Control Glands Research Articles

The roles of mTORC1 in parathyroid gland function in chronic kidney disease-induced secondary hyperparathyroidism: Evidence from male genetic mousemodels and clinical data.

Secondary hyperparathyroidism (SHP) associated with chronic kidney disease (CKD) contributes to morbidity and mortality, yet the related parathyroid signaling pathways are not fully understood. Previous studies have indicated that the parathyroid mTORC1 pathway is activated in both experimental CKD and hypocalcemia-induced SHP. Furthermore, mice with parathyroid-specific mTOR deficiency (PT-mTOR-/-) exhibit disrupted parathyroid glands, but maintain normal serum PTH levels. Conversely, PT-Tsc1-/- mice, with mTORC1 hyperactivation, have enlarged glands and high serum PTH and calcium levels. We now uncover links between mTORC1 function, parathyroid gland morphology, and the response to CKD. Despite impaired gland structure, PT-mTOR-/- mice increased serum PTH to levels similar to controls in response to CKD, but not to acute kidney injury (AKI), highlighting the adaptability of their parathyroid glands to chronic but not acute stimulation. PT-Tsc1-/- mice, with enlarged glands also exhibited a CKD-induced rise in serum PTH comparable to controls, but with a reduced magnitude, suggesting compromised secretion capacity. Parathyroid glands from PT-Tsc1-/- mice displayed sustained high PTH secretion in culture, with no further increase when exposed to calcium-depleted media, unlike control glands. Complementing these findings, human data from 106 healthcare organizations demonstrated that drug-induced mTORC1 inhibition is associated with reduced serum PTH and a lower incidence of SHP in kidney transplant recipients. Collectively, our findings underscore the complex interplay between mTORC1 signaling and gland structure in the pathogenesis of SHP.

Will Nigella sativa oil protect parotid glands of rats against cranium gamma irradiation? Histological and immunohistochemical evaluation

BackgroundRadiation plays an essential role in treating malignancies. Radiation exposure of salivary glands often results in permanent loss of their functions; therefore, their protection against radiation is crucial. Nigella sativa oil (NSO) is a useful antioxidant against free radicals. The purpose of this study was to investigate the radio-protective effect of NSO on oxidative injury of parotid glands of gamma-irradiated rats. Methods: Twenty-eight male albino rats were divided into four groups (n = 7): Group 1: Neither NSO nor radiation, Group 2: Rats received NSO 400 mg/kg, Group 3: Rats received 15 Gy cranium gamma irradiation & Group 4: Rats received gamma irradiation and NSO. Rats were sacrificed two weeks after the last NSO dose. Histological sections of parotid glands were stained with H&E, Masson’s trichrome and anti-TGF-β antibodies. Area percentage of Masson’s trichrome and TGF-β expression was morphometrically examined. Results: Parotid glands of control and NSO groups revealed normal morphology. Gamma-irradiated glands showed loss of normal acinar architecture and slight acinar shrinkage. NSO treatment of gamma-irradiated glands preserved acinar outline and architecture. Masson’s trichrome stained samples revealed trace amounts of collagen fibers in control and NSO groups, and excessive amounts of collagen fibers in gamma-irradiated group, in addition to few collagen fibers for gamma-irradiated glands treated with NSO. Additionally, control and NSO groups showed negative TGF-β expression. Gamma-irradiated group showed high TGF-β expression, while NSO treated gamma-irradiated group showed moderate TGF-β expression. Conclusions: Gamma-irradiation adversely affected parotid glands, and in contrast, NSO seemed to positively counteract this adverse effect.

