Advancing downstream purification of cell and gene therapy medicinal products

The advent of advanced therapies in the biopharmaceutical industry has moved the spotlight into complex products such as viral vectors or stem cells, holding great promise in a myriad of clinical targets. Currently, the challenge for a widespread application of these new biopharmaceuticals is the development of cost-effective bioprocesses while maintaining product\u27s bioactivity and quality attributes. This presentation will focus on the latest advancements on downstream purification of cell and gene therapy medicinal products, supported by process innovation and the flexibility of old, but robust technologies such as tangential flow filtration (TFF) and chromatography. Improvement of purification yields of virus based biopharmaceuticals can be achieved through the rational development of alternative strategies, combining different modes of operation, such as flow through purification or multi-column chromatography, together with the recent developments of chromatographic media and fundamental understanding of the adsorption phenomena as reported for the case of gene therapy medicinal products. Critical quality attributes of cell based medicinal products cannot be compromised by the processing route chosen. The use of the already established TFF technology has the potential to improve the purity of cell based products, with the evaluation of critical process parameters of cell concentration and washing being of paramount importance. The purity of such products can also be incremented with the use of negative mode expanded bed adsorption chromatography with a new multimodal prototype matrix based on core–shell bead technology as demonstrated for the case of human mesenchymal stem cells. In summary, the advancement of the purification of complex biopharmaceutical entities, such as the ones here reported, can be described as an incremental process, but still with space for inn

Thyroid Hormones within the Normal Range and Cardiac Function in the General Population: The EPIPorto Study

Background: Hypothyroidism and hyperthyroidism are associated with marked changes in cardiac structure and function. However, the association of thyroid function within the normal range with cardiac structure and function in the general population remains uncertain. Methods: Eight hundred thirty-five subjects aged ≥45 years from the EPIPorto cohort (evaluation between 2006 and 2008) were cross-sectionally analyzed. We excluded participants with TSH, free T4 (FT4), or free T3 (FT3) outside of the reference range or with self-reported cardiovascular or thyroid disease. Cardiac structure and function were evaluated by echocardiography. We used linear regression models unadjusted and adjusted for sex and age (model 1), and sex, age, BMI, diabetes, hypertension, and smoking (model 2). Nonlinear associations were assessed using restricted cubic splines. Results: The mean age was 61.5 years (SD 10.5); 61.1% of the patients were women. In the adjusted model 2, heart rate was positively associated with FT3; diastolic blood pressure was positively associated with TSH; LV end-diastolic and end-systolic volumes were inversely associated with TSH, and ejection fraction was nonlinearly associated with FT3, with higher ejection fractions near the limits of the reference range. Left ventricle (LV) posterior wall thickness was nonlinearly associated with FT4 in the adjusted model 1, with a greater thickness near the limits of the reference range. Regarding diastolic function, no significant associations were observed in adjusted models. Conclusions: Thyroid function within the reference range was associated with heart rate, blood pressure, cardiac structure, and function. Increasing thyroid function (lower TSH, higher FT4, or higher FT3) was associated with a higher heart rate, a lower diastolic blood pressure, and larger LV volumes. LV wall thickness and ejection fraction had a U-shaped association with thyroid hormones.This work was supported by the DOCnet project (NORTE-01-0145-FEDER-000003), supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and the NETDIAMOND project (POCI-01-0145-FEDER-016385), supported by European Structural and Investment Funds, Lisbon’s Regional Operational Program 2020, and national funds from the Portuguese Foundation for Science and Technology – both projects through the Cardiovascular Research Center (UnIC, FCT 51/94) – and by the Portuguese Foundation for Science and Technology (grant POCI/SAU-ESP/61492/2004) and the Unidade de Investigação em Epidemiologia – Instituto de Saúde Pública da Universidade do Porto (EPIUnit) (POCI-01-0145-FEDER-006862, ref. info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UID/DTP/04750/2013/PT)

Serum Levels of Vitamin D and Dental Caries in 7-Year-Old Children in Porto Metropolitan Area

Vitamin D deficiency has been associated with significant changes in dental structures. In children, it can induce enamel and dentin defects, which have been identified as risk factors for caries. This study aimed to assess the association between low serum 25-hydroxyvitamin D (25(OH) D) levels ( 0), and advanced caries (d3–6mft/D3–6MF > 0). Serum 25(OH) D was measured using a competitive electrochemiluminescence immunoassay protein-binding assay. Bivariate analysis and multivariate logistic regression were used. Advanced caries in permanent teeth was significantly associated with children’s vitamin D levels <30 ng/mL, gastrointestinal disorders, higher daily intake of cariogenic food, and having had a dental appointment at ≤7 years old. Optimal childhood levels of vitamin D may be considered an additional preventive measure for dental caries in the permanent dentition.Generation XXI was funded by Programa Operacional de Saúde (Regional Department of Ministry of Health). It has support from the Portuguese Foundation for Science and Technology (FCT) and from the Calouste Gulbenkian Foundation. Ana Cristina Santos holds a FCT Investigator contract info:eu-repo/grantAgreement/FCT/Investigador FCT/IF/01060/2015/CP1319/CT0001/PT. The authors received no specific funding for this work

