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IScience Jun 2024The developing mouse pancreas is surrounded by mesoderm compartments providing signals that induce pancreas formation. Most pancreatic organoid protocols lack this...
The developing mouse pancreas is surrounded by mesoderm compartments providing signals that induce pancreas formation. Most pancreatic organoid protocols lack this mesoderm niche and only partially capture the pancreatic cell repertoire. This work aims to generate pancreatic aggregates by differentiating mouse embryonic stem cells (mESCs) into mesoderm progenitors (MPs) and pancreas progenitors (PPs), without using Matrigel. First, mESCs were differentiated into epiblast stem cells (EpiSCs) to enhance the PP differentiation rate. Next, PPs and MPs aggregated together giving rise to various pancreatic cell types, including endocrine, acinar, and ductal cells, and to endothelial cells. Single-cell RNA sequencing analysis revealed a larger endocrine population within the PP + MP aggregates, as compared to PPs alone or PPs in Matrigel aggregates. The PP + MP aggregate gene expression signatures and its endocrine population percentage closely resembled those of the endocrine population found in the mouse embryonic pancreas, which holds promise for studying pancreas development.
PubMed: 38832019
DOI: 10.1016/j.isci.2024.109959 -
Investigative Ophthalmology & Visual... Jun 2024Loss of function of the lacrimal gland (LG), which produces the aqueous tear film, is implicated in age-related dry eye. To better understand this deterioration, we...
PURPOSE
Loss of function of the lacrimal gland (LG), which produces the aqueous tear film, is implicated in age-related dry eye. To better understand this deterioration, we evaluated changes in lipid metabolism and inflammation in LGs from an aging model.
METHODS
LG sections from female C57BL/6J mice of different ages (young, 2-3 months; intermediate, 10-14 months; old, ≥24 months) were stained with Oil Red-O or Toluidine blue to detect lipids. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blotting of LG lysates determined differences in the expression of genes and proteins related to lipid metabolism. A photobleaching protocol to quench age-related autofluorescence was used in LG sections to evaluate changes in immunofluorescence associated with NPC1, NPC2, CTSL, and macrophages (F4/80, CD11b) with age using confocal fluorescence microscopy.
RESULTS
Old LGs showed increased lipids prominent in basal aggregates in acinar cells and in extra-acinar sites. LG gene expression of Npc1, Npc2, Lipa, and Mcoln2, encoding proteins involved in lipid metabolism, was increased with age. NPC1 was also significantly increased in old LGs by western blotting. In photobleached LG sections, confocal fluorescence microscopy imaging of NPC1, NPC2, and CTSL immunofluorescence showed age-associated enrichment in macrophages labeled to detect F4/80. Although mononuclear macrophages were detectable in LG at all ages, this novel multinucleate macrophage population containing NPC1, NPC2, and CTSL and enriched in F4/80 and some CD11b was increased with age at extra-acinar sites.
CONCLUSIONS
Lipid-metabolizing proteins enriched in F4/80-positive multinucleated macrophages are increased in old LGs adjacent to sites of lipid deposition in acini.
Topics: Animals; Female; Mice, Inbred C57BL; Aging; Mice; Lipid Metabolism; Macrophages; Blotting, Western; Lacrimal Apparatus; Real-Time Polymerase Chain Reaction; Microscopy, Confocal; Disease Models, Animal; Dry Eye Syndromes
PubMed: 38829671
DOI: 10.1167/iovs.65.6.1 -
Heliyon Jun 2024Acute pancreatitis (AP) is an inflammatory disease characterized by localized pancreatic injury and a systemic inflammatory response. Fatty acids (FAs), produced during... (Review)
Review
Acute pancreatitis (AP) is an inflammatory disease characterized by localized pancreatic injury and a systemic inflammatory response. Fatty acids (FAs), produced during the breakdown of triglycerides (TGs) in blood and peripancreatic fat, escalate local pancreatic inflammation to a systemic level by damaging pancreatic acinar cells (PACs) and triggering M1 macrophage polarization. This paper provides a comprehensive analysis of lipases' roles in the onset and progression of AP, as well as the effects of long-chain fatty acids (LCFAs) on the function of pancreatic acinar cells (PACs). Abnormalities in the function of PACs include Ca overload, premature trypsinogen activation, protein kinase C (PKC) expression, endoplasmic reticulum (ER) stress, and mitochondrial and autophagic dysfunction. The study highlights the contribution of long-chain saturated fatty acids (LC-SFAs), especially palmitic acid (PA), to M1 macrophage polarization through the activation of the NLRP3 inflammasome and the NF-κB pathway. Furthermore, we investigated lipid lowering therapy for AP. This review establishes a theoretical foundation for pro-inflammatory mechanisms associated with FAs in AP and facilitating drug development.