Changes in Ocular Surface Parameter After Intense Pulsed Light Therapy in Ocular Demodicosis: A Case Report

Introduction : Demodex infestation may manifest as blepharitis and dry eye syndrome. Intense Pulsed Light (IPL) therapy is an emerging option for managing ocular demodicosis.
 Case Illustration : A 77-years-old woman complained of itch and watery eyes since four years ago. Meibomian pouting, telangiectasias, foamy secretions, and sleeves were identified in both eyelids. Right eye examination showed 52% and 42% meibomian dropout in the superior and inferior eyelid, respectively, Schirmer test 6 mm, lipid layer thickness (LLT) 43 nm, tear meniscus height (TMH) 0.36 mm, and non-invasive tear break-up time (NIBUT) 9.6 seconds. Left eye examination showed 65% and 56% meibomian dropout in the superior and inferior eyelid, respectively, Schirmer test 5 mm, LLT 66 nm, TMH 0.16 mm, and NIBUT 5.8 seconds. Demodex mites were identified during eyelash wet mount examination. She underwent four sessions of IPL. At the two-month follow-up, complaints were alleviated. Signs of blepharitis were subsided. Right eye examination showed 36% and 34% meibomian dropout in the superior and inferior eyelid, respectively, Schirmer's test 9 mm, LLT 68 nm, TMH 0.49 mm, and NIBUT 10.7 seconds. Left eye examination showed 25% and 35% meibomian dropout in the superior and inferior eyelid, respectively, Schirmer's test 9 mm, LLT >100 nm, TMH 0.9 mm, and NIBUT 6.2 seconds.
 Discussion : IPL has a role in managing ocular demodicosis by local inflammation control, meibum liquefaction, and meibomian glands photomodulation. IPL is also thought to directly eradicate Demodex mites.
 Conclusion : Improvement of ocular surface parameters was observed after IPL therapy in ocular demodicosis.

Abstract A084: DEK overexpression in the murine mammary gland promotes epithelial hyperplasia

Abstract Introduction: Breast cancer (BC) remains a leading cause of cancer-associated mortality for women in the USA, which makes it critically important to identify and study molecular drivers of pathogenesis. We have previously identified the chromatin modifying DEK protein to have oncogenic properties. DEK is a histone H3 chaperone that is over-expressed in over 60% of breast cancers, particularly triple negative breast cancers, and is associated with poor survival. To date, all studies regarding the role of DEK in BC have manipulated DEK expression in cultured immortalized MCF10A human mammary epithelial cells and BC cell lines. However, the oncogenic functions of DEK in vivo and in normal mammary epithelium have remained unexplored. Methods: We utilized a tetracycline-off regulated, DEK overexpressing (DEK-OE) transgenic mouse model that targets mammary epithelium through the mouse mammary tumor virus (MMTV) promoter. We subsequently performed bulk RNA-Seq transcriptomic analyses, western blotting, and both histological and morphological assessments of control vs. DEK-OE mammary gland tissue to identify the oncogenic effects of DEK over-expression in vivo. Results: Bulk RNA sequencing and gene ontology analyses identified cell cycle regulation as a significantly upregulated biological process in DEK-OE versus control glands. Subsequent studies confirmed upregulation of proteins that promote the G1-S cell cycle transition including cyclins A and D and CDKs 2, 4, and 6 as well as increased numbers of Ki67+ cells. We also observed down-regulation of genes that inhibit entry the G1-S transition, such as p21 and p27. This pro-proliferative molecular status correlated with the development of mammary gland hyperplasia. Importantly, in silico analyses demonstrated gene correlations between DEK and cell cycle regulators that were also evident in human breast cancers. One potential mechanism for this transcriptional deregulation is that we identified increased histone H3K27me3 epigenetic modifications in DEK-OE cells, in both mouse and human models, which may occur through EZH2 activity. Conclusions: Combined, our work indicates that DEK is not a tumor initiator, but its oncogenic activities are likely due to its ability to promote proliferation of mammary epithelial cells. Furthermore, we report a novel function for DEK in the epigenetic regulation of histone H3 K27 trimethylation, which is mediated by the well-known EZH2 oncogene in triple negative breast cancers. Citation Format: Megan Johnstone, Ashley Leck, Taylor Lange, Lisa Privette Vinnedge. DEK overexpression in the murine mammary gland promotes epithelial hyperplasia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A084.

