The glycan processing and site occupancy of recombinant Thy-1 is markedly affected by the presence of a glycosylphosphatidylinositol anchor

Thy-1 is a cell surface glycoprotein containing three N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The effect of the anchor on its N-linked glyco-sylation was investigated by comparing the glycosylation of soluble recombinant Thy-1 (sThy-1) with that of recombinant GPI anchored Thy-1, both expressed in Chinese hamster ovary cells. The sThy-1 was produced in a variety of isoforms including some which lacked carbohydrate on all three sequons whereas the GPI anchored form appeared fully glycosylated like native Thy-1. This was surprising as the attachment of N-linked sugars occurs cotranslationally and it was not expected that the presence of a C-terminal GPI anchor signal sequence would affect sequon occupancy. Furthermore sThy-1 lacking glycosylation could be produced with the inhibitor tunicamycin but in contrast cell surface expression of unglycosylated GPI anchored Thy-1 could not be obtained. The GPI anchored form appeared less processed with almost 4-fold more oligo-mannose oligosaccharides than in sThy-1 and also with less sialylated and core fucosylated biantennary glycans. Possible mechanisms whereby the anchor or the anchor signal sequence, control site occupancy and maturation are discussed.     

Factors affecting the presentation of exogenous hepatitis B virus core antigen

Hepatitis B virus core antigen (HBcAg) plays a critical role in terminating acute Hepatitis B virus infection and may be used as a potential vaccine candidate. The cell surface major histocompatibility complex (MHC) class 1 molecules are thought to be involved in the presentation of HBcAg. Surface MHC class 1 HLA A2 heavy chain (HC) and trimeric molecules were characterized on transfected Hela cells used as antigen presenting cells (APC) for the presentation of HBcAg. The results show that antibodies against HC HLA A2 and trimeric HLA-A2 molecules resulted in increased activation of HBcAg 18-27 minimal peptide specific cytotoxic T lymphocytes (CTLs), while the addition of exogenous beta2-microglobulin decreased the activation of HBcAg specific CTLs. Further, specific CD8+ T cells were activated only when Hela cells as APCs were primed with HBcAg (peptide, soluble or embedded on virosomes) at pH 6.5.     

Implementation of Organ Culture storage of donor corneas: a 3 year study of its impact on the corneal transplant wait list at the Lions New South Wales Eye Bank

Organ Culture corneal storage offers an extended storage time and increased donor pool and tissue assessment opportunities. In September 2011, the Lions New South Wales Eye Bank (LNSWEB) moved from hypothermic storage to Organ Culture corneal storage. This study evaluates the impact of implementation of Organ Culture on donor eye retrieval and the corneal transplant waiting list over a 3 year period in NSW, Australia. Retrospective review of the LNSWEB data from September 2011 to August 2014. Tissue collection, waiting list and tissue utilization data were recorded. The data from September 2008 to August 2011 for Optisol-GS storage was used for comparison. The annual donor and cornea collection rate increased 35 % and 44 % respectively with Organ Culture compared to Optisol-GS storage. The utilization rate of corneal tissue increased from 73.4 % with hypothermic storage to 77.2 % with Organ Culture storage. The transplant wait list decreased by 77.3 % from September 2011 to August 2014 and correlated with the increased rate of corneal transplantation (r = ?0.9381, p < 0.0001). No other factors impacting the wait list changed over this period. Corneas not used from either storage method were due to unacceptable endothelial cell density/viability. The contamination rate of corneas stored in Organ Culture medium was low at 1.74 %. The Organ Culture storage method increases the corneal donor pool available to Eye banks. The practical benefits of the extended storage time and increased donor assessment opportunities have directly led to an increase in corneal utilization rate and a significant decrease in recipient wait list time.     

Quantitative imaging of pO2 in orthotopic murine gliomas: hypoxia correlates with resistance to radiation

Hypoxia is considered one of the microenvironmental factors associated with the malignant nature of glioblastoma. Thus, evaluating intratumoural distribution of hypoxia would be useful for therapeutic planning as well as assessment of its effectiveness during the therapy. Electron paramagnetic resonance imaging (EPRI) is an imaging technique which can generate quantitative maps of oxygen in vivo using the exogenous paramagnetic compound, triarylmethyl and monitoring its line broadening caused by oxygen. In this study, the feasibility of EPRI for assessment of oxygen distribution in the glioblastoma using orthotopic U87 and U251 xenograft model is examined. Heterogeneous distribution of pO₂ between 0 and 50?mmHg was observed throughout the tumours except for the normal brain tissue. U251 glioblastoma was more likely to exhibit hypoxia than U87 for comparable tumour size (median pO₂; 29.7 and 18.2?mmHg, p?=?.028, in U87 and U251, respectively). The area with pO₂ under 10?mmHg on the EPR oximetry (HF10) showed a good correlation with pimonidazole staining among tumours with evaluated size. In subcutaneous xenograft model, irradiation was relatively less effective for U251 compared with U87. In conclusion, EPRI is a feasible method to evaluate oxygen distribution in the brain tumour.     

