Erratum: Physiological lentiviral vectors for the generation of improved CAR-T cells.

[This corrects the article DOI: 10.1016/j.omto.2022.05.003.].

Physiological lentiviral vectors for the generation of improved CAR-T cells.

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms. However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%-50% of the treated patients. Most CAR-T cells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of their moderate and TCR-like expression profile. Compared with CAR-T cells generated with human elongation factor α (EF1α)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-α) and interferon (IFN)-É£ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo. Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing of AW-CAR-T cells in guanosine monophosphate (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products.

High-dose corticosteroid pulse therapy increases the survival rate in COVID-19 patients at risk of hyper-inflammatory response.

OBJECTIVE: Test whether high dose corticosteroid pulse therapy (HDCPT) with either methylprednisolone or dexamethasone is associated with increased survival in COVID-19 patients at risk of hyper-inflammatory response. Provide some initial diagnostic criteria using laboratory markers to stratify these patients. METHODS: This is a prospective observational study, 318 met the inclusion criteria. 64 patients (20.1%) were treated with HDCPT by using at least 1.5mg/kg/24h of methylprednisolone or dexamethasone equivalent. A multivariate Cox regression (controlling for co-morbidities and other therapies) was carried out to determine whether HDCPT (among other interventions) was associated with decreased mortality. We also carried out a 30-day time course analysis of laboratory markers between survivors and non-survivors, to identify potential markers for patient stratification. RESULTS: HDCPT showed a statistically significant decrease in mortality (HR = 0.087 [95% CI 0.021-0.36]; P < 0.001). 30-day time course analysis of laboratory marker tests showed marked differences in pro-inflammatory markers between survivors and non-survivors. As diagnostic criteria to define the patients at risk of developing a COVID-19 hyper-inflammatory response, we propose the following parameters (IL-6 > = 40 pg/ml, and/or two of the following: C-reactive protein > = 100 mg/L, D-dimer > = 1000 ng/ml, ferritin > = 500 ng/ml and lactate dehydrogenase > = 300 U/L). CONCLUSIONS: HDCPT can be an effective intervention to increase COVID-19 survival rates in patients at risk of developing a COVID-19 hyper-inflammatory response, laboratory marker tests can be used to stratify these patients who should be given HDCPT. This study is not a randomized clinical trial (RCT). Future RCTs should be carried out to confirm the efficacy of HDCPT to increase the survival rates of COVID-19.

Randomised, double-blind, placebo-controlled clinical trial for evaluating the efficacy of intracoronary injection of autologous bone marrow mononuclear cells in the improvement of the ventricular function in patients with idiopathic dilated myocardiopathy: a study protocol.

BACKGROUND: Cellular therapies have been increasingly applied to diverse human diseases. Intracoronary infusion of bone marrow-derived mononuclear cells (BMMNC) has demonstrated to improve ventricular function after acute myocardial infarction. However, less information is available about the role of BMMNC therapy for the treatment of dilated myocardiopathies (DCs) of non-ischemic origin. This article presents the methodological description of a study aimed at investigating the efficacy of intracoronary injection of autologous BMMNCs in the improvement of the ventricular function of patients with DC. METHODS: This randomised, placebo-controlled, double-blinded phase IIb clinical trial compares the improvement on ventricular function (measured by the changes on the ejection fraction) of patients receiving the conventional treatment for DC in combination with a single dose of an intracoronary infusion of BMMNCs, with the functional recovery of patients receiving placebo plus conventional treatment. Patients assigned to both treatment groups are monitored for 24 months. This clinical trial is powered enough to detect a change in Left Ventricular Ejection Fraction (LVEF) equal to or greater than 9%, although an interim analysis is planned to re-calculate sample size. DISCUSSION: The study protocol was approved by the Andalusian Coordinating Ethics Committee for Biomedical Research (Comité Coordinador de Ética en Investigación Biomédica de Andalucia), the Spanish Medicines and Medical Devices Agency (Agencia Española de Medicamentos y Productos Sanitarios), and is registered at the EU Clinical Trials Register (EudraCT: 2013-002015-98). The publication of the trial results in scientific journals will be performed in accordance with the applicable regulations and guidelines to clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT02033278 (First Posted January 10, 2014): https://clinicaltrials.gov/ct2/show/NCT02033278 ; EudraCT number: 2013-002015-98, EU CT Register: https://www.clinicaltrialsregister.eu/ctr-search/search?query=2013-002015-98 . Trial results will also be published according to the CONSORT statement at conferences and reported peer-reviewed journals.

