to immune tolerance. Although the environment of EC is rich in TGFB and in FOXP3+, CD4+ T cells, the mechanisms leading to tolerance have not been elucidated. Recently, we discovered that equine trophoblast cells secrete pregnancy-specific glycoproteins (PSGs). Since human and murine PSGs activate latent TGFB, we hypothesized that equine PSGs may have a similar activity. We performed plasmon surface resonance experiments to show that equine PSG CEACAM49 can directly bind to the latency-associated peptide (LAP) of both TGFB1 and TGFB2. We then found that the binding of CEACAM49 leads to the activation of TGFB1 as determined by both ELISA and cell-based assays. Furthermore, the activation of TGFB is a unique function of PSGs within the human CEA family, because CEACAM1, 3, 5, 6, 8 do not activate this cytokine. This finding further strengthens the classification of CEACAM49 as an equine PSG. Based on our results, we hypothesize that activation of latent TGFB in the EC environment by equine PSGs secreted by invasive trophoblast cells, could contribute to the generation of regulatory T cells (Tregs) to maintain immune tolerance.

Recombinant Pregnancy-Specific Glycoprotein 1 Has a Protective Role in a Murine Model of Acute Graft-versus-Host Disease.

Acute graft-versus-host disease (aGVHD) is an immune-mediated reaction that can occur after hematopoietic stem cell transplantation in which donor T cells recognize the host antigens as foreign, destroying host tissues. Establishment of a tolerogenic immune environment while preserving the immune response to infectious agents is required for successful bone marrow transplantation. Pregnancy-specific glycoprotein 1 (PSG1), which is secreted by the human placenta into the maternal circulation throughout pregnancy, likely plays a role in maintaining immunotolerance to prevent rejection of the fetus by the maternal immune system. We have previously shown that PSG1 activates the latent form of transforming growth factor ß1 (TGF-ß), a cytokine essential for the differentiation of tolerance-inducing CD4+FoxP3+ regulatory T cells (Tregs). Consistent with this observation, treatment of naïve murine T cells with PSG1 resulted in a significant increase in FoxP3+ cells that was blocked by a TGF-ß receptor I inhibitor. We also show here that PSG1 can increase the availability of active TGF-ß in vivo. As the role of CD4+FoxP3+ cells in the prevention of aGVHD is well established, we tested whether PSG1 has beneficial effects in a murine aGHVD transplantation model. PSG1-treated mice had reduced numbers of tissue-infiltrating inflammatory CD3+ T cells and had increased expression of FoxP3 in T cells compared with vehicle-treated mice. In addition, administration of PSG1 significantly inhibited aGVHD-associated weight loss and mortality. On the other hand, administration of PSG1 was less effective in managing aGVHD in the presence of an alloimmune reaction against a malignancy in a graft-versus-leukemia experimental model. Combined, this data strongly suggests that PSG1 could be a promising treatment option for patients with aGVHD following bone marrow transplantation for a nonmalignant condition, such as an autoimmune disorder or a genetic immunodeficiency.

Association of Continuity of Primary Care and Statin Adherence.

PURPOSE: Deficiencies in medication adherence are a major barrier to effectiveness of chronic condition management. Continuity of primary care may promote adherence. We assessed the association of continuity of primary care with adherence to long-term medication as exemplified by statins. RESEARCH DESIGN: We linked data from a prospective study of 267,091 Australians aged 45 years and over to national data sets on prescription reimbursements, general practice claims, hospitalisations and deaths. For participants having a statin dispense within 90 days of study entry, we computed medication possession ratio (MPR) and usual provider continuity index (UPI) for the subsequent two years. We used multivariate Poisson regression to calculate the relative risk (RR) and 95% confidence interval (CI) for the association between tertiles of UPI and MPR adjusted for socio-demographic and health-related patient factors, including age, gender, remoteness of residence, smoking, alcohol intake, fruit and vegetable intake, physical activity, prior heart disease and speaking a language other than English at home. We performed a comparison approach using propensity score matching on a subset of the sample. RESULTS: 36,144 participants were eligible and included in the analysis among whom 58% had UPI greater than 75%. UPI was significantly associated with 5% increased MPR for statin adherence (95% CI 1.04-1.06) for highest versus lowest tertile. Dichotomised analysis using a cut-off of UPI at 75% showed a similar effect size. The association between UPI and statin adherence was independent of socio-demographic and health-related factors. Stratification analyses further showed a stronger association among those who were new to statins (RR 1.33, 95% CI 1.15-1.54). CONCLUSIONS: Greater continuity of care has a positive association with medication adherence for statins which is independent of socio-demographic and health-related factors.