Abstract A035: An analysis of the contribution of the glutathione and thioredoxin antioxidant pathways to triple negative breast cancer development

Abstract The accumulation of reactive oxygen species (ROS), also known as oxidative stress, occurs during tumorigenesis, for example because of changes in cell metabolism or in a cell’s microenvironment. Since elevated ROS can cause senescence or cell death, cells with increased ROS-buffering capacity are selected for during tumor initiation and progression. It is unknown, however, at which stage during carcinogenesis oxidative stress develops, which oxidative response programs are engaged, and whether there is a spatial distribution of oxidative stress within the tissue. Our lab developed a murineTrp53loxp/loxp Brca1loxp/loxp model of triple negative breast cancer (TNBC) that histologically recapitulates the disease progression of human, progressing from normal epithelium, to abnormal proliferation, before advancing to adenocarcinoma. We are applying cyclic immunofluorescence and spatial transcriptomics to study relevant antioxidant pathways in situ. These studies are complemented by analyses of previously published scRNAseq datasets from similar models. Our initial observations have shown that antioxidant expression peaks at a stage of tumorigenesis prior to frank tumor formation, suggesting that nascent tumor cells must overcome oxidative stress to progress. Inhibition of a key antioxidant pathway, the glutathione pathway, did not significantly decrease the Trp53-/- Brca1-/- cell population, and preliminary spatial transcriptomics data do not support an induction of the major players of this pathway during tumorigenesis. A closer examination of the expression patterns of these genes within the mammary gland demonstrates that they are not expressed in the cell of origin of TNBC. In contrast, members of the thioredoxin (TXN) pathway are expressed in the precursor cells of TNBC. One member of the TXN pathway, Txnrd1, is upregulated in Trp53-/- Brca1-/- glands, but not contralateral control glands. This upregulation occurs around the time of epithelial cell expansion post tumor suppressor loss. Furthermore, glutathione pathway inhibition drives Txnrd1 protein expression up in the mutant, but not wildtype glands. Together, our data support an important role for the thioredoxin pathway in reducing oxidative stress during tumorigenesis. Ongoing work aims to assess whether the thioredoxin pathway may represent a viable treatment option to prevent or slow the development of breast cancer in the Trp53/Brca1-deficient background. Insights into vulnerabilities of Brca1-deficient cells may provide BRCA1 germline mutation carriers new approaches for cancer prevention that have otherwise been limited to mastectomy and oophorectomy. Citation Format: Nomeda Girnius, Aylin Z Henstridge, Francesca Silvestri, Carman Man Chung Li, Joan S Brugge. An analysis of the contribution of the glutathione and thioredoxin antioxidant pathways to triple negative breast cancer development [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A035.

Method for evaluating milk production in mouse mammary gland.

Lactation is a characteristic physiological function of mammals and is important for nourishing infants and the dairy industry; however, the molecular mechanisms underlying the function remain to be elucidated. A technique to directly evaluate the quantity and quality of milk in mice is necessary for the study of the lactation mechanism in vivo. By measuring the changes in milk amount after different durations of milk accumulation (0-24 h) using a ductal cannulation technique and oxytocin supplementation, we estimated the milk production rate at a single mammary gland level. In addition, collected milk was available to assess milk quality, including creamatocrit, osmolarity, and concentrations of ions, lactose, and total protein. Moreover, as a proof of principle, the effects of intraductal administration of a hypertonic solution to the abdominal mammary gland were examined. This stimulation increased milk amount, possibly by osmosis, compared with the contralateral control gland. These results demonstrated that this method is useful for examining the lactation ability and mechanisms in vivo. Studies using this method will contribute to the further understanding of lactation mechanisms in mammals.

Lysosome-Associated Membrane Protein 3 Induces Lysosome-Dependent Cell Death by Impairing Autophagic Caspase 8 Degradation in the Salivary Glands of Individuals With Sjögren's Disease.

