Human lymph node fibroblastic reticular cells maintain heterogeneous characteristics in culture.

Fibroblastic reticular cells (FRCs) are mesenchymal stromal cells in human lymph nodes (LNs) playing a pivotal role in adaptive immunity. Several FRC subsets have been identified, yet it remains to be elucidated if their heterogeneity is maintained upon culture. Here, we established a protocol to preserve and culture FRCs from human LNs and characterized their phenotypic profile in fresh LN suspensions and upon culture using multispectral flow cytometry. We found nine FRC subsets in fresh human LNs, independent of donor, of which four persisted in culture throughout several passages. Interestingly, the historically FRC-defining marker podoplanin (PDPN) was not present on all FRC subsets. Therefore, we propose that CD45negCD31neg human FRCs are not restricted by PDPN expression, as we found CD90, BST1, and CD146/MCAM to be more widely expressed. Together, our data provide insight into FRC heterogeneity in human LNs, enabling further investigation into the function of individual FRC subsets.

Unbiased method for spectral analysis of cells with great diversity of autofluorescence spectra.

Autofluorescence is an intrinsic feature of cells, caused by the natural emission of light by photo-excitatory molecular content, which can complicate analysis of flow cytometry data. Different cell types have different autofluorescence spectra and, even within one cell type, heterogeneity of autofluorescence spectra can be present, for example, as a consequence of activation status or metabolic changes. By using full spectrum flow cytometry, the emission spectrum of a fluorochrome is captured by a set of photo detectors across a range of wavelengths, creating an unique signature for that fluorochrome. This signature is then used to identify, or unmix, that fluorochrome's unique spectrum from a multicolor sample containing different fluorescent molecules. Importantly, this means that this technology can also be used to identify intrinsic autofluorescence signal of an unstained sample, which can be used for unmixing purposes and to separate the autofluorescence signal from the fluorophore signals. However, this only works if the sample has a singular, relatively homogeneous and bright autofluorescence spectrum. To analyze samples with heterogeneous autofluorescence spectral profiles, we setup an unbiased workflow to more quickly identify differing autofluorescence spectra present in a sample to include as "autofluorescence signatures" during the unmixing of the full stained samples. First, clusters of cells with similar autofluorescence spectra are identified by unbiased dimensional reduction and clustering of unstained cells. Then, unique autofluorescence clusters are determined and are used to improve the unmixing accuracy of the full stained sample. Independent of the intensity of the autofluorescence and immunophenotyping of cell subsets, this unbiased method allows for the identification of most of the distinct autofluorescence spectra present in a sample, leading to less confounding autofluorescence spillover and spread into extrinsic phenotyping markers. Furthermore, this method is equally useful for spectral analysis of different biological samples, including tissue cell suspensions, peripheral blood mononuclear cells, and in vitro cultures of (primary) cells.

Pancreatic cancer-associated fibroblasts modulate macrophage differentiation via sialic acid-Siglec interactions.

Despite recent advances in cancer immunotherapy, pancreatic ductal adenocarcinoma (PDAC) remains unresponsive due to an immunosuppressive tumor microenvironment, which is characterized by the abundance of cancer-associated fibroblasts (CAFs). Once identified, CAF-mediated immune inhibitory mechanisms could be exploited for cancer immunotherapy. Siglec receptors are increasingly recognized as immune checkpoints, and their ligands, sialic acids, are known to be overexpressed by cancer cells. Here, we unveil a previously unrecognized role of sialic acid-containing glycans on PDAC CAFs as crucial modulators of myeloid cells. Using multiplex immunohistochemistry and transcriptomics, we show that PDAC stroma is enriched in sialic acid-containing glycans compared to tumor cells and normal fibroblasts, and characterized by ST3GAL4 expression. We demonstrate that sialic acids on CAF cell lines serve as ligands for Siglec-7, -9, -10 and -15, distinct from the ligands on tumor cells, and that these receptors are found on myeloid cells in the stroma of PDAC biopsies. Furthermore, we show that CAFs drive the differentiation of monocytes to immunosuppressive tumor-associated macrophages in vitro, and that CAF sialylation plays a dominant role in this process compared to tumor cell sialylation. Collectively, our findings unravel sialic acids as a mechanism of CAF-mediated immunomodulation, which may provide targets for immunotherapy in PDAC.

