Current approaches to develop "off-the-shelf" chimeric antigen receptor (CAR)-T cells for cancer treatment: a systematic review.

Chimeric antigen receptor (CAR)-T cell therapy is one of the most promising advances in cancer treatment. It is based on genetically modified T cells to express a CAR, which enables the recognition of the specific tumour antigen of interest. To date, CAR-T cell therapies approved for commercialisation are designed to treat haematological malignancies, showing impressive clinical efficacy in patients with relapsed or refractory advanced-stage tumours. However, since they all use the patient´s own T cells as starting material (i.e. autologous use), they have important limitations, including manufacturing delays, high production costs, difficulties in standardising the preparation process, and production failures due to patient T cell dysfunction. Therefore, many efforts are currently being devoted to contribute to the development of safe and effective therapies for allogeneic use, which should be designed to overcome the most important risks they entail: immune rejection and graft-versus-host disease (GvHD). This systematic review brings together the wide range of different approaches that have been studied to achieve the production of allogeneic CAR-T cell therapies and discuss the advantages and disadvantages of every strategy. The methods were classified in two major categories: those involving extra genetic modifications, in addition to CAR integration, and those relying on the selection of alternative cell sources/subpopulations for allogeneic CAR-T cell production (i.e. γδ T cells, induced pluripotent stem cells (iPSCs), umbilical cord blood T cells, memory T cells subpopulations, virus-specific T cells and cytokine-induced killer cells). We have observed that, although genetic modification of T cells is the most widely used approach, new approaches combining both methods have emerged. However, more preclinical and clinical research is needed to determine the most appropriate strategy to bring this promising antitumour therapy to the clinical setting.

MRGPRX2 signaling involves the Lysyl-tRNA synthetase and MITF pathway.

MRGPRX2, a G-protein-coupled-seven transmembrane domain receptor, is mainly expressed in mast cells and neurons and is involved in skin immunity and pain. It is implicated in the pathophysiology of non-IgE-mediated immediate hypersensitivity and has been related to adverse drug reactions. Moreover, a role has been proposed in asthma, atopic dermatitis, contact dermatitis, and chronic spontaneous urticaria. Although it has a prominent role in disease, its signaling transduction is poorly understood. This study shows that MRGPRX2 activation with substance P increased Lysyl t-RNA synthetase (LysRS) translocation to the nucleus. LysRS is a moonlighting protein with a dual role in protein translation and IgE signaling in mast cells. Upon allergen- IgE-FcεRI crosslinking, LysRS is translocated to the nucleus and activates microphthalmia-associated transcription factor (MITF) activity. In this study, we found that MRGPRX2 triggering led to MITF phosphorylation and increased MITF activity. Therefore, overexpression of LysRS increased MITF activity after MRGPRX2 activation. MITF silencing reduced MRGPRX2-dependent calcium influx and mast cell degranulation. Furthermore, a MITF pathway inhibitor, ML329, impaired MITF expression, calcium influx, and mast cell degranulation. Moreover, drugs such as atracurium, vancomycin, and morphine, reported to induce MRGPRX2-dependent degranulation, increased MITF activity. Altogether, our data show that MRGPRX2 signaling enhances MITF activity, and its abrogation by silencing or inhibition resulted in defective MRGPRX2 degranulation. We conclude that MRGPRX2 signaling involves the LysRS and MITF pathway. Thus, MITF and MITF-dependent targets may be considered therapeutic approaches to treat pathologies where MRGPRX2 is implicated.

MITF Downregulation Induces Death in Human Mast Cell Leukemia Cells and Impairs IgE-Dependent Degranulation.

Activating mutations in KIT (CD117) have been associated with several diseases, including gastrointestinal stromal tumors and mastocytosis. Rapidly progressing pathologies or drug resistance highlight the need for alternative treatment strategies. Previously, we reported that the adaptor molecule SH3 binding protein 2 (SH3BP2 or 3BP2) regulates KIT expression at the transcriptional level and microphthalmia-associated transcription factor (MITF) expression at the post-transcriptional level in human mast cells and gastrointestinal stromal tumor (GIST) cell lines. Lately, we have found that the SH3BP2 pathway regulates MITF through miR-1246 and miR-5100 in GIST. In this study, miR-1246 and miR-5100 were validated by qPCR in the SH3BP2-silenced human mast cell leukemia cell line (HMC-1). MiRNA overexpression reduces MITF and MITF-dependent target expression in HMC-1. The same pattern was observed after MITF silencing. In addition, MITF inhibitor ML329 treatment reduces MITF expression and affects the viability and cell cycle progression in HMC-1. We also examine whether MITF downregulation affected IgE-dependent mast cell degranulation. MiRNA overexpression, MITF silencing, and ML329 treatment reduced IgE-dependent degranulation in LAD2- and CD34+-derived mast cells. These findings suggest MITF may be a potential therapeutic target for allergic reactions and deregulated KIT mast-cell-mediated disorders.