The molecular epidemiology of HIV-1 in the Comunidad Valenciana (Spain): analysis of transmission clusters.

HIV infections are still a very serious concern for public heath worldwide. We have applied molecular evolution methods to study the HIV-1 epidemics in the Comunidad Valenciana (CV, Spain) from a public health surveillance perspective. For this, we analysed 1804 HIV-1 sequences comprising protease and reverse transcriptase (PR/RT) coding regions, sampled between 2004 and 2014. These sequences were subtyped and subjected to phylogenetic analyses in order to detect transmission clusters. In addition, univariate and multinomial comparisons were performed to detect epidemiological differences between HIV-1 subtypes, and risk groups. The HIV epidemic in the CV is dominated by subtype B infections among local men who have sex with men (MSM). 270 transmission clusters were identified (>57% of the dataset), 12 of which included ≥10 patients; 11 of subtype B (9 affecting MSMs) and one (n = 21) of CRF14, affecting predominately intravenous drug users (IDUs). Dated phylogenies revealed these large clusters to have originated from the mid-80s to the early 00 s. Subtype B is more likely to form transmission clusters than non-B variants and MSMs to cluster than other risk groups. Multinomial analyses revealed an association between non-B variants, which are not established in the local population yet, and different foreign groups.

Health care provider responses to initial parental reports of autism spectrum disorder symptoms: results from a nationally representative sample.

BACKGROUND: Health care provider (HCP) responses to initial parental report of autism spectrum disorder (ASD) symptoms were examined in relation to latency to diagnosis and child chronological age at diagnosis. METHOD: Secondary data analyses were conducted for a sample of 1384 parents of children with ASD utilizing data from the National Survey of Children with Special Health Care Needs (NS-CSHCN, 2009-2010 National Survey of Children with Special Health Care Needs, 2009) and the Centers for Disease Control and Prevention Pathways to Diagnosis and Services (CDC PDS, Survey of pathways to diagnosis and services, 2011). RESULTS: Approximately 44% of the sample experienced predominantly delayed HCP responses, 38% experienced predominantly proactive responses, while the remaining 18% experienced a relatively even mix of delayed and proactive responses across HCPs. With regard to outcomes correlated with the type of HCP response, individuals exposed to proactive HCPs were diagnosed with ASD almost a year earlier for child chronological age than individuals exposed to mixed HCPs. This difference increased beyond a year between individuals receiving proactive HCPs versus individuals experiencing delayed HCPs. Finally, after controlling for socioeconomic status, parent-reported severity of ASD symptoms, and age at time of referral, proactive HCP was correlated with decreased time to diagnosis from parental first report of ASD symptoms. CONCLUSIONS: Results are discussed with regard to increasing proactive HCP responses to parental first concerns of ASD symptomology versus a mix or delayed responses.

Predictors of Elopement Exhibited by School-Aged Children With Special Health Care Needs: Towards the Development of a Screening Instrument for Elopement.

Elopement exhibited by school-aged children with special health care needs is a relatively low frequency problem behavior with the potential for severe negative consequences for the child and family. Using data from the Centers for Disease Control and Prevention (CDC) Survey of Pathways to Diagnosis and Services, our results represent one of the first empirical studies of variables that may be associated with children with special health care needs engaging in elopement. Using data from a nationally representative sample of children with special health care needs, our results revealed two variables that were statistically significant predictors of parent-reported elopement in the past year: (1) the child's chronological age, and (2) the presence of an autism spectrum disorder (ASD) diagnosis. We found that the likelihood of an elopement event was inversely related to age, but positively associated with the presence of an ASD diagnosis. Using parent-response items from the CDC data set, we selected a set of questions to screen for risk of elopement and analyzed their psychometric properties. We discuss the need for future research to validate this screening instrument for school-aged youth with special health care needs. Our study provides an initial psychometric analysis to support a potential screening instrument for elopement events among school-aged youth that needs to be validated by a longitudinal study of its predictive validity.

Immunoassays for trifloxystrobin analysis. Part II. Assay development and application to residue determination in food.