In-depth immune profiling of peripheral blood mononuclear cells in patients with pancreatic ductal adenocarcinoma reveals discriminative immune subpopulations.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis with a 5-year survival of less than 10%. More knowledge of the immune response developed in patients with PDAC is pivotal to develop better combination immune therapies to improve clinical outcome. In this study, we used mass cytometry time-of-flight to undertake an in-depth characterization of PBMCs from patients with PDAC and examine the differences with healthy controls and patients with benign diseases of the biliary system or pancreas. Peripheral blood mononuclear cells from patients with PDAC or benign disease are characterized by the increase of pro-inflammatory cells, as CD86+ classical monocytes and memory T cells expressing CCR6+ and CXCR3+, associated with T helper 1 (Th1) and Th17 immune responses, respectively. However, PBMCs from patients with PDAC present also an increase of CD39+ regulatory T cells and CCR4+CCR6-CXCR3- memory T cells, suggesting Th2 and regulatory responses. Concluding, our results show PDAC develops a multifaceted immunity, where a proinflammatory component is accompanied by regulatory responses, which could inhibit potential antitumor mechanisms.

Beyond lectures and practical courses: Teaching pharmacology using imaginative pedagogical tools.

Pharmacology has broadened its scope considerably in recent decades. Initially, it was of interest to chemists, doctors and pharmacists. In recent years, however, it has been incorporated into the teaching of biologists, molecular biologists, biotechnologists, chemical engineers and many health professionals, among others. Traditional teaching methods, such as lectures or laboratory work, have been superseded by the use of new pedagogical approaches to enable a better conceptualization and understanding of the discipline. In this article, we present several new methods that have been used in Spanish universities. Firstly, we describe a teaching network that has allowed the sharing of pedagogical innovations in Spanish universities. A European experience to improve prescribing safety is described in detail. The use of popular films and medical TV series in biomedical students shows how these audiovisual resources can be helpful in teaching pharmacology. The use of virtual worlds is detailed to introduce this new approach to teaching. The increasingly important area of the social aspects of pharmacology is also considered in two sections, one devoted to social pharmacology and the other to the use of learning based on social services to improve understanding of this important area. Finally, the use of Objective Structured Clinical Evaluation in pharmacology allows to know how this approach can help to better evaluate clinical pharmacology students. In conclusion, this article allows to know new pedagogical methods resources used in some Spanish universities that may help to improve the teaching of pharmacology.

The role of CD56bright NK cells in neurodegenerative disorders.

Emerging evidence suggests a potential role for natural killer (NK) cells in neurodegenerative diseases, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. However, the precise function of NK cells in these diseases remains ambiguous. The existence of two NK cell subsets, CD56bright and CD56dim NK cells, complicates the understanding of the contribution of NK cells in neurodegeneration as their functions within the context of neurodegenerative diseases may differ significantly. CD56bright NK cells are potent cytokine secretors and are considered more immunoregulatory and less terminally differentiated than their mostly cytotoxic CD56dim counterparts. Hence, this review focusses on NK cells, specifically on CD56bright NK cells, and their role in neurodegenerative diseases. Moreover, it explores the mechanisms underlying their ability to enter the central nervous system. By consolidating current knowledge, we aim to provide a comprehensive overview on the role of CD56bright NK cells in neurodegenerative diseases. Elucidating their impact on neurodegeneration may have implications for future therapeutic interventions, potentially ameliorating disease pathogenesis.

