Dynamic CD8+ T cell responses to cancer immunotherapy in human regional lymph nodes are disrupted in metastatic lymph nodes.

CD8+ T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment, recent studies in mice identified responses in lymph nodes (LNs) as essential; however, the role of LNs in human cancer patients remains unknown. We examined CD8+ T cells in human head and neck squamous cell carcinomas, regional LNs, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8+ T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the tumor. After anti-PD-L1 immunotherapy, Tpex in uninvolved LNs reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8+ T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LNs, these response hallmarks were impaired, with immunosuppressive cellular niches. Our results identify important roles for LNs in anti-tumor immune responses in humans.

A Structured Tumor-Immune Microenvironment in Triple Negative Breast Cancer Revealed by Multiplexed Ion Beam Imaging.

The immune system is critical in modulating cancer progression, but knowledge of immune composition, phenotype, and interactions with tumor is limited. We used multiplexed ion beam imaging by time-of-flight (MIBI-TOF) to simultaneously quantify in situ expression of 36 proteins covering identity, function, and immune regulation at sub-cellular resolution in 41 triple-negative breast cancer patients. Multi-step processing, including deep-learning-based segmentation, revealed variability in the composition of tumor-immune populations across individuals, reconciled by overall immune infiltration and enriched co-occurrence of immune subpopulations and checkpoint expression. Spatial enrichment analysis showed immune mixed and compartmentalized tumors, coinciding with expression of PD1, PD-L1, and IDO in a cell-type- and location-specific manner. Ordered immune structures along the tumor-immune border were associated with compartmentalization and linked to survival. These data demonstrate organization in the tumor-immune microenvironment that is structured in cellular composition, spatial arrangement, and regulatory-protein expression and provide a framework to apply multiplexed imaging to immune oncology.

Mass cytometry reveals a conserved immune trajectory of recovery in hospitalized COVID-19 patients.

While studies have elucidated many pathophysiological elements of COVID-19, little is known about immunological changes during COVID-19 resolution. We analyzed immune cells and phosphorylated signaling states at single-cell resolution from longitudinal blood samples of patients hospitalized with COVID-19, pneumonia and/or sepsis, and healthy individuals by mass cytometry. COVID-19 patients showed distinct immune compositions and an early, coordinated, and elevated immune cell signaling profile associated with early hospital discharge. Intra-patient longitudinal analysis revealed changes in myeloid and T cell frequencies and a reduction in immune cell signaling across cell types that accompanied disease resolution and discharge. These changes, together with increases in regulatory T cells and reduced signaling in basophils, also accompanied recovery from respiratory failure and were associated with better outcomes at time of admission. Therefore, although patients have heterogeneous immunological baselines and highly variable disease courses, a core immunological trajectory exists that defines recovery from severe SARS-CoV-2 infection.

Innate type 2 immunity controls hair follicle commensalism by Demodex mites.

Demodex mites are commensal parasites of hair follicles (HFs). Normally asymptomatic, inflammatory outgrowth of mites can accompany malnutrition, immune dysfunction, and aging, but mechanisms restricting Demodex outgrowth are not defined. Here, we show that control of mite HF colonization in mice required group 2 innate lymphoid cells (ILC2s), interleukin-13 (IL-13), and its receptor, IL-4Ra-IL-13Ra1. HF-associated ILC2s elaborated IL-13 that attenuated HFs and epithelial proliferation at anagen onset; in their absence, Demodex colonization led to increased epithelial proliferation and replacement of gene programs for repair by aberrant inflammation, leading to the loss of barrier function and HF exhaustion. Humans with rhinophymatous acne rosacea, an inflammatory condition associated with Demodex, had increased HF inflammation with decreased type 2 cytokines, consistent with the inverse relationship seen in mice. Our studies uncover a key role for skin ILC2s and IL-13, which comprise an immune checkpoint that sustains cutaneous integrity and restricts pathologic infestation by colonizing HF mites.

Single-cell analysis by mass cytometry reveals metabolic states of early-activated CD8+ T cells during the primary immune response.

