Evaluation of cell culture in microfluidic chips for application in monoclonal antibody production

Microfluidic chips are useful devices for cell culture that allow cell growth under highly controlled conditions, as is required for production of therapeutic recombinant proteins. To understand the optimal conditions for growth of cells amenable of recombinant protein expression in these devices,we culturedHEK-293T cells under different microfluidic experimental conditions. The cells were cultured in polymethyl methacrylate (PMMA) and polydi-methylsiloxane (PDMS)microdevices, in the absence or presence of the cell adhesion agent poly-D-lysine. Different microchannel geometries and thicknesses, as well as the influence of the flow rate have also been tested, showing their great influence in cell adhesion and growth. Results show that the presence of poly-D-lysine improves the adhesion and viability of the cells in continuous or discontinuous flow. Moreover, the optimal adhesion of cells was observed in the corners of themicrochannels, as well as in wide channels possibly due to the decrease in the flow rate in these areas. These studies provide insight into the optimal architecture of microchannels for long-term culture of adherent cells in order to use microfluidics devices as bioreactors for monoclonal antibodies production.Fil: Peñaherrera Pazmiño, Ana Belén. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Payés, Cristian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Sierra Rodero, Marina. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vega, M.. Universidad Tecnológica Nacional; ArgentinaFil: Rosero, G.. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; ArgentinaFil: Helguera, Gustavo Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pérez, M. S.. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; Argentin

Tendency in the growth of creole kids in extensive systems

The present investigation was developed at the State of Puebla, Mexico, to describe the tendency in the growth of Creole kids by means of the evaluation of the average daily weight gain (ADWG). A total of 161 kids were controlled from the birth to the 102 days of age. Every 15 days the kids were weighed, previous fasting with separation of the mother from afternoon of the previous day. The variables included were date and weight at birth, sex, childbirth type and weights every 15 days. The results show that the average weight to the birth was 2.562 ± 0.583 kg, the males surpassing females (p<0.01) in 9.57 percent (0.232 kg). Differences were observed in favour of those of simple childbirth. The final weight was 10.186 ± 2.723 kg for the females and 11.508 ± 3.616 kg for the males. The males from simple childbirth reached a greater weight (12.229 kg). The average daily weight gain (g) for all the animals was of 82.37 ± 26.9 g. The weight at the first month reached 106.2 g; at the second 95.5 g and 53.9 g at third. The smaller gains are observed in the females born from double childbirth with 64.01 ± 17.1 g, whereas the best growth behavior was find in males from simple childbirth with 94.34 ± 30.31 g. The better adjustment of the growth was obtained with potential type equations for the total of kids (Y= 3.23X0.262); second degree polynomial equations were better for the females (Y= 3.35 + 0.07497X -0.00007085 X2); potential equations for males (Y= 4.23X0.21); and also, potential for kids of simple childbirth (Y= 3.91X0.23) and second degree polynomial equations for kids of double childbirth (Y= 3.26 + 0.0947X - 0.00002069 X2).En el Estado de Puebla, México, se desarrolló la presente investigación con el objetivo de describir la tendencia en el crecimiento de cabritos criollos mediante la evaluación de la ganancia media diaria de peso (GMDp). Se dio seguimiento individual a 161 cabritos desde el nacimiento hasta los 102 días de edad, pesándose cada 15 días, previo ayuno con separación de la madre desde la tarde del día anterior. Las variables consideradas fueron fecha y peso de nacimiento, sexo, tipo de parto y pesos quincenales. Los resultados muestran que el peso promedio al nacimiento es de 2,562 ± 0,583 kg, superando los machos a las hembras (p<0,01) en un 9,57 p.100 (0,232 kg). Los de parto simple tuvieron mejor comportamiento. El peso final fue de 10,186 ± 2,723 kg para las hembras y de 11,508 ± 3,616 kg para los machos. La ganancia media diaria de peso (g) para el total de los animales fue de 82,37 ± 26,9 g en promedio. Por períodos, al primer mes alcanzan 106,2 g; en el segundo 95,5 g y 53,9 en el tercero. Las menores ganancias se observan en las hembras nacidas de parto gemelar, con 64,01 ± 17,1 g, mientras que el mejor comportamiento lo tienen los machos simples con 94,34 ± 30,31g. Las ecuaciones de mejor ajuste del crecimiento fueron de tipo potencial para el total de cabritos (Y= 3,23X 0,262); polinomial de segundo grado para las hembras (Y= 3,35 + 0,07497X - 0,00007085X2); potencial para machos (Y= 4,23X 0,21); potencial para cabritos de parto simple (Y =3,91X 0,23) y polinomial de segundo grado para cabritos de parto doble (Y= 3,26 + 0,0947X - 0,00002069X2)

