Analysis of the impact of handling and culture on the expansion and functionality of NK cells.

Natural killer (NK) cells are lymphocytes of the innate immune system that play a key role in the elimination of tumor and virus-infected cells. Unlike T cells, NK cell activation is governed by their direct interaction with target cells via the inhibitory and activating receptors present on their cytoplasmic membrane. The simplicity of this activation mechanism has allowed the development of immunotherapies based on the transduction of NK cells with CAR (chimeric antigen receptor) constructs for the treatment of cancer. Despite the advantages of CAR-NK therapy over CAR-T, including their inability to cause graft-versus-host disease in allogenic therapies, a deeper understanding of the impact of their handling is needed in order to increase their functionality and applicability. With that in mind, the present work critically examines the steps required for NK cell isolation, expansion and storage, and analyze the response of the NK cells to these manipulations. The results show that magnetic-assisted cell sorting, traditionally used for NK isolation, increases the CD16+ population of NK cultures only if the protocol includes both, antibody incubation and passage through the isolation column. Furthermore, based on the importance of surface potential on cellular responses, the influence of surfaces with different net surface charge on NK cells has been evaluated, showing that NK cells displayed higher proliferation rates on charged surfaces than on non-charged ones. The present work highlights the relevance of NK cells manipulation for improving the applicability and effectiveness of NK cell-based therapies.

Natural killer cell subsets in endometrial fluid: a pilot study of their association with the endometrial cycle and reproductive parameters.

PURPOSE: To investigate if there are natural killer (NK) cells in endometrial fluid (EF) and their relationship with the endometrial cycle and reproductive parameters. METHODS: The population under study consisted of 43 women aged 18-40 undergoing infertility workup at our University Hospital in 2021-2022. The EF samples were obtained at the first visit to our unit, on occasion of the mock embryo transfer. The day of the cycle was considered only in cycles of 27-29 days. An immunophenotype study of NK in EF was performed by flow cytometry analysis. In a subgroup of women, on the same day, NK was studied in EF and peripheral blood. RESULTS: Our study is the first to evidence NK cells in EF. None of the NK cells observed corresponded to a mature peripheral blood NK cell population (stages 4-5), and neither endometrial nor decidual uNK cells were detected. Nevertheless, we found 2 patient groups with an NK cell subset with a higher expression of CD16+, which could belong to an intermediate or transient stage between the uNK and pbNK NK cell population in the EF. We found that CD16 was significantly increased in the mid-late luteal phase and its correlation with the day of the cycle. The NK immunophenotype was different in EF and peripheral blood. CONCLUSION: We described a new component of the EF, the NK cells, whose CD16 activity is closely correlated with the day of the cycle. These cells could play a role in implantation/implantation failure.

Sedación administrada por médicos generales para procedimientos endoscópicos de baja complejidad: experiencia en una unidad de endoscopia de una clínica de alta complejidad en Cali / Sedation Administered by General Practitioners for Low Complexity Endoscopic Procedures: Experience in an Endoscopy Unit of a Tertiary Referral Hospital in Cali

Resumen Objetivos: en Colombia se ha venido implementando la sedación por médicos no anestesiólogos para procedimientos endoscópicos fuera del quirófano. Se describió la experiencia en la unidad de gastroenterología de una clínica de alto nivel de atención en Cali, Colombia. Materiales y métodos: estudio observacional, de tipo cohorte analítica para describir la frecuencia y el tipo de eventos adversos asociados a los procedimientos de sedación por médicos generales, y evaluar los factores asociados a su ocurrencia en pacientes que acudieron a la unidad de endoscopia de la Fundación Valle del Lili para la realización de estudios endoscópicos bajo sedación intravenosa que, por ser de bajo riesgo, fue aplicada por un médico no anestesiólogo entre noviembre de 2018 y junio de 2019. Se realizó análisis descriptivo, se calcularon mediana y rango intercuartílico para las variables numéricas, y frecuencias para las variables cualitativas. Resultados: se incluyeron 1506 participantes, 59,4 % ASA I y 40,6 % ASA II. En promedio, la dosis inicial de propofol fue de 60 mg y la dosis total, de 140 mg. Se registraron eventos adversos no serios en 46 pacientes (3,05 %) y el más común fue la desaturación transitoria (80,4 %). Ningún paciente presentó eventos adversos serios. El puntaje inicial promedio de la escala de Aldrete fue 8, mientras que al alta el puntaje promedio fue de 10. Conclusiones: la sedación para procedimientos endoscópicos dada por médicos no anestesiólogos es segura, siempre y cuando sea realizado por personal entrenado que realice una adecuada valoración de los antecedentes (cardiovasculares, gastrointestinales y neurológicos) y factores de riesgo del paciente dentro del marco de los lineamientos institucionales vigentes.

