Delta Cells Research Articles

The leptin receptor has no role in delta-cell control of beta-cell function in the mouse.

Leptin inhibits insulin secretion from isolated islets from multiple species, but the cell type that mediates this process remains elusive. Several mouse models have been used to explore this question. Ablation of the leptin receptor (Lepr) throughout the pancreatic epithelium results in altered glucose homeostasis and ex vivo insulin secretion and Ca2+ dynamics. However, Lepr removal from neither alpha nor beta cells mimics this result. Moreover, scRNAseq data has revealed an enrichment of LEPR in human islet delta cells. We confirmed LEPR upregulation in human delta cells by performing RNAseq on fixed, sorted beta and delta cells. We then used a mouse model to test whether delta cells mediate the diminished glucose-stimulated insulin secretion in response to leptin. Ablation of Lepr within mouse delta cells did not change glucose homeostasis or insulin secretion, whether mice were fed a chow or high-fat diet. We further show, using a publicly available scRNAseq dataset, that islet cells expressing Lepr lie within endothelial cell clusters. In mice, leptin does not influence beta-cell function through delta cells.

Pathogenic mechanisms and potential therapeutic targets for Parkinson disease revealed by bioinformatic analysis of necroptosis and immune cell infiltration.

Parkinson disease (PD) is an age-dependent neurodegenerative disease with very high prevalence by age 80 years. Necroptosis is a newly identified form of programmed cell death implicated in neurodegenerative diseases, but has not yet been conclusively associated with PD. This study examined the contributions of necroptosis to PD using bioinformatics analysis. Datasets GSE26927, GSE49036, and GSE54536 from the gene expression omnibus database were analyzed for differentially expressed genes (DEGs). These DEGs were then subjected to gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to identify associated functions and signaling mechanisms. Necroptosis-related differentially expressed genes (NRDEGs) were then identified by the overlap of DEGs and the necroptosis gene set hsa04217. The STRING database and Cytoscape software were then used to build and visualize a protein-protein interaction network and identify hubs and key functional modules among NRDEGs. In addition, immune cell type abundance was analyzed based on DEGs using ImmuCellAI. The identified DEGs, KEGG pathway enrichment terms, and protein-protein interaction network structures of NRDEGs were validated using an independent dataset (GSE54536). The necroptosis pathway was significantly enriched and activated in PD samples. Thirteen NRDEGs were identified in the GSE26927 and GSE49036 datasets, including receptor interacting serine/threonine kinase 1, CASP8 and FADD like apoptosis regulator, TNFRSF1A associated via death domain, and interleukin 1 beta, of which 6 were validated in the GSE54536 dataset. According to gene ontology and KEGG analyses, these NRDEGs are involved in necroptosis-related processes, apoptosis, B cell receptor signaling pathways, and NOD-like receptor signaling pathways. Analysis of DEGs also revealed significant increases in CD8 + T cell and Tex cell infiltration and significant decreases in B cell and T gamma delta cell infiltration within the PD brain. Necroptosis pathways are active in PD and associated with immune cell infiltration. The factors controlling necroptotic signaling and immune infiltration identified in this study may be valuable diagnostic markers and therapeutic targets for PD.

PyCreas: a tool for quantification of localization and distribution of endocrine cell types in the islets of Langerhans.

Manifest diabetes, but also conditions of increased insulin resistance such as pregnancy or obesity can lead to islet architecture remodeling. The contributing mechanisms are as poorly understood as the consequences of altered cell arrangement. For the quantification of the different cell types but also the frequency of different cell-cell contacts within the islets, different approaches exist. However, few methods are available to characterize islet cell distribution in a statistically valid manner. Here we describe PyCreas, an open-source tool written in Python that allows semi-automated analysis of islet cell distribution based on images of pancreatic sections stained by immunohistochemistry or immunofluorescence. To ensure that the PyCreas tool is suitable for quantitative analysis of cell distribution in the islets at different metabolic states, we studied the localization and distribution of alpha, beta, and delta cells during gestation and prediabetes. We compared the islet cell distribution of pancreatic islets from metabolically healthy NMRI mice with that of New Zealand obese (NZO) mice, which exhibit impaired glucose tolerance (IGT) both preconceptionally and during gestation, and from C57BL/6N (B6) mice, which acquire this IGT only during gestation. Since substrain(s) of the NZO mice are known to show a variant in the Abcc8 gene, we additionally examined preconceptional SUR1 knock-out (SUR1-KO) mice. PyCreas provided quantitative evidence that alterations in the Abcc8 gene are associated with an altered distribution pattern of islet cells. Moreover, our data indicate that this cannot be a consequence of prolonged hyperglycemia, as islet architecture is already altered in the prediabetic state. Furthermore, the quantitative analysis suggests that states of transient IGT, such as during common gestational diabetes mellitus (GDM), are not associated with changes in islet architecture as observed during long-term IGT. PyCreas provides the ability to systematically analyze the localization and distribution of islet cells at different stages of metabolic disease to better understand the underlying pathophysiology.