Lysosome-associated membrane protein 3 (LAMP3) overexpression is implicated in the development and progression of Sjögren's disease (SjD) by inducing lysosomal membrane permeabilization (LMP) and apoptotic cell death in salivary gland epithelium. The aim of this study was to clarify the molecular details of LAMP3-induced lysosome-dependent cell death and to test lysosomal biogenesis as a therapeutic intervention. Human labial minor salivary gland biopsies were analyzed using immunofluorescence staining for LAMP3 expression levels and galectin-3 puncta formation, a marker of LMP. Expression level of caspase 8, an initiator of LMP, was determined by Western blotting in cell culture. Galectin-3 puncta formation and apoptosis were evaluated in cell cultures and a mouse model treated with glucagon-like peptide 1 receptor (GLP-1R) agonists, a known promoter of lysosomal biogenesis. Galectin-3 puncta formation was more frequent in the salivary glands of SjD patients compared to control glands. The proportion of galectin-3 puncta-positive cells was positively correlated with LAMP3 expression levels in the glands. LAMP3 overexpression increased caspase 8 expression, and knockdown of caspase 8 decreased galectin-3 puncta formation and apoptosis in LAMP3-overexpressing cells. Inhibition of autophagy increased caspase 8 expression, while restoration of lysosomal function using GLP-1R agonists decreased caspase 8 expression, which reduced galectin-3 puncta formation and apoptosis in both LAMP3-overexpressing cells and mice. LAMP3 overexpression induced lysosomal dysfunction, resulting in lysosome-dependent cell death via impaired autophagic caspase 8 degradation, and restoring lysosomal function using GLP-1R agonists could prevent this. These findings suggested that LAMP3-induced lysosomal dysfunction is central to disease development and is a target for therapeutic intervention in SjD.

Development and accuracy validation of a fat fraction imaging biomarker for sialadenitis in the parotid gland

BackgroundThe diagnosis of sialadenitis, the most frequent disease of the salivary glands, is challenging when the symptoms are mild. In such cases, biomarkers can be used as definitive diagnostic indicators. Recently, biomarkers have been developed by extracting and analyzing pathological and morphological features from medical imaging. This study aimed to establish a diagnostic reference for sialadenitis based on the quantitative magnetic resonance imaging (MRI) biomarker IDEAL-IQ and assess its accuracy.MethodsPatients with sialadenitis (n = 46) and control subjects (n = 90) that underwent MRI were selected. Considering that the IDEAL-IQ value is a sensitive fat fractional marker to the body mass index (BMI), all subjects were also categorized as under-, normal-, and overweight. The fat fraction of parotid gland in the control and sialadenitis groups were obtained using IDEAL-IQ map. The values from the subjects in the control and sialadenitis groups were compared in each BMI category. For comparison, t-tests and receiver operating characteristic (ROC) curve analyses were performed.ResultsThe IDEAL-IQ fat faction of the control and sialadenitis glands were 38.57% and 23.69%, respectively, and the differences were significant. The values were significantly lower in the sialadenitis group (P), regardless of the BMI types. The area under the ROC curve (AUC) was 0.83 (cut-off value: 28.72) in patients with sialadenitis. The AUC for under-, normal-, and overweight individuals were 0.78, 0.81, and 0.92, respectively.ConclusionsThe fat fraction marker based on the IDEAL-IQ method was useful as an objective indicator for diagnosing sialadenitis. This marker would aid less-experienced clinicians in diagnosing sialadenitis.

Evaluating the transcriptional landscape and cell-cell communication networks in chronically irradiated parotid glands

Understanding the transcriptional landscape that results in chronic salivary hypofunction after irradiation will help identify injury mechanisms and develop regenerative therapies. We present scRNA-seq analysis from control and irradiated murine parotid glands collected 10months after irradiation. We identify a population of secretory cells defined by specific expression of Etv1, which may be an acinar cell precursor. Acinar and Etv1+ secretory express Ntrk2 and Erbb3, respectively while the ligands for these receptors are expressed in myoepithelial and stromal cells. Furthermore, our data suggests that secretory cells and CD4+CD8+T-cells are the most transcriptionally affected during chronic injury with radiation, suggesting active immune involvement. Lastly, evaluation of cell-cell communication networks predicts that neurotrophin, neuregulin, ECM, and immune signaling are dysregulated after irradiation, and thus may play a role in the lack of repair. This resource will be helpful to understand cell-specific pathways that may be targeted to repair chronic damage in irradiated glands.