Abstract Objectives: in Colombia, sedation by non-anesthesiologists for endoscopic procedures outside the operating room has been implemented. A description of an experience in the gastroenterology unit of a tertiary referral hospital in Cali, Colombia, was conducted. Materials and methods: an analytical cohort observational study to describe the frequency and type of adverse events associated with sedation procedures performed by general practitioners and evaluate the factors related to their occurrence in patients who attended the endoscopy unit of Fundación Valle del Lili for endoscopic studies under intravenous sedation. Between November 2018 and June 2019, non-anesthesiologist physicians performed this procedure due to the minimal risk implied. A descriptive analysis was completed, and the median and interquartile range were calculated for numerical variables and frequencies for qualitative variables. Results: There were 1506 participants, 59.4% ASA I and 40.6% ASA II in this study. On average, the starting dose of propofol was 60 mg, and the total dose was 140 mg. Forty-six patients (3.05%) reported non-severe adverse events; the most common occurrence was transient desaturation (80.4%). No patients experienced severe adverse events. The average initial Aldrete scale score was 8, while at discharge, the average score was 10. Conclusions: sedation for endoscopic procedures performed by non-anesthesiologists is safe provided that it is performed by trained personnel conducting a correct assessment of the patient's (cardiovascular, gastrointestinal, and neurological) history and risk factors within the framework of the current institutional guidelines.

A Comparative Study of Cell Culture Conditions during Conversion from Primed to Naive Human Pluripotent Stem Cells.

The successful reprogramming of human somatic cells into induced pluripotent stem cells (hiPSCs) represented a turning point in the stem cell research field, owing to their ability to differentiate into any cell type with fewer ethical issues than human embryonic stem cells (hESCs). In mice, PSCs are thought to exist in a naive state, the cell culture equivalent of the immature pre-implantation embryo, whereas in humans, PSCs are in a primed state, which is a more committed pluripotent state than a naive state. Recent studies have focused on capturing a similar cell stage in human cells. Given their earlier developmental stage and therefore lack of cell-of-origin epigenetic memory, these cells would be better candidates for further re-differentiation, use in disease modeling, regenerative medicine and drug discovery. In this study, we used primed hiPSCs and hESCs to evaluate the successful establishment and maintenance of a naive cell stage using three different naive-conversion media, both in the feeder and feeder-free cells conditions. In addition, we compared the directed differentiation capacity of primed and naive cells into the three germ layers and characterized these different cell stages with commonly used pluripotent and lineage-specific markers. Our results show that, in general, naive culture NHSM medium (in both feeder and feeder-free systems) confers greater hiPSCs and hESCs viability and the highest naive pluripotency markers expression. This medium also allows better cell differentiation cells toward endoderm and mesoderm.

Identifying SARS-CoV-2 'memory' NK cells from COVID-19 convalescent donors for adoptive cell therapy.