Beta cell primary cilia mediate somatostatin responsiveness via SSTR3

ABSTRACT Somatostatin is a paracrine modulator of insulin secretion and beta cell function with pleotropic effects on glucose homeostasis. The mechanism of somatostatin-mediated communication between delta and beta cells is not well-understood, which we address in this study via the ciliary somatostatin receptor 3 (SSTR3). Primary cilia are membrane organelles that act as signaling hubs in islets by virtue of their subcellular location and enrichment in signaling proteins such as G-protein coupled receptors (GPCRs). We show that SSTR3, a ciliary GPCR, mediates somatostatin suppression of insulin secretion in mouse islets. Quantitative analysis of calcium flux using a mouse model of genetically encoded beta cell-specific GCaMP6f calcium reporter shows that somatostatin signaling alters beta cell calcium flux after physiologic glucose stimulation, an effect that depends on endogenous SSTR3 expression and the presence of intact primary cilia on beta cells. Comparative in vitro studies using SSTR isoform antagonists demonstrate a role for SSTR3 in mediating somatostatin regulation of insulin secretion in mouse islets. Our findings support a model in which ciliary SSTR3 mediates a distinct pathway of delta-to-beta cell regulatory crosstalk and may serve as a target for paracrine modulation.

Exploring the Dynamic Role of Gamma-Delta T Cells in Clinical Pathology: A Systematic Review of Current Knowledge and Future Perspectives

Gamma-delta [γδ] T cells are a significant subset of T cells with ability to recognize many antigens without the guidance of major histocompatibility complex (MHC) molecules. A lot of studies have been conducted on them ever since they were discovered 36 years ago. Advances have been made about their structure and function. Thus, there is a need for an up-to-date review about their significance as well as their roles in Multiple Sclerosis. The study aimed to provide an up-to-date knowledge concerning gamma delta T cells in pathogenesis of Multiple Sclerosis (MS). A systematic review methodology was used to scope information from recent studies about gamma delta T cells. The review was carried out in PubMed database and it followed the PRISMA guidelines. The computerized review yielded 60 peer-reviewed articles published between 2011 and 2020. The results showed that gamma delta cells play a critical role in protection against infections and the fight against malignant cells. Most of the reviewed studies highlighted recent advances in research. It was noted that the subset of T cells plays a role in pathogenesis of MS. Several studies have highlighted the protective and detrimental effects of γδ T cells. On the one hand, the cells contribute to the pathogenesis of MS, while, on the other hand, they also help in immunoregulation. However, there is a dearth of literature on their specific role and their mechanism of action. Therefore, further studies are needed about γδ T cells in both human and animal model studies. Based on the findings of the reviewed studies, there is great promise in the use of γδ T cells for MS immunotherapy

1748-P: Assessing the Presence of Delta-Cell–Mediated Electrical Coupling in the Islet

Somatostatin-producing delta cells play an important role in the regulation of paracrine cross talk in the islet through inhibition of insulin and glucagon secretion by beta and alpha cells, respectively. However, recent studies have suggested the presence of electrical coupling via gap junctions between delta and beta cells. Electrical coupling mediated cross-talk would support the correlation between pulsatile insulin and somatostatin release and reveal a structural source of cell-cell communication within the islet. To test whether beta and delta cells are gap junction coupled, we measured the permeability of cell to cell connections and the relative dynamics of Ca2+. Islets of Sst-Cre;Rosa-LSL-tdTomato mice were used which show delta-cell specific labelling. They were then loaded with the cationic dye rhodamine-123 at 5mM glucose to measure performance of fluorescence recovery after photobleaching to assess gap junction coupling of delta cells and non-delta cells. Calcium dynamics were assessed using calcium dye Fluo-4 upon stimulating the islet from low glucose (2mM or 5 mM) to high glucose(11mM). No significant differences between recovery rate were observed when comparing delta cells and beta cells (p>0.05), which indicates some level of gap junction permeability among delta cells exists. The calcium activity of a population of delta cells was similar to those of neighboring beta cells, indicating a functional connection exists between these cell types. Furthermore, first-responder beta cells showed a preferential proximity to delta cells suggesting an impact of delta cells on functional sub-populations of nearby beta-cells. These results suggest a possible feedback system exists in the islet that is governed by electrical coupling of delta cells, which is important for both the function of beta cells and insulin secretion, and may also impact secretion of other islet hormones. Disclosure C.H.Levitt: None. R.K.Benninger: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (P30DK116073, R01DK102950, R01DK106412)