COVID-19 outbreaks in a transmission control scenario: challenges posed by social and leisure activities, and for workers in vulnerable conditions, Spain, early summer 2020

Severe acute respiratory syndrome coronavirus 2 community-wide transmission declined in Spain by early May 2020, being replaced by outbreaks and sporadic cases. From mid-June to 2 August, excluding single household outbreaks, 673 outbreaks were notified nationally, 551 active (>6,200 cases) at the time. More than half of these outbreaks and cases coincided with: (i) social (family/friends’ gatherings or leisure venues) and (ii) occupational (mainly involving workers in vulnerable conditions) settings. Control measures were accordingly applied

KRAS p.G12C mutation occurs in 1% of EGFR-mutated advanced non-small-cell lung cancer patients progressing on a first-line treatment with a tyrosine kinase inhibitor

Background: KRAS is mutated in ∼30% of non-small-cell lung cancer (NSCLC) but it has also been identified as one of the mechanisms underlying resistance to tyrosine kinase inhibitors (TKIs) in EGFR-positive NSCLC patients. Novel KRAS inhibitors targeting KRAS p.G12C mutation have been developed recently with promising results. The proportion of EGFR-positive NSCLC tumours harbouring the KRAS p.G12C mutation upon disease progression is completely unexplored. Materials and methods: Plasma samples from 512 EGFR-positive advanced NSCLC patients progressing on a first first-line treatment with a TKI were collected. The presence of KRAS p.G12C mutation was assessed by digital PCR. Results: Overall, KRAS p.G12C mutation was detected in 1.17% of the samples (n = 6). In two of these cases, we could confirm that the KRAS p.G12C mutation was not present in the pre-treatment plasma samples, supporting its role as an acquired resistance mutation. According to our data, KRASG12C patients showed similar clinicopathological characteristics to those of the rest of the study cohort and no statistically significant associations between any clinical features and the presence of the mutation were found. However, two out of six KRASG12C tumours harboured less common EGFR driver mutations (p.G719X/p.L861Q). All KRASG12C patients tested negative for the presence of p.T790M resistance mutation. Conclusions: The KRAS p.G12C mutation is detected in 1% of EGFR-positive NSCLC patients who progress on a first line with a TKI. All KRASG12C patients were negative for the presence of the p.T790M mutation and they did not show any distinctive clinical featureThis work was supported by CLARIFY project (https://www.clarify2020.eu/). ES-H was supported by the Consejería de Ciencia, Universidades e Innovación of the Comunidad de Madrid (Doctorados Industriales of the Comunidad de Madrid) [grant number IND2019/BMD-17258]. EG-V was supported by AECC (Asociación española Contra el Cáncer) grant ‘Programa de Prácticas de Laboratorio de curso académico AECC 2020’ (no grant number)

Clinical and molecular parameters associated to pneumonitis development in non-small-cell lung cancer patients receiving chemoimmunotherapy from NADIM trial