PubMed: 38828311
DOI: 10.1016/j.heliyon.2024.e31296 -
BioRxiv : the Preprint Server For... May 2024Oncogenic mutations in KRAS are present in approximately 95% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) and are considered the initiating event...
Oncogenic mutations in KRAS are present in approximately 95% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) and are considered the initiating event of pancreatic intraepithelial neoplasia (PanIN) precursor lesions. While it is well established that KRAS mutations drive the activation of oncogenic kinase cascades during pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (PP2A) has been implicated in suppressing KRAS-driven cellular transformation. However, low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRAS induces the expression of both an endogenous inhibitor of PP2A activity, Cancerous Inhibitor of PP2A (CIP2A), and the PP2A substrate, c-MYC. Consistent with these findings, KRAS sequestered the specific PP2A subunit responsible for c-MYC degradation, B56α, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation , knockout of B56α promoted KRAS tumorigenesis by accelerating acinar-to-ductal metaplasia (ADM) and the formation of PanIN lesions. The process of ADM was attenuated in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (SMAPs). Together, our results suggest that suppression of PP2A-B56α through KRAS signaling can promote the MYC-driven initiation of pancreatic tumorigenesis.
PubMed: 38826439
DOI: 10.1101/2023.07.01.547283 -
BDJ Open May 2024Salivary gland (SG) hypofunction is a common clinical condition arising from radiotherapy to suppress head and neck cancers. The radiation often destroys the SG... (Review)
Review
OBJECTIVE
Salivary gland (SG) hypofunction is a common clinical condition arising from radiotherapy to suppress head and neck cancers. The radiation often destroys the SG secretory acini, and glands are left with limited regenerative potential. Due to the complex architecture of SG acini and ducts, three-dimensional (3D) bioprinting platforms have emerged to spatially define these in vitro epithelial units and develop mini-organs or organoids for regeneration. Due to the limited body of evidence, this comprehensive review highlights the advantages and challenges of bioprinting platforms for SG regeneration.
METHODS
SG microtissue engineering strategies such as magnetic 3D bioassembly of cells and microfluidic coaxial 3D bioprinting of cell-laden microfibers and microtubes have been proposed to replace the damaged acinar units, avoid the use of xenogeneic matrices (like Matrigel), and restore salivary flow.
RESULTS
Replacing the SG damaged organ is challenging due to its complex architecture, which combines a ductal network with acinar epithelial units to facilitate a unidirectional flow of saliva. Our research group was the first to develop 3D bioassembly SG epithelial functional organoids with innervation to respond to both cholinergic and adrenergic stimulation. More recently, microtissue engineering using coaxial 3D bioprinting of hydrogel microfibers and microtubes could also supported the formation of viable epithelial units. Both bioprinting approaches could overcome the need for Matrigel by facilitating the assembly of adult stem cells, such as human dental pulp stem cells, and primary SG cells into micro-sized 3D constructs able to produce their own matrix and self-organize into micro-modular tissue clusters with lumenized areas. Furthermore, extracellular vesicle (EV) therapies from organoid-derived secretome were also designed and validated ex vivo for SG regeneration after radiation damage.
CONCLUSION
Magnetic 3D bioassembly and microfluidic coaxial bioprinting platforms have the potential to create SG mini-organs for regenerative applications via organoid transplantation or organoid-derived EV therapies.
PubMed: 38816372
DOI: 10.1038/s41405-024-00219-2 -
BioRxiv : the Preprint Server For... Jun 2024Saliva contains antimicrobial peptides considered integral components of host innate immunity, and crucial for protection against colonizing microbial species. Most...