1781-P: Leptin Regulates Delta-Cell Somatostatin Secretion from Human and Mouse Islets

The delta cell is uniquely positioned in the islet to integrate local signals and circulating nutrient cues to regulate alpha and beta cell function through somatostatin (SST) secretion. In type 2 diabetes (T2D), delta cell responses to ambient glucose are disproportionate and consequently alter insulin and glucagon secretion leading to dysglycemia. However, we know relatively little about the mechanisms influencing delta cells. Thus, understanding factors that regulate delta cell SST secretion may reveal pathogenic mechanisms contributing to T2D and direct new therapies to achieve glucose homeostasis. Leptin is a circulating hormone that potently inhibits insulin and glucagon secretion, but notably, the leptin receptor (LepR) is exclusively expressed on delta cells of human islets. Our preliminary studies demonstrate for the first time that leptin stimulates SST secretion from human islets. Leptin-induced SST corresponded with decreased glucagon and insulin secretion. Leptin effects were conserved in mouse islets and required delta cell specific LepR expression. Our single-cell RNA-seq studies revealed Stat3 target genes become depleted in delta cells that lack LepR, suggesting canonical leptin signals regulate transcriptional activity in delta cells. Along these lines, delta cell specific Stat3 KO islets (Sst-Cre+/wt;Stat3fl/fl) do not respond to leptin. On-going studies in human islets employ pharmacologic and genetic interventions with live functional imaging and unbiased approaches that will reveal the mechanisms of leptin action in human delta cells. These studies are the first to describe leptin effects on delta cells and uncover a unifying mechanism whereby leptin acts indirectly on alpha and beta cells through paracrine SST signaling. As such, our findings are expected to reveal new signaling mechanisms governing islet function that potentially exert therapeutic benefits for T2D. Disclosure S.M.Hartig: None. A.Cox: None.

1777-P: Three-Dimensional Imaging Reveals Pancreatic Islet Fast Calcium Wave Origin Formation

Pulsatile insulin secretion triggered by global calcium waves within pancreatic β cells is essential to blood glucose homeostasis. The calcium wave propagation forms by gap junction coupling among beta cells, but the emergence of wave origins is still unclear. We developed a 3D islet calcium imaging system and adapted data processing methods to observe and analyze islet calcium wave propagation quantitatively. A global fast-traveling calcium wave identifies in all kinds of oscillation modes. The calcium waves could initiate by multiple conserved β-cell clusters. We showed that the beta cell subpopulation, which first responded to high glucose stimulation, would partially contribute to calcium wave origin formation. To determine whether the spatial distribution of alpha or delta cells affects wave origin formation, we developed transgenetic mice that all islet cells were directly labeled with GCaMP6f, and alpha cells or delta cells were marked with tdTomato by Cre-Loxp strategy. Our data suggested that delta cells in the periphery modulated wave origins by electrical coupling and inhibitory paracrine interaction. Population oscillator models based on natural islet 3D structures reproduce the wave propagation observed in experiments. Interestingly, 3D imaging showed only local calcium wave bursts in ob/ob mice islets, and Immunofluorescence staining showed those sporadic bursts also correlated to delta cells. Disclosure Y.Yu: None.

