Mechanistic insights into the biological activity of S-Sulfocysteine in CHO cells using a multi-omics approach.

S-Sulfocysteine (SSC), a bioavailable L-cysteine derivative (Cys), is known to be taken up and metabolized in Chinese hamster ovary (CHO) cells used to produce novel therapeutic biological entities. To gain a deeper mechanistic insight into the SSC biological activity and metabolization, a multi-omics study was performed on industrially relevant CHO-K1 GS cells throughout a fed-batch process, including metabolomic and proteomic profiling combined with multivariate data and pathway analyses. Multi-layered data and enzymatical assays revealed an intracellular SSC/glutathione mixed disulfide formation and glutaredoxin-mediated reduction, releasing Cys and sulfur species. Increased Cys availability was directed towards glutathione and taurine synthesis, while other Cys catabolic pathways were likewise affected, indicating that cells strive to maintain Cys homeostasis and cellular functions.

A reflection on the improvement of Chinese hamster ovary cell-based bioprocesses through advances in proteomic techniques.

Chinese hamster ovary (CHO) cells are the preferred mammalian host for the large-scale production of recombinant proteins in the biopharmaceutical industry. Research endeavors have been directed to the optimization of CHO-based bioprocesses to increase protein quantity and quality, often in an empirical manner. To provide a rationale for those achievements, a myriad of CHO proteomic studies has arisen in recent decades. This review gives an overview of significant advances in LC-MS-based proteomics and sheds light on CHO proteomic studies, with a particular focus on CHO cells with superior bioprocessing phenotypes (growth, viability, titer, productivity and cQA), that have exploited novel proteomic or sub-omic techniques. These proteomic findings expand the current knowledge and understanding about the underlying protein clusters, protein regulatory networks and biological pathways governing such phenotypic changes. The proteomic studies, highlighted herein, will help in the targeted modulation of these cell factories to the desired needs.

Use of 5-Thio-L-Fucose to modulate binding affinity of therapeutic proteins.

The reduction of antibody core-fucosylation is known to enhance antibody-dependent cellular cytotoxicity (ADCC). In this study, 5-Thio-l-Fucose (ThioFuc) was investigated as a media and feed supplement for modulating the fucosylation profile of therapeutic proteins and, thereby, improving the resulting effector functions. Glycan analysis of five different therapeutic proteins produced by a diverse set of Chinese hamster ovary cell lines demonstrated a clone dependent impact of ThioFuc treatment. Using rituximab as a model, an efficient dose- and time-dependent reduction of core-fucosylation up to a minimum of 5% were obtained by ThioFuc. Besides a concomitant increase in the afucosylation level up to 48%, data also revealed up to 47% incorporation of ThioFuc in place of core-fucosylation. In accordance with the glycan data, antibodies produced in the presence of ThioFuc revealed an enhanced FcγRIIIa binding up to 7.7-fold. Furthermore, modified antibodies subjected to a cell-based ADCC reporter bioassay proved to exert both a 1.5-fold enhanced ADCC efficacy and 2.6-fold enhancement in potency in comparison to their native counterparts-both of which contribute to an improvement in the ADCC activity. In conclusion, ThioFuc is a potent fucose derivative with potential applications in drug development processes.

A systematic review of the impact of antifungal stewardship interventions in the United States.

BACKGROUND: Antimicrobial resistance is a widely recognized public health threat, and stewardship interventions to combat this problem are well described. Less is known about antifungal stewardship (AFS) initiatives and their influence within the United States. The purpose of this study was to evaluate evidence on the impact of AFS interventions on clinical and performance measures. METHODS: A systematic review of English language studies identified in the PubMed and EMBASE databases was performed through November 2017. The review was conducted in accordance with PRISMA. Search terms included antifungal stewardship, antimicrobial stewardship, Candida, candidemia, candiduria, and invasive fungal disease. Eligible studies were those that described an AFS program or intervention occurring in the US and evaluated clinical or performance measures. RESULTS: Fifty-four articles were identified and 13 were included. Five studies evaluated AFS interventions and reported clinical outcomes (mortality and length of stay) and performance measures (appropriate antifungal choice and time to therapy). The remaining eight studies evaluated general stewardship interventions and reported data on antifungal consumption. All studies were single center, quasi-experimental with varying interventions across studies. AFS programs had no impact on mortality (3 of 3 studies), with an overall rate of 27% in the intervention group and 23% in the non-intervention group. Length of stay (5 of 5) was also similar between groups (range, 9-25 vs. 11-22). Time to antifungal therapy improved in 2 of 5 studies, and appropriate choice of antifungal increased in 2 of 2 studies. Antifungal consumption was significantly blunted or reduced following stewardship initiation (8 of 8), although a direct comparison between studies was not possible due to a lack of common units. CONCLUSION: The available evidence suggests that AFS interventions can improve performance measures and decrease antifungal consumption. Although this review did not detect improvements in clinical outcomes, significant adverse outcomes were not reported.