The effects of health-related food taxes on the environmental impact of consumer food purchases: secondary analysis of data from a randomised controlled trial in a virtual supermarket.

OBJECTIVE: To measure the effects of health-related food taxes on the environmental impact of consumer food purchases in a virtual supermarket. DESIGN: This is a secondary analysis of data from a randomised controlled trial in which participants were randomly assigned to a control condition with regular food prices (n 152), an experimental condition with a sugar-sweetened beverage (SSB) tax (n 131) or an experimental condition with a nutrient profiling tax based on Nutri-Score (n 112). Participants were instructed to undertake their typical weekly grocery shopping for their households. Primary outcome measures were three environmental impact indicators: greenhouse gas (GHG) emissions, land use and blue water use per household per week. Data were analysed using linear regression analyses. SETTING: Three-dimensional virtual supermarket. PARTICIPANTS: Dutch adults (≥ 18 years) who were responsible for grocery shopping in their household (n 395). RESULTS: GHG emissions (-7·6 kg CO2-eq; 95 % CI -12·7, -2·5) and land use (-3·9 m2/year; 95 % CI -7·7, -0·2) were lower for the food purchases of participants in the nutrient profiling tax condition than for those in the control condition. Blue water use was not affected by the nutrient profiling tax. Moreover, the SSB tax had no significant effect on any of the environmental impact indicators. CONCLUSIONS: A nutrient profiling tax based on Nutri-Score reduced the environmental impact of consumer food purchases. An SSB tax did not affect the environmental impact in this study.

Cancer-on-chip models for metastasis: importance of the tumor microenvironment.

Cancer-on-chip (CoC) models, based on microfluidic chips harboring chambers for 3D tumor-cell culture, enable us to create a controlled tumor microenvironment (TME). CoC models are therefore increasingly used to systematically study effects of the TME on the various steps in cancer metastasis. Moreover, CoC models have great potential for developing novel cancer therapies and for predicting patient-specific response to cancer treatments. We review recent developments in CoC models, focusing on three main TME components: (i) the anisotropic extracellular matrix (ECM) architectures, (ii) the vasculature, and (iii) the immune system. We aim to provide guidance to biologists to choose the best CoC approach for addressing questions about the role of the TME in metastasis, and to inspire engineers to develop novel CoC technologies.

Towards Full Thickness Small Intestinal Models: Incorporation of Stromal Cells.

INTRODUCTION: Since small intestine is one of the major barriers of the human body, there is a need to develop reliable in vitro human small intestinal models. These models should incorporate both the epithelial and lamina propria compartments and have similar barrier properties compared to that of the human tissue. These properties are essential for various applications, such as studying cell-cell interaction, intestinal diseases and testing permeability and metabolism of drugs and other compounds. The small intestinal lamina propria contains multiple stromal cell populations with several important functions, such as secretion of extracellular matrix proteins and soluble mediators. In addition, stromal cells influence the intestinal epithelial barrier, support the intestinal stem cell niche and interact with immune cells. METHODS: In this review, we provide an extensive overview on the different types of lamina propria stromal cells found in small intestine and describe a combination of molecular markers that can be used to distinguish each different stromal cell type. We focus on studies that incorporated stromal cells into human representative small intestine models cultured on transwells. RESULTS AND CONCLUSION: These models display enhanced epithelial morphology, increased cell proliferation and human-like barrier properties, such as low transepithelial electrical resistance (TEER) and intermediate permeability, thus better mimicking the native human small intestine than models only consisting of an epithelium which generally show high TEER and low permeability.

An Organotypic Human Lymph Node Model Reveals the Importance of Fibroblastic Reticular Cells for Dendritic Cell Function.