196-OR: The Long-Chain Fatty-Acid Receptor FFA4 Stimulates Insulin Secretion via Inhibition of Somatostatin Release from the Delta Cell

The long-chain fatty-acid receptor FFA4 exerts beneficial effects on glucose homeostasis and insulin secretion and is considered a potential therapeutic target for type 2 diabetes. FFA4 is expressed in islets, but its precise mechanism of action remains unknown. Previous studies from our group suggest that FFA4 is expressed in delta cells and regulates somatostatin secretion. The objective of this study was to test the hypothesis that FFA4 agonists indirectly stimulate insulin secretion via inhibition of somatostatin release. In 1-h static incubations of isolated, wild-type mouse islets, the FFA4 agonist CpdA dose-dependently potentiated glucose-induced insulin secretion from 2.2±0.2 to 3.4±0.2 % of insulin content at the concentration of 50 μM (n=8; p<0.001), and simultaneously decreased somatostatin secretion from 28.4±2.1 to 10.5±0.9 pM (n=8; p<0.0001). No effect of CpdA on insulin or somatostatin secretion was observed in islets from mice that do not express somatostatin (insulin: 3.2±0.1 vs 2.9±0.4, n=6, NS; somatostatin: 11.4±1.2 vs 11.9±1.2, n=6, NS). No sex differences were observed. Likewise, in delta-cell deficient islets isolated from diphtheria toxin-treated male mice expressing the diphtheria toxin receptor under the somatostatin promoter, no effect of CpdA was observed on insulin or somatostatin release (insulin: 4.9±0.9 vs 4.1±0.8, n=3, NS; somatostatin: 10.1±4.9 vs 9.2±5.3, n=6, NS). We conclude that FFA4 stimulation of insulin secretion is exclusively mediated by inhibition of somatostatin secretion from delta cells. Disclosure L.Reininger: None. M.Ethier: None. C.Tremblay: None. J.Ghislain: None. M.Huising: Consultant; AstraZeneca, Research Support; Crinetics Pharmaceuticals, Inc. V.Poitout: Consultant; AstraZeneca. M.Flisher: None. Funding National Institutes of Health (R01DK132597)

195-OR: To Which Degree Do Alpha Cells Shape the Role of the Beta Cells First Responders?

Calcium (Ca2+) uptake drives glucose-stimulated insulin secretion from the pancreatic β-cells. The “1st responder” subpopulation of β-cells disproportionally control the Ca2+ uptake during the first phase, which is disrupted with ageing and in diabetes. Here we determine whether alpha and delta cells play a formative role in the 1st responder β-cells state. We used MIP-CreER GCaMP6s mouse model which expresses Ca2+-sensitive GFP specifically in β-cells. We performed simultaneous recording of Ca2+ dynamics and gap junction permeability in individual islets. We stimulated islets with glucose, and with the GLP1-R antagonist, exendin 9-39 (Ex9). Based on the first-phase Ca2+ dynamics we defined the “1st responder” and the “last responder” cells. We tested their consistency under repeated glucose stimulation with and without the Ex9. We next stained them for insulin, glucagon, and somatostatin and measured distances in 3D from all β-cells to alpha and delta cells. The Ex9 disrupted the 1st responder consistency (only half of the original 1st responders remained such under repeated glucose stimulation), compared to the control experiment. Ex9 increased the islet's first-to-last cell response time by 150 +/- 74 seconds making islet's response to glucose two times more heterogeneous compared to control (p=0.0517). On average 1st responders were located closer to the alpha cells, and last responders - further from them (p=0.0039). At the same time, first responders had significantly less delta cell neighbors compared to the last responders (p=0.0137). Our gap junction permeability data showed no gap junction activity in alpha-beta cell couples. In conclusion, 1st responder beta cells have more alpha cell and less delta cell neighbors than last responders. The role of the alpha cell in the formation of the 1st responder cell state is likely realized via paracrine interaction. Disclosure V.Kravets: None. C.H.Levitt: None. R.K.Benninger: None. Funding Burroughs Wellcome Fund (25B1756 to V.K.); National Institutes of Health (R01DK102950, R01DK106412 to R.K.B.); Human Islet Research Network (UC24DK104162 to V.K.)