Acinar Cell Proliferation Promoted by BMP2 in Injured Mouse Parotid Gland: BMP2 Promotes Cell Proliferation in Parotid Gland.

To identify factors that affect salivary gland recovery, we investigated the expression and function of bone morphogenetic protein 2 (BMP2) in mice. Using a micro clip, mice parotid glands were removed 7 days after the ligation of the unilateral parotid excretory duct. Thereafter, they were weighed and stained with hematoxylin and eosin, and BMP2 expression was examined via real-time reverse transcription-polymerase chain reaction. Primary cultures of parotid glands were prepared, and BMP2 protein was added to the culture medium for 48 hr to examine its effect on cell proliferation. E-cadherin and vimentin expression was examined using western blotting. Finally, immunohistochemical staining using an anti-Ki67 antibody was performed. Duct-ligated parotid glands weighed less than those that were collected after sham surgery and showed acinar cell atrophy. They also showed higher BMP2 expression than control glands. Primary-cultured parotid acinar cells supplemented with BMP2 showed higher proliferative potential than control cells. Furthermore, they showed E-cadherin, but not vimentin, expression, and their percentage of Ki67-positive cells were higher than that corresponding to the controls. Injury to salivary glands by excretory duct ligation increased BMP2 expression, which may be involved in maintaining salivary gland function by inducing acinar cell proliferation.

Separate and combined effects of advanced age and obesity on mammary adipose inflammation, immunosuppression and tumor progression in mouse models of triple negative breast cancer.

Advanced age and obesity are independent risk and progression factors for triple negative breast cancer (TNBC), which presents significant public health concerns for the aging population and its increasing burden of obesity. Due to parallels between advanced age- and obesityrelated biology, particularly adipose inflammation, we hypothesized that advanced age and obesity each accelerate mammary tumor growth through convergent, and likely interactive, mechanisms. To test this hypothesis, we orthotopically transplanted murine syngeneic TNBC cells into the mammary glands of young normoweight control (7 months), young diet-induced obese (DIO), aged normoweight control (17 months), and aged DIO female C57BL/6J mice. Here we report accelerated tumor growth in aged control and young DIO mice, compared with young controls. Transcriptional analyses revealed, with a few exceptions, overlapping patterns of mammary tumor inflammation and tumor immunosuppression in aged control mice and young DIO mice, relative to young controls. Moreover, aged control and young DIO tumors, compared with young controls, had reduced abundance ofcytotoxic CD8 T cells. Finally, DIO in advanced age exacerbated mammary tumor growth, inflammation and tumor immunosuppression. These findings demonstrate commonalities in the mechanisms driving TNBC in aged and obese mice, relative to young normoweight controls. Moreover, we found that advanced age and DIO interact to accelerate mammary tumor progression. Given the US population is getting older and more obese, age- and obesity-related biological differences will need to be considered when developing mechanism-based strategies for preventing or controlling breast cancer.

Comprehensive RNA-seq profiling to evaluate the rabbit mammary gland transcriptome after mastitis.

Mastitis is a relatively common disease in rabbit does. The aim of this study was to investigate a relationship between the severity of clinical signs and pathological observations, and to analyze differentially expressed genes (DEGs) in the mammary gland with mastitis versus healthy mammary gland. The result showed that rectal temperatures of the rabbits with both mild mastitis and severe mastitis were higher than that of control. Cell counting result showed that the somatic cell count (SCC) only in milk of the rabbit with severe mastitis was significantly higher than that in the control group. However, the number of heterophils in the histological sections of mammary gland with mild mastitis was significantly higher than that of control. A total of 1096 DEGs between the control and mastitis mammary gland was identified by RNA-sequencing (RNA-seq). Gene ontology (GO) showed that most of up-regulated genes were enriched in terms such as Response to stimulus, Signal transduction, and Cell communication. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these genes were mostly enriched in the pathways such as Rap1 signaling pathway, Proteoglycans in cancer, and PI3K-Akt signaling pathway. However, the downregulated genes were mainly enriched in metabolic process and significantly involved in Metabolic pathway. The data provides useful information to further dissect the molecular genetic mechanisms underlying rabbit mastitis, which is a prerequisite for designing effective intervention strategies.