Association of thyroid function, within the euthyroid range, with cardiovascular risk: The EPIPorto study

Background: Thyroid hormones are important modulators of cardiovascular function. Both hypothyroidism and hyperthyroidism are known to contribute to an increased cardiovascular risk. It remains uncertain whether thyroid hormones level within the euthyroid range are associated with cardiometabolic risk. We aimed to evaluate the association between thyroid function levels within the euthyroid range and cardiovascular risk in a population-based cohort. Methods: Eight hundred thirty-five subjects aged ≥45 years from the EPIPorto population-based cohort were included. We excluded participants with TSH, free T4 (FT4), or free T3 (FT3) outside of the reference range, or with previous cardiovascular or thyroid disease. The associations between thyroid function, cardiovascular risk factors and the 10-year estimated risk of cardiovascular events (using SCORE2 and SCORE2-OP) were evaluated in linear and logistic regression models, crudely and adjusting for age, sex, BMI, diabetes, and smoking. Results: The mean age of the participants was 61.5 (SD 10.5) years and 38.9% were men. Eleven percent of the participants had diabetes, 47.8% had dyslipidemia, and 54.8% had hypertension. The mean body mass index (BMI) was 27.4 (SD 4.6) kg/m2, and the median (percentile25-75) 10-year risk of cardiovascular events was 5.46% (2.92, 10.11). Participants with higher BMI, larger waist circumference and higher hs-CRP had higher levels of FT3 and FT3/FT4 ratio. Lower FT3/FT4 ratio and higher FT4 levels were associated with higher prevalence of diabetes and more adverse lipid profile. Higher TSH, lower FT3 and lower FT3/FT4 ratio were associated with lower eGFR. Lower FT3, lower FT3/FT4 ratio and higher FT4 were associated with an increased 10-year risk of cardiovascular events. Conclusions: In a population-based study, variations of thyroid function within the euthyroid range were associated with cardiovascular risk factors. On one hand, individuals with higher BMI, larger waist circumference and higher hs-CRP had higher levels of FT3 and FT3/FT4 ratio. On the other hand, a decreased conversion of T4 to T3 (lower FT3, lower FT3/FT4 ratio and/or higher FT4) was associated with a higher prevalence of diabetes, a more adverse lipid profile, a lower eGFR and an increased 10-year risk of cardiovascular events. Copyright © 2022 Neves, Fontes-Carvalho, Borges-Canha, Leite, von Hafe, Vale, Martins, Guimarães, Carvalho, Leite-Moreira and Azevedo.This work was supported by the DOCnet project (NORTE-01-0145-FEDER-000003), supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and the NETDIAMOND project (POCI-01-0145-FEDER-016385), supported by European Structural and Investment Funds, Lisbon’s Regional Operational Program 2020, and national funds from the Portuguese Foundation for Science and Technology – both projects through the Cardiovascular Research Center (UnIC, FCT 51/94) – and by the Portuguese Foundation for Science and Technology (grant POCI/SAU-ESP/61492/2004) and the Unidade de Investigação em Epidemiologia – Instituto de Saúde Pública da Universidade do Porto (EPIUnit) (POCI-01-0145-FEDER-006862, ref. UID/DTP/04750/2013). This work is financed by national funds through the FCT - Foundation for Science and Technology, I.P., within the scope of projects UIDB/04750/2020 and LA/P/0064/2020

Capilia™ TB-Neo assay: a new tool for rapid distinction between tuberculous and non-tuberculous mycobacteria

SETTING: The ability to rapidly distinguish between Mycobacterium tuberculosis complex (MTC) and non-tuberculous mycobacteria (NTM) is critical in clinical practice. OBJECTIVE: To evaluate the usefulness of an immunochromatographic (IC) assay to distinguish between MTC and NTM. DESIGN: We analysed a panel of 145 cultures from 128 patients. The routine molecular identification approaches, such as the AccuProbe™ Mycobacterium tuberculosis complex culture identification test and GenoType(®) Mycobacterium assays, were used as reference methods. RESULTS: Of the 101 positive cultures, 98 were correctly identified using the Capilia™ TB-Neo Assay. Of the three discordant isolates, one was identified as M. bovis bacille Calmette-Guérin (BCG) and two as M. tuberculosis. Although we have not performed the sequencing of these strains, some false-negative results have been described due to mutations in the mpb64 gene or with some M. bovis BCG strains. We did not observe false-positive results or any cross-reaction with 22 NTM strains, 12 non-mycobacterial micro-organisms and 10 negative cultures. CONCLUSION: We report good overall performance (sensitivity 97%, specificity 100%, positive predictive value 100% and negative predictive value 96%) of this rapid assay that is easy to perform and interpret and does not require sample preparation, trained technicians or expensive equipment