Saliva contains antimicrobial peptides considered integral components of host innate immunity, and crucial for protection against colonizing microbial species. Most notable is histatin-5 which is exclusively produced in salivary glands with uniquely potent antifungal activity against the opportunistic pathogen . Recently, SARS-CoV-2 was shown to replicate in salivary gland acinar cells eliciting local immune cell activation. In this study, we performed mechanistic and clinical studies to investigate the implications of SARS-CoV-2 infection on salivary histatin-5 production and colonization. Bulk RNA-sequencing of parotid salivary glands from COVID-19 autopsies demonstrated statistically significant decreased expression of histatin genes. hybridization, coupled with immunofluorescence for co-localization of SARS-CoV-2 spike and histatin in salivary gland cells, showed that histatin was absent or minimally present in acinar cells with replicating viruses. To investigate the clinical implications of these findings, salivary histatin-5 levels and oral burden in saliva samples from three independent cohorts of mild and severe COVID-19 patients and matched healthy controls were evaluated. Results revealed significantly reduced histatin-5 in SARS-CoV-2 infected subjects, concomitant with enhanced prevalence of . Analysis of prospectively recovered samples indicated that the decrease in histatin-5 is likely reversible in mild-moderate disease as concentrations tended to increase during the post-acute phase. Importantly, salivary cytokine profiling demonstrated correlations between activation of the Th17 inflammatory pathway, changes in histatin-5 concentrations, and subsequent clearance of in a heavily colonized subject. The importance of salivary histatin-5 in controlling the proliferation of was demonstrated using an assay where was able to proliferate in COVID-19 saliva with low histatin-5, but not with high histatin-5. Taken together, the findings from this study provide direct evidence implicating SARS-CoV-2 infection of salivary glands with compromised oral innate immunity, and potential predisposition to oral candidiasis.
PubMed: 38798323
DOI: 10.1101/2024.05.13.593942 -
Cancers May 2024We conducted a pilot study to analyze the differential methylation status of 20 primary acinar adenocarcinomas of the lungs. These adenocarcinomas had to be wild type in...
We conducted a pilot study to analyze the differential methylation status of 20 primary acinar adenocarcinomas of the lungs. These adenocarcinomas had to be wild type in mutation analysis and had either high (TPS > 50%; = 10) or negative (TPS < 1%; = 10) PD-L1 status to be integrated into our study. To examine the methylation of 866,895 specific sites, we utilized the Illumina Infinium EPIC bead chip array. Both hypermethylation and hypomethylation play significant roles in tumor development, progression, and metastasis. They also impact the formation of the tumor microenvironment, which plays a decisive role in tumor differentiation, epigenetics, dissemination, and immune evasion. The gained methylation patterns were correlated with PD-L1 expression. Our analysis has identified distinct methylation patterns in lung adenocarcinomas with high and negative PD-L1 expression. After analyzing the correlation between the methylation results of genes and promoters with their pathobiology, we found that tumors with high expression of PD-L1 tend to exhibit oncogenic effects through hypermethylation. On the other hand, tumors with negative PD-L1 expression show loss of their suppressor functions through hypomethylation. The suppressor functions of hypermethylated genes and promoters are ineffective compared to simultaneously activated dominant oncogenic mechanisms. The tumor microenvironment supports tumor growth in both groups.
PubMed: 38791918
DOI: 10.3390/cancers16101841 -
Current Issues in Molecular Biology May 2024Acute pancreatitis (AP) is characterized by elevated intracellular Ca concentrations, mitochondrial dysfunction, and oxidative stress in pancreatic acinar cells. Algal...