Amino Acid Substitutions within HLA-B*27-Restricted T Cell Epitopes Prevent Recognition by Hepatitis Delta Virus-Specific CD8+ T Cells.

Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection.IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.

Trends in pneumococcal meningitis hospitalizations following the introduction of the 13-valent pneumococcal conjugate vaccine in the United States.

BACKGROUND: The 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in 2010 in the U.S. and its impact on pneumococcal meningitis (PM) is unknown. We assessed the impact of PCV13 on PM hospitalization rates 4years after the vaccine was introduced. METHODS: This was a retrospective analysis of the National Inpatient Sample from 2008-2014. Patients with an ICD-9-CM code for PM (320.1) were identified and rates calculated using US Census data as the denominator. Data weights were used to derive national estimates. We examined three time periods: 2008-2009 (late post-PCV7), 2010 (transition year), and 2011-2014 (post-PCV13). RESULTS: During the study period, there were 10,493 hospitalizations due to PM in the U.S. Overall, PM incidence decreased from 0.62 to 0.38 cases per 100,000 over this time (39% decrease; P<0.01). Among children <2years, the average annualized PM rate decreased by 45% from 2.19 to 1.20 per 100,000 (P=0.10). Annual PM rates decreased in those aged 18-39years (0.25-0.15 cases per 100,000; P=0.02) and 40-64years (0.95-0.54 cases per 100,000; P=0.03). A total of 1016 deaths were due to PM, and the case fatality rate was variable over the study period (8.3%-11.2%; P=0.96). CONCLUSION: Following the introduction of PCV13, hospitalization rates for PM decreased significantly with no subsequent improvements in case-fatality rate.

"I can do it": does confidence and perceived ability in learning new ICT skills predict pre-service health professionals' attitude towards engaging in e-healthcare?

BACKGROUND: There are many factors affecting health professionals' willingness to engage in e-health. One of these factors is whether health professionals perceive themselves to be able to learn new skills, and have the confidence in mastering these new Information and Communication Technology (ICT) skills. OBJECTIVE: This study examined how health students' confidence and perceived ability for learning new ICT skills affect their attitude towards engaging in e-health. METHODS: A survey was conducted to explore students' attitude towards using e-health and their perceived self-efficacy and confidence to learn new ICT skills. Multiple regression analysis was used to examine the relationship between confidence and self-efficacy, and attitude towards engaging in e-health controlling for participants' age, gender, and prior IT learning experience. RESULTS: The three scales measuring attitude, confidence and self-efficacy showed good internal consistency with respective Cronbach's Alpha scores of 0.835, 0.761 and 0.762. Multiple regression analysis showed a significant relationship between confidence, self-efficacy and prior IT learning experiences with attitude towards e-health after adjusting for the effect of each other (F3,350=17.20,p<0.001). CONCLUSION: Self-efficacy and confidence in learning new ICT skills together with previous ICT training either at or outside their university studies are significant factors associated with students' attitude towards using e-health. Enhancing students' level of self-efficacy in learning new ICT skills may be the key to the success of implementation of e-health initiatives.

HLA micropolymorphisms strongly affect peptide-MHC multimer-based monitoring of antigen-specific CD8+ T cell responses.

Peptide-MHC (pMHC) multimers have become one of the most widely used tools to measure Ag-specific T cell responses in humans. With the aim of understanding the requirements for pMHC-based personalized immunomonitoring, in which individuals expressing subtypes of the commonly studied HLA alleles are encountered, we assessed how the ability to detect Ag-specific T cells for a given peptide is affected by micropolymorphic differences between HLA subtypes. First, analysis of a set of 10 HLA-A*02:01-restricted T cell clones demonstrated that staining with pMHC multimers of seven distinct subtypes of the HLA-A*02 allele group was highly variable and not predicted by sequence homology. Second, to analyze the effect of minor sequence variation in a clinical setting, we screened tumor-infiltrating lymphocytes of an HLA-A*02:06 melanoma patient with either subtype-matched or HLA-A*02:01 multimers loaded with 145 different melanoma-associated Ags. This revealed that of the four HLA-A*02:06-restricted melanoma-associated T cell responses observed in this patient, two responses were underestimated and one was overlooked when using subtype-mismatched pMHC multimer collections. To our knowledge, these data provide the first demonstration of the strong effect of minor sequence variation on pMHC-based personalized immunomonitoring, and they provide tools to prevent this issue for common variants within the HLA-A*02 allele group.

E-health as a life long learning process: how to prepare health professionals for this journey.