Immunochemical assays constitute complementary analytical methods for small organic molecule determination. We herein describe the characterisation and optimisation of two competitive enzyme-linked immunosorbent assays in different formats using monoclonal antibodies to the Quinone outside inhibitor (QoI) fungicide trifloxystrobin. Antibody selectivity was evaluated using a variety of agrochemicals and the main trifloxystrobin metabolite. Acceptable tolerance of the immunoassay to methanol, ethanol, and acetonitrile was observed in all cases, whereas a dissimilar influence of buffer pH and ionic strength was found. Moreover, the influence of Tween 20 over the analytical parameters was studied. The limits of detection of the optimised assays were below 0.1 µg L(-1). Excellent recoveries, even at 10 µg kg(-1), were obtained when strawberry, tomato, and cucumber samples spiked with trifloxystrobin were analysed. Finally, statistical agreement was found between immunoassay and reference chromatographic results using blind-spiked and in-field treated samples.

Sensitive monoclonal antibody-based immunoassays for kresoxim-methyl analysis in QuEChERS-based food extracts.

Kresoxim-methyl is nowadays widely used to combat a diversity of common diseases affecting high-value crops. In this article, we report the development and characterization of two novel immunoassays for the analysis of this pioneer strobilurin fungicide, and for the first time, a validation study with food samples was performed. A direct and an indirect competitive immunoassay based on a new anti-kresoxim-methyl monoclonal antibody were developed for sensitive and specific chemical analysis. Optimized assays showed limits of detection of 0.1 µg/L. Fruit and vegetable samples were extracted with acetonitrile by the QuEChERS procedure and analyzed by the developed immunoassays after a simple dilution in buffer, affording limits of quantification below US and European maximum residue limits. Immunochemical results of samples from kresoxim-methyl-sprayed strawberry fields demonstrated good statistical agreement with gas chromatography coupled to mass spectrometry as reference technique.

Immunoassays for trifloxystrobin analysis. Part I. Rational design of regioisomeric haptens and production of monoclonal antibodies.

Trifloxystrobin is one of the main active principles belonging to the strobilurin family of crop protection compounds. In this article, the synthesis of a battery of regioisomeric functionalized derivatives of trifloxystrobin is described. The same aliphatic linear carboxylated chain was introduced as spacer arm in all of the synthesized haptens, but it was located at different positions of the parent molecule. N,N'-Disuccinimidyl carbonate was employed for hapten activation, so the resulting N-hydroxysuccinimyl ester could be readily purified and efficiently coupled to proteins. After immunization and hybridoma generation, a collection of 20 mouse monoclonal antibodies from different immunizing haptens was obtained. The analytical performance of these immunoreagents was evaluated in terms of affinity and selectivity with the aim to develop rapid and practical immunochemical procedures for trifloxystrobin determination.

Structure-immunogenicity relationship of kresoxim-methyl regioisomeric haptens.

Kresoxim-methyl was one of the two first strobilurins to be discovered, and nowadays it is widely used as an antifungal agent in crop protection. Because of its low molecular weight and negligible structural complexity, the generation of antibodies to kresoxim-methyl noticeably requires the preparation of functionalized haptens. In this study, the introduction of a hydrocarbon spacer arm at the aromatic moieties of the target molecule was carried out by a convergent strategy based on the Sonogashira cross-coupling reaction, and functionalized linkers of the same length were also tethered to the aliphatic toxophore group by the O-alkylation reaction. Evaluation of the immune response, in terms of antibody affinity, showed a differential behavior among five synthesized haptens whose sole dissimilarity was the derivatization site. The characteristic (methoxyimino)acetate moiety of strobilurins was revealed as the optimum linker position for high-affinity polyclonal and monoclonal antibody production. However, good monoclonal antibodies were isolated from mice immunized with a hapten carrying the linker at an opposite site, which otherwise generated a poor polyclonal response in rabbits. Site-heterology was confirmed as a feasible approach for the improvement of the apparent affinity, particularly with polyclonal antibodies. Several of the monoclonal antibodies generated in the context of this project could be proper binders for kresoxim-methyl immunosensing over different analytical platforms.

Perinatal undernutrition programmes thyroid function in the adult rat offspring.