199-OR: Optogenetic Control of Pancreatic Delta Cells Reveals Their Central Role in Hormone Release from Pancreatic Islets

Diabetes mellitus is a metabolic disorder resulting from insufficient insulin release and associated with dysregulation of glucagon secretion. Somatostatin secreted from pancreatic δ-cells is a strong paracrine factor for both insulin and glucagon secretion in islets. Here, we developed an optogenetic approach, through specific expressing light-sensitive channels/pumps in δ-cells, to investigate the paracrine control of hormone release from islets. We generated mouse models that express either a light-sensitive cation channel, channelrhodopsin-2 (ChR2), or a chloride pump, halorhodopsin (HR), specifically in the pancreatic δ-cells (SST-ChR2 and SST-HR mouse). Their specific expression was confirmed by immunostaining. Optoactivation of ChR2 at 1mM glucose rapidly depolarized δ-cells by 37mV (n=5) and stimulated electrical activity and somatostatin release. At high glucose or tolbutamide, light activation of HR repolarized δ-cells by 15 mV (n=3), suppressing action potentials. Optoactivation of SST-ChR2 δ-cells suppressed, after a variable delay depending on the distance from δ-cells, action potential firing in neighbouring α-cells, inhibited exocytosis by 30% (n=5) and glucagon secretion by 55% at 1mM glucose (n=7). In a subset (35%) α-cells with no spontaneous action potentials, opto-inhibition of SST-HR δ-cells caused a 9mV depolarization (n=7) and induced action potential firing, which may account for the stimulation of glucagon secretion. Neither optoactivation, nor opto-inhibition of δ-cells affected insulin secretion at hypo- and hyperglycaemia. However, at normoglycaemia (6 mM glucose), optoactivation of δ-cells depolarized neighbouring β-cells (via gap-junctions) and evoked action potential firing, leading to a paradoxical 160% stimulation of insulin secretion (n=9). We conclude that δ-cells play a central role in the regulation of both insulin and glucagon release but that the effects are more complex than appreciated to date. Disclosure H.Dou: None. C.A.Miranda: None. J.Tolö: None. Q.Zhang: None. P.Rorsman: None. Funding Swedish Research Council

Chromatin accessibility differences between alpha, beta, and delta cells identifies common and cell type-specific enhancers

BackgroundHigh throughput sequencing has enabled the interrogation of the transcriptomic landscape of glucagon-secreting alpha cells, insulin-secreting beta cells, and somatostatin-secreting delta cells. These approaches have furthered our understanding of expression patterns that define healthy or diseased islet cell types and helped explicate some of the intricacies between major islet cell crosstalk and glucose regulation. All three endocrine cell types derive from a common pancreatic progenitor, yet alpha and beta cells have partially opposing functions, and delta cells modulate and control insulin and glucagon release. While gene expression signatures that define and maintain cellular identity have been widely explored, the underlying epigenetic components are incompletely characterized and understood. However, chromatin accessibility and remodeling is a dynamic attribute that plays a critical role to determine and maintain cellular identity.ResultsHere, we compare and contrast the chromatin landscape between mouse alpha, beta, and delta cells using ATAC-Seq to evaluate the significant differences in chromatin accessibility. The similarities and differences in chromatin accessibility between these related islet endocrine cells help define their fate in support of their distinct functional roles. We identify patterns that suggest that both alpha and delta cells are poised, but repressed, from becoming beta-like. We also identify patterns in differentially enriched chromatin that have transcription factor motifs preferentially associated with different regions of the genome. Finally, we not only confirm and visualize previously discovered common endocrine- and cell specific- enhancer regions across differentially enriched chromatin, but identify novel regions as well. We compiled our chromatin accessibility data in a freely accessible database of common endocrine- and cell specific-enhancer regions that can be navigated with minimal bioinformatics expertise.ConclusionsBoth alpha and delta cells appear poised, but repressed, from becoming beta cells in murine pancreatic islets. These data broadly support earlier findings on the plasticity in identity of non-beta cells under certain circumstances. Furthermore, differential chromatin accessibility shows preferentially enriched distal-intergenic regions in beta cells, when compared to either alpha or delta cells.

Presence of immunogenic alternatively spliced insulin gene product in human pancreatic delta cells

