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Spray drying is increasingly being applied to process biopharmaceuticals, particularly monoclonal antibodies (mAbs). However, due to their protein nature, mAbs are susceptible to ...degradation when subjected to various stresses during a drying process. Despite extensive research in this domain, identifying the appropriate formulation composition and spray drying conditions remains a complex challenge, requiring further studies to enhance the understanding on how process and formulation parameters impact mAb stability in reconstituted solutions. This research aims to explore spray drying as technique for producing pharmaceutical mAbs-based powders intended for reconstitution and subcutaneous injection. In the initial phase of this study, using a model mAb (mAb-A), the influence of dissociated and coupled process stresses on protein stability after solution reconstitution was investigated. The findings revealed a detrimental interplay of mechanical, interfacial, and thermal/dehydration stresses on mAb-A stability, notably characterized by an increase in protein aggregation. Subsequently, in a second phase, the study delved into the impact of spray drying processing conditions, the level of excipients, and protein concentration on mAb-A aggregation in reconstituted solutions. The obtained results highlighted the critical role of formulation composition as a parameter deserving further study, specifically concerning the selection of type and concentration of stabilizers to be added in the liquid mAb-A solution to be dried.
Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, ...chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior.
assays are excellent approaches to extrapolate to
situations and study live cells behavior. Here we present four
protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on
wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and
spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.
The analysis of tumor interstitial fluid (TIF) composition is a valuable procedure to identify antimetastatic targets, and different laboratories have set up techniques for TIF isolation and ...proteomic analyses. However, those methods had never been compared in samples from the same tumor and patient. In this work, we compared the two most used methods, elution and centrifugation, in pieces of the same biopsy samples of cutaneous squamous cell carcinoma (cSCC). First, we established that high G-force (10 000g) was required to obtain TIF from cSCC by centrifugation. Second, we compared the centrifugation method with the elution method in pieces of three different cSCC tumors. We found that the mean protein intensities based in the number of peptide spectrum matches was significantly higher in the centrifuged samples than in the eluted samples. Regarding the robustness of the methods, we observed higher overlapping between both methods (77–80%) than among samples (50%). These results suggest that there exists an elevated consistence of TIF composition independently of the method used. However, we observed a 3-fold increase of extracellular proteins in nonoverlapped proteome obtained by centrifugation. We therefore conclude that centrifugation is the method of choice to study the proteome of TIF from cutaneous biopsies.
Germinal center follicular T helper (GCTfh) cells are essential players in the differentiation of B cells. Circulating follicular T helper (cTfh) cells share phenotypic and functional properties with ...GCTfh cells. Distinct subpopulations of cTfh with different helper capabilities toward B cells can be identified: cTfh1 (CXCR3
CCR6
), cTfh2 (CXCR3
CCR6
), and cTfh17 (CXCR3
CCR6
). Alterations in cTfh function and/or distribution have been associated with autoimmunity, infectious diseases, and more recently, with several monogenic immunodeficiencies. Common variable immunodeficiency (CVID) disease is the commonest symptomatic primary immunodeficiency with a genetic cause identified in only 2-10% of patients. Although a heterogeneous disease, most patients show a characteristic defective B cell differentiation into memory B cells or antibody-secreting cells. We investigated if alterations in CVID cTfh cells frequency or distribution into cTfh1, cTfh2, and cTfh17 subpopulations and regulatory follicular T (Tfr) cells could be related to defects in CVID B cells. We found increased percentages of cTfh exhibiting higher programmed death-1 expression and altered subpopulations distribution in smB
CVID patients. In contrast to smB
patients and controls, cTfh from smB
CVID patients show increased cTfh1 and decreased cTfh17 subpopulation percentages and increased CXCR3
CCR6
cTfh, a population analogous to the recently described pathogenic Th17.1. Moreover, Tfr cells are remarkably decreased only in smB
CVID patients. In conclusion, increased cTfh17.1 and cTfh1/cTfh17 ratio in CVID patients could influence B cell fate in smB
CVID patients, with a more compromised B cell compartment, and the decrease in Tfr cells may lead to high risk of autoimmune conditions in CVID patients.
T-type calcium channels (TTCCs) are overexpressed in several cancers. In this review, we summarize the recent advances and new insights into TTCC biology, tumor progression, and prognosis biomarker ...and therapeutic potential in the melanoma field. We describe a novel correlation between the Cav3.1 isoform and the increased basal autophagy in BRAF
-mutant melanomas and after acquired resistance to BRAF inhibitors. Indeed, TTCC blockers reduce melanoma cell viability and migration/invasion in vitro and tumor growth in mice xenografts in both BRAF-inhibitor-sensitive and -resistant scenarios. These studies open a new, promising therapeutic approach for disseminated melanoma and improved treatment in BRAFi relapsed melanomas, but further validation and clinical trials are needed for it to become a real therapeutic option.