Memory T cells are thought to rely on oxidative phosphorylation and short-lived effector T cells on glycolysis. Here, we investigated how T cells arrive at these states during an immune response. To understand the metabolic state of rare, early-activated T cells, we adapted mass cytometry to quantify metabolic regulators at single-cell resolution in parallel with cell signaling, proliferation, and effector function. We interrogated CD8+ T cell activation in vitro and in response to Listeria monocytogenes infection in vivo. This approach revealed a distinct metabolic state in early-activated T cells characterized by maximal expression of glycolytic and oxidative metabolic proteins. Cells in this transient state were most abundant 5 days post-infection before rapidly decreasing metabolic protein expression. Analogous findings were observed in chimeric antigen receptor (CAR) T cells interrogated longitudinally in advanced lymphoma patients. Our study demonstrates the utility of single-cell metabolic analysis by mass cytometry to identify metabolic adaptations of immune cell populations in vivo and provides a resource for investigations of metabolic regulation of immune responses across a variety of applications.

Influence of Self-MHC Class I Recognition on the Dynamics of NK Cell Responses to Cytomegalovirus Infection.

Although interactions between inhibitory Ly49 receptors and their self-MHC class I ligands in C57BL/6 mice are known to limit NK cell proliferation during mouse CMV (MCMV) infection, we created a 36-marker mass cytometry (CyTOF) panel to investigate how these inhibitory receptors impact the NK cell response to MCMV in other phenotypically measurable ways. More than two thirds of licensed NK cells (i.e., those expressing Ly49C, Ly49I, or both) in uninfected mice had already differentiated into NK cells with phenotypes indicative of Ag encounter (KLRG1+Ly6C-) or memory-like status (KLRG1+Ly6C+). These pre-existing KLRG1+Ly6C+ NK cells resembled known Ag-specific memory NK cell populations in being less responsive to IL-18 and IFN-α stimulation in vitro and by selecting for NK cell clones with elevated expression of a Ly49 receptor. During MCMV infection, the significant differences between licensed and unlicensed (Ly49C-Ly49I-) NK cells disappeared within both CMV-specific (Ly49H+) and nonspecific (Ly49H-) responses. This lack of heterogeneity carried into the memory phase, with only a difference in CD16 expression manifesting between licensed and unlicensed MCMV-specific memory NK cell populations. Our results suggest that restricting proliferation is the predominant effect licensing has on the NK cell population during MCMV infection, but the inhibitory Ly49-MHC interactions that take place ahead of infection contribute to their limited expansion by shrinking the pool of licensed NK cells capable of robustly responding to new challenges.

Successful treatment of thrombotic microangiopathy associated with dengue infection: A case report and literature review.

Dengue infection has been associated with multiple renal complications, including glomerulonephritis, acute tubular necrosis, tubulointerstitial nephritis, and thrombotic microangiopathy (TMA), this last one being a rare complication of dengue, with only a few reported cases. TMA associated with dengue can be explained by an alteration in the activity of the enzyme ADAMTS13, leading to thrombotic thrombocytopenic purpura; or it can be secondary to direct or indirect endothelial injury by the virus, which leads to hemolytic uremic syndrome. Here, we present a case of severe TMA, not related to ADAMTS13, which was clearly associated with dengue infection.

Microplate quantification of total phenolic content from plant extracts obtained by conventional and ultrasound methods.

INTRODUCTION: There is increasing interest in phenolic compounds around the world because of their potential positive impact on human health. Phenolic compounds are largely found in fruits and vegetables. Extraction of phenolic compounds is a very important step in their recovery. The newly developed technique of ultrasound-assisted extraction (UAE) appears to be an advantageous alternative compared with conventional techniques, because it is simple and environmental friendly. The potential of UAE needs to be evaluated in each plant in order to demonstrate its efficiency. OBJECTIVE: The objective of the present study was to compare a conventional method and UAE on the extraction efficiency of phenolic compounds from Jatropha dioica, Fluorensia cernua, Turnera diffusa and Eucalyptus camaldulensis plants and evaluate the in vitro anti-oxidant potential. METHODS: Validation of the new method was carried out using mixed-model methodology and regression analysis. Feasibility of this new method was shown and applied using several plants extracts obtained by different extraction methods from semi-arid Mexican plants, which were characterised by high levels of polyphenols. Additionally, the anti-oxidant potential of these extracts was determined by 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. RESULTS: Results showed that the new microplate method can be used to determine total phenolic content in plant extracts. Additionally, an alternative extraction method by ultrasound was less efficient compared with the conventional method. CONCLUSION: The tested plants are good candidates to obtain nutraceuticals and functional food ingredients.