Aims/hypothesisTranscriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets.MethodsAlternative splicing of insulin pre-mRNA was determined by PCR analysis performed on human islet RNA and single-cell RNA-seq analysis. Antisera were generated to detect insulin variants in human pancreatic tissue using immunohistochemistry, electron microscopy and single-cell western blot to confirm the expression of insulin variants. Cytotoxic T lymphocyte (CTL) activation was determined by MIP-1β release.ResultsWe identified an alternatively spliced INS product. This variant encodes the complete insulin signal peptide and B chain and an alternative C-terminus that largely overlaps with a previously identified defective ribosomal product of INS. Immunohistochemical analysis revealed that the translation product of this INS-derived splice transcript was detectable in somatostatin-producing delta cells but not in beta cells; this was confirmed by light and electron microscopy. Expression of this alternatively spliced INS product activated preproinsulin-specific CTLs in vitro. The exclusive presence of this alternatively spliced INS product in delta cells may be explained by its clearance from beta cells by insulin-degrading enzyme capturing its insulin B chain fragment and a lack of insulin-degrading enzyme expression in delta cells.Conclusions/interpretationOur data demonstrate that delta cells can express an INS product derived from alternative splicing, containing both the diabetogenic insulin signal peptide and B chain, in their secretory granules. We propose that this alternative INS product may play a role in islet autoimmunity and pathology, as well as endocrine or paracrine function or islet development and endocrine destiny, and transdifferentiation between endocrine cells. INS promoter activity is not confined to beta cells and should be used with care when assigning beta cell identity and selectivity.Data availabilityThe full EM dataset is available via www.nanotomy.org (for review: http://www.nanotomy.org/OA/Tienhoven2021SUB/6126-368/). Single-cell RNA-seq data was made available by Segerstolpe et al [13] and can be found at https://sandberglab.se/pancreas. The RNA and protein sequence of INS-splice was uploaded to GenBank (BankIt2546444 INS-splice OM489474).Graphical abstract

Molecular phenotyping of single pancreatic islet leader beta cells by “Flash-Seq”

AimsSpatially-organized increases in cytosolic Ca2+ within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including “leaders” which initiate Ca2+ waves. Whether leader cells possess unique molecular features, or localisation, is unknown. Main methodsHigh speed confocal Ca2+ imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected “hub” cells. To explore transcriptomic differences between beta cell sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing (“Flash-Seq”). Key findingsDistinct Ca2+ wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29 % of islets, though “hub” cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padj < 0.05) between leader and follower cells included genes involved in cilium biogenesis and transcriptional regulation. Providing some support for these findings, ADCY6 immunoreactivity tended to be higher in leader than follower cells, whereas cilia number and length tended to be lower in the former. Finally, leader cells were located significantly closer to delta, but not alpha, cells in Euclidian space than were follower cells. SignificanceThe existence of both a discrete transcriptome and unique localisation implies a role for these features in defining the specialized function of leaders. These data also raise the possibility that localised signalling between delta and leader cells contributes to the initiation and propagation of islet Ca2+ waves.

An unusual lupus like malar rash pattern with hepatosplenomegaly as a presentation of gamma delta t cell lymphoma

About 12% of all lymphoid tumors worldwide are peripheral t cell lymphomas, which are uncommon subtypes of non-hodgkin lymphoma. they have a poor prognosis and are more common in populations and countries in asia. T cell lymphomas with gamma delta t cell receptors expressed in them are very rare (less than 1% of lymphoid neoplasms) and extremely aggressive; they originate from gamma delta t cells; a small subset of peripheral t cells with direct antigen recognition capability acting at the interface between innate and adaptive immunity .&#x0D; Lymphoid tissue, skin, gastrointestinal tract, and the red pulp of the spleen are where gamma delta t lymphocytes tend to cocentrate, representing only 1-5% of the total circulating lymphocytes, but accounting for up to 30% of the entire t cell population.&#x0D; In contrast to alpha beta t cells, gamma delta t lymphocytes develop from cd4/cd8 thymic precursors in the bone marrow and usually lack the major histocompatibility complex restriction.&#x0D; These lymphocytes conduct as cytotoxic cells similar to natural killer cells in their activity as early effectors of innate, non-specific immune response and are capable of t cell receptor rearrangement as well as phagocytosis.&#x0D; Hepatosplenic t cell lymphoma and primary cutanous gamma delta cell lymphoma are recognized as two different gamma delta t cell lymphoma entities.&#x0D; A well characterized extranodal lymphoma, hepatosplenic t cell lymphoma has a disguised onset, secondary to intrasinusoidal infiltrationn of the spleen, liver, and bone marrow, has a rapidly progressive course, is poorly responsive to chemotherapy.&#x0D; A panniculitis-like clinical picture with prominent epidermal involvement can be a presentation of primary cutaneous gamma delta t cell lymphoma.&#x0D; In this case study , the authors will discuss a case of 19 year old male patient present with a lupus like malar rash and hepatosplenomegaly and who had been diagnosed with gamma delta t cell lymphoma.