Sublethal cadmium exposure in the freshwater snail Lymnaea stagnalis meets a deficient, poorly responsive metallothionein system while evoking oxidative and cellular stress

The Great Pond snail Lymnaea stagnalis (Gastropoda, Hygrophila) is a wide-spread freshwater gastropod, being considered as a model organism for research in many fields of biology, including ecotoxicology. The aim of the present study was to explore the Cd sensitivity of L. stagnalis through the measurement of a biomarker battery for oxidative, toxic and cellular stress. The interpretation of biomarker parameters occurred against the background of a truncated metallothionein protein with a limited Cd-binding capacity. Individuals of L. stagnalis were exposed through 14 days to uncontaminated water (controls) or to low (30 μg · L−1) or high (50 μg · L−1) Cd concentrations. The digestive gland of control and low-Cd exposed snails was processed for transcriptional analysis of the Metallothionein (MT) gene expression, and for determination of biomarkers for oxidative stress, toxicity and cellular stress. Digestive gland supernatants of high-Cd exposed snails were subjected to chromatography and subsequent analysis by spectrophotometry. It was shown that the MT system of L. stagnalis is functionally deficient, with a poor Cd responsiveness at both, the transcriptional and the protein expression levels. Instead, L. stagnalis appears to rely on alternative detoxification mechanisms such as Cd binding by phytochelatins and metal inactivation by compartmentalization within the lysosomal system. In spite of this, however, traces of Cd apparently leak out of the pre-determined detoxification pathways, leading to adverse effects, which is clearly indicated by biomarkers of oxidative and cellular stress.

Change in Morphological Features of Enlarged Subarachnoid Spaces Following Treatment in Idiopathic Normal Pressure Hydrocephalus.

Focally enlarged sulci (FES) are areas of proposed extraventricular fluid entrapment that may occur within idiopathic normal pressure hydrocephalus (iNPH) with radiographic evidence of disproportionately enlarged subarachnoid-space hydrocephalus (DESH), and should be differentiated from atrophy. To evaluate for change in FES size and pituitary height after shunt placement in iNPH. Retrospective. A total of 125 iNPH patients who underwent shunt surgery and 40 age-matched controls. 1.5 T and 3T. Axial T2w FLAIR, 3D T1w MPRAGE, 2D sagittal T1w. FES were measured in three dimensions and volume was estimated by assuming an ellipsoid shape. Pituitary gland height was measured in the mid third of the gland in iNPH patients and controls. Wilcoxon signed-rank test for comparisons between MRI measurements; Wilcoxon rank sum test for comparison ofcases/controls. Significance level was P < 0.05. Fifty percent of the patients had FES. FES volume significantly decreased between the pre and first postshunt MRI by a median of 303 mm3 or 30.0%. Pituitary gland size significantly increased by 0.48 mm or 14.4%. FES decreased significantly by 190 mm3 or 23.1% and pituitary gland size increased significantly by 0.25 mm or 6% between the first and last postshunt MRI. Decrease in size of FES after shunt placement provides further evidence that these regions are due to disordered cerebrospinal fluid (CSF) dynamics and should not be misinterpreted as atrophy. A relatively smaller pituitary gland in iNPH patients that normalizes after shunt is a less-well recognized feature of altered CSF dynamics. 3 TECHNICAL EFFICACY: Stage 2.

In Vivo Transfection of Rat Salivary Glands With Fluorescently Tagged Aquaporin-5 Channel DNA.