Cell Therapy for Colorectal Cancer: The Promise of Chimeric Antigen Receptor (CAR)-T Cells.

Colorectal cancer (CRC) is a global public health problem as it is the third most prevalent and the second most lethal cancer worldwide. Major efforts are underway to understand its molecular pathways as well as to define the tumour-associated antigens (TAAs) and tumour-specific antigens (TSAs) or neoantigens, in order to develop an effective treatment. Cell therapies are currently gaining importance, and more specifically chimeric antigen receptor (CAR)-T cell therapy, in which genetically modified T cells are redirected against the tumour antigen of interest. This immunotherapy has emerged as one of the most promising advances in cancer treatment, having successfully demonstrated its efficacy in haematological malignancies. However, in solid tumours, such as colon cancer, it is proving difficult to achieve the same results due to the shortage of TSAs, on-target off-tumour effects, low CAR-T cell infiltration and the immunosuppressive microenvironment. To address these challenges in CRC, new approaches are proposed, including combined therapies, the regional administration of CAR-T cells and more complex CAR structures, among others. This review comprehensively summarises the current landscape of CAR-T cell therapy in CRC from the potential tumour targets to the preclinical studies and clinical trials, as well as the limitations and future perspectives of this novel antitumour strategy.

Characterization of a complex phenotype (fever-dependent recurrent acute liver failure and osteogenesis imperfecta) due to NBAS and P4HB variants.

We report the clinical, biochemical and genetic findings from a Spanish boy of Caucasian origin who presented with fever-dependent RALF (recurrent acute liver failure) and osteogenesis imperfecta (OI). Whole-exome sequencing (WES) uncovered two compound heterozygous variants in NBAS (c.[1265 T > C];[1549C > T]:p.[(Leu422Pro)];[(Arg517Cys)]), and a heterozygous variant in P4HB (c.[194A > G];[194=]:p.[(Lys65Arg)];[(Lys65=)]) that was transmitted from the clinically unaffected mother who was mosaic carrier of the variant. Variants in NBAS protein have been associated with ILFS2 (infantile liver failure syndrome-2), SOPH syndrome (short stature, optic nerve atrophy, and Pelger-Huët anomaly syndrome), and multisystem diseases. Several patients showed clinical manifestations affecting the skeletal system, such as osteoporosis, pathologic fractures and OI. Experiments in the patient's fibroblasts demonstrated that mutated NBAS protein is overexpressed and thermally unstable, and reduces the expression of MGP, a regulator of bone homeostasis. Variant in PDI (protein encoded by P4HB) has been associated with CLCRP1 (Cole-Carpenter syndrome-1), a type of severe OI. An increase of COL1A2 protein retention was observed in the patient's fibroblasts. In order to study if the variant in P4HB was involved in the alteration in collagen trafficking, overexpression experiments of PDI were carried out. These experiments showed that overexpression of mutated PDI protein produces an increase in COL1A2 retention. In conclusion, these results corroborate that the variants in NBAS are responsible for the liver phenotype, and demonstrate that the variant in P4HB is involved in the bone phenotype, probably in synergy with NBAS variants.

Utilidad de la ecografía pulmonar precoz en bronquiolitis aguda leve-moderada: estudio piloto / Usefulness of early lung ultrasound in acute mild-moderate acute bronchiolitis. A pilot study