The tumor microenvironment of benign and malignant salivary gland tumors.

BACKGROUND: Treatment of salivary gland tumors (SGTs) remains challenging. Little is known about the immune landscape of SGTs. We aimed to characterize the tumor microenvironment in benign and malignant SGTs. METHODS: Eleven benign and nine malignant tumors were collected from patients undergoing curative intent surgery. Specimens were analyzed using mass cytometry by time-of-flight. Immune cell populations were manually gated, and T cells were clustered using the FlowSOM algorithm. Population frequencies were compared between high-grade and low-grade malignancies, corrected for multiple hypothesis testing. RESULTS: There were trends towards increased CD4+ and CD8+ T cells among malignant tumors. High-grade malignancies exhibited trends towards higher frequencies of CD8+ PD-1+ CD39+ CD103+ exhausted T cells, CD4+ FoxP3+ TCF-1+ CD127- Tregs, and CD69+ CD25- CD4+ T cells compared to low-grade malignancies. CONCLUSION: SGTs exhibit significant immunologic diversity. High-grade malignancies tended to have greater infiltration of exhausted CD8+ T cells and Tregs, which may guide future studies for immunotherapy strategies.

A mass cytometry approach to track the evolution of T cell responses during infection and immunotherapy by paired T cell receptor repertoire and T cell differentiation state analysis.

T cell receptor (TCR) recognition followed by clonal expansion is a fundamental feature of adaptive immune responses. Here, we developed a mass cytometric (CyTOF) approach combining antibodies specific for different TCR Vα- and Vß-chains with antibodies against T cell activation and differentiation proteins to identify antigen-specific expansions of T cell subsets and assess aspects of cellular function. This strategy allowed for the identification of expansions of specific Vß and Vα chain expressing CD8+ and CD4+ T cells with varying differentiation states in response to Listeria monocytogenes, tumors, and respiratory influenza infection. Expanded Vß chain expressing T cells could be directly linked to the recognition of specific antigens from Listeria, tumor cells, or influenza. In the setting of influenza infection, we showed that the common therapeutic approaches of intramuscular vaccination or convalescent serum transfer altered the clonal diversity and differentiation state of responding T cells. Thus, we present a new method to monitor broad changes in TCR specificity paired with T cell differentiation during adaptive immune responses.

Surfactant therapy using vibrating-mesh nebulizers in adults with COVID-19-induced ARDS: A case series.

Coronavirus adult respiratory distress syndrome, characterized by decreased surfactant due to lysis of type II pneumocytes and hyaline membrane formation, contributes to severe hypoxemia. The administration of surfactant via high-flow nasal cannula (HFNC) may positively affect lung structure and function in this context. In this study, we report on five clinical cases, encompassing patients aged 40-60 years of both sexes, who tested positive for coronavirus disease 2019 via real-time polymerase chain reaction and exhibited significant pulmonary compromise with elevated inflammatory biomarkers. These patients were treated with aerosol therapy using surfactant delivered through vibrating-mesh nebulizers alongside HFNC. Of these patients, four demonstrated positive responses to the treatment, suggesting that aerosol therapy with surfactant through vibrating-mesh nebulizers could be a viable rescue therapy in adults receiving HFNC oxygen therapy for hypoxemic respiratory failure caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Unfortunately, one patient had a negative outcome and succumbed. The findings from these cases indicate that the use of aerosol therapy with vibrating-mesh nebulizers as rescue therapy might offer an alternative approach for managing adults with hypoxemic respiratory failure due to SARS-CoV-2, as evidenced by the positive outcomes in four out of the five cases presented.

HPLC-ESI-IT-MS/MS analysis and biological activity of triterpene glycosides from the Colombian marine sponge Ectyoplasia ferox.

The marine sponge Ectyoplasia ferox produces antipredatory and allelopathic triterpenoid glycosides as part of its chemical defense repertoire against predators, competitors, and fouling organisms. These molecules are responsible for the pharmacological potential found in the glycosides present in this species. In order to observe the glycochemical diversity present in E. ferox, a liquid chromatography coupled to a tandem mass spectrometry approach to analyse a complex polar fraction of this marine sponge was performed. This gave valuable information for about twenty-five compounds three of which have been previously reported and another three which were found to be composed of known aglycones. Furthermore, a group of four urabosides, sharing two uncommon substitutions with carboxyl groups at C-4 on the terpenoid core, were identified by a characteristic fragmentation pattern. The oxidized aglycones present in this group of saponins can promote instability, making the purification process difficult. Cytotoxicity, cell cycle modulation, a cell cloning efficiency assay, as well as its hemolytic activity were evaluated. The cytotoxic activity was about IC50 40 µg/mL on Jurkat and CHO-k1 cell lines without exhibiting hemolysis. Discussion on this bioactivity suggests the scanning of other biological models would be worthwhile.