Background The acinar cells of salivary glands are responsible for most saliva production and are, unfortunately, h­­ighly radiosensitive. As such, dry mouth or xerostomia is an adverse effect experienced by half of head and neck cancer patients treated with radiation. We evaluate a novel method of gene transfection of aquaporin channels to rat salivary glands.Materials and methodsA green fluorescent protein (GFP)-tagged human Aquaporin-5 (AQP5) cDNA sequence cloned into a pCMV6-AC-GFP vector was complexed with lipofectamine 2000. One submandibular gland of the anesthetized rats was injected with the complexed cDNA and lipid solution under ultrasound guidance, while the opposite gland was injected with the vehicle control. The animals were sacrificed between 24 to 48 hours post-injection. The salivary glands were removed and evaluated via fluorescence imaging. Western blot assays were also performed to determine AQP5 cDNA expression.Results In the experiments, the submandibular glands were identified and injected under ultrasound guidance. Four control glands and eight experimental glands were evaluated. The cDNA was expressed successfully and variably within the experimental glands, noting greater intensity along the cell surface consistent with appropriate trafficking of the AQP5 channel. Western blot analysis demonstrated variable expression in the experimental sample with no expression in the control sample. Several glands across the groups showed mild to moderate interstitial edema or inflammation.Conclusion In this study, we demonstrate an alternative in vivo transfection method via lipofection and demonstrate the successful expression of the AQP5 channel in rat salivary gland tissue.

Contribution of Interleukin‐4–Induced Epithelial Cell Senescence to Glandular Fibrosis in IgG4‐Related Sialadenitis

IgG4-related sialadenitis (IgG4-RS) is a chronic fibroinflammatory disease characterized by glandular fibrosis and hyposalivation. This study was undertaken to explore the role of cellular senescence in the pathogenesis of IgG4-RS-related fibrosis. The expression of senescence markers and proinflammatory cytokines in the submandibular glands (SMGs) of IgG4-RS patients (n = 18) and controls (n = 14) was determined by proteomics, real-time polymerase chain reaction, Western blotting, and immunohistochemistry. After interleukin-4 (IL-4) treatment, high-throughput RNA sequencing was performed to identify the differentially expressed genes in SMG-C6 cells. A glandular fibrosis model was established by the intraglandular injection of IL-4 into mouse SMGs (n = 8 per group). Salivary acinar and ductal epithelial cells underwent senescence in IgG4-RS patients, as indicated by the elevated activity of senescence-associated β-galactosidase, lipofuscin accumulation, enhanced expression of senescence markers (p53 and p16INK4A ), and up-regulation of senescence-associated secretory phenotype factors. Moreover, there was a significant increase in IL-4 levels in SMGs from IgG4-RS patients (P < 0.01), which positively correlated with p16INK4A expression and the fibrosis score. Incubation with IL-4 exacerbated salivary epithelial cell senescence by increasing the expression of p16INK4A through the reactive oxygen species (ROS)/p38 MAPK pathway. Supernatant collected from IL-4-induced senescent SMG-C6 cells enhanced fibroblast activation and matrix protein production (P < 0.05). Furthermore, injecting mice with IL-4 promoted fibrosis and senescence phenotypes in SMGs in vivo. The cellular senescence induced by IL-4 through the ROS/p38 MAPK-p16INK4A pathway promotes fibrogenesis in IgG4-RS. Our data suggest that cellular senescence could serve as a novel therapeutic target for treating IgG4-RS.

SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

SUMMARYInherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.

Analysis of the Human Pineal Proteome by Mass Spectrometry.

The human pineal gland regulates the day-night dynamics of multiple physiological processes, especially through the secretion of melatonin. Recently, using mass spectrometry-based proteomics and dedicated analysis tools, we have identified regulated proteins and signaling pathways that differ between day and night and/or between control and autistic pineal glands. This large-scale proteomic approach is the method of choice to study proteins in a biological system globally. This chapter proposes a protocol for large-scale analysis of the pineal gland proteome.