Pancreatic Islet Cells Response to IFNγ Relies on Their Spatial Location within an Islet

Type 1 diabetes (T1D) is an auto-immune disease characterized by the progressive destruction of insulin-producing pancreatic beta cells. While beta cells are the target of the immune attack, the other islet endocrine cells, namely the alpha and delta cells, can also be affected by the inflammatory milieu. Here, using a flow cytometry-based strategy, we compared the impact of IFNγ, one of the main cytokines involved in T1D, on the three endocrine cell subsets isolated from C57BL/6 mouse islets. RNA-seq analyses revealed that alpha and delta cells exposed in vitro to IFNγ display a transcriptomic profile very similar to that of beta cells, with an increased expression of inflammation key genes such as MHC class I molecules, the CXCL10 chemokine and the programmed death-ligand 1 (PD-L1), three hallmarks of IFNγ signaling. Interestingly, at low IFNγ concentration, we observed two beta cell populations (responders and non-responders) based on PD-L1 protein expression. Our data indicate that this differential sensitivity relies on the location of the cells within the islet rather than on the existence of two different beta cells subsets. The same findings were corroborated by the in vivo analysis of pancreatic islets from the non-obese diabetic mouse model of T1D, showing more intense PD-L1 staining on endocrine cells close to immune infiltrate. Collectively, our work demonstrates that alpha and delta cells are as sensitive as beta cells to IFNγ, and suggests a gradual diffusion of the cytokine into an islet. These observations provide novel insights into the in situ inflammatory processes occurring in T1D progression.

Single-cell RNA sequencing combined with single-cell proteomics identifies the metabolic adaptation of islet cell subpopulations to high-fat diet in mice.

Islets have complex heterogeneity and subpopulations. Cell surface markers representing alpha, beta and delta cell subpopulations are urgently needed for investigations to explore the compositional changes of each subpopulation in obesity progress and diabetes onset, and the adaptation mechanism of islet metabolism induced by a high-fat diet (HFD). Single-cell RNA sequencing (scRNA-seq) was applied to identify alpha, beta and delta cell subpopulation markers in an HFD-induced mouse model of glucose intolerance. Flow cytometry and immunostaining were used to sort and assess the proportion of each subpopulation. Single-cell proteomics was performed on sorted cells, and the functional status of each alpha, beta and delta cell subpopulation in glucose intolerance was deeply elucidated based on protein expression. A total of 33,999 cells were analysed by scRNA-seq and clustered into eight populations, including alpha, beta and delta cells. For alpha cells, scRNA-seq revealed that the Ace2low subpopulation had downregulated expression of genes related to alpha cell function and upregulated expression of genes associated with beta cell characteristics in comparison with the Ace2high subpopulation. The impaired function and increased fragility of ACE2low alpha cells exposure to HFD was further suggested by single-cell proteomics. As for beta cells, the CD81high subpopulation may indicate an immature signature of beta cells compared with the CD81low subpopulation, which had robust function. We also found differential expression of Slc2a2 in delta cells and a potentially stronger cellular function and metabolism in GLUT2low delta cells than GLUT2high delta cells. Moreover, an increased proportion of ACE2low alpha cells and CD81low beta cells, with a constant proportion of GLUT2low delta cells, were observed in HFD-induced glucose intolerance. We identified ACE2, CD81 and GLUT2 as surface markers to distinguish, respectively, alpha, beta and delta cell subpopulations with heterogeneous maturation and function. The changes in the proportion and functional status of islet endocrine subpopulations reflect the metabolic adaptation of islets to high-fat stress, which weakened the function of alpha cells and enhanced the function of beta and delta cells to bring about glycaemic homeostasis. Our findings provide a fundamental resource for exploring the mechanisms maintaining each islet endocrine subpopulation's fate and function in health and disease. The scRNA-seq analysis datasets from the current study are available in the Gene Expression Omnibus (GEO) repository under the accession number GSE203376.

Identification of Two CDK5R1-Related Subtypes and Characterization of Immune Infiltrates in Alzheimer's Disease Based on an Integrated Bioinformatics Analysis.