Reconstruction and flux-balance analysis of the Plasmodium falciparum metabolic network

In the paper we present a metabolic reconstruction and flux-balance analysis (FBA) of Plasmodium falciparum, the primary agent of malaria. The compartmentalized metabolic network of the parasite accounts for 1001 reactions and 616 metabolites. Enzyme–gene associations were established for 366 genes and 75% of all enzymatic reactions.The model was able to reproduce phenotypes of experimental gene knockout and drug inhibition assays with up to 90% accuracy. The model also can be used to efficiently integrate mRNA-expression data to improve the accuracy of metabolic predictions.Using FBA of the reconstructed metabolic network, we identified 40 enzymatic drug targets (i.e. in silico essential genes) with no or very low sequence identity to human proteins.We experimentally tested one of the identified drug targets, nicotinate mononucleotide adenylyltransferase, using a recently discovered small-molecule inhibitor

BCG Infection after bladder cancer treatment – 3 clinical case reports

Immunotherapy with Bacillus Calmette-Guérin (BCG) to treat non-muscle invasive bladder cancer has become an effective and superior alternative to chemotherapy. Intravesical treatment with BCG appears to be relatively safe; however, occasionally BCG infection complicates such treatment. In the present work we describe three patients in whom BCG infection occurred after intravesical BCG therapy. All patients had positive urine culture forMycobacterium tuberculosis complex, using AccuProbe culture identification and then Genotype Mycobacterium MTBC test identified Mycobacterium bovis BCG. The diagnosis is difficult and microbiologic study is usually negative, so high index of suspicion is essential

A fully human anti-IL-7Rα antibody promotes antitumor activity against T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer for which treatment options often result in incomplete therapeutic efficacy and long-term side-effects. Interleukin 7 (IL-7) and its receptor IL-7Rα promote T-ALL development and mutational activation of IL-7Rα associates with very high risk in relapsed disease. Using combinatorial phage-display libraries and antibody reformatting, we generated a fully human IgG1 monoclonal antibody (named B12) against both wild-type and mutant human IL-7Rα, predicted to form a stable complex with IL-7Rα at a different site from IL-7. B12 impairs IL-7/IL-7R-mediated signaling, sensitizes T-ALL cells to treatment with dexamethasone and can induce cell death per se. The antibody also promotes antibody-dependent natural killer-mediated leukemia cytotoxicity in vitro and delays T-cell leukemia development in vivo, reducing tumor burden and promoting mouse survival. B12 is rapidly internalized and traffics to the lysosome, rendering it an attractive vehicle for targeted intracellular delivery of cytotoxic cargo. Consequently, we engineered a B12-MMAE antibody-drug conjugate and provide proof-of-concept evidence that it has increased leukemia cell killing abilities as compared with the naked antibody. Our studies serve as a stepping stone for the development of novel targeted therapies in T-ALL and other diseases where IL-7Rα has a pathological role

The emotional movie database (EMDB): a self-report and psychophysiological study

Film clips are an important tool for evoking emotional responses in the laboratory. When compared with other emotionally potent visual stimuli (e.g., pictures), film clips seem to be more effective in eliciting emotions for longer periods of time at both the subjective and physiological levels. The main objective of the present study was to develop a new database of affective film clips without auditory content, based on a dimensional approach to emotional stimuli (valence, arousal and dominance). The study had three different phases: (1) the pre-selection and editing of 52 film clips (2) the self-report rating of these film clips by a sample of 113 participants and (3) psychophysiological assessment [skin conductance level (SCL) and the heart rate (HR)] on 32 volunteers. Film clips from different categories were selected to elicit emotional states from different quadrants of affective space. The results also showed that sustained exposure to the affective film clips resulted in a pattern of a SCL increase and HR deceleration in high arousal conditions (i.e., horror and erotic conditions). The resulting emotional movie database can reliably be used in research requiring the presentation of non-auditory film clips with different ratings of valence, arousal and dominance.Portuguese Foundation for Science and Technology with individual grants (SFRH/BD/41484/2007 and SFRH/BD/64355/2009

Mechanism of action of probiotics

The modern diet doesn't provide the required amount of beneficial bacteria. Maintenance of a proper microbial ecology in the host is the main criteria to be met for a healthy growth. Probiotics are one such alternative that are supplemented to the host where by and large species of Lactobacillus, Bifidobacterium and Saccharomyces are considered as main probiotics. The field of probiotics has made stupendous strides though there is no major break through in the identification of their mechanism of action. They exert their activity primarily by strengthening the intestinal barrier and immunomodulation. The main objective of the study was to provide a deep insight into the effect of probiotics against the diseases, their applications and proposed mechanism of action