OBJETIVO: Evaluar la correlación entre hallazgos de la ecografía pulmonar realizada precozmente con las escalas de gravedad clínica y su asociación con la evolución posterior en la bronquiolitis aguda (BA) leve-moderada. PACIENTES Y MÉTODOS: Estudio observacional prospectivo. Se incluyó a lactantes con BA leve-moderada evaluados mediante ecografía pulmonar en las primeras 24 h tras la atención hospitalaria. Se graduó la afectación pulmonar (rango 0-50 puntos) sobre la base de un score ecográfico (ScECO). Se evaluó la correlación entre el ScECO y 2 escalas clínicas de uso habitual: escala de Wood Downes Ferres modificada (WDFM) y escala del Hospital Sant Joan de Déu (HSJD). Así mismo se valoró la asociación entre el ScECO y la evolución clínica posterior (ingreso en la Unidad de Cuidados Intensivos Pediátricos [UCIP], días de hospitalización y días de oxigenoterapia). RESULTADOS: Se incluyó a 59 pacientes con una edad mediana de 90 días (RIQ: 30-270 días). La puntuación mediana del ScECO fue de 6 puntos (2-8) en los pacientes que no requirieron ingreso, 9 (5-13,7) en los ingresados en planta y 17 (14,5-18) en los pacientes que precisaron traslado de planta a la UCIP (p = 0,001). El ScECO tuvo una correlación lineal moderada con la escala de WDFM (rho = 0,504, p < 0,001) y HSJD (rho = 0,518; p < 0,001). El ScECO se asoció al ingreso en UCIP (OR 2,5 [IC del 95%: 1,1-5,9]; p = 0,035), mayor estancia hospitalaria (1,2 días (IC del 95%: 0,55, 1,86); p = 0,001) y duración de oxigenoterapia (0,87 días (IC del 95%: 0,26, 1,48); p = 0,006). CONCLUSIONES: La ecografía pulmonar precoz se correlaciona de forma moderada con la gravedad de la BA evaluada por escalas clínicas y guarda cierta relación con la evolución clínica

OBJECTIVE: To determine the correlation between the findings seen in early lung ultrasound with the clinical severity scales, and its association with the subsequent progression of the mild-moderate acute bronchiolitis (AB). PATIENTS AND METHODS: An observational prospective study conducted on infants with mild-moderate BA, using lung ultrasound in the first 24 hours of hospital care. The lung involvement was graded (range 0-50 points) based on an ultrasound score (ScECO) and 2 routinely used clinical scales: the modified Wood Downes Ferres (WDFM), and the Hospital Sant Joan de Deu (HSJD). The relationship between the ScECO and the subsequent clinical progression (admission to the Paediatric Intensive Care Unit (PICU), days in hospital, and days of oxygen therapy), was also determined. RESULTS: The study included a total of 59 patients, with a median age of 90 days (IQR: 30-270 days). The median ScECO score was 6 points (2-8) in the patients that did not require hospital admission, with 9 points (5-13.7) admitted to the ward, and 17 (14.5-18) in the patients who needed to be transferred from the ward to the PICU (P = .001). The ScECO had a moderate lineal association with the WDFM scale (rho = 0.504, P<.001) and the HSJD (rho = 0.518; P<.001). The ScECO was associated with admission to PICU (OR 2.5 (95% CI: 1.1-5.9); P=.035), longer hospital stay (1.2 days 95% CI: 0.55-1.86); P=.001] and duration of oxygen therapy [0.87 days (95% CI: 0.26-1.48); P=.006). CONCLUSIONS: There is a moderate correlation between early lung ultrasound findings with the severity of the AB evaluated by the clinical scales, as well as some relationship with the clinical progression

[Usefulness of early lung ultrasound in acute mild-moderate acute bronchiolitis. A pilot study]. / Utilidad de la ecografía pulmonar precoz en bronquiolitis aguda leve-moderada: estudio piloto.

OBJECTIVE: To determine the correlation between the findings seen in early lung ultrasound with the clinical severity scales, and its association with the subsequent progression of the mild-moderate acute bronchiolitis (AB). PATIENTS AND METHODS: An observational prospective study conducted on infants with mild-moderate BA, using lung ultrasound in the first 24hours of hospital care. The lung involvement was graded (range 0-50 points) based on an ultrasound score (ScECO) and 2routinely used clinical scales: the modified Wood Downes Ferres (WDFM), and the Hospital Sant Joan de Deu (HSJD). The relationship between the ScECO and the subsequent clinical progression (admission to the Paediatric Intensive Care Unit (PICU), days in hospital, and days of oxygen therapy), was also determined. RESULTS: The study included a total of 59 patients, with a median age of 90 days (IQR: 30-270 days). The median ScECO score was 6 points (2-8) in the patients that did not require hospital admission, with 9 points (5-13.7) admitted to the ward, and 17 (14.5-18) in the patients who needed to be transferred from the ward to the PICU (P=.001). The ScECO had a moderate lineal association with the WDFM scale (rho=0.504, P<.001) and the HSJD (rho=0.518; P<.001). The ScECO was associated with admission to PICU [OR 2.5 (95% CI: 1.1-5.9); P=.035], longer hospital stay [1.2 days 95% CI: 0.55-1.86); P=.001] and duration of oxygen therapy [0.87 days (95% CI: 0.26-1.48); P=.006]. CONCLUSIONS: There is a moderate correlation between early lung ultrasound findings with the severity of the AB evaluated by the clinical scales, as well as some relationship with the clinical progression.