Alzheimer's disease (AD) is a neurodegenerative disorder and the major cause of senile dementia. The Reelin pathway has been involved in both learning and AD pathogenesis. However, the specific Reelin-related gene signature during the pathological process remains unknown. Reelin-related gene (CDK5R1) expression was analyzed using the GEO datasets. The relevant genes of CDK5R1 were identified using differential expression analysis and weighted gene correlation network analysis (WGCNA) based on the GSE43850 dataset. ConsensusClusterPlus analysis was applied to identify subtypes (C1 and C2) of AD. The CIBERSORT algorithm was used to assess the immune cell infiltration between the two AD subtypes. CDK5R1 was downregulated in AD. 244 differentially expressed CDK5R1-related genes (DECRGs) between the two subgroups were mainly enriched in GABAergic synapse, neuroactive ligand-receptor interaction, synapse organization, neurotransmitter transport, etc. Furthermore, the GSVA results indicated that immune-related pathways were significantly enriched in the C1 subgroup. Interestingly, 10 Reelin pathway-related genes (CRK, DAB2IP, LRP8, RELN, STAT5A, CDK5, CDK5R1, DAB1, FYN, and SH3KBP1) were abnormally expressed between the two subgroups. The proportion of T cell gamma delta, monocytes, macrophage M2, and dendritic cells activated decreased from C1 to C2, while the proportion of plasma cells, T cell follicular helper, and NK cells activated increased. Two CDK5R1-related subtypes of AD were identified, helping us to better understand the role of CDK5R1 in the pathological process of AD.

Transmission electron microscopic investigation of the dark and light pancreatic acinar beta-cells of young-domesticated pig (Sus Suidae, Erxleben 1777).

The current study was designed to perform a transmission electron microscopic investigation focusing on the dark and light pancreatic acinar β-cells of young domesticated pig (Sus Suidae). This study depended on the fresh pancreatic specimens from 5 healthy young (2-month-old) pigs that were collected immediately after they were slaughtered at the abattoir of Abdelkader Alexandria, Egypt. In our findings, the acinar pancreas was formed of pyramidal pancreatic acinar cells with large spherical nuclei of condensed heterochromatin at the periphery and prominent eccentric nucleoli. Zymogen granules were observed at the apical region of the acinar cells, and they appear as electron dense bodies. Numerous mitochondria and Golgi complexes observed in the acinar cell cytoplasm. The electron dense acinar cells were joined by junctional complexes. The rough endoplasmic reticulum was more prominent in the electron-dense acinar cells than did electron-lucent acinar cells. There was no connective tissue capsule separate the acinar portion of pancreas from the pancreatic islets. The pancreatic islets mainly formed of β-cells. The irregular α-cells possess numerous small granules. The cytoplasmic β-cells granules were surrounded by hallow area and enclosed by a limiting membrane. Delta cells were generally polygonal in shape and found in clumps throughout the islet and they were also identified in between β-cells. Their granules were of moderate electron density and were generally smaller than β-cells' granules. The limiting membrane was tightly enclosed the delta cells granules and the hallow area around the granule were found similar to the granules of β-cells.

Data-driven analysis to identify prognostic immune-related biomarkers in BRAF mutated cutaneous melanoma microenvironment.

Skin cutaneous melanoma is one of the deadly diseases, and more than 50% of the patients have BRAF gene mutations. Evidence suggests that oncogenic BRAF modulates the immune system's ability to recognize SKCM cells. Due to the complexity of the tumor microenvironment (TME) and a lack of a rational mechanistic basis, it is urgent to investigate the immune infiltration and identify prognostic biomarkers in BRAF mutated SKCM patients. Multiple methods including ESTIMATE algorithm, differential gene analysis, prognostic analysis and immune infiltration analysis were performed to investigate the tumor microenvironment. Based on the patient's immune score and stromal score, immune-related genes DEGs were identified. Functional analysis revealed that these genes were mainly enriched in biological processes such as immune response, defense response and positive regulation of immune system. Furthermore, we analyzed the immune infiltrating cell components of BRAF mutated patients and revealed 4 hub genes associated with overall survival time. Several cells (Monocyte, Macrophage and Gamma delta cells) have been found to be significantly decreased in immune-high BRAF mutated SKCM group. While CD4+T, CD8+T, CD4 naïve, Tr1, Th2 and many T cell subsets were significantly increased in immune-high group. These immune cells and genes were closely related to each other. This study revealed that the dysregulation of immune function and immune cells may contribute to the poor outcomes of BRAF mutated patients. It is of great significance to our further understanding of the TME and immune dysfunction in BRAF mutated SKCM.