Exploring metabolic demands of high density CHO-cell cultures

As the world population continues to grow and age, access to medical therapies, like therapeutic monoclonal antibodies (mAb), increases in importance. Chinese hamster ovary (CHO) cells are the preferred expression platform for biopharmaceutical production, such as mAb, due to their ability to generate human-like post-translational modifications and strong legislation background. Unfortunately, biopharmaceuticals production using CHO cells is costly leading to an expensive product. Detailed knowledge of their metabolic demands is still lacking. Hence, increasing understanding would help to develop rational approaches to enhance expression, lower production costs and subsequently make therapies more accessible. Industrial large scale CHO cell cultures are typically run in fed-batch mode, which is limited by the accumulation of inhibitory by-products. Driven by higher volumetric productivities, shorter residence time of products, industry is shifting towards perfusion cultures. This shift in cultivation mode is accompanied by new metabolic conditions for the cell in form of higher cell densities and different exometabolite levels. Comparison of both culturing modes, as well as different perfusion dilution rates, revealed differences in growth and productivity patterns. Cell densities and cell specific productivities increased from fed-batch to perfusion and from low to high dilution rates. Aiming to progress from pattern observations to a systems biology view, the metabolome and proteome profiles of both cultures modes will be analysed and mapped onto the CHO genome-scale model. Thus, protein expression and metabolite levels will be compared on a genome wide level and provide a metabolic profile, specific for the culture condition. Alterations between metabolic profiles of different culture conditions can then be identified, understood and utilized to reverse engineer variations by using genetic engineering approaches or media design and process optimization. Overall, this study aims to establish a systems biology approach to better understand the CHO cell metabolism in the aspect of emerging perfusion systems based on comparison of different culture modes

Second-trimester amniotic fluid proteins changes in subsequent spontaneous preterm birth

IntroductionThe global sequence of the pathogenesis of preterm labor remains unclear. This study aimed to compare amniotic fluid concentrations of extracellular matrix-related proteins (procollagen, osteopontin and IL-33), and of cytokines (IL-19, IL-6, IL-20, TNF alpha, TGF beta, and IL-1 beta) in asymptomatic women with and without subsequent spontaneous preterm delivery. Material and methodsWe used amniotic fluid samples of singleton pregnancy, collected by amniocentesis between 16 and 20 weeks' gestation, without stigmata of infection (i.e., all amniotic fluid samples were tested with broad-range 16 S rDNA PCR to distinguish samples with evidence of past bacterial infection from sterile ones), during a randomized, double-blind, placebo-controlled trial to perform a nested case-control laboratory study. Cases were women with a spontaneous delivery before 37 weeks of gestation (preterm group). Controls were women who gave birth at or after 39 weeks (full term group). Amniotic fluid concentrations of the extracellular matrix-related proteins and cytokines measured by immunoassays were compared for two study groups. : NCT00718705. ResultsBetween July 2008 and July 2011, in 12 maternal-fetal medicine centers in France, 166 women with available PCR-negative amniotic fluid samples were retained for the analysis. Concentrations of procollagen, osteopontin, IL-19, IL-6, IL-20, IL-33, TNF alpha, TGF beta, and IL-1 beta were compared between the 37 who gave birth preterm and the 129 women with full-term delivery. Amniotic fluid levels of procollagen, osteopontin, IL-19, IL-33, and TNF alpha were significantly higher in the preterm than the full-term group. IL-6, IL-20, TGF beta, and IL-1 beta levels did not differ between the groups. ConclusionsIn amniotic fluid 16 S rDNA PCR negative samples obtained during second-trimester amniocentesis, extracellular matrix-related protein concentrations (procollagen, osteopontin and IL-33), together with IL-19 and TNF alpha, were observed higher at this time in cases of later spontaneous preterm birth

Factors associated with unprotected anal intercourse among men who have sex with men in Douala, Cameroon

Objectives Research on men who have sex with men (MSM) in sub-Saharan Africa was neglected for a long time. The objective of this study was to understand factors associated with unprotected anal intercourse (UAI) with male partners among a group of MSM living in the city of Douala, Cameroon. Methods In 2008, a survey on the sexual activity and practices of MSM was set up in Douala in collaboration with a local community-based organisation. Data were collected among a convenience sample of 168 MSM during face-to-face interviews with trained interviewers. Results A total of 142 individuals reported sexual activity during the previous 6 months, among whom 80 (57%) reported UAI with male partners. In a multivariate logistic regression model adjusted for the frequency of sexual intercourse, not having had access to prevention interventions and not knowing any HIV-infected person were both independently associated with a higher risk of UAI. Other factors associated with this higher risk included having had a stable male partnership at some point in one's life and not having been out of Douala for more than 4 weeks during the previous year. Conclusions This community-based research is the first study of MSM in Cameroon and the HIV transmission risks they face. Results show the importance of HIV prevention interventions from peers, and underline the need to maintain efforts to develop specific interventions targeting MSM more efficiently in the African context