COVID-19 disease is the manifestation of syndrome coronavirus 2 (SARS-CoV-2) infection, which is causing a worldwide pandemic. This disease can lead to multiple and different symptoms, being lymphopenia associated with severity one of the most persistent. Natural killer cells (NK cells) are part of the innate immune system, being fighting against virus-infected cells one of their key roles. In this study, we determined the phenotype of NK cells after COVID-19 and the main characteristic of SARS-CoV-2-specific-like NK population in the blood of convalescent donors. CD57+ NKG2C+ phenotype in SARS-CoV-2 convalescent donors indicates the presence of 'memory'/activated NK cells as it has been shown for cytomegalovirus infections. Although the existence of this population is donor dependent, its expression may be crucial for the specific response against SARS-CoV-2, so that, it gives us a tool for selecting the best donors to produce off-the-shelf living drug for cell therapy to treat COVID-19 patients under the RELEASE clinical trial (NCT04578210).

Uneven metabolic and lipidomic profiles in recovered COVID-19 patients as investigated by plasma NMR metabolomics.

COVID-19 is a systemic infectious disease that may affect many organs, accompanied by a measurable metabolic dysregulation. The disease is also associated with significant mortality, particularly among the elderly, patients with comorbidities, and solid organ transplant recipients. Yet, the largest segment of the patient population is asymptomatic, and most other patients develop mild to moderate symptoms after SARS-CoV-2 infection. Here, we have used NMR metabolomics to characterize plasma samples from a cohort of the abovementioned group of COVID-19 patients (n = 69), between 3 and 10 months after diagnosis, and compared them with a set of reference samples from individuals never infected by the virus (n = 71). Our results indicate that half of the patient population show abnormal metabolism including porphyrin levels and altered lipoprotein profiles six months after the infection, while the other half show little molecular record of the disease. Remarkably, most of these patients are asymptomatic or mild COVID-19 patients, and we hypothesize that this is due to a metabolic reflection of the immune response stress.

The Race of CAR Therapies: CAR-NK Cells for Fighting B-Cell Hematological Cancers.

Acute lymphoblastic leukemia (ALL) and Chronic lymphocytic leukemia (CLL) are the most common leukemias in children and elderly people, respectively. Standard therapies, such as chemotherapy, are only effective in 40% of ALL adult patients with a five-year survival rate and therefore new alternatives need to be used, such as immunotherapy targeting specific receptors of malignant cells. Among all the options, CAR (Chimeric antigen receptor)-based therapy has arisen as a new opportunity for refractory or relapsed hematological cancer patients. CARs were designed to be used along with T lymphocytes, creating CAR-T cells, but they are presenting such encouraging results that they are already in use as drugs. Nonetheless, their side-effects and the fact that it is not possible to infuse an allogenic CAR-T product without causing graft-versus-host-disease, have meant using a different cell source to solve these problems, such as Natural Killer (NK) cells. Although CAR-based treatment is a high-speed race led by CAR-T cells, CAR-NK cells are slowly (but surely) consolidating their position; their demonstrated efficacy and the lack of undesirable side-effects is opening a new door for CAR-based treatments. CAR-NKs are now in the field to stay.

Diagnostic delay of associated interstitial lung disease increases mortality in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease whose main extra-articular organ affected is the lung, sometimes in the form of diffuse interstitial lung disease (ILD) and conditions the prognosis. A multicenter, observational, descriptive and cross-sectional study of consecutive patients diagnosed with RA-ILD. Demographic, analytical, respiratory functional and evolution characteristics were analyzed to evaluate the predictors of progression and mortality. 106 patients were included. The multivariate analysis showed that the diagnostic delay was an independent predictor of mortality (HR 1.11, CI 1.01-1.23, p = 0.035). Also, age (HR 1.33, 95% CI 1.09-1.62, p = 0.0045), DLCO (%) (HR 0.85, 95% CI 0.73-0.98, p = 0.0246), and final SatO2 (%) in the 6MWT (HR 0.62, 95% CI 0.39-0.99, p = 0.0465) were independent predictor variables of mortality, as well as GAP index (HR 4.65, 95% CI 1.59-13.54, p = 0.0051) and CPI index (HR 1.12, 95% CI 1.03-1.22, p = 0.0092). The withdrawal of MTX or LFN after ILD diagnosis was associated with disease progression in the COX analysis (HR 2.18, 95% CI 1.14-4.18, p = 0.019). This is the first study that highlights the diagnostic delay in RA-ILD is associated with an increased mortality just like happens in IPF.