Increasing evidence suggests that alterations in early nutrition programme physiological changes in adulthood. In the present study, we determined the effects of undernutrition during gestation and lactation on the programming of thyroid function in adult rat offspring. Perinatal undernutrition was achieved by a 40% food restriction in female Wistar rats from the mating day to weaning. On postpartum day 21, the offspring of the control and food-restricted dams were weaned and given free access to a commercial diet until adulthood. The results showed that undernourished rats exhibited decreased 3,5,3'-triiodothyronine (T3) levels but had normal thyroxine (T4) and thyrotropin (TSH) levels at weaning; on day 90, these rats displayed a significant flip, exhibiting normalised T3 (total and free) and total T4 levels, but low free T4 and persistently higher TSH levels, which were maintained even on postnatal day 140. This profile was accompanied by a scarce fat depot, a lower RMR and an exacerbated sympathetic brown adipose tissue (BAT) tone (deiodinase type 2 expression) in basal conditions. Moreover, when a functional challenge (cold exposure) was applied, the restricted group exhibited partial changes in TSH (29 v. 100%) and T4 (non-response v. 17%) levels, a significant decrease in leptin levels (75 v. 32%) and the maintenance of a sympathetic BAT over-response (higher noradrenaline levels) in comparison with the control group. The findings of the present study suggest that undernutrition during the perinatal period produces permanent changes in the hypothalamus-pituitary-thyroid axis with consequent low body weight and decreased RMR and facultative thermogenesis. We hypothesise that these changes predispose individuals to exhibiting adult subclinical hypothyroidism.

Influencia de la inmigración en los patrones de transmisión de la tuberculosis en Castellón (2004–2007) / Influence of immigration on tuberculosis transmission patterns in Castellón, Spain (2004–2007)

Objetivo: Describir los patrones de transmisión de la tuberculosis en Castellón en un periodo de importantes cambios demográficos. Métodos: Estudio prospectivo descriptivo de los pacientes con cultivo positivo en la provincia de Castellón entre 2004 y2007. Se describen los pacientes por año y nacionalidad, y se cotejan con los casos declarados a la Dirección General de Salud Pública (DGSP). Se estudia la población con patrón molecular disponible por RFLP (restriction fragment length polymorphism) y se analizan las variables de los pacientes agrupados (clusters) a partir de los datos de la DGSP y del Programa de Gestión del Laboratorio.Resultados: Según la DGSP, la tasa global de tuberculosis por 100.000 habitantes en la provincia de Castellón fue de 15,7 en 2004, 19,9 en 2005, 18,2 en 2006 y 17,5 en 2007. En nuestro laboratorio se identificaron las cepas de 301 pacientes, que suponen el 77% (301/390) de los casos declarados y el94% (301/321) aquellos con cultivo positivo. El porcentaje de tuberculosis en extranjeros aumentó hasta superar el 50% en 2007. Se disponía de estudio molecular en el 95% de los casos (286), con un 58% deespañoles y un 42% de extranjeros. El porcentaje de agregación fue del 40%, con un 30% de clusters mixtos.Según el estudio convencional de contactos, el 85% de los pacientes en cluster habían sido considerados casos aislados. Conclusiones: El aumento de la tasa de tuberculosis en Castellón se debe, principalmente, al creciente número de los casos en extranjeros. Disponer del estudio molecular de todos los pacientes con cultivo positivo nos ha permitido analizar cómo y dónde se transmite la tuberculosis. El 40% de los pacientes se agruparon en clusters, y eran mixtos un tercio de ellos, lo que indica una elevada integración de los inmigrantes (AU)

Background: This study aimed to identify tuberculosis transmission patterns in Castellón in a period ofmajor demographic changes. Methods: A prospective study of patients with positive culture in the province of Castellon over a 4-yearperiod (2004–2007) was carried out. Cases were described by year and nationality and were compared with those reported to the Department of Public Health. We studied the population with available molecular patterns, identified through restriction fragment length polymorphism (RFLP) and analyzed the variables from patient clusters, based on data collected in surveys of the Department of Health and the Laboratory Management Program. Results: According to data from the Department of Public Health, the overall rate of tuberculosis per100,000 inhabitants in the province of Castellón was 15.7 in 2004, 19.9 in 2005, 18.2 in 2006 and 17.5 in 2007. In our laboratory, strains were identified from 301 patients, representing 77% (301/390) of reported cases and 94% (301/321) of reported cases with a positive culture. The percentage of tuberculosis amongforeigners increased with age, exceeding 50% in 2007. Molecular studies were available in 95% of patients (286); 58% were Spanish and 42% were foreigners, of whom 54% were Romanians. The cluster percentage was 40%, with 30% of mixed clusters. According to conventional contact studies, 85% of patients in clusters had been considered isolated cases.Conclusions: The increased rate of tuberculosis in Castellón was mainly due to the increasing numberof cases among foreigners, mostly Romanians. The availability of molecular studies in all patients with apositive culture allowed us to analyze how and where tuberculosis is transmitted in our province. Forty percent of the patients were grouped into clusters; of these, mixed clusters accounted for one third,indicating the high integration of immigrants in our area (AU)