BACKGROUND: The e-health environment is a rapidly changing one. To effectively engage with technology for healthcare delivery, health professionals must be able to adapt to this constantly evolving environment very quickly. Learning and adapting to new e-health technologies is a life-long learning process. OBJECTIVE: This study examined the effectiveness of incorporating self-directed and transformative learning approaches to introduce health sciences students to e-health concepts and skills. METHODS: Two surveys were conducted to measure students' e-health knowledge and their perceived self-efficacy in using commonly available software to complete tasks required for an assessment in the unit of study. These surveys were conducted at the beginning and the end of the semester. Paired t-tests with Bonferroni adjustment were used to examine the effect of the teaching approach on students' self-perceived efficacy. RESULTS: It was found that students showed significant improvement in their knowledge of, and perceived efficacy in using, commonly available software to carry out spreadsheet, database and data manipulation operations after intervention. CONCLUSION: This study shows that the combined self-directed and transformative teaching and learning approach is effective in helping students to identify their learning needs and develop skills to seek out resources that enable them to learn new e-health skills and concepts in a self-directed manner.

Fatal Mycobacterium colombiense/cytomegalovirus coinfection associated with acquired immunodeficiency due to autoantibodies against interferon gamma: a case report.

BACKGROUND: Reports of acquired immunodeficiency due to autoantibodies against interferon gamma in the adult population are increasing. The interleukin-12-dependent interferon-gamma axis is a major regulatory pathway of cell-mediated immunity and is critical for protection against a few intracellular organisms, including non-tuberculous mycobacteria and Salmonella spp. We report the first case of a fatal disseminated Mycobacterium colombiense/cytomegalovirus coinfection in an adult woman associated with the acquisition of autoantibodies against interferon-gamma. CASE PRESENTATION: A 49-year-old woman, born to nonconsanguineous parents in Laos, but who had lived in Canada for the past 30 years, presented with a 1-month history of weight loss, fatigue, cough, and intermittent low-grade fever. A thoracic computed tomography scan revealed an 8 × 7 cm irregular mass impacting the right superior lobar bronchus along with multiple mediastinal and hilar adenopathies. On the fourth day of admission, the patient developed fever with purulent expectorations. Treatment for a post-obstructive bacterial pneumonia was initiated while other investigations were being pursued. Almost every culture performed during the patient's hospitalization was positive for M. colombiense. Given the late presentation of symptoms - at the age of 49 years - and the absence of significant family or personal medical history, we suspected an acquired immunodeficiency due to the presence of anti-interferon-gamma autoantibodies. This was confirmed by their detection at high levels in the plasma and a STAT1 phosphorylation assay on human monocytes. The final diagnosis was immunodeficiency secondary to the production of autoantibodies against interferon-gamma, which resulted in a post-obstructive pneumonia and disseminated infection of M. colombiense. The clinical course was complicated by the presence of a multiresistant Pseudomonas aeruginosa post-endobronchial ultrasound mediastinitis, cytomegalovirus pneumonitis with dissemination, and finally, susceptible P. aeruginosa ventilator-associated pneumonia with septic shock and multiple organ failure, leading to death despite appropriate antibacterial and anti-mycobacterial treatment. CONCLUSIONS: Although rare, acquired immunodeficiency syndromes should be considered in the differential diagnosis of patients with severe, persistent, or recurrent infections. Specifically, severe non-tuberculous mycobacteria or Salmonella infections in adults without any other known risk factors may warrant examination of autoantibodies against interferon-gamma because of their increasing recognition in the literature.

Generation of precursor cell lines from preneoplastic fields surrounding head and neck cancers.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) develops in the mucosal linings of the upper aerodigestive tract. HNSCC may develop in large preneoplastic fields, which are in most cases invisible, but can be detected microscopically and by genetic analysis. METHODS: Cells of mucosal tissue biopsies were cultured and genetically analyzed. Genetic changes in established preneoplastic cultures were compared to the corresponding tumor and surgical margins. RESULTS: Of 45 mucosal tissue biopsies taken from primary tumor resection specimen, 26 were successfully cultured and could be genetically analyzed. In 1 culture, genetic changes were found and an immortalized preneoplastic cell line was obtained with genetic changes that were also found in a surgical margin of the corresponding specimen. CONCLUSION: Our data show that noninvasive fields surrounding HNSCC may consist of immortalized preneoplastic cell clones. Our preneoplastic cell line is a valuable tool to develop and test treatment strategies for precursor fields in patients with HNSCC.

The effect of e-health contents on health science students' attitude toward the efficiency of health ICT in care provision.