Purification, Culture, and CD19-CAR Lentiviral Transduction of Adult and Umbilical Cord Blood NK Cells.

Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system. The role that NK cells play is analogous to that of cytotoxic T cells in that they provide rapid responses to virus-infected cells and responses to tumor formation. Unmodified NK cells have long been used in various immunotherapies to treat different tumors, with only marginal success. However, in the last few years, NK cells modified to express chimeric antigen receptors (CAR-NK cells) have emerged as particularly ideal cellular platforms for antigen-specific antitumor agents. Unlike CAR-T cells, they do not elicit allogeneic responses or graft-versus-host disease and therefore can be administered to recipients with differing MHC expression. This article outlines protocols to obtain CD19-CAR-NK cells, focusing on the importance of obtaining and culturing a purified NK cell population and how to attain good transfection efficiency. © 2020 Wiley Periodicals LLC Basic Protocol 1: Purification and culture of adult peripheral blood and umbilical cord blood NK cells Basic Protocol 2: CD19-CAR lentiviral transduction of adult peripheral blood or umbilical cord blood NK cells Support Protocol: Production of lentiviral supernatant.

Cytotoxic Natural Killer Subpopulations as a Prognostic Factor of Malignant Pleural Effusion.

BACKGROUND: Malignant pleural effusion (MPE) is a sign of advanced disease of poor prognosis. As natural killer (NK) cells are involved in the first line of tumour defence, we aimed to validate a new diagnostic and prognostic indicator for MPE based on NK subpopulations of pleural fluid (PF) and peripheral blood (PB). METHODS: NK subpopulations were determined in PF and PB in 71 patients with malignant, paramalignant or benign pleural effusion. The receiver operating characteristic (ROC) curves, Kaplan-Meier, multivariable Cox model and decision trees created with the CHAID (Chi-square automatic interaction detector) methodology were employed. RESULTS: We demonstrated that the PF/PB ratios of the CD56 bright CD16- and CD56 dim CD16- NK subpopulations were higher (p = 0.013 and p = 0.003, respectively) in MPEs and paramalignant pleural effusions (PPEs) than in benign ones, with an AUC of 0.757 and 0.741, respectively. The PF/PB ratio of CD16+ NK and CD57+ NK obtained a higher hazard ratio (HR) in the crude Cox's regression analysis. In the adjusted Cox's regression analysis, the PF/PB ratio of CD16+ NK gave the highest HR (HR 6.1 [1.76-21.1]) (p = 0.004). In the decision tree created for the MPE prognosis, we observed that the main predictor variable among the studied clinical, radiological, and analytical variables was lung mass, and that 92.9% of the patients who survived had a PF/PB ratio of the CD56 dim CD16+ NK subpopulation ≤ 0.43. CONCLUSIONS: Our data suggest that both the PF/PB ratios of cytotoxic subpopulations CD57+ NK and CD16+ NK are useful as a prognostic factor of MPE. Other subpopulations (CD56 bright CD16- and CD56 dim CD16- NK) could help to diagnose MPE.

Anatomic Association of the Proximal Fingernail Matrix to the Extensor Pollicis Longus Tendon: A Morphological and Histological Study.

BACKGROUND: Extensor tendon disorders may cause severe functional impairments, and there is a lack of knowledge about their anatomic associations with the proximal fingernail matrix. OBJECTIVE: To delineate the association between the distal extensor pollicis longus tendon (EPLT) insertion and the limit of the fingernail matrix in the thumb. METHODS: The limit of the fingernail matrix and the distal bony insertion of the EPLT were identified in five thumbs from fresh-frozen human cadavers. An additional five thumbs were fixed and the longitudinal thumb sections were histologically analyzed. RESULTS: The terminal limit of the matrix and fingernail was dorsal and overlapped to the EPL tendon, which was located between the fingernail matrix and the phalanx, and extended dorsally to the distal section of the terminal phalanx in all ten thumb bodies. CONCLUSION: The fingernail matrix is not directly inserted into the periosteum of the dorsal section of the base to the distal phalanx, because this anatomic relationship is separated by the deep fibers of the EPLT.