The neglected hepatitis C virus genotypes 4, 5 and 6: an international consensus report

Hepatitis C virus (HCV) genotypes 4, 5 and 6 represent >20% of all HCV cases worldwide. HCV-4 is mainly seen in Egypt, where it represents 90% of all HCV cases. Antischistosomal therapy was the main cause of contamination there, followed by procedures performed by informal providers and traditional healers such as dental care, wound treatment, circumcision, deliveries, excision and scarification. It is also highly prevalent in sub-Saharan Africa and in the Middle East. In Europe, its prevalence has recently increased particularly among intravenous drug users and in immigrants. HCV-5 is mainly found in South Africa, where it represents 40% of all HCV genotypes, but four pockets of HCV-5 were found in France, Spain, Syria and Belgium and sporadic cases were found elsewhere. The mode of transmission is mainly iatrogenic and transfusion. HCV-6 is found in Hong Kong, Vietnam, Thailand and Myanmar and also in American and Australian from Asian origin. The response to treatment in HCV-4 is intermediate between HCV-1 and HCV-2 and HCV-3. A sustained viral response is achieved in 43-70% with pegylated interferon and ribavirin. It is higher in Egyptians than Europeans and Africans and is negatively related to insulin resistance and to the severity of fibrosis. It increases to >80% with 24 weeks of therapy only if a rapid virological response is achieved. In HCV-5, a sustained virological response is achieved in >60% with 48 weeks of therapy. HCV-6 is also considered an easy-to-treat genotype, leading to a response in 60-85% of cases. © 2009 John Wiley & Sons A/S.link_to_subscribed_fulltex

Addressing ethical challenges in the Genetics Substudy of the National Eye Survey of Trinidad and Tobago (GSNESTT).

BACKGROUND: The conduct of international collaborative genomics research raises distinct ethical challenges that require special consideration, especially if conducted in settings that are research-naïve or resource-limited. Although there is considerable literature on these issues, there is a dearth of literature chronicling approaches taken to address these issues in the field. Additionally no previous ethical guidelines have been developed to support similar research in Trinidad and Tobago. METHODS: A literature review was undertaken to identify strategies used to address common ethical issues relevant to human genetics and genomics research in research-naïve or resource-limited settings. Strategies identified were combined with novel approaches to develop a culturally appropriate, multifaceted strategy to address potential challenges in the Genetics Substudy of the National Eye Survey of Trinidad and Tobago (GSNESTT). RESULTS: Regarding the protection of study participants, we report a decision to exclude children as participants; the use of a Community Engagement and Sensitization Strategy to increase the genetic literacy of the target population; the involvement of local expertise to ensure cultural sensitivity and to address potential comprehension barriers in informed consent; and an audit of the informed consent process to ensure valid consent. Concerning the regulation of the research, we report on ethics approvals from relevant authorities; a Materials Transfer Agreement to guide sample ownership and export; and a Sample Governance Committee to oversee data use and data access. Finally regarding the protection of the interests of scientists from the host country, we report on capacity building efforts to ensure that local scientists have access to data collected through the project and appropriate recognition of their contributions in future publications. CONCLUSION: This paper outlines an ethical framework for the conduct of population-based genetics and genomics research in Trinidad and Tobago; highlights common issues arising in the field and strategies to address these

The cost-effectiveness of long-term antiviral therapy in the management of HBeAg-positive and HBeAg-negative chronic hepatitis B in Singapore

The purpose of this study was the economic evaluation of short-duration treatments of chronic hepatitis B (CHB) and longer duration antiviral treatment for up to 5 years. Two 10-health state Markov models were developed for hepatitis B e antigen (HBeAg)-positive and HBeAg-negative CHB patients respectively. The perspective of this economic evaluation was the Singapore healthcare system and CHB patient. The models followed cohorts of HBeAg-positive and HBeAg-negative CHB patients, respectively, over a period of 40 years, by which time the majority of the cohorts would have died if left untreated. Costs and benefits were discounted at 5% per annum. Annual rates of disease progression and the magnitude of treatment effects were obtained from the literature, with a focus on data obtained in Asian patients and meeting the criteria for therapy as described in internationally recognized management guidelines. Short-course therapy with α-interferon, or 1-year treatment with pegylated interferon α-2a, lamivudine or adefovir had limited impact on disease progression. In contrast, treatment of CHB with antiviral therapy for 5 years substantially decreased the rate of disease progression. Treatment with lamivudine for 1-year is highly cost-effective compared with no treatment of CHB but has limited effect on reducing the rate of disease progression. Compared with 1-year treatment with lamivudine, sequential antiviral therapies for up to 5 years (i.e. lamivudine plus adefovir on emergence of lamivudine resistance or adefovir plus lamivudine on emergence of adefovir resistance) are highly cost-effective by international standards. These conclusions are robust to uncertainties in model inputs and are consistent with the findings of other recently published studies

Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B

BACKGROUND: Available treatments for hepatitis B e antigen (HBeAg)-negative chronic hepatitis B are associated with poor sustained responses. As a result, nucleoside and nucleotide analogues are typically continued indefinitely, a strategy associated with the risk of resistance and unknown long-term safety implications. METHODS: We compared the efficacy and safety of peginterferon alfa-2a (180 microg once weekly) plus placebo, peginterferon alfa-2a plus lamivudine (100 mg daily), and lamivudine alone in 177, 179, and 181 patients with HBeAg-negative chronic hepatitis B, respectively. Patients were treated for 48 weeks and followed for an additional 24 weeks. RESULTS: After 24 weeks of follow-up, the percentage of patients with normalization of alanine aminotransferase levels or hepatitis B virus (HBV) DNA levels below 20,000 copies per milliliter was significantly higher with peginterferon alfa-2a monotherapy (59 percent and 43 percent, respectively) and peginterferon alfa-2a plus lamivudine (60 percent and 44 percent) than with lamivudine monotherapy (44 percent, P=0.004 and P=0.003, respectively; and 29 percent, P=0.007 and P=0.003, respectively). Rates of sustained suppression of HBV DNA to below 400 copies per milliliter were 19 percent with peginterferon alfa-2a monotherapy, 20 percent with combination therapy, and 7 percent with lamivudine alone (P<0.001 for both comparisons with lamivudine alone). Loss of hepatitis B surface antigen occurred in 12 patients in the peginterferon groups, as compared with 0 patients in the group given lamivudine alone. Adverse events, including pyrexia, fatigue, myalgia, and headache, were less frequent with lamivudine monotherapy than with peginterferon alfa-2a monotherapy or combination therapy. CONCLUSIONS: Patients with HBeAg-negative chronic hepatitis B had significantly higher rates of response, sustained for 24 weeks after the cessation of therapy, with peginterferon alfa-2a than with lamivudine. The addition of lamivudine to peginterferon alfa-2a did not improve post-therapy response rates. Copyright 2004 Massachusetts Medical Societypublished_or_final_versio

Viral Resistance in Hepatitis B: Prevalence and Management

Hepatitis B is a DNA virus affecting hundreds of millions of individuals worldwide. As the clinical sequelae of cirrhosis and hepatocellular cancer are increasingly recognized to be related to viral levels, the impetus increases to offer treatment to those previously not treated. With the development of more robust antivirals with reasonable safety profiles, long-term treatment is becoming more common. The oral nucleos(t)ide analogs have become the preferred first-line therapies for most genotypes of hepatitis B. Five are now available, all with different potencies and resistance profiles. Long-term data spanning several years are now available for most compounds in this arena. This article focuses on the common natural variants and those secondary to nucleos(t)ide therapy, as well as diagnostic methods to detect resistance

Protecting and Evaluating Genomic Privacy in Medical Tests and Personalized Medicine

In this paper, we propose privacy-enhancing technologies for medical tests and personalized medicine methods that use patients' genomic data. Focusing on genetic disease-susceptibility tests, we develop a new architecture (between the patient and the medical unit) and propose a "privacy-preserving disease susceptibility test" (PDS) by using homomorphic encryption and proxy re-encryption. Assuming the whole genome sequencing to be done by a certified institution, we propose to store patients' genomic data encrypted by their public keys at a "storage and processing unit" (SPU). Our proposed solution lets the medical unit retrieve the encrypted genomic data from the SPU and process it for medical tests and personalized medicine methods, while preserving the privacy of patients' genomic data. We also quantify the genomic privacy of a patient (from the medical unit's point of view) and show how a patient's genomic privacy decreases with the genetic tests he undergoes due to (i) the nature of the genetic test, and (ii) the characteristics of the genomic data. Furthermore, we show how basic policies and obfuscation methods help to keep the genomic privacy of a patient at a high level. We also implement and show, via a complexity analysis, the practicality of PDS