Clonal selection and characterization of epigenetic variation in Pichia pastoris

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2010.Page 71 blank. Cataloged from PDF version of thesis.Includes bibliographical references (p. 67-70).Recombinant proteins produced by different host organisms have been broadly used as therapeutics. Considering the demand for large quantities of protein drugs, methods are needed to increase reactor titers in a timely and cost-effective manner. We used random chemical mutagenesis to modify a wild-type strain of the heterologous protein production host Pichia pastoris, which resulted in overall improvement of the secretion rate of the mutated population. More than 4000 single-cells were simultaneously screened for high secretion of a human Fc fragment using microengraving and the top-producing clones were retrieved. Future characterization of these improved clones by transcript profiling should yield information about networks of genes central in heterologous protein secretion in the yeast P. pastoris.by Vasiliki Panagiotou.S.M

Protein engineering approaches for direct antigen targeting in CD8+ T cell inducing vaccines

Effective vaccine design and public vaccination programs have led to the eradication of several diseases and protected millions of people from deadly infections. However, intracellular infections remain a challenge and their eradication requires the activation of cytolytic mechanisms. CD8+ T cells promote effector mechanisms for infected cell killing subsequent to their priming by antigen-presenting cells. Their activation occurs upon detection of intracellular antigen in a pathway that many current subunit vaccine technologies are trying to approach by cross-presentation. Advances in CD8+ T cell-inducing subunit vaccines include direct targeting of antigens to cross-presenting cells and are studied in this thesis. Antigen delivery to the appropriate cell type for cross-presentation to CD8+ T cells can occur with fusion antibodies. Our laboratory has previously demonstrated that antigens bearing an erythrocyte-binding antibody domain (TER119) target the liver. We used this technology to fuse TER119 and ovalbumin (OVA) antigen with the BBOX domain of the high mobility group box 1 (HMGB1) danger signal and co-administer it with CpG-B adjuvant in a hepatic vaccine context. In liver vaccines, activation of local cytotoxic responses represents an important challenge. Erythrocyte-mediated OVA delivery with adjuvants resulted in cytotoxic CD8+ T lymphocyte (CTL) activation with memory formation in the liver and protection was provided after infection with Listeria monocytogenes. Another method for antigen targeting is through a cross-presenting dendritic cell (DC) subset, called CD8+ DCs. Recent studies in mice showed successful vaccination against OVA fused to XCL1 ligand, which binds CD8+ DCs. To attract CD8+ DCs to the site of injection for increased antigen uptake, we co-injected XCL1-OVA with a fusion protein consisting of XCL1 and the extracellular matrix (ECM) binding domain of placenta growth factor-2 (PlGF-2123-144), called XCL1-PlGF. XCL1-PlGF binds extracellular matrix at the injection site and attracts CD8+ DCs locally. In the context of prophylactic and therapeutic vaccination, the combination of two fusion proteins (XCL1-OVA and XCL1-PlGF) enhanced cytotoxicity and prolonged survival against B16-OVA melanoma. Finally, antigen transport via biomaterial delivery platforms was evaluated as an alternative method to deliver antigens to immunologically relevant sites. Our laboratory and others have shown that nanoscale carriers promote CTL responses in vaccination. Here we present a cationic micelle vaccination platform in which OVA and adjuvant (CpG-B and/or MPLA) loading is mediated by non-covalent molecular encapsulation and adsorption. Following a prime and double boost vaccination, CTL responses were raised in the spleen and lymph nodes of vaccinated mice, along with antigen-specific antibody production. These findings highlight the advantages of the micelle carrier platform in vaccine applications. Overall, this thesis presents novel techniques to target antigens to the appropriate cell compartments, facilitating CTL activation in vaccination either by direct targeting of antigen fused to antibodies and chemokines, or antigen delivery with polymeric vehicles. These platforms for optimized delivery seek to improve current approaches and impact the design of new strategies in CD8+ T cell inductive vaccination

Systematic Single-Cell Analysis of Pichia pastoris Reveals Secretory Capacity Limits Productivity

Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity

High-dose bevacizumab for radiation-induced brain necrosis: a case report

Radiation-induced brain necrosis (RIBN) is a common adverse event from radiation therapy. We present a case of a 56-year-old man, diagnosed with non-small-cell lung cancer with brain metastases 2 years prior, for which he had received whole brain radiotherapy and brain stereotactic radiosurgery, who presented to the oncology unit with headache, dizziness and abnormal gait. MRI of the brain revealed radiological worsening of a cerebellar mass, including edema and mass effect. After a multidisciplinary tumor board meeting, the patient was diagnosed with RIBN and received 4 cycles of high-dose bevacizumab, with complete symptom resolution and significant radiological response. We report the successful use of a high-dose, shorter-duration treatment protocol of bevacizumab for RIBN

The Epidemiology of Pulmonary Nontuberculous Mycobacteria: Data from a General Hospital in Athens, Greece, 2007–2013

Background. The epidemiology of pulmonary nontuberculous mycobacteria (NTM) in Greece is largely unknown. Objectives. To determine the incidence and the demographic, microbiological, and clinical characteristics of patients with pulmonary NTM infection and pulmonary NTM disease. Methods. A retrospective review of the demographic, microbiological, and clinical characteristics of patients with NTM culture-positive respiratory specimens from January 2007 to May 2013. Results. A total of 120 patients were identified with at least one respiratory NTM isolate and 56 patients (46%) fulfilled the microbiological ATS/IDSA criteria for NTM disease. Of patients with adequate data, 16% fulfilled the complete ATS/IDSA criteria for NTM disease. The incidence of pulmonary NTM infection and disease was 18.9 and 8.8 per 100.000 inpatients and outpatients, respectively. The spectrum of NTM species was high (13 species) and predominated by M. avium-intracellulare complex (M. avium (13%), M. intracellulare (10%)), M. gordonae (14%), and M. fortuitum (12%). The ratio of isolation of NTM to M. tuberculosis in all hospitalized patients was 0.59. Conclusions. The first data on the epidemiology of pulmonary NTM in Athens, Greece, are presented. NTM infection is common in patients with chronic respiratory disease. However, only a significantly smaller proportion of patients fulfill the criteria for NTM disease

The Epidemiology of Pulmonary Nontuberculous Mycobacteria: Data from a General Hospital in Athens, Greece, 2007-2013

Background. The epidemiology of pulmonary nontuberculous mycobacteria (NTM) in Greece is largely unknown. Objectives. To determine the incidence and the demographic, microbiological, and clinical characteristics of patients with pulmonary NTM infection and pulmonary NTM disease. Methods. A retrospective review of the demographic, microbiological, and clinical characteristics of patients with NTM culture-positive respiratory specimens from January 2007 to May 2013. Results. A total of 120 patients were identified with at least one respiratory NTM isolate and 56 patients (46%) fulfilled the microbiological ATS/IDSA criteria for NTM disease. Of patients with adequate data, 16% fulfilled the complete ATS/IDSA criteria for NTM disease. The incidence of pulmonary NTM infection and disease was 18.9 and 8.8 per 100.000 inpatients and outpatients, respectively. The spectrum of NTM species was high (13 species) and predominated by M. avium-intracellulare complex (M. avium (13%), M. intracellulare (10%)), M. gordonae (14%), and M. fortuitum (12%). The ratio of isolation of NTM to M. tuberculosis in all hospitalized patients was 0.59. Conclusions. The first data on the epidemiology of pulmonary NTM in Athens, Greece, are presented. NTM infection is common in patients with chronic respiratory disease. However, only a significantly smaller proportion of patients fulfill the criteria for NTM disease