BACKGROUND: Human lymph node (HuLN) models have emerged with invaluable potential for immunological research and therapeutic application given their fundamental role in human health and disease. While fibroblastic reticular cells (FRCs) are instrumental to HuLN functioning, their inclusion and recognition of importance for organotypic in vitro lymphoid models remain limited. METHODS: Here, we established an in vitro three-dimensional (3D) model in a collagen-fibrin hydrogel with primary FRCs and a dendritic cell (DC) cell line (MUTZ-3 DC). To study and characterise the cellular interactions seen in this 3D FRC-DC organotypic model compared to the native HuLN; flow cytometry, immunohistochemistry, immunofluorescence and cytokine/chemokine analysis were performed. RESULTS: FRCs were pivotal for survival, proliferation and localisation of MUTZ-3 DCs. Additionally, we found that CD1a expression was absent on MUTZ-3 DCs that developed in the presence of FRCs during cytokine-induced MUTZ-3 DC differentiation, which was also seen with primary monocyte-derived DCs (moDCs). This phenotype resembled HuLN-resident DCs, which we detected in primary HuLNs, and these CD1a- MUTZ-3 DCs induced T cell proliferation within a mixed leukocyte reaction (MLR), indicating a functional DC status. FRCs expressed podoplanin (PDPN), CD90 (Thy-1), CD146 (MCAM) and Gremlin-1, thereby resembling the DC supporting stromal cell subset identified in HuLNs. CONCLUSION: This 3D FRC-DC organotypic model highlights the influence and importance of FRCs for DC functioning in a more realistic HuLN microenvironment. As such, this work provides a starting point for the development of an in vitro HuLN.

Unraveling the impact of sialic acids on the immune landscape and immunotherapy efficacy in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Despite the successful application of immune checkpoint blockade in a range of human cancers, immunotherapy in PDAC remains unsuccessful. PDAC is characterized by a desmoplastic, hypoxic and highly immunosuppressive tumor microenvironment (TME), where T-cell infiltration is often lacking (immune desert), or where T cells are located distant from the tumor islands (immune excluded). Converting the TME to an immune-inflamed state, allowing T-cell infiltration, could increase the success of immunotherapy in PDAC. METHOD: In this study, we use the KPC3 subcutaneous PDAC mouse model to investigate the role of tumor-derived sialic acids in shaping the tumor immune landscape. A sialic acid deficient KPC3 line was generated by genetic knock-out of the CMAS (cytidine monophosphate N-acetylneuraminic acid synthetase) enzyme, a critical enzyme in the synthesis of sialic acid-containing glycans. The effect of sialic acid-deficiency on immunotherapy efficacy was assessed by treatment with anti-programmed cell death protein 1 (PD-1) and agonistic CD40. RESULT: The absence of sialic acids in KPC3 tumors resulted in increased numbers of CD4+ and CD8+ T cells in the TME, and reduced frequencies of CD4+ regulatory T cells (Tregs) within the T-cell population. Importantly, CD8+ T cells were able to infiltrate the tumor islands in sialic acid-deficient tumors. These favorable alterations in the immune landscape sensitized sialic acid-deficient tumors to immunotherapy, which was ineffective in sialic acid-expressing KPC3 tumors. In addition, high expression of sialylation-related genes in human pancreatic cancer correlated with decreased CD8+ T-cell infiltration, increased presence of Tregs, and poorer survival probability. CONCLUSION: Our results demonstrate that tumor-derived sialic acids mediate T-cell exclusion within the PDAC TME, thereby impairing immunotherapy efficacy. Targeting sialic acids represents a potential strategy to enhance T-cell infiltration and improve immunotherapy outcomes in PDAC.

Spatial immune composition of tumor microenvironment in patients with pancreatic cancer.