Induction and activation of latent transforming growth factor-ß1 are carried out by two distinct domains of pregnancy-specific glycoprotein 1 (PSG1).

Pregnancy-specific glycoproteins (PSGs) are a family of Ig-like proteins secreted by specialized placental cells. The PSG1 structure is composed of a single Ig variable region-like N-terminal domain and three Ig constant region-like domains termed A1, A2, and B2. Members of the human and murine PSG family have been shown to induce anti-inflammatory cytokines from monocytes and macrophages and to stimulate angiogenesis. We recently showed that recombinant forms of PSG1 (PSG1-Fc and PSG1-His) and PSG1 purified from the serum of pregnant women are associated with the immunoregulatory cytokine TGF-ß1 and activated latent TGF-ß1. Here, we sought to examine the requirement of specific PSG1 domains in the activation of latent TGF-ß1. Plasmon surface resonance studies showed that PSG1 directly bound to the small latent complex and to the latency-associated peptide of TGF-ß1 and that this binding was mediated through the B2 domain. Furthermore, the B2 domain alone was sufficient for activating the small latent complex. In separate experiments, we found that the PSG1-mediated induction of TGF-ß1 secretion in macrophages was dependent on the N-terminal domain. Mutagenesis analysis revealed that four amino acids (LYHY) of the CC' loop of the N-terminal domain were required for induction of latent TGF-ß1 secretion. Together, our results show that two distinct domains of PSG1 are involved in the regulation of TGF-ß1 and provide a mechanistic framework for how PSGs modulate the immunoregulatory environment at the maternal-fetal interface for successful pregnancy outcome.

Factors influencing adherence in long-term use of statins.

PURPOSE: To assess the factors influencing adherence in long-term medication use as exemplified by statins. METHODS: Data from an in-depth survey of Australians aged 45 years and over were linked to national prescription reimbursement data to assess medication possession ratio (MPR) for statins for the middle two years of a four-year period of statin possession. Poisson regression was used to calculate the relative risk (RR) for adherence (MPR ≥ 80%) for patient characteristics and factors related to access to and need for health care services. Separate models were fit for patients receiving healthcare concession subsidies and those who do not ('general beneficiaries'). RESULTS: In the analysis, 42 492 concessional and 16 110 general beneficiary patients were included, with 80.1% and 56.7% showing MPR ≥ 80%, respectively. In both models, RR for adherence was significantly elevated for older (age 65+) and less healthy (worse self-rated health, pre-existing heart condition or obese) individuals, and for those who had private health insurance. Significantly lower RR (i.e. more non-adherence) was found for individuals reporting speaking a language other than English at home, who were smokers, were employed, and had higher levels of education, and for those who reported psychological distress. Income had no significant relationship with adherence, and the pattern of adherence by remoteness of area of residence was inconsistent. CONCLUSIONS: Poor adherence in long-term use of statins is commonplace, but a number of key predictors-including age, language other than English spoken at home, smoking status and psychological distress-are readily assessable by the prescribing practice.

The eCHAT program to facilitate healthy changes in New Zealand primary care.

This article describes eCHAT (electronic case-finding and help assessment tool), designed to improve health and well-being through systematic screening and intervention for modifiable lifestyle and mental health issues in primary care populations and monitoring to inform continuous quality improvement. eCHAT allows patients to identify unhealthy behaviors (risky substance use, gambling, being subject to abuse, physical inactivity) and negative mood states (depression, anxiety, anger) with which they would like help before a visit using an iPad in the waiting room or via the Internet in the community. Family physicians access summarized results, including scores and interpretations of screening tests at the point of care. eCHAT stimulates conversations between patients and clinicians about life changes they might make, encouraging active participation in decision making and engagement in self-management. Stepped-care clinical decision support tools offer interventions through self-management options to primary care interventions through to secondary care referral. As well as systematically screening and intervening in individual practice populations, anonymous collated and encrypted data also can be used to measure the mental health and lifestyle risk factors and interventions provided at practice network, regional, and national levels to monitor system and organizational performance improvements, identify regional and national variations, benchmark service delivery, and support quality improvement.

Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects.

Insect steroid hormones (ecdysteroids) are important for female reproduction in many insect species and are required for the initiation and coordination of vital developmental processes. Ecdysteroids are also important for adult male physiology and behavior, but their exact function and site of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. We have examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis of ecdysone (E), the precursor of the molting hormone 20-hydroxyecdysone (20E), are expressed in the tubular accessory glands (TAGs) of adult males. In contrast, expression of the gene encoding the enzyme mediating 20E synthesis was detected in the ovaries of females. Further, Spookiest (Spot), an enzyme presumably required for endowing tissues with competence to produce ecdysteroids, is male specific and predominantly expressed in the TAGs. We also show that prothoracicotropic hormone (PTTH), a regulator of E synthesis during larval development, regulates ecdysteroid levels in the adult stage in Drosophila melanogaster and the gene for its receptor Torso seems to be expressed specifically in the accessory glands of males. The composite results suggest strongly that the accessory glands of adult male insects are the main source of E, but not 20E. The finding of a possible male-specific source of E raises the possibility that E and 20E have sex-specific roles analogous to the vertebrate sex steroids, where males produce primarily testosterone, the precursor of estradiol. Furthermore this study provides the first evidence that PTTH regulates ecdysteroid synthesis in the adult stage and could explain the original finding that some adult insects are a rich source of PTTH.

Early expression of pregnancy-specific glycoprotein 22 (PSG22) by trophoblast cells modulates angiogenesis in mice.

Mouse and human pregnancy-specific glycoproteins (PSG) are known to exert immunomodulatory functions during pregnancy by inducing maternal leukocytes to secrete anti-inflammatory cytokines that promote a tolerogenic decidual microenvironment. Many such anti-inflammatory mediators also function as proangiogenic factors, which, along with the reported association of murine PSG with the uterine vasculature, suggest that PSG may contribute to the vascular adaptations necessary for successful implantation and placental development. We observed that PSG22 is strongly expressed around the embryonic crypt on Gestation Day 5.5, indicating that trophoblast giant cells are the main source of PSG22 during the early stages of pregnancy. PSG22 treatment up-regulated the secretion of transforming growth factor beta 1 and vascular endothelial growth factor A (VEGFA) in murine macrophages, uterine dendritic cells, and natural killer cells. A possible role of PSGs in uteroplacental angiogenesis is further supported by the finding that incubation of endothelial cells with PSG22 resulted in the formation of tubes in the presence and absence of VEGFA. We determined that PSG22, like human PSG1 and murine PSG17 and PSG23, binds to the heparan sulfate chains in syndecans. Therefore, our findings indicate that despite the independent evolution and expansion of human and rodent PSG, members in both families have conserved functions that include their ability to induce anti-inflammatory cytokines and proangiogenic factors as well as to induce the formation of capillary structures by endothelial cells. In summary, our results indicate that PSG22, the most abundant PSG expressed during mouse early pregnancy, is likely a major contributor to the establishment of a successful pregnancy.

Human pregnancy specific beta-1-glycoprotein 1 (PSG1) has a potential role in placental vascular morphogenesis.