Development of the long head of the biceps brachial tendon: A possible explanation of the anatomical variations.

The anatomical variations of the proximal portion of the long head of the biceps brachii tendon (LHBT) are rarely observed in clinical practice. However, an increase in the rate of shoulder arthroscopic surgery has led to an increase in the observation of anatomical variations of this region. The aim of this work was to analyze the development of the LHBT in 23 human embryos ranging from the 6th to 8th weeks of development. The LHBT develops from the glenohumeral interzonal mesenchyme in the 6th week. By week 7, the myotendinous junction of the LHBT develops. The anlage of the LHBT is separated from that of the glenohumeral capsule during week 8. Our results suggest that the most important period for the LHBT development occurs between the 6th and 8th weeks of embryonic development. Alterations during this critical period may cause anatomical variations of the LHBT. An additional case report from our own experience is provided as Supplementary material.

Evaluation of Lip Prints on Different Supports Using a Batch Image Processing Algorithm and Image Superimposition.

This study aimed to develop and to assess an algorithm to facilitate lip print visualization, and to digitally analyze lip prints on different supports, by superimposition. It also aimed to classify lip prints according to sex. A batch image processing algorithm was developed, which facilitated the identification and extraction of information about lip grooves. However, it performed better for lip print images with a uniform background. Paper and glass slab allowed more correct identifications than glass and the both sides of compact disks. There was no significant difference between the type of support and the amount of matching structures located in the middle area of the lower lip. There was no evidence of association between types of lip grooves and sex. Lip groove patterns of type III and type I were the most common for both sexes. The development of systems for lip print analysis is necessary, mainly concerning digital methods.

OP9 Feeder Cells Are Superior to M2-10B4 Cells for the Generation of Mature and Functional Natural Killer Cells from Umbilical Cord Hematopoietic Progenitors.

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of mature and functional NK cells. An option for future immunotherapy treatments is to use large amounts of NK cells derived and differentiated from umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), mainly because UCB is one of the most accessible HSC sources. In our study, we compared the potential of two stromal cell lines, OP9 and M2-10B4, for in vitro generation of mature and functional CD56+ NK cells from UCB CD34+ HSC. We generated higher number of CD56+ NK cells in the presence of the OP9 cell line than when they were generated in the presence of M2-10B4 cells. Furthermore, higher frequency of CD56+ NK cells was achieved earlier when cultures were performed with the OP9 cells than with the M2-10B4 cells. Additionally, we studied in detail the maturation stages of CD56+ NK cells during the in vitro differentiation process. Our data show that by using both stromal cell lines, CD34+ HSC in vitro differentiated into the terminal stages 4-5 of maturation resembled the in vivo differentiation pattern of human NK cells. Higher frequencies of more mature NK cells were reached earlier by using OP9 cell line than M2-10B4 cells. Alternatively, we observed that our in vitro NK cells expressed similar levels of granzyme B and perforin, and there were no significant differences between cultures performed in the presence of OP9 cell line or M2-10B4 cell line. Likewise, degranulation and cytotoxic activity against K562 target cells were very similar in both culture conditions. The results presented here provide an optimal strategy to generate high numbers of mature and functional NK cells in vitro, and point toward the use of the OP9 stromal cell line to accelerate the culture procedure to obtain them. Furthermore, this method could establish the basis for the generation of mature NK cells ready for cancer immunotherapy.

Predicting Malignant and Paramalignant Pleural Effusions by Combining Clinical, Radiological and Pleural Fluid Analytical Parameters.