This study examined the composition of the immune microenvironment at different sites within resected pancreas specimens from patients with pancreatic ductal adenocarcinoma (PDAC). Therefore, single-cell suspensions were made from fresh tumor and non-tumorous tissue. Fourteen patients were included from whom twelve PDAC and five non-tumorous samples were obtained. These samples were analyzed with a nineteen marker panel on the Aurora spectral flow cytometer. Furthermore, slides from formalin-fixed paraffine PDACs of eight additional patients were stained with eight markers and analyzed by multispectral imaging. These corresponded to central tumor, periphery of the tumor, i.e., invasive front and resected lymph node and were divided into tumor and adjacent tissue. In the single-cell suspension, a decreased ratio between lymphoid and myeloid cells and between M1 and M2 macrophages was observed in the tumor tissue compared to non-tumorous tissue. Furthermore, an increase in CD169 + macrophages in patients undergoing neoadjuvant therapy was found. Using immunofluorescence, more macrophages compared to T cells were observed, as well as a lower ratio of CD8 to M2 macrophage, a higher ratio of CD4-CD8 T cells and a higher ratio of immune-suppressive cells to pro-inflammatory cells in the PDAC area compared to the adjacent non-tumorous tissue. Finally, there were more immune-suppressive cells in the central tumor area compared to the invasive front. In conclusion, we show a gradient in the immune-suppressive environment in PDAC from most suppressive in the central tumor to least suppressive in distant non-tumorous tissue.

The immune microenvironment after neoadjuvant therapy compared to upfront surgery in patients with pancreatic cancer.

BACKGROUND: Patients with resectable and borderline resectable pancreatic ductal adenocarcinoma increasingly receive neoadjuvant therapy prior to surgery. However, the effect of neoadjuvant therapy on the immune microenvironment remains largely unknown. We analyzed the immune microenvironment in pancreatic cancer tumor tissue samples from patients treated with neoadjuvant therapy compared to patients after upfront surgery to gain knowledge about the immunological environment after therapy. METHODS: Multispectral imaging was performed on tissue from resected specimens from patients with PDAC who underwent upfront surgery (n = 10), neoadjuvant FOLFIRINOX (n = 10) or gemcitabine + radiotherapy (gem-RT) (n = 9) followed by surgery. The samples were selected by a dedicated pancreas pathologist from both the central part and the invasive front of the tumor (by the resected vein or venous surface) and subsequently analyzed using the Vectra Polaris. RESULTS: Patients receiving neoadjuvant FOLFIRINOX display a more pro-inflammatory immune profile, with less regulatory T cells and more CD8 T cells in the tumor tissue compared to patients receiving neoadjuvant gem-RTgem-RT or undergoing upfront surgery. Furthermore, CD163+ macrophages were decreased, and a higher CD163- macrophages versus CD163+ macrophages ratio was found in patients with neoadjuvant FOLFIRINOX. In all treatment groups, percentage of FoxP3+ B cells was significantly higher in tumor tissue compared to adjacent tissue. Furthermore, an increase in regulatory T cells in the tumor tissue was found in patients undergoing upfront surgery or receiving neoadjuvant gem-RT. In the gem-RT group, less CD8 T cells and a higher CD163+ macrophages to CD8 ratio were noted in the tumor tissue, suggesting a more immune suppressive profile in the tumor tissue. CONCLUSION: Patients receiving neoadjuvant FOLFIRINOX display a more pro-inflammatory immune profile compared to patients receiving neoadjuvant gem-RT or undergoing upfront surgery. Furthermore, in all treatment groups, a more immune suppressive microenvironment was found in the tumor tissue compared to the adjacent non-tumorous tissue.

Different Levels of Ultraprocessed Food and Beverage Consumption and Associations with Environmental Sustainability and All-cause Mortality in EPIC-NL.