Ulososides and urabosides--triterpenoid saponins from the Caribbean marine sponge Ectyoplasia ferox.

Three new triterpene glycosides, named ulososide F (1), urabosides A (2) and B (3), together with the previously reported ulososide A (4), were isolated from the Caribbean marine sponge Ectyoplasia ferox. Their structures were elucidated using extensive interpretation of 1D and 2D-NMR data, as well as HRESIMS. The aglycon of all compounds is a rare 30-norlonastane and the sugar residues were identified after acid hydrolysis and GC analyses. Cytotoxicities of the isolated compounds were evaluated against Jurkat and CHO cell lines by a MTT in vitro assay as well as a hemolysis assay. Unexpectedly, all these saponin derivatives showed very low activity in our bioassays.

The Effect of Participatory Heat Education on Agricultural Worker Knowledge.

OBJECTIVES: Farmworkers disproportionately experience preventable adverse health effects from heat exposure. We sought to evaluate the effect of participatory heat education on farmworker knowledge. METHODS: We conducted a parallel, comparison group intervention study to investigate the effectiveness of a Spanish/English participatory, culturally-tailored, heat education-based intervention on farmworker heat knowledge in the Summer 2019. We used convenience sampling to recruit adult outdoor farmworkers from Central/Eastern Washington State, USA. Crews were randomized to receive the intervention (n = 40 participants) versus not receive the intervention (n = 43 participants). We assessed changes in heat knowledge, scored on a scale from 0 to 11, between baseline, immediate post-intervention, and post-season, which was approximately three months after baseline, using the Wilcoxon signed-rank test. We compared differences in knowledge scores from baseline to post-season between groups using analysis of variance. RESULTS: Average knowledge scores improved from 4.6 (standard deviation [sd] 1.5) to 6.3 (sd 2.0) pre to post season in the intervention group (p < 0.001). There was a greater improvement in pre-post knowledge scores in the intervention (average difference 1.6, sd 2.0) versus the comparison group (average difference 0.41, sd 1.7) (p = 0.04). CONCLUSIONS: Participatory heat training was effective in improving farmworker heat knowledge over the course of a summer season. Results of this study will be used to guide heat prevention efforts for farmworkers. TRIAL REGISTRATION: ClinicalTrials.gov Registration Number: NCT04234802.

An Unusual Case of Severe Pneumonia Caused Due Candida Tropicalis With a Favorable Clinical Response to Antifungals in a Nonimmunocompromised Patient From the Community.

Severe pneumonia due to Candida tropicalis infection mainly occurs in immunosuppressed patients or those currently receiving broad-spectrum antibiotics. Herein, we report a case of severe pneumonia caused due to C tropicalis in an elderly patient. A 72-year-old man with a previous history of hypertension, ischemic stroke, and facial paralysis sequelae treated with the botulinic toxin, was admitted to the hospital for dyspnea. Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) infection was negative. Computed tomography of the chest revealed bilateral consolidation with left predominance. A bronchoalveolar lavage sample was sent to molecular biology, but no microorganisms were detected using a FilmArray respiratory panel. However, mamanocandidas test for candida was 166 pg/mL (positive), and fungal structures were identified by the MALDI-TOF Biotyper mass spectrometry and attributed to C tropicalis. Antifungal therapy was started using caspofungin 75 mg as the initial dose followed by 50 mg daily. After 10 days of treatment, ventilatory weaning was achieved. By day 14, the patient was decannulated from the tracheostomy. Oral antifungal treatment with voriconazole was continued, and he was discharged from intensive care in good clinical condition. Severe pneumonia due to C tropicalis might occur in specific cases, especially in those patients with risk factors, and must thus be considered when approaching such cases.

Use of threshold PEP with an adult non-rebreather oxygen mask plus prone positioning in acute hypoxemic respiratory failure due to SARS-CoV-2 infection during the collapse of the health system in a low-income country.