URAT1 and GLUT9 mutations in Spanish patients with renal hypouricemia.

BACKGROUND: Renal hypouricemia (RHUC), a rare inherited disorder characterized by impaired uric acid (UA) reabsorption in the proximal tubule, is caused by mutations in SLC22A12 or SLC2A9. Most mutations have been identified in Japanese patients, and only a few have been detected in Europeans. METHODS: We report clinical, biochemical and genetics findings of fourteen Spanish patients, six Caucasians and eight of Roma ethnia, diagnosed with idiopathic RHUC. Two of the patients presented exercise-induced acute renal failure and another one had several episodes of nephrolithiasis and four of them had progressive deterioration of renal function, while the rest were asymptomatic. RESULTS: Molecular analysis revealed SLC22A12 mutations in ten of the patients, and SLC2A9 mutations in the other four. A new heterozygous SLC22A12 missense mutation, c.1427C>A (p.A476D), was identified in two affected members of the same family. The rest of the patients presented homozygous, heterozygous or compound heterozygous mutations that have been previously identified in patients with RHUC; SLC22A12 p.T467M and p.L415_G417del, and SLC2A9 p.T125M. Expression studies in Xenopus oocytes revealed that c.1427C>A reduced UA transport but did not alter the location of URAT1 protein on the plasma membrane. CONCLUSIONS: The biochemical and clinical features of our patients together with the genetic analysis results confirmed the diagnosis of RHUC. This is the first report describing SLC22A12 and SLC2A9 mutations in Spanish patients.

Enuresis secundaria como presentación de feocromocitoma en una niña de 13 años / Pheochromocytoma presenting with secondary enuresis in a 13-year-old girl

El feocromocitoma es un tumor raro, infrecuente en la edad pediátrica. Los síntomas clásicos derivados del exceso de catecolaminas son cefalea, sudoración y palpitaciones, aunque los niños pueden tener una clínica más atípica. La hipertensión arterial suele ser un signo constante en la mayoría de los pacientes. Existen pocos casos descritos de poliuria como forma de presentación de feocromocitoma. Se presenta el caso de una niña de 13 años remitida a consulta de Nefrología Pediátrica por enuresis secundaria de un año de evolución. La tensión arterial clínica tomada durante la exploración era superior al percentil 99 para su edad y talla, motivo por el que se decidió el ingreso para su estudio y tratamiento.

Pheochromocytoma is a rare tumor which is infrequent in children. Although the clinical presentation in children can be atypical, the classic symptoms are headache, sweating and tachycardia. Hypertension is often a constant sign in most patients. There are few cases in literature reporting pheochromocytoma presented with polyuria. We present a 13-year-old girl who came to the Pediatric Nephrologist due to a year of evolution of secondary enuresis. When her blood pressure was taken, she was above the 99th percentile that corresponds to her age and her height that is why she was admitted for treatment and diagnostic study.

[Pheochromocytoma presenting with secondary enuresis in a 13-year-old girl]. / Enuresis secundaria como presentación de feocromocitoma en una niña de 13 años.

Pheochromocytoma is a rare tumor which is infrequent in children. Although the clinical presentation in children can be atypical, the classic symptoms are headache, sweating and tachycardia. Hypertension is often a constant sign in most patients. There are few cases in literature reporting pheochromocytoma presented with polyuria. We present a 13-year-old girl who came to the Pediatric Nephrologist due to a year of evolution of secondary enuresis. When her blood pressure was taken, she was above the 99th percentile that corresponds to her age and her height that is why she was admitted for treatment and diagnostic study.

El feocromocitoma es un tumor raro, infrecuente en la edad pediátrica. Los síntomas clásicos derivados del exceso de catecolaminas son cefalea, sudoración y palpitaciones, aunque los niños pueden tener una clínica más atípica. La hipertensión arterial suele ser un signo constante en la mayoría de los pacientes. Existen pocos casos descritos de poliuria como forma de presentación de feocromocitoma. Se presenta el caso de una niña de 13 años remitida a consulta de Nefrología Pediátrica por enuresis secundaria de un año de evolución. La tensión arterial clínica tomada durante la exploración era superior al percentil 99 para su edad y talla, motivo por el que se decidió el ingreso para su estudio y tratamiento.

Raquitismo hipofosfatémico ligado a X por mutaciones en PHEX: variabilidad clínica y evolutiva / X-linked hypophosphatemic rickets due to mutations in PHEX: Clinical and evolutionary variability

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Cystinuria in a patient with 19q12q13.1 deletion.