Natura 2000 habitat mapping in Cyprus using high resolution orthophoto maps

The Natura 2000 network is an essential tool for the protection and conservation of habitats and species throughout Europe. Each member state is responsible for the designation and management of Special Areas of Conservation (SACs), according to the provisions of the Habitats Directive (92/43/EEC). One of the key components for the conservation of these areas and their natural habitats is monitoring to ensure their long-term protection and maintenance. In this study, high resolution orthophotos were used to update the habitat maps of 17 SACs of the Natura 2000 network in Cyprus (areas under the effective control of the Government of the Republic of Cyprus) and also to map two (2) new candidate Sites of Community Importance (cSCIs). Habitat mapping involved an initial photo-interpretation using orthophotos and, subsequently, the aggregation of information from supplementary data (i.e. Corine Land Cover, crosswalks between EUNIS habitats Classification and Corine Land Cover, Google earth imagery from different years and seasons, etc.) to produce a map with all available information (spatial and descriptive) regarding habitat types and their cover area. Validation of the updated habitat mapping was performed by habitat and flora experts, combined with extensive field work. Additionally, 100 monitoring protocols were used to record and assess the conservation degree of 13 different habitat types inside these 19 areas. The study demonstrates that high-resolution orthophotos, combined with field work, significantly contributed to the improvement of the Natura 2000 habitat mapping. Remote sensing applications are powerful tools for identifying, mapping and monitoring of the Natura 2000 habitats

25-hydroxyvitamin D in malignant pleural disease: a prospective cohort study

IntroductionGrowing evidence suggests a role of vitamin D in various cancers but the significance of vitamin D in malignant pleural disease remains unexplored. We sought to investigate the concentration and diagnostic role of 25-hydroxyvitamin D (25(OH)D) in malignant pleural effusions. Materials and MethodsProspective study of consecutive treatment-naive patients with a new diagnosis of pleural effusion. ResultsSeventy-eight patients were studied, 45 of whom had malignant pleural effusions. Concentration of 25(OH)D in pleural fluid was significantly higher than serum in both malignant (15.2 ng/mL (9.7, 25.6) versus 10.2 ng/mL (6.4, 17.7), P<.001) and benign (11.4 ng/mL (8.4, 23.6) versus 7.9 (5.9, 16.1), P<.001) pleural disease. Pleural fluid 25(OH)D was almost significantly higher in exudates compared to transudates (P=.050) but it did not differ significantly between malignant and benign effusions (P=.217) and it was not diagnostic for malignant pleural disease (area under the ROC curve .58, 95% CI .45-.71). ConclusionsIn subjects with unselected pleural effusions, 25(OH)D in pleural fluid was not diagnostic for malignant pleural disease. The novel finding of convincingly and consistently higher 25(OH)D in pleural fluid than serum suggests a role for vitamin D in pleural disease and merits further research

Characterization of relationships between intracellular and secreted proteins for single cells of <i>P. pastoris</i>.

<p>Density plots of the relative rates of eGFP secretion by single cells analyzed by microengraving with respect to the relative amount of intracellular eGFP determined by fluorescence microscopy for clones containing (A) one eGFP gene copy under pGAPDH or (B) two eGFP gene copies under pAOX1. Dashed line indicates the limit of detection for secreted eGFP in microengraving (background+2σ). The median amounts of internal eGFP for cells above and below this limit of detection are marked (X) and are significantly different (Mann-Whitney test, p≪0.001 for both pGAPDH and pAOX strains). (C) Density plot of the relative rates of secretion analyzed by microengraving for the glycosylated Fc fragment and eGFP produced simultaneously in single cells at two different loci using pGAPDH. Pearson's correlation coefficient for secretion of these two proteins as shown is 0.79.</p

Effects of altering relative rates of secretion and degradation on modeled distribution of intracellular and secreted protein.

<p>Density plot of the relative rates of protein secretion by single cells against the relative amount of intracellular protein for model data sets under three conditions: 1) where median k_ERAD≥k_sec (purple), 2) where median k_sec≪k_sec in Condition 1 (light pink), and 3) where median k_sec≪k_sec in Condition 1 and median k_ERAD>k_ERAD and/or k_expexp in Condition 1 (dark pink). The median amount of intracellular protein for populations in Conditions 1 and 3 are marked (X). The secretion-inhibited population (light pink) was generated by increasing t_sec to 400 min (standard deviation 40 min) while keeping all other parameters the same as the initial population (purple) derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037915#pone-0037915-g004" target="_blank">Figure 4C</a>. The stress-induced population (dark pink) was generated by reducing k_exp or decreasing t_ERAD to obtain a similar median level of protein in the ER as the original population.</p