BACKGROUND: The adverse health effects of high ultraprocessed food and drink (UPFD) consumption are well documented. However, the environmental impact remains unclear, and the separate effects of ultraprocessed foods (UPFs) and drinks (UPDs) on all-cause mortality have not been studied previously. OBJECTIVES: To assess the association between levels of UPFD, UPF, and UPD consumption and diet-related environmental impacts and all-cause mortality in Dutch adults. METHODS: Habitual diets were assessed by a Food Frequency Questionnaire (FFQ) from 1993-1997 in 38,261 participants of the Dutch European Prospective Investigation into Cancer and Nutrition cohort. The mean follow-up time was 18.2 y (SD = 4.1); 4,697 deaths occurred. FFQ items were categorized according to the NOVA classification. Associations between quartiles of UPFD, UPF, and UPD consumption and environmental impact indicators were analyzed using general linear models and all-cause mortality by Cox proportional hazard models. The lowest UPFD, UPF, and UPD consumption quartiles were used as comparator. RESULTS: The average UPFD consumption was 181 (SD = 88) g/1000 kcal. High UPF consumption was statistically significantly inversely associated with all environmental impact indicators (Q4vsQ1: -13.6% to -3.0%), whereas high UPD consumption was, except for land use, statistically significant positively associated with all environmental impact indicators (Q4vsQ1: 1.2% to 5.9%). High UPFD consumption was heterogeneously associated with environmental impacts (Q4vsQ1: -4.0% to 2.6%). After multivariable adjustment, the highest quartiles of UPFD and UPD consumption were significantly associated with all-cause mortality (HRQ4vsQ1: 1.17, 95%CI: 1.08, 1.28 and HRQ4vsQ1: 1.16, 95%CI: 1.07, 1.26, respectively). UPF consumption of Q2 and Q3 were associated with a borderline significant lower risk of all-cause mortality (HRQ2vsQ1: 0.93, 95% CI: 0.85, 1.00; HRQ3vsQ1: 0.91, 95% CI: 0.84, 0.99) whereas Q4 was not statistically significant (HRQ4vsQ1: 1.06, 95% CI: 0.97, 1.15). CONCLUSIONS: Reducing UPD consumption may lower environmental impact and all-cause mortality risk; however, this is not shown for UPFs. When categorizing food consumption by their degree of processing, trade-offs are observed for human and planetary health aspects.

Adherence to the EAT-Lancet Healthy Reference Diet in Relation to Risk of Cardiovascular Events and Environmental Impact: Results From the EPIC-NL Cohort.

Background The Healthy Reference Diet (HRD) was created to formulate dietary guidelines that would be healthy and sustainable. We aimed to construct a diet score measuring adherence to the HRD and to explore its association with cardiovascular events and environmental impact. Methods and Results We included 35 496 participants from the population-based EPIC-NL (European Prospective Investigation into Cancer and Nutrition-Netherlands) study. HRD scores were calculated using data from food frequency questionnaires (0-140). Data on morbidity and mortality were retrieved through linkage with national and death registries. Data on environmental impact indicators were obtained from life cycle assessments. Associations between adherence to the HRD and cardiovascular events were estimated with Cox proportional hazard models. Linear regression analysis was conducted for the adherence to the HRD and each environmental indicator. High adherence to the HRD was associated with 14%, 12%, and 11% lower risks of cardiovascular disease (hazard ratio [HR]Q4vsQ1, 0.86 [95% CI, 0.78-0.94]), coronary heart disease (HRQ4vsQ1, 0.88 [95% CI, 0.78-1.00]), and total stroke (HRQ4vsQ1, 0.89 [95% CI, 0.72-1.10]), respectively. High HRD adherence was associated with 2.4% (95% CI, -5.0 to 0.2) lower greenhouse gas emissions, 3.9% (95% CI, -5.2 to -2.6) less land use, 0.5% (95% CI, -2.6 to 1.6), less freshwater eutrophication, 3.3% (95% CI, -5.8 to -0.8), less marine eutrophication, 7.7% (95% CI, -10.8 to -4.6), less terrestrial acidification, and 32.1 % (95% CI, 28.5-35.7) higher blue water use. Conclusions High adherence to the HRD was associated with lower risk of cardiovascular disease, coronary heart disease, and modestly lower levels of most environmental indicators but a higher level of blue water use.

