New Approaches for Basophil Activation Tests Employing Dendrimeric Antigen-Silica Nanoparticle Composites.

In vitro cell activation through specific IgE bound to high-affinity receptors on the basophil surface is a widely used strategy for the evaluation of IgE-mediated immediate hypersensitivity reactions to betalactams. Cellular activation requires drug conjugation to a protein to form a large enough structure displaying a certain distance between haptens to allow the cross-linking of two IgE antibodies bound to the basophil's surface, triggering their degranulation. However, no information about the size and composition of these conjugates is available.

Involvement of autologous myeloid dendritic cells in the evaluation of immediate hypersensitivity reactions to betalactams.

Background: Amoxicillin (AX) and clavulanic acid (CLV) are the betalactam antibiotics (BLs) most used to treat bacterial infections, although they can trigger immediate hypersensitivity reactions (IDHRs). The maturation analysis of monocyte-derived dendritic cells (moDCs) and their capacity to induce proliferative response of lymphocytes are useful to test the sensitisation to a drug, although without optimal sensitivity. Nevertheless, this can be improved using directly isolated DCs such as myeloid DCs (mDCs).

Epigenetic targets to enhance antitumor immune response through the induction of tertiary lymphoid structures.

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates found in sites of chronic inflammation such as tumors and autoimmune diseases. The discovery that TLS formation at tumor sites correlated with good patient prognosis has triggered extensive research into various techniques to induce their formation at the tumor microenvironment (TME). One strategy is the exogenous induction of specific cytokines and chemokine expression in murine models.

Diagnosis of immediate reactions to amoxicillin: Comparison of basophil activation markers CD63 and CD203c in a prospective study.

Background: Amoxicillin (AX) combined or not with clavulanic acid (CLV) is frequently involved in IgE-mediated reactions. Drug provocation test (DPT) is considered as the gold standard for diagnosis, although contraindicated in high-risk patients. Basophil activation test (BAT) can help diagnose immediate reactions to beta-lactams, although controversy exists regarding the best activation marker.

Technical challenges for complete implementation of automated growth-based methods for microbiological examination of advanced therapy medicinal products. What's wrong with Candida albicans?

Background: Automated growth-based methods for sterility testing of cell-therapy products should be qualified to demonstrate that they are equivalent to, or better than, the conventional reference method. The aim of the present study was to assess the ability of the BACTEC FX40 system to detect low microbial contamination and to confirm the suitability of the method in the presence of seven different human mesenchymal cell-based products, according to Ph. Eur.

The Evolution of Clinically Aggressive Triple-Negative Breast Cancer Shows a Large Mutational Diversity and Early Metastasis to Lymph Nodes.

In triple-negative breast cancer (TNBC), only 30% of patients treated with neoadjuvant chemotherapy achieve a pathological complete response after treatment and more than 90% die due to metastasis formation. The diverse clinical responses and metastatic developments are attributed to extensive intrapatient genetic heterogeneity and tumor evolution acting on this neoplasm. In this work, we aimed to evaluate genomic alterations and tumor evolution in TNBC patients with aggressive disease.

Comprehensive Genomic Profile of Heterogeneous Long Follow-Up Triple-Negative Breast Cancer and Its Clinical Characteristics Shows DNA Repair Deficiency Has Better Prognostic.

Triple-negative breast cancer (TNBC) presents a marked diversity at the molecular level, which promotes a clinical heterogeneity that further complicates treatment. We performed a detailed whole exome sequencing profile of 29 Mexican patients with long follow-up TNBC to identify genomic alterations associated with overall survival (OS), disease-free survival (DFS), and pathologic complete response (PCR), with the aim to define their role as molecular predictive factors of treatment response and prognosis. We detected 31 driver genes with pathogenic mutations in (53%), (27%), (9%), (9%), and (9%), and 16 operative mutational signatures.

AR-V7 as a Biomarker for Resistance to Treatment with Abiraterone/Enzalutamide in Three Latin American Countries: A Hypothetical Cost-Saving Analysis.