Developing the Concepts of Homeostasis, Homeorhesis, Allostasis, Elasticity, Flexibility and Plasticity of Brain Function

I discuss some concepts advanced for the understanding of the complex dynamics of brain functions, and relate them to approaches in affective, cognitive and action neurosciences. These functions involve neuro-glial interactions in a dynamic system that receives sensory signals from the outside of the central nervous system, processes information in frequency, amplitude and phase-modulated electrochemical waves, and control muscles and glands to generate behavioral patterns. The astrocyte network is in charge of controlling global electrochemical homeostasis, and Hodgkin–Huxley dynamics drive the bioelectric homeostasis of single neurons. In elastic processes, perturbations cause instability, but the system returns to the basal equilibrium. In allostatic processes, perturbations elicit a response from the system, reacting to the deviation and driving the system to stable states far from the homeostatic equilibrium. When the system does not return to a fixed point or region of the state space, the process is called homeorhetic, and may present two types of evolution: (a) In flexible processes, there are previously existing “attractor” stable states that may be achieved after the perturbation, depending on context; (b) In plastic processes, the homeostatic set point(s) is(are) changed; the system is in a process of adaptation, in which the allostatic forces do not drive it back to the previous set point, but project to the new one. In the temporal phase from the deviant state to the recovery of stability, the system generates sensations that indicate if the recovery is successful (pleasure-like sensations) or if there is a failure (pain-like sensations).

Dosimetric, Volumetric, and Positional Stability After Submandibular Gland Transfer

Xerostomia and its effects are significant challenges for patients receiving radiation therapy (RT) for head and neck cancers (HNC). Submandibular gland (SMG) transfer has demonstrated promise in reduction of subjective and objective xerostomia. However, SMG transfer investigations have largely employed conventional RT or adjuvant RT, with little evaluation in patients treated with definitive modern RT. Furthermore, there has been no assessment of whether anatomic changes during RT may affect the potential benefits of SMG. We sought to characterize the preliminary dosimetric, volumetric, and positional stability of SMG transfer on an ongoing prospective case-control study.Previously untreated patients with oropharyngeal HNC, N0-N2b (unilateral neck disease) were enrolled. SMG transfers were performed by experienced surgeons per the Jha and Seikaly technique, transposing the gland anteriorly into the submental neck. Definitive VMAT plans were optimized according to institutional guidelines (goal mean SMG dose < 26 Gy), patients were treated to 70 Gy in 33-35 fractions. To assess the stability of planned dose and change in SMG volume and position, repeat CT simulation was performed at week 4. These were rigidly registered to the initial simulation centered about the mandible and hyoid, with the original dose superimposed. Glands were meticulously re-contoured on both scans. Statistical comparisons were by Wilcoxon paired rank tests or Mann-Whitney U tests.To date, 11 patients with SMG transfer and 5 controls have been treated, all male. In 3 cases, no re-simulation was available and a week 4 cone-beam CT was used. The mean time from gland transfer to RT start was 28.3 days. From simulation to week 4, transposed SMGs shrunk by a mean of 3.49 cc (27.6%), no different than corresponding contralateral SMGs at 2.37 cc (23.5%) or control SMGs (SMGs in control patients on the N0 side) at 1.71 cc (22.8%). There were no associations between SMG volume change and dose by linear regression or Spearman's rank correlation. Transposed SMGs had an average mean dose of 28.2 Gy on initial sim. At week 4, extrapolated to a full course, this increased to 32.8 Gy (P = 0.01). Average mean dose to control glands was 46 Gy, statistically greater than transposed glands (P = 0.03), with a non-significant 1.5 Gy increase at week 4. Mean change in geometric position was 0.45 cm for transposed glands, 0.37 cm for contralateral glands (P = .19), and 0.32 for control glands (P = .37).SMG transfer achieved significant reduction in dose compared to controls. While SMG shrinkage and movement were not separately different from contralateral or control glands, the higher dose at re-simulation indicates these factors may together have an effect, calling for careful SMG delineation, attention to daily position and image guidance, and planned re-simulation in order to maximize dosimetric benefits. Our trial is ongoing and clinical and QoL endpoints will be examined with these findings in mind.