Acute pancreatitis (AP) is characterized by elevated intracellular Ca concentrations, mitochondrial dysfunction, and oxidative stress in pancreatic acinar cells. Algal oil (AO) has demonstrated antioxidant and anti-inflammatory properties. This study aims to explore the effects of algal oil on the microenvironment of AP. Rat pancreatic acinar AR42J cells were pretreated with AO containing 0, 50, 100, or 150 μM of docosahexaenoic acid (DHA) 2 h prior to AP induction using sodium taurocholate (STC). After 1 h of STC treatment, AR42J cells exhibited a significant increase in intracellular Ca concentration and the production of amylase, lipase, reactive oxygen species, and pro-inflammatory mediators, including tumor necrosis factor-α and interleukin-6. These STC-induced increases were markedly reduced in cells pretreated with AO. In comparison to cells without AO, those treated with a high dose of AO before STC exposure demonstrated a significant increase in mitochondrial membrane potential and a decrease in lipid peroxidation. Furthermore, STC-activated nuclear factor kappa-B (NF-κB) was attenuated in AO-pretreated cells, as evidenced by a significant decrease in activated NF-κB. In conclusion, AO may prevent damage to pancreatic acinar cells by alleviating intracellular Ca overload, mitigating mitochondrial dysfunction, reducing oxidative stress, and attenuating NF-κB-targeted inflammation.
PubMed: 38785535
DOI: 10.3390/cimb46050267 -
JCI Insight May 2024Acute Pancreatitis (AP) is among the most common hospital gastrointestinal diagnosis; understanding the mechanisms underlying the severity of AP are critical for...
Acute Pancreatitis (AP) is among the most common hospital gastrointestinal diagnosis; understanding the mechanisms underlying the severity of AP are critical for development of new treatment options for this disease. Here, we evaluate the biological function of phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in AP pathogenesis in two independent genetically engineered mouse models of AP. PFKFB3 is elevated in AP and severe AP (SAP) and knockout of Pfkfb3 abrogates the severity of alcoholic SAP (FAEE-SAP). Using a combination of genetic, pharmacological, and molecular studies we define the interaction of PFKFB3 with inositol 1,4,5-trisphosphate receptor (IP3R) as a key event mediating this phenomenon. Further analysis demonstrated that the interaction between PFKFB3 and IP3R promotes FAEE-SAP severity by altering intracellular calcium homeostasis in acinar cells. Together our results support a PFKFB3-driven mechanism controlling AP pathobiology and define this enzyme as a therapeutic target to ameliorate the severity of this dismal condition.
PubMed: 38781030
DOI: 10.1172/jci.insight.169481 -
Modern Pathology : An Official Journal... May 2024The new grading system for lung adenocarcinoma proposed by the International Association for the Study of Lung Cancer (IASLC) defines prognostic subgroups on the basis...
The new grading system for lung adenocarcinoma proposed by the International Association for the Study of Lung Cancer (IASLC) defines prognostic subgroups on the basis of histologic patterns observed on surgical specimens. This study sought to provide novel insights into the IASLC grading system, with particular focus on recurrence-specific survival (RSS) and lung cancer-specific survival among patients with stage I adenocarcinoma. Under the IASLC grading system, tumors were classified as grade 1 (lepidic predominant with <20% high-grade patterns [micropapillary, solid, and complex glandular]), grade 2 (acinar or papillary predominant with <20% high-grade patterns), or grade 3 (≥20% high-grade patterns). Kaplan-Meier survival estimates, pathologic features, and genomic profiles were investigated for patients whose disease was reclassified into a higher grade under the IASLC grading system on the basis of the hypothesis that they would strongly resemble patients with predominant high-grade tumors. Overall, 423 (29%) of 1443 patients with grade 1 or 2 tumors classified based on the predominant pattern-based grading system had their tumors upgraded to grade 3 based on the IASLC grading system. The RSS curves for patients with upgraded tumors were significantly different from those for patients with grade 1 or 2 tumors (log-rank P < .001) but not from those for patients with predominant high-grade patterns (P = .3). Patients with upgraded tumors had a similar incidence of visceral pleural invasion and spread of tumor through air spaces as patients with predominant high-grade patterns. In multivariable models, the IASLC grading system remained significantly associated with RSS and lung cancer-specific survival after adjustment for aggressive pathologic features such as visceral pleural invasion and spread of tumor through air spaces. The IASLC grading system outperforms the predominant pattern-based grading system and appropriately reclassifies tumors into higher grades with worse prognosis, even after other pathologic features of aggressiveness are considered.
PubMed: 38777035
DOI: 10.1016/j.modpat.2024.100520