OBJECTIVE: This study aimed to examine the effects of e-health education content on the attitude of undergraduate health science students towards the efficiency of health ICT in healthcare provision. METHODS: A cross-sectional survey design was used. Participants were Health Sciences students attending The University of Sydney. Students were divided into three groups: junior students enrolled in a subject with non e-health content; senior students enrolled in a subject with non e-health content; and students enrolled in a subject with e-health content. Students' attitude towards the efficiency of ICT in healthcare provision was measured by a modified version of the Information Technology Attitude Scales for Health (ITASH). RESULTS: Students enrolled in the subject with e-health content had a significantly higher average baseline attitude score than the other two groups (T198=-3.47, p=0.001; T93=-2.43, p=0.017). The repeat measures analysis yielded a result with significant interaction between survey time and student group (F2, 267=4.99, p=0.007) suggesting that changes of score was dependent on student group status. CONCLUSION: Subjects rich in e-health content significantly enhanced student attitudes, even with a group of students with a rather positive initial attitude. To facilitate the uptake and utilisation of health ICT by the future health workforce, it is important for tertiary educational institutes to provide students with sufficient exposure to specific health-related ICT training, via specifically designed subjects delivering both generic and specific e-health content.

Outpatient penicillin use after negative skin testing and drug challenge in a pediatric population.

The practice of elective penicillin skin testing could be compromised by the fact that patients, their parents, or their physicians remain reluctant to reuse penicillin-class antibiotics (PCAs) despite a negative evaluation by an allergist. This study addresses reuse of PCAs in a pediatric population after negative penicillin skin testing and drug challenge and factors associated with its reluctance. All children evaluated for a history of penicillin allergy at the CHU Sainte-Justine Allergy Clinic between January 1998 and June 2000 with negative skin testing and drug challenge were included in the study. A telephone survey was conducted between May and October 2002 to assess the perception of the initial reaction by the parents, subsequent use of antibiotics, and antibiotic-related adverse reactions. Among the 200 children selected, parents of 170 (85%) children completed the survey. Since the allergist evaluation, 130 (76%) children had received antibiotics. PCA was used in 59 (45%) children. Parents of 24 (18%) children refused PCAs because they still feared an adverse reaction. They were more likely to have been very frightened by their child's allergic reaction than other parents whose children had used PCAs (p = 0.008). Although elective penicillin skin testing is useful and safe in the pediatric population, a significant proportion of parents still refuse PCAs even though they are needed. Identification of parents that were very frightened by their children's allergic reactions and additional reassurance could improve this situation.

Comparing online and offline self-disclosure: a systematic review.

Disclosure of personal information is believed to be more frequent in online compared to offline communication. However, this assumption is both theoretically and empirically contested. This systematic review examined existing research comparing online and offline self-disclosure to ascertain the evidence for current theories of online communication. Studies that compared online and offline disclosures in dyadic interactions were included for review. Contrary to expectations, disclosure was not consistently found to be greater in online contexts. Factors such as the relationship between the communicators, the specific mode of communication, and the context of the interaction appear to moderate the degree of disclosure. In relation to the theories of online communication, there is support for each theory. It is argued that the overlapping predictions of each theory and the current state of empirical research highlights a need for an overarching theory of communication that can account for disclosure in both online and offline interactions.

Complement factor 7 gene mutations in relation to meningococcal infection and clinical recurrence of meningococcal disease.

Meningococcal disease is caused by Neisseria meningitidis which is associated with high morbidity and mortality. Recurrences of meningococcal infection have been observed in patients with terminal complement component defects, because of the inefficient formation of the lytic membrane attack complex (MAC), C5b-9. Complement component C7 is one of the five plasma proteins to form the MAC. The gene C7 may carry mutations that cause functional abnormalities or the mere absence of the C7 protein. More than 200 patients were screened for aberrant C7 protein by isoelectric focusing (C7 IEF). These were compared with patients in whom recurrent meningococcal infection had resulted in the diagnosis of complete C7 absence (C7Q0). A higher proportion of C7 IEF variants were found in meningitis cases compared to controls (p=0.03). In contrast to C7Q0 patients, recurrent meningococcal infection was never observed in C7 IEF cases. Whereas C7Q0 sera were defective in meningococcal serogroup B and W135 killing assays, the sera of patients with C7 IEF variants were only defective in complement-mediated killing when classical pathway activation by (endogenous) anti-meningococcal antibodies was blocked. Upon sequence analysis we characterized the genetic background of the C7*6 and C7*8 IEF pattern and identified three novel C7 gene mutations in 13 C7Q0 patients. In conclusion, C7 IEF variants can determine meningococcal killing in the early stage of infection when antibody-independent killing prevails. The results endorse the lack of clinical recurrences once antibodies are present, whereas in C7Q0 patients the anti-meningococcal antibodies may not suffice to protect from recurrent meningococcal infection.