A synthetic human 3D in vitro lymphoid model enhancing B-cell survival and functional differentiation.

To investigate B-cell differentiation and maturation occurring in the germinal center (GC) using in vitro culture systems, key factors and interactions of the GC reaction need to be accurately simulated. This study aims at improving in vitro GC simulation using 3D culture techniques. Human B-cells were incorporated into PEG-4MAL hydrogels, to create a synthetic extracellular matrix, supported by CD40L cells, human tonsil-derived lymphoid stromal cells, and cytokines. The differentiation and antibody production of CD19+B-cells was best supported in a 5.0%-PEG-4MAL, 2.0 mM-RGD-peptide composition. The 3D culture significantly increased plasmablast and plasma cell numbers as well as antibody production, with less B-cell death compared to 2D cultures. Class switching of naive CD19+IgD+B-cells toward IgG+ and IgA+B-cells was observed. The formation of large B-cell clusters indicates the formation of GC-like structures. In conclusion, a well-characterized and controllable hydrogel-based human 3D lymphoid model is presented that supports enhanced B-cell survival, proliferation, differentiation, and antibody production.

Factors that influence the pancreatic and duodenal microbiome in patients undergoing pancreatic surgery.

BACKGROUND/OBJECTIVES: This study examined the correlation between pancreatic microbiome and patients characteristics. Furthermore, we compared different duodenal materials to examine their reflection of the pancreatic microbiome. METHODS: Patients undergoing pancreatic surgery were included in the study. Characteristics of those patients were prospectively registered and sterile pancreatic biopsies were collected during surgery. After completion of the resection, duodenal fluid, -tissue and -swab were collected. Bacterial DNA was extracted and analyzed with IS-pro assay. RESULTS: Paired samples of 51 patients were available for evaluation, including pancreatic biopsies from all patients, 22 duodenal fluids, 21 duodenal swabs and 11 duodenal tissues. The pancreatic microbiome consisted mostly of Proteobacteria followed by Firmicutes, Actinobacteria, Fusobacteria and Verrucomicrobia (FAFV) and Bacteroidetes. On species level, Enterococcus faecalis, Escherichia coli, and Enterobacter-Klebsiella were most abundant. In pancreatic biopsies, the total bacterial load and Proteobacteria load were significantly higher in patients with biliary drainage (54618.0 vs 5623.5; 9119.0 vs 2067.1). Patients who used proton pump inhibitors had a significantly higher total bacterial load (115964.7 vs 8495.8), more FAFV (66862.9 vs 1890.1), more Proteobacteria (24245.9 vs 2951.4) and more Bacteroidetes (542.5 vs 25.8). The head of the pancreas contained significantly more bacteria (21193.4 vs 2096.8) and more FAFV (5225.7 vs 19.0) compared to the tail, regardless of biliary drainage. Furthermore, the microbiome of all duodenal materials showed a weak correlation with the pancreatic microbiome. CONCLUSION: Biliary drainage, use of proton pump inhibitors, and anatomic location of the pancreatic biopsy influence the pancreatic microbiome. Furthermore, the duodenal microbiome does not suffice as a surrogate for the pancreatic microbiome.

Fcα Receptor-1-Activated Monocytes Promote B Lymphocyte Migration and IgA Isotype Switching.