BACKGROUND: The usefulness of clinical, radiological and pleural fluid analytical parameters for diagnosing malignant and paramalignant pleural effusion is not clearly stated. Hence this study aimed to identify possible predictor variables of diagnosing malignancy in pleural effusion of unknown aetiology. METHODS: Clinical, radiological and pleural fluid analytical parameters were obtained from consecutive patients who had suffered pleural effusion of unknown aetiology. They were classified into three groups according to their final diagnosis: malignant, paramalignant and benign pleural effusion. The CHAID (Chi-square automatic interaction detector) methodology was used to estimate the implication of the clinical, radiological and analytical variables in daily practice through decision trees. RESULTS: Of 71 patients, malignant (n = 31), paramalignant (n = 15) and benign (n = 25), smoking habit, dyspnoea, weight loss, radiological characteristics (mass, node, adenopathies and pleural thickening) and pleural fluid analytical parameters (pH and glucose) distinguished malignant and paramalignant pleural effusions (all with a p < 0.05). Decision tree 1 classified 77.8% of malignant and paramalignant pleural effusions in step 2. Decision tree 2 classified 83.3% of malignant pleural effusions in step 2, 73.3% of paramalignant pleural effusions and 91.7% of benign ones. CONCLUSIONS: The data herein suggest that the identified predictor values applied to tree diagrams, which required no extraordinary measures, have a higher rate of correct identification of malignant, paramalignant and benign effusions when compared to techniques available today and proved most useful for usual clinical practice. Future studies are still needed to further improve the classification of patients.

Forensic Facial Reconstruction: Relationship Between the Alar Cartilage and Piriform Aperture.

During forensic facial reconstruction, facial features may be predicted based on the parameters of the skull. This study evaluated the relationships between alar cartilage and piriform aperture and nose morphology and facial typology. Ninety-six cone beam computed tomography images of Brazilian subjects (49 males and 47 females) were used in this study. OsiriX software was used to perform the following measurements: nasal width, distance between alar base insertion points, lower width of the piriform aperture, and upper width of the piriform aperture. Nasal width was associated with the lower width of the piriform aperture, sex, skeletal vertical pattern of the face, and age. The current study contributes to the improvement of forensic facial guides by identifying the relationships between the alar cartilages and characteristics of the biological profile of members of a population that has been little studied thus far.

Differentiation between palatal rugae patterns of twins by means of the Briñón method and an improved technique

Palatal rugae patterns are anatomic structures considered unique to each person. Monozygotic twins present similarities, however, Rugoscopy in particular, may contribute to their individualization for forensic purposes. The aims of this study were: to study the palatal rugae classifications of Briñón; to propose improvements to facilitate use of this method, if pertinent; and to characterize palatal rugae in a sample of Brazilian monozygotic twins and singletons. Precise reproducibility of the two methods of Briñón, from 1982 and 2011, was prevented by poor intra-examiner agreement (70% and 13% respectively). Our proposed improvements to these methods, although preliminary, were associated with better results. The most common palatal rugae patterns were types A, M, and Q. Palatal rugae were confirmed to be unique to each individual, even in monozygotic twins. Furthermore, twins did not exhibit any special patterns that might facilitate their differentiation from singletons.

Comparison Among Manual Facial Approximations Conducted by Two Methodological Approaches of Face Prediction.

This study verified the difference between two methods of forensic facial approximation (FFA) regarding recognition and resemblance rates. Three-dimensional models of skulls were obtained from computerized tomography (CT) scans of two subjects (targets). Two manual FFAs were performed for each target, by applying two different guidelines for the facial structures (what we called "American method" (AM) and "Combined method" (CM)). Unfamiliar assessors evaluated the sculptures by recognition and resemblance tests. The AM was that which allowed more correct responses of recognition and higher resemblance's scores for the male target (p < 0.001). Regarding guidelines for modeling characteristics of the face, the ones that are practical and easily performed for sculptures, such as the length of the anterior nasal spine multiplied by 3 for nose prediction, may offer better results in terms of resemblance.