Fruit, vegetables, cereals, and olive oil are common elements of the Mediterranean diet (MD), but each country in the Mediterranean basin has its own gastronomic customs influenced by socio-cultural, ...religious, and economic factors. This study compared the dietary habits of three Mediterranean populations with different cultures and lifestyles, a total of 600 adults (61.9% females) between 25 and 70 yrs from Spain, Morocco, and Palestine. All participants completed a self administered questionnaire, including sociodemographic and anthropometric items, a validated semi-quantitative food frequency questionnaire adapted to the foods consumed in each country, and three 24-h recalls. MD adherence was estimated with the MD Serving Score (MDSS). All populations showed a moderate adherence to the Mediterranean dietary pattern. In comparison to the Palestine population, MDSS-assessed adherence to the MD was 6.36-fold higher in the Spanish population and 3.88-fold higher in the Moroccan population. Besides the country of origin, age was another predictive factor of MD adherence, which was greater (higher MDSS) in participants aged over 50 yrs than in those aged 30 yrs or younger. This preliminary study contributes initial data on dietary differences between European and non-European countries in the Mediterranean basin. The Spanish diet was shown to be closer to MD recommendations than the diet of Morocco or Palestine. Given the impact of good dietary habits on the prevention of chronic non-transmittable diseases, health policies should focus on adherence to a healthy diet, supporting traditional dietary patterns in an era of intense commercial pressures for change.
Melanoma is a malignant neoplasia that is highly resistant to chemotherapy and radiotherapy and is associated with poor prognosis in advanced stage. Targeting melanoma that harbors the common ...BRAFV600E mutation with kinase inhibitors, such as vemurafenib, reduces tumor burden, but these tumors frequently acquire resistance to these drugs. We previously proposed that T-type calcium channel (TTCC) expression may serve as a biomarker for melanoma progression and prognosis, and we showed that TTCC blockers reduce migration and invasion rates because of autophagy blockade only in BRAFV600E-mutant melanoma cells. Here, we demonstrated that high expression of the TTCC Cav3.1 isoform is related to autophagic status in vemurafenib-resistant BRAFV600E-mutant melanoma cells and human biopsies, and in silico analysis revealed an enrichment of Cav3.1 expression in post-treatment melanomas. We also demonstrated that the TTCC blocker mibefradil induces apoptosis and impairs migration and invasion via inhibition of autophagy in resistant melanoma cells and mouse xenograft models. Moreover, we identified an association between PTEN status and Cav3.1 expression in these cells as a marker of sensitivity to combination therapy in resistant cells. Together, our results suggest that TTCC blockers offer a potential targeted therapy in resistant BRAFV600E-mutant melanoma and a therapeutic strategy to reduce progression toward BRAF inhibitor resistance.
Background: Cutaneous melanoma shows high variability regarding clinicopathological presentation, evolution and prognosis. Methods: Next generation sequencing was performed to analyze hotspot ...mutations in different areas of primary melanomas (MMp) and their paired metastases. Clinicopathological features were evaluated depending on the degree of variation of the BRAFV600E mutant allele frequency (MAF) in MMp. Results: In our cohort of 14 superficial spreading, 10 nodular melanomas and 52 metastases, 17/24 (71%) melanomas had a BRAFV600E mutation and 5/24 (21%) had a NRASQ61 mutation. We observed a high variation of BRAFV600E MAF (H-BRAFV600E) in 7/17 (41%) MMp. The H-BRAFV600E MMp were all located on the trunk, had lower Breslow and mitotic indexes and predominantly, a first nodal metastasis. Regions with spindled tumor cells (Spin) and high lymphocytic infiltrate (HInf) were more frequent in the H-BRAFV600E patients (4/7; 57%), whereas regions with epithelial tumor cells (Epit) and low lymphocytic infiltrate (LInf) were predominant (6/10; 60%) and exclusive in the low BRAFV600E MAF variation tumors (L-BRAFV600E). The H-BRAFV600E/Spin/HInf MMp patients had better prognostic features and nodal first metastasis. Conclusions: The H-BRAFV600E MMp were located on the trunk, had better prognostic characteristics, such as lower Breslow and mitotic indexes as well as high lymphocytic infiltrate.
Summary
Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that ...T‐type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3‐II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E).
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