Oligosaccharide analysis and molecular modeling of soluble forms of glycoproteins belonging to the Ly-6, scavenger receptor, and immunoglobulin superfamilies expressed in Chinese hamster ovary cells

Most cell surface molecules are glycoproteins consisting of linear arrays of globular domains containing stretches of amino acid sequence with similarities to regions in other proteins. These conserved regions form the basis for the classification of proteins into superfamilies. Recombinant soluble forms of six leukocyte antigens belonging to the Ly-6 (CD59), scavenger receptor (CD5), and immunoglobulin (CD2, CD48, CD4, and Thy-1) superfamilies were expressed in the same Chinese hamster ovary cell line, thus providing an opportunity to examine the extent to which N-linked oligosaccharide processing might vary in a superfamily-, domain-, or protein-dependent manner in a given cell. While we found no evidence for superfamily-specific modifications of the glycans, marked differences were seen in the types of oligosaccharides attached to individual proteins within a given superfamily. The relative importance of local protein surface properties versus the overall tertiary structure of the molecules in directing this protein-specific variation was examined in the context of molecular models. These were constructed using the 3D structures of the proteins, glycan data from this study, and an oligosaccharide structural database. The results indicated that both the overall organization of the domains and the local protein structure can have a large bearing on site-specific glycan modification of cells in stasis. This level of control ensures that the surface of a single cell will display a diverse repertoire of glycans and precludes the presentation of multiple copies of a single oligosaccharide on the cell surface. The glycans invariably shield large regions of the protein surfaces although, for the glycoproteins examined here, these did not hinder the known active sites of the molecules. The models also indicated that sugars are likely to play a role in the packing of the native cell surface glycoproteins and to limit nonspecific protein-protein interactions. In addition, glycans located close to the cell membrane are likely to affect crucially the orientation of the glycoproteins to which they are attached.     

Pyruvate Induces Transient Tumor Hypoxia by Enhancing Mitochondrial Oxygen Consumption and Potentiates the Anti-Tumor Effect of a Hypoxia-Activated Prodrug TH-302

Background

TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.

Methodology/Results

The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O₂), with minimal effect under aerobic conditions (21% O₂). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm³. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm³), significantly delayed tumor growth.

Conclusions/Significance

Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.     

High annual risk of tuberculosis infection among nursing students in South India: a cohort study

Background

Nurses in developing countries are frequently exposed to infectious tuberculosis (TB) patients, and have a high prevalence of TB infection. To estimate the incidence of new TB infection, we recruited a cohort of young nursing trainees at the Christian Medical College in Southern India. Annual tuberculin skin testing (TST) was conducted to assess the annual risk of TB infection (ARTI) in this cohort.

Methodology/Principal Findings

436 nursing students completed baseline two-step TST testing in 2007 and 217 were TST-negative and therefore eligible for repeat testing in 2008. 181 subjects completed a detailed questionnaire on exposure to tuberculosis from workplace and social contacts. A physician verified the questionnaire and clinical log book and screened the subjects for symptoms of active TB. The majority of nursing students (96.7%) were females, almost 84% were under 22 years of age, and 80% had BCG scars. Among those students who underwent repeat testing in 2008, 14 had TST conversions using the ATS/CDC/IDSA conversion definition of 10 mm or greater increase over baseline. The ARTI was therefore estimated as 7.8% (95%CI: 4.3–12.8%). This was significantly higher than the national average ARTI of 1.5%. Sputum collection and caring for pulmonary TB patients were both high risk activities that were associated with TST conversions in this young nursing cohort.

Conclusions

Our study showed a high ARTI among young nursing trainees, substantially higher than that seen in the general Indian population. Indian healthcare providers and the Indian Revised National TB Control Programme will need to implement internationally recommended TB infection control interventions to protect its health care workforce.     

Electron paramagnetic resonance imaging of tumor pO₂

Electron paramagnetic resonance imaging (EPRI) can be used to noninvasively and quantitatively obtain three-dimensional maps of tumor pO?. The paramagnetic tracer triarylmethyl (TAM), a substituted trityl radical moiety, is not toxic to animals and provides narrow isotropic spectra, which is ideal for in vivo EPR imaging experiments. From the oxygen-induced spectral broadening of TAM, pO? maps can be derived using EPRI. The instrumentation consists of an EPRI spectrometer and 7T magnetic resonance imaging (MRI) system both operating at a common radiofrequency of 300 MHz. Anatomic images obtained by MRI can be overlaid with pO? maps obtained from EPRI. With imaging times of less than 3 min, it was possible to monitor the dynamics of oxygen changes in tumor and distinguish chronically hypoxic regions from acutely hypoxic regions. In this article, the principles of pO? imaging with EPR and some relevant examples of tumor imaging are reviewed.