Patients with inflammatory bowel disease (IBD) produce enhanced immunoglobulin A (IgA) against the microbiota compared to healthy individuals, which has been correlated with disease severity. Since IgA complexes can potently activate myeloid cells via the IgA receptor FcαRI (CD89), excessive IgA production may contribute to IBD pathology. However, the cellular mechanisms that contribute to dysregulated IgA production in IBD are poorly understood. Here, we demonstrate that intestinal FcαRI-expressing myeloid cells (i.e., monocytes and neutrophils) are in close contact with B lymphocytes in the lamina propria of IBD patients. Furthermore, stimulation of FcαRI-on monocytes triggered production of cytokines and chemokines that regulate B-cell differentiation and migration, including interleukin-6 (IL6), interleukin-10 (IL10), tumour necrosis factor-α (TNFα), a proliferation-inducing ligand (APRIL), and chemokine ligand-20 (CCL20). In vitro, these cytokines promoted IgA isotype switching in human B cells. Moreover, when naïve B lymphocytes were cultured in vitro in the presence of FcαRI-stimulated monocytes, enhanced IgA isotype switching was observed compared to B cells that were cultured with non-stimulated monocytes. Taken together, FcαRI-activated monocytes produced a cocktail of cytokines, as well as chemokines, that stimulated IgA switching in B cells, and close contact between B cells and myeloid cells was observed in the colons of IBD patients. As such, we hypothesize that, in IBD, IgA complexes activate myeloid cells, which in turn can result in excessive IgA production, likely contributing to disease pathology. Interrupting this loop may, therefore, represent a novel therapeutic strategy.

Electrokinetic and dielectric response of a concentrated salt-free colloid: Different approaches to counterion finite-size effects.

In the present work, a general model is developed for the electrokinetics and dielectric response of a concentrated salt-free colloid that takes into account the finite size of the counterions released by the particles to the solution. The effects associated with the counterion finite size have been addressed using a hard-sphere model approach elaborated by Carnahan and Starling [N. F. Carnahan and K. E. Starling, Equation of state for nonattracting rigid spheres, J. Chem. Phys. 51, 635 (1969)0021-960610.1063/1.1672048]. A more simple description of the finite size of the counterions based on that by Bikerman has also been considered for comparison. The studies carried out in this work include predictions on the effect of the finite counterion size on the equilibrium properties of the colloid and its electrokinetic and dielectric response when it is subjected to constant or alternating electric fields. The results show how important the counterion finite-size effects are for most of the electrokinetic and dielectric properties of highly charged and concentrated colloids, mainly for the static and dynamic electrophoretic mobilities. Furthermore, new insights are provided on the counterion condensation effect when counterions are allowed to have finite size. Focus is placed on the changes undergone by their concentration in the condensation layer for low-salt and highly charged colloids.

A Saccharomyces cerevisiae model and screen to define the functional consequences of oncogenic histone missense mutations.

Somatic missense mutations in histone genes turn these essential proteins into oncohistones, which can drive oncogenesis. Understanding how missense mutations alter histone function is challenging in mammals as mutations occur in a single histone gene. For example, described oncohistone mutations predominantly occur in the histone H3.3 gene, despite the human genome encoding 15 H3 genes. To understand how oncogenic histone missense mutations alter histone function, we leveraged the budding yeast model, which contains only 2 H3 genes, to explore the functional consequences of oncohistones H3K36M, H3G34W, H3G34L, H3G34R, and H3G34V. Analysis of cells that express each of these variants as the sole copy of H3 reveals that H3K36 mutants show different drug sensitivities compared to H3G34 mutants. This finding suggests that changes to proximal amino acids in the H3 N-terminal tail alter distinct biological pathways. We exploited the caffeine-sensitive growth of H3K36-mutant cells to perform a high copy suppressor screen. This screen identified genes linked to histone function and transcriptional regulation, including Esa1, a histone H4/H2A acetyltransferase; Tos4, a forkhead-associated domain-containing gene expression regulator; Pho92, an N6-methyladenosine RNA-binding protein; and Sgv1/Bur1, a cyclin-dependent kinase. We show that the Esa1 lysine acetyltransferase activity is critical for suppression of the caffeine-sensitive growth of H3K36R-mutant cells while the previously characterized binding interactions of Tos4 and Pho92 are not required for suppression. This screen identifies pathways that could be altered by oncohistone mutations and highlights the value of yeast genetics to identify pathways altered by such mutations.