Cystinuria is a genetic cause of kidney stones with a prevalence of 1 in 7000 births. So far, two genes have been described responsible for this disorder (SLC3A1 and SLC7A9). We report a patient with an SLC7A9 gene mutation located in 19q13.1 on one allele and with a 19q12q13 region deletion on the other allele. The characteristic clinical features of the 19q13.1 microdeletion syndrome include facial dysmorphism, signs of ectodermal dysplasia, growth retardation, neurologic features and genitourinary anomalies. Cystinuria has not yet been described as part of this syndrome, although one of its responsible genes (SLC7A9) is in the same genomic location. The index case is a 6-year-old male presented with distinctive facial features, cutis aplasia of the scalp, rudimentary teeth, microcephaly, intrauterine and postnatal growth retardation, psychomotor developmental delay, speech delay, epilepsy, inguinal hernias and cystinuria. An array-CGH analysis was performed, finding a large deletion of the 19q12q13.11 cytobands, which affects 19 genes. Two of them are involved in the 19q13.11 deletion syndrome and another affected gene is SLC7A9, responsible for type B cystinuria. Sanger sequencing was performed as well, detecting a heterozygous mutation of the SLC7A9 gene, located in 19q13.1. As far as we know, this is the first described case of cystinuria in a patient with SLC7A9 gene mutation located in 19q13.1 on one allele and with 19q12q13 region deletion on the other allele. Although this patient can be classified as a type B heterozygote and, therefore, his renal prognosis is not severe, the occasional nephrolithiasis found in such patients justifies a close follow-up with regular testing of urinary cystine excretion. We suggest that the recessive behavior of this case, explains the clinical features regarding cystinuria. We propose that in the face of patients affected of a phenotype matchable with 19q13.11 syndrome and cystinuria, a mutational or sequencing study of the SLC7A9 gene should be performed to allow an early onset of diagnosis and treatment.

TRPC6 mutational analysis in a large cohort of patients with focal segmental glomerulosclerosis.

BACKGROUND: Mutations in the TRPC6 gene have been reported in six families with adult-onset (17-57 years) autosomal dominant focal segmental glomerulosclerosis (FSGS). Electrophysiology studies confirmed augmented calcium influx only in three of these six TRPC6 mutations. To date, the role of TRPC6 in childhood and adulthood non-familial forms is unknown. METHODS: TRPC6 mutation analysis was performed by direct sequencing in 130 Spanish patients from 115 unrelated families with FSGS. An in silico scoring matrix was developed to evaluate the pathogenicity of amino acid substitutions, by using the bio-physical and bio-chemical differences between wild-type and mutant amino acid, the evolutionary conservation of the amino acid residue in orthologues, homologues and defined domains, with the addition of contextual information. RESULTS: Three new missense substitutions were identified in two clinically non-familial cases and in one familial case. The analysis by means of this scoring system allowed us to classify these variants as likely pathogenic mutations. One of them was detected in a female patient with unusual clinical features: mesangial proliferative FSGS in childhood (7 years) and partial response to immunosupressive therapy (CsA + MMF). Asymptomatic carriers of this likely mutation were found within her family. CONCLUSIONS: We describe for the first time TRPC6 mutations in children and adults with non-familial FSGS. It seems that TRPC6 is a gene with a very variable penetrance that may contribute to glomerular diseases in a multi-hit setting.

Gas6 promotes inflammation by enhancing interactions between endothelial cells, platelets, and leukocytes.

The role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart transplantation). Thus, Gas6 amplifies EC activation, thereby playing a key role in enhancing the interactions between ECs, platelets, and leukocytes during inflammation.

Volumetric properties of sunflower methyl ester oil at high pressure.

Biodiesel is an alternative to diesel oil (DO), because it is a fuel obtained from renewable resources that has lower emissions than DO. Biomass production should promote agricultural activity to obtain fuels for the transport sector. The study of the behavior of biodiesel at varying pressure and temperature is very interesting because diesel engines are mechanical systems that work with fuels submitted to high pressure. The specific volume, isothermal compressibility, and cubic expansion coefficients of refined sunflower methyl ester oil (SMEO) and unrefined sunflower methyl ester oil (URSMEO) were obtained and compared with those of DO from 0.1 to 350 MPa and 288.15 to 328.15 K. This work shows that oil refinement did not significantly modify any of the properties studied of the final biodiesel. Compared with DO, both SMEOs were about 6% denser, whereas isothermal compressibility and cubic expansion coefficients were bigger or smaller for DO depending on pressure and temperature.