[Influence of immigration on tuberculosis transmission patterns in Castellón, Spain (2004-2007)]. / Influencia de la inmigración en los patrones de transmisión de la tuberculosis en Castellón (2004-2007).

BACKGROUND: This study aimed to identify tuberculosis transmission patterns in Castellón in a period of major demographic changes. METHODS: A prospective study of patients with positive culture in the province of Castellon over a 4-year period (2004-2007) was carried out. Cases were described by year and nationality and were compared with those reported to the Department of Public Health. We studied the population with available molecular patterns, identified through restriction fragment length polymorphism (RFLP) and analyzed the variables from patient clusters, based on data collected in surveys of the Department of Health and the Laboratory Management Program. RESULTS: According to data from the Department of Public Health, the overall rate of tuberculosis per 100,000 inhabitants in the province of Castellón was 15.7 in 2004, 19.9 in 2005, 18.2 in 2006 and 17.5 in 2007. In our laboratory, strains were identified from 301 patients, representing 77% (301/390) of reported cases and 94% (301/321) of reported cases with a positive culture. The percentage of tuberculosis among foreigners increased with age, exceeding 50% in 2007. Molecular studies were available in 95% of patients (286); 58% were Spanish and 42% were foreigners, of whom 54% were Romanians. The cluster percentage was 40%, with 30% of mixed clusters. According to conventional contact studies, 85% of patients in clusters had been considered isolated cases. CONCLUSIONS: The increased rate of tuberculosis in Castellón was mainly due to the increasing number of cases among foreigners, mostly Romanians. The availability of molecular studies in all patients with a positive culture allowed us to analyze how and where tuberculosis is transmitted in our province. Forty percent of the patients were grouped into clusters; of these, mixed clusters accounted for one third, indicating the high integration of immigrants in our area.

Tumor necrosis factor-α-secreting CD16+ antigen presenting cells are effectively removed by granulocytapheresis in ulcerative colitis patients.

BACKGROUND AND AIM: In human blood, two main subsets of antigen-presenting-cells (APCs) have been described: plasmocytoid dendritic cells (pDC) and myeloid dendritic cells (mDC) which are further subdivided in CD11c-mDC and CD16-mDC DC. In ulcerative colitis patients (UC) peripheral blood APCs express significant levels of the activation and lack immature-tolerogeneic APCs. Adacolumn selective granulocytapheresis (GCAP) has been associated with clinical efficacy in patients with UC. In the present study we sought the effect of sequential GCAP procedures in peripheral blood APCs in patients with UC and the effect on soluble cytokines. METHODS: We used multiparametric flow cytometry to quantify peripheral blood APCs and serum cytokines in 210 samples obtained from seven patients with steroid-dependent or steroid resistant UC undergoing GCAP treatment. Samples were drawn before, after 30 and 60 min of each session. RESULTS: Each GCAP session resulted in a dramatic tenfold reduction of peripheral blood CD16-mDC (P < 0.01), pDC decreased twofold (P = 0.05) but CD11c-mDC remained unchanged. This depletion was reached after 30 min and maintained at 60 min. The depletion of CD16-mDC and monocytes was associated with a reduction of serum tumor necrosis factor levels and a raise in interleukin-10 levels, although no statistical difference was reached. CONCLUSION: The effect of GCAP in peripheral blood APC consisted mainly on a significant depletion of tumor necrosis factor-α secreting CD16-mDC. This finding could suggest a potential mechanism of GCAP beneficial effect that must be confirmed in larger series.

Respuesta de los autores: Nocardiosis cutánea primaria por Nocardia brasiliensis en España / Author’s reply: Primary nocardiosis by Nocardia brasiliensis in Spain

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