Background: Prostate cancer is the most incident and one of the deadliest male cancers in Latin America. Treatment for patients with metastatic castration-resistant prostate cancer (mCRPC) includes androgen receptor signaling inhibitors such as abiraterone and enzalutamide, for which androgen receptor splice variant 7 (AR-V7) has emerged as a biomarker for primary resistance. Our study sought to analyze the potential economic impact of the use of AR-V7 detection as a treatment indicator in patients with mCRPC in three Latin American countries.

Latin American Study of Hereditary Breast and Ovarian Cancer : A Genomic Epidemiology Approach.

Hereditary Breast and Ovarian Cancer (HBOC) syndrome is responsible for ~5-10% of all diagnosed breast and ovarian cancers. Breast cancer is the most common malignancy and the leading cause of cancer-related mortality among women in Latin America (LA). The main objective of this study was to develop a comprehensive understanding of the genomic epidemiology of HBOC throughout the establishment of The Latin American consortium for HBOC-LACAM, consisting of specialists from 5 countries in LA and the description of the genomic results from the first phase of the study.

Comprehensive Analysis of Germline Variants in Mexican Patients with Hereditary Breast and Ovarian Cancer Susceptibility.

Hereditary breast and ovarian cancer syndrome (HBOC) represents 5⁻10% of all patients with breast cancer and is associated with high-risk pathogenic alleles in genes, but only for 25% of cases. We aimed to find new pathogenic alleles in a panel of 143 cancer-predisposing genes in 300 Mexican cancer patients with suspicion of HBOC and 27 high-risk patients with a severe family history of cancer, using massive parallel sequencing. We found pathogenic variants in 23 genes, including .

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent.

Hematopoietic stem cell (HSC)-based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein-displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34 (hCD34) progenitor cells upon a single application.

Cancer Genomic Resources and Present Needs in the Latin American Region.

In Latin America (LA), cancer is the second leading cause of death, and little is known about the capacities and needs for the development of research in the field of cancer genomics. In order to evaluate the current capacity for and development of cancer genomics in LA, we collected the available information on genomics, including the number of next-generation sequencing (NGS) platforms, the number of cancer research institutions and research groups, publications in the last 10 years, educational programs, and related national cancer control policies. Currently, there are 221 NGS platforms and 118 research groups in LA developing cancer genomics projects.

Expression of the epidermodysplasia verruciformis-associated genes EVER1 and EVER2 is activated by exogenous DNA and inhibited by LMP1 oncoprotein from Epstein-Barr virus.

EVER1 and EVER2 are mutated in epidermodysplasia verruciformis patients, who are susceptible to human betapapillomavirus (HPV) infection. It is unknown whether their products control the infection of other viruses. Here, we show that the expression of both genes in B cells is activated immediately after Epstein-Barr virus (EBV) infection, whereas at later stages, it is strongly repressed via activation of the NF-κB signaling pathway by latent membrane protein 1 (LMP1).

Baboon envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early-cytokine-stimulated and resting HSCs.

Hematopoietic stem cell (HSC)-based gene therapy holds promise for the cure of many diseases. The field is now moving toward the use of lentiviral vectors (LVs) as evidenced by 4 successful clinical trials. These trials used vesicular-stomatitis-virus-G protein (VSV-G)-LVs at high doses combined with strong cytokine-cocktail stimulation to obtain therapeutically relevant transduction levels; however, they might compromise the HSC character.

Epstein-Barr virus down-regulates tumor suppressor DOK1 expression.

The DOK1 tumor suppressor gene encodes an adapter protein that acts as a negative regulator of several signaling pathways. We have previously reported that DOK1 expression is up-regulated upon cellular stress, via the transcription factor E2F1, and down-regulated in a variety of human malignancies due to aberrant hypermethylation of its promoter. Here we show that Epstein Barr virus (EBV) infection of primary human B-cells leads to the down-regulation of DOK1 gene expression via the viral oncoprotein LMP1.

Lentiviral vectors displaying modified measles virus gp overcome pre-existing immunity in in vivo-like transduction of human T and B cells.

Gene transfer into quiescent T and B cells is important for gene therapy and immunotherapy approaches. Previously, we generated lentiviral vectors (LVs) pseudotyped with Edmonston (Ed) measles virus (MV) hemagglutinin (H) and fusion (F) glycoproteins (H/F-LVs), which allowed efficient transduction of quiescent human T and B cells. However, a major obstacle in the use of H/F-LVs in vivo is that most of the human population is vaccinated against measles.

A novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice.

In vivo lentiviral vector (LV)-mediated gene delivery would represent a great step forward in the field of gene therapy. Therefore, we have engineered a novel LV displaying SCF and a mutant cat endogenous retroviral glycoprotein, RDTR. These RDTR/SCF-LVs outperformed RDTR-LVs for transduction of human CD34(+) cells (hCD34(+)).

In vivo gene delivery into hCD34+ cells in a humanized mouse model.

In vivo targeted gene delivery to hematopoietic stem cells (HSCs) would mean a big step forward in the field of gene therapy. This would imply that the risk of cell differentiation and loss of homing/-engraftment is reduced, as there is no need for purification of the target cell. In vivo gene delivery also bypasses the issue that no precise markers that permit the isolation of a primitive hHSC exist up to now.

Measles virus glycoprotein-pseudotyped lentiviral vector-mediated gene transfer into quiescent lymphocytes requires binding to both SLAM and CD46 entry receptors.

Gene transfer into quiescent T and B cells is of importance for gene therapy and immunotherapy approaches to correct hematopoietic disorders. Previously, we generated lentiviral vectors (LVs) pseudotyped with the Edmonston measles virus (MV) hemagglutinin and fusion glycoproteins (Hgps and Fgps) (H/F-LVs), which, for the first time, allowed efficient transduction of quiescent human B and T cells. These target cells express both MV entry receptors used by the vaccinal Edmonston strain, CD46 and signaling lymphocyte activation molecule (SLAM).

Advances in the field of lentivector-based transduction of T and B lymphocytes for gene therapy.

Efficient gene transfer into quiescent T and B lymphocytes for gene therapy or immunotherapy purposes may allow the treatment of several genetic dysfunctions of the hematopoietic system, such as immunodeficiencies, and the development of novel therapeutic strategies for cancers and acquired diseases. Lentiviral vectors (LVs) can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T and B cells. In T cells, completion of reverse transcription (RT), nuclear import, and subsequent integration of the vesicular stomatitis virus G protein pseudotyped LV (VSVG-LV) genome does not occur efficiently unless they are activated via the T-cell receptor (TCR) or by survival-cytokines inducing them to enter into the G(1b) phase of the cell cycle.

Efficient and stable transduction of resting B lymphocytes and primary chronic lymphocyte leukemia cells using measles virus gp displaying lentiviral vectors.

Up to now, no lentiviral vector (LV) tool existed to govern efficient and stable gene delivery into quiescent B lymphocytes, which hampers its application in gene therapy and immunotherapy areas. Here, we report that LVs incorporating measles virus (MV) glycoproteins, H and F, on their surface allowed transduction of 50% of quiescent B cells, which are not permissive to VSVG-LV transduction. This high transduction level correlated with B-cell SLAM expression and was not at cost of cell-cycle entry or B-cell activation.

Was cDNA sequences modulate transgene expression of was promoter-driven lentiviral vectors.

Abstract The development of vectors that express a therapeutic transgene efficiently and specifically in hematopoietic cells (HCs) is an important goal for gene therapy of hematological disorders. We have previously shown that a 500-bp fragment from the proximal Was gene promoter in a lentiviral vector (LV) was sufficient to achieve more than 100-fold higher levels of Wiskott-Aldrich syndrome protein in HCs than in nonhematopoietic cells (non-HCs). We show now that this differential was reduced up to 10 times when the enhanced green fluorescent protein gene (eGFP) was expressed instead of Was in the same LV backbone.

Strategies for targeting lentiviral vectors.

Vectors derived from retroviruses such as lentiviruses and onco-retroviruses are probably among the most suitable tools to achieve a long-term gene transfer since they allow stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors should be preferred gene delivery vehicles over vectors derived from onco-retroviruses (MLV) since in contrast to the latter they can transduce non-proliferating target cells. Moreover, lentiviral vectors that have the capacity to deliver transgenes into specific tissues are expected to be of great value for various gene transfer approaches in vivo.