Previous studies suggest that human pregnancy specific beta-1-glycoproteins (PSGs) play immunomodulatory roles during pregnancy; however, other possible functions of PSGs have yet to be explored. We have observed that PSGs induce transforming growth factor beta 1 (TGFB1), which among its other diverse functions inhibits T-cell function and has proangiogenic properties. The present study investigates a potential role for PSG1, the most abundant PSG in maternal serum, as a possible inducer of proangiogenic growth factors known to play an important role in establishment of the vasculature at the maternal-fetal interface. To this end, we measured TGFB1, vascular endothelial growth factors (VEGFs) A and C, and placental growth factor (PGF) protein levels in several cell types after PSG1 treatment. In addition, tube formation and wound healing assays were performed to investigate a possible direct interaction between PSG1 and endothelial cells. PSG1 induced up-regulation of both TGFB1 and VEGFA in human monocytes, macrophages, and two human extravillous trophoblast cell lines. We did not observe induction of VEGFC or PGF by PSG1 in any of the cells tested. PSG1 treatment resulted in endothelial tube formation in the presence and absence of VEGFA. Site-directed mutagenesis was performed to map the essential regions within the N-domain of PSG1 required for functional activity. We found that the aspartic acid at position 95, previously believed to be required for binding of PSGs to cells, is not required for PSG1 activity but that the amino acids implicated in the formation of a salt bridge within the N-domain are essential for PSG1 function.

Studies on the Black Box: incorporation of 3-oxo-7-dehydrocholesterol into ecdysteroids by Drosophila melanogaster and Manduca sexta.

It has long been hypothesized that the oxidation of 7-dehydrocholesterol (7dC), made from dietary cholesterol (C), to 3-oxo-7dC (3-oxo-Delta(5,7)C) immediately precedes the unknown "Black Box" oxidations that lead to the formation of the first up-stream intermediate exhibiting the highly characteristic ecdysteroid structure of the steroid molting hormone of insects, crustaceans and some other arthropods. Perhaps rate-limiting and under the control of the prothoracicotropic hormone (PTTH), the biosynthesis of 3-oxo-7dC and its subsequent oxidative modifications have been difficult to study because of their apparent instability, i.e. no intermediates between 7dC and the diketol (3-oxo-25,22,2-trideoxyecdysone) have ever been observed or identified in insect prothoracic gland incubations with radiolabelled precursors. However, we show that 3-oxo-7dC can be converted into lipophilic, photosensitive, ketone-blocked (PSKB) ketal derivatives which will release 3-oxo-7dC when and where desired following brief irradiation with innocuous long-wave (365 nm) UV-light both in vivo and in vitro. In this manner, 3-oxo-7dC is quickly and efficiently incorporated into ecdysteroids by adult male and female Drosophila raised on a diet containing the PSKB ketals and in prothoracic glands of Manduca sexta incubated with the ketals emulsified into media. The instability of 3-oxo-7dC and its spontaneous transformation into extensively electron-delocalized intermediates will be discussed in relation to a possible mechanism of the Black Box oxidations eventually leading to the production of the active molting hormone 20-hydroxyecdysone (20E).

N-glycosylation is required for binding of murine pregnancy-specific glycoproteins 17 and 19 to the receptor CD9.

PROBLEM: Murine pregnancy-specific glycoproteins (PSGs) are encoded by 17 different genes. Different family members have different expression levels at different stages of embryonic development. It is currently unknown whether all members of this family of placentally secreted proteins have the same function and bind to the same receptor. Furthermore, the requirement of post-translational modifications for the activity of these highly glycosylated proteins remains undetermined. METHOD OF STUDY: Recombinant PSG17 and PSG19 were generated and purified by affinity chromatography. An expression library was screened to identify the receptor for mouse PSG19. Binding to the receptor by proteins generated in different expression systems and mapping of the binding domain were analyzed by pull-down assays. Analysis of the carbohydrate composition of the receptor-binding domain was performed with the DIG glycan differentiation kit. RESULTS: PSG19 binds to the tetraspanin CD9, specifically to extra cellular loop 2 and can induce secretion of TGFbeta1 by a macrophage cell line. The receptor-binding domain of PSG17 and PSG19 is post-translationally modified by the addition of N-linked carbohydrates and, when expressed in CHO cells, terminal sialic acids are detected. PSGs produced in bacteria do not bind CD9. CONCLUSION: PSG19, as previously determined for PSG17, binds to the second extracellular loop 2 of the tetraspanin CD9. The first immunoglobulin variable-like domain of PSG19 is sufficient for receptor binding and function. Analysis of receptor usage by the remaining 15 murine PSGs will most likely require that the proteins be generated in eukaryotic expression systems, as we have demonstrated that the addition of carbohydrates is essential for PSG-receptor interaction.