During the coronavirus disease 2019 pandemic, Ecuador reported a collapse of the healthcare system, in which intensive care unit beds were lacking. Therefore, we sought to determine whether the use of threshold expiratory positive pressure with an adult non-rebreather oxygen mask plus prone positioning is useful for improving oxygenation. Twelve patients were included. Eight patients (66.7%) survived, while four patients (33.3%) died. Baseline arterial oxygen saturation (%) prior placement median (interquartile range) 85.5% (80%-89%) and arterial oxygen saturation (%) post placement of the device was median (interquartile range) (93%-96%) (P = 0.0001). Respiratory rate before placement was median (interquartile range) 38 (36-42) and post placement of the device was median (interquartile range) 24 (22-30) (P = 0.0005). The use of an adapted device might be useful for the management of acute hypoxemic respiratory failure due to severe acute respiratory syndrome coronavirus 2 pneumonia, particularly when mechanical ventilators and high-flow oxygen systems are unavailable.

Quality of red tilapia viscera oil (Oreochromis sp.) as a function of extraction methods.

This study aimed to propose a simple and efficient heating-freezing method for oil recovery from red tilapia (Oreochromis sp.) viscera, suitable for industrial application and that does not affect its composition. Three methodologies for oil extraction were studied: a) direct heating (69 °C and 29 min) of samples followed by separation of the oil by decantation, b) direct heating with subsequent freezing and c) solvent extraction assisted by ultrasound. For the oil obtained by each methodology, the following factors were determined: peroxide and iodine values, oxidative stability index, yield percentages and fatty acid profile and, to evaluate the changes thereof, a thermal analysis by differential scanning calorimetry was performed. An oil extracted by centrifugation from fresh viscera was used as control. Results showed yields of 92,126%, 60,99% and 55,36% for the oil obtained by heating and freezing, heating and decanting and solvent extraction, respectively, the other evaluated parameters were similar among each other. The content of PUFA was not affected by heating when compared to the control oil, although a decrease was observed in the solvent extracted oil. This behavior was corroborated with the thermal analysis, which showed that the higher PUFA content, the lower the melting temperatures of the oils and the energy required for phase change. A principal component analysis allowed determining that while there are no differences in the abundance of fatty acids C20:1, 14:0, 18:0, 16:1 and C16:0, there are differences for fatty acids C18:1 and C18:2 depending on the method of extraction used in the oil obtention. The results of this study show that the heating-freezing extraction method is a good alternative for acquiring value-added products and facilitates their implementation in rural areas. Furthermore, allows obtaining a product with high content of polyunsaturated fatty acids (at least a third of the total content).

Noninvasive Mechanical Ventilation with Average Volume-Assured Pressure Support versus BiPAP S/T in De Novo Hypoxemic Respiratory Failure.

Background: Bilevel positive airway pressure in spontaneous/time and average volume-assured pressure support (BiPAP·S/T-AVAPS) could maintain an adequate tidal volume by reducing the patient's inspiratory effort; however, this ventilatory strategy has not been compared with other ventilatory modes, especially the conventional BiPAP S/T mode, when noninvasive mechanical ventilation (NIMV) is used. The primary objective of this study was to determine the rate of success and failure of the use of BiPAP·S/T-AVAPS versus BiPAP·S/T alone in patients with mild-to-moderate "de novo" hypoxemic respiratory failure. Methods: This was a matched-cohort study. Subjects with mild-to-moderate de novo hypoxemic respiratory failure were divided into two groups according to the ventilatory strategy used. The subjects in the BiPAP·S/T group were paired with those in the BiPAP·S/T-AVAPS group. Results: A total of 58 subjects were studied. Twenty-nine subjects in the BiPAP·S/T group were paired with 29 subjects in the BiPAP·S/T-AVAPS group. Twenty patients (34.5%) presented with "failure of NIMV," while 38 (65.5%) patients did not. In addition, 13 (22.4%) patients died, while 45 (77.6%) recovered. No differences were found in the percentage of intubation (P=0.44) and mortality (P=0.1). Conclusion: The BiPAP S/T-AVAPS ventilator mode was not superior to the BiPAP·S/T mode. A high mortality rate was observed in patients with NIMV failure in both modes. This trial is registered with https://doi.org/10.1186/ISRCTN17904857.