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER); Bristol-Myers Squibb (BMS); Grupo Español de Cáncer de Pulmón (GECP); European Social Fund (ESF) i Comunidad de Madrid (PEJD-2019-PRE/BMD-17006, PEJ16/MED/AI-1972, PEJD-2018-PRE/SAL-8641).Background Pneumonitis (Pn) is one of the main immune-related adverse effects, having a special importance in lung cancer, since they share affected tissue. Despite its clinical relevance, Pn development remains an unpredictable treatment adverse effect, whose mechanisms are mainly unknown, being even more obscure when it is associated to chemoimmunotherapy. Methods In order to identify parameters associated to treatment related Pn, we analyzed clinical variables and molecular parameters from 46 patients with potentially resectable stage IIIA non-small-cell lung cancer treated with neoadjuvant chemoimmunotherapy included in the NADIM clinical trial (NCT03081689). Pn was defined as clinical or radiographic evidence of lung inflammation without alternative diagnoses, from treatment initiation to 180 days. Results Among 46 patients, 12 developed Pn (26.1%). Sex, age, smoking status, packs-year, histological subtype, clinical or pathological response, progression-free survival, overall survival and number of nivolumab cycles, were not associated to Pn development. Regarding molecular parameters at diagnosis, Pn development was not associated to programmed death ligand 1, TPS, T cell receptor repertoire parameters, or tumor mutational burden. However, patients who developed Pn had statistically significant lower blood median levels of platelet to monocyte ratio (p=0.012) and teratocarcinoma-derived growth factor 1 (p=0.013; area under the curve (AUC) 0.801), but higher median percentages of natural killers (NKs) (p=0.019; AUC 0.786), monocytes (p=0.017; AUC 0.791), MSP (p=0.006; AUC 0.838), PARN (p=0.017; AUC 0.790), and E-Cadherin (p=0.022; AUC 0.788). In addition, the immune scenario of Pn after neoadjuvant treatment involves: high levels of neutrophils and NK cells, but low levels of B and T cells in peripheral blood; increased clonality of intratumoral T cells; and elevated plasma levels of several growth factors (EGF, HGF, VEGF, ANG-1, PDGF, NGF, and NT4) and inflammatory cytokines (MIF, CCL16, neutrophil gelatinase-associated lipocalin, BMP-4, and u-PAR). Conclusions Although statistically underpowered, our results shed light on the possible mechanisms behind Pn development, involving innate and adaptative immunity, and open the possibility to predict patients at high risk. If confirmed, this may allow the personalization of both, the surveillance strategy and the therapeutic approaches to manage Pn in patients receiving chemoimmunotherapy

Pretreatment tissue TCR repertoire evenness is associated with complete pathologic response in patients with NSCLC receiving neoadjuvant chemoimmunotherapy

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER); Comunidad de Madrid. Consejería de Ciencia, Universidades e Innovación (PEJD-2019-PRE/BMD-17006).Purpose: Characterization of the T-cell receptor (TCR) repertoire may be a promising source for predictive biomarkers of pathologic response to immunotherapy in locally advanced non-small cell lung cancer (NSCLC). Experimental Design: In this study, next-generation TCR sequencing was performed in peripheral blood and tissue samples of 40 patients with NSCLC, before and after neoadjuvant chemoimmunotherapy (NADIM clinical trial, NCT03081689), considering their complete pathologic response (CPR) or non-CPR. Beyond TCR metrics, tissue clones were ranked by their frequency and spatiotemporal evolution of top 1% clones was determined. Results: We have found a positive association between an uneven TCR repertoire in tissue samples at diagnosis and CPR at surgery. Moreover, TCR most frequently ranked clones (top 1%) present in diagnostic biopsies occupied greater frequency in the total clonal space of CPR patients, achieving an AUC ROC to identify CPR patients of 0.967 (95% confidence interval, 0.897-1.000; P ¼ 0.001), and improving the results of PD-L1 tumor proportion score (TPS; AUC = 0.767; P = 0.026) or tumor mutational burden (TMB; AUC = 0.550; P = 0.687). Furthermore, tumors with high pretreatment top 1% clonal space showed similar immune cell populations but a higher immune reactive gene expression profile. Finally, the selective expansion of pretreatment tissue top 1% clones in peripheral blood of CPR patients suggests also a peripheral immunosurveillance, which could explain the high survival rate of these patients. Conclusions: We have identified two parameters derived from TCR repertoire analysis that could outperform PD-L1 TPS and TMB as predictive biomarkers of CPR after neoadjuvant chemoimmunotherapy, and unraveled possible mechanisms of CPR involving enhanced tumor immunogenicity and peripheral immunosurveillance