Nuclei Of Epithelial Cells Research Articles

Cancer of unknown primary with gastrointestinal profiles: A distinct CUP subset.

3606 Background: Cancer of unknown primary (CUP) with a “gastrointestinal (GI) profile” appears to be a clinically distinct subset, based on immunophenotyping, specifically staining with cytokeratins 20 (CK20) and 7 (CK7), a type I and II keratin, respectively and caudal type homeobox 2 (CDX2) protein, a transcription factor expressed in nuclei of intestinal epithelial cells. However, only a limited clinicomolecular account of this entity exists. Comprehensive profiling is needed to substantially impact personalized therapeutics and improve prognosis. Methods: We identified 401 pts evaluated at MD Anderson Cancer Center. Pts were classified into 3 cohorts based on immunohistochemistry: lower GI profile CUP (LGI-CUP), other-GI profile CUP (OGI-CUP), or non-GI CUP (NGI-CUP). A control group of known lower GI primary cancers was derived from MSK-IMPACT data (cBioPortal, 1075 patients, LGI-KP). Clinical and pathological data including molecular profiling, therapy and survival were logged. Fisher-exact test was used. Kaplan-Meier method was used to estimate overall survival (OS) and compared with log-rank test. Results: Among 748 pts, 401 (53.6%) had adequate immunostaining for analysis. Of these, 72 (18.0%), 226 (56.4%) and 103 (25.7%) were classified as LGI-CUP, OGI-CUP, and NGI-CUP, respectively. LGI-CUP were enriched for adenocarcinoma (88% v 86% v 35%, P < 0.0001) and good risk Culine score (59% v 51% v 37%, P = 0.02), compared to OGI-CUP and NGI-CUP, respectively. While NGI-CUP had greater proportions of poorly differentiated histology (91% v 55% vs 66%, P < 0.0001) and bone metastases (30% vs 7% v 24%, P = 0.0001) compared to LGI-CUP and OGI-CUP, respectively. Comparison of key mutations is shown in table. Median OS of LGI-CUP (14.6 months [95%CI: 12.0 – 17.2]) was similar to OGI-CUP (14.3 months [95%CI: 10.7 – 18.0]) and NGI-CUP (16.2 months [95%CI: 12.4 – 20.0]), P = 0.75, but significantly lower than LGI-KP cohort (28.6 months, [95%CI 24.4 – 32.7], p < 0.001). LGI-CUP was treated preferentially with fluoropyrimidine-based GI regimen [5-FU/capecitabine +/- oxaliplatin or irinotecan] (77% vs 43% v 5%) compared to OGI-CUP and NGI-CUP, respectively. However, median OS for LGI-CUP treated with 5-FU-based regimens was higher (16.0, 95%CI 13.0 – 19.0) than those treated with non-5-FU-based treatments (10.9, 95%CI 5.6 – 16.2), P = 0.002. Conclusions: LGI-CUP appears to be a molecularly distinct entity from and NGI-CUP, as well as lower GI CUP from known entities. LGI-CUP patients treated with 5-FU-based regimens appear to have improved survival. [Table: see text]

Immunoexpression of SIRT1, 6, and 7 in oral leukoplakia and oral squamous cell carcinoma.

Sirtuins (SIRTs) are a family of proteins involved in the metabolic process responsible for extending the lifespan. The role of SIRT1, 6, and 7 in oral squamous cell carcinoma (OSCC) and oral leukoplakia (OLP), one of its precursors, is still elusive. In this study, 82 OLP and 77 OSCC were immunohistochemically examined for SIRT1, 6, and 7. Stained sections were thoroughly scanned and evaluated using a digital image analysis program. The SIRT1, 6, and 7 expressions were detected in the nuclei of epithelial and carcinoma cells in various degrees. Afterward, any correlations among SIRTs, including associations with clinicopathological features and the Kaplan-Meier curves were analyzed. OSCC demonstrated significantly higher SIRT1 expression than OLP, while non-dysplastic lesions showed significantly higher SIRT6 expression than other lesions. A strong correlation was observed between SIRT6 and 7 in OLP, SIRT1 and 6 in in OSCC and in SIRT6 and 7 when all lesion types were considered. There were no significant differences between SIRTs reactivity and the clinical features in OLP. For OSCC, SIRT1 and 6 was found to be directly associated with site of the lesion, while SIRT7 showed a direct relationship between gender, stromal lymphocytic infiltration, and depth of the invasion. OSCC with high SIRT7 expression revealed a slightly lower survival probability, although not statistically significant (p = 0.1019). Our findings suggest that SIRT1, 6, and 7 may play correlated and diverse roles in the development and advancement of OSCC.

IL-33 promotes inflammatory responses and exacerbates lung pathology in a mouse model of SARS-CoV-2 infection

Abstract The ongoing COVID-19 pandemic caused by SARS-CoV-2 has resulted in more than 6.6 million deaths worldwide as of the end of 2022. Clinical data revealed that dysregulated host immune responses may contribute to disease severity and poor recovery outcomes; however, the mechanisms that underlie immune dysregulation and disease severity are relatively unknown. Interleukin-33 (IL-33), a damage-associated molecular pattern (DAMP) molecule, is mainly expressed in nucleus of epithelial and endothelial cells. Elevated serum IL-33 levels have been recently reported in COVID-19 patients and might be associated with adverse outcomes. In this study, we infected B6 mice with a mouse-adapted SARS-CoV-2 MA10 and found that IL-33 was significantly increased in the lungs at day 2 post-infection, accompanied by highly upregulated inflammatory chemokines CXCL1, CXCL2 and CCL2. Next, we used IL-33 −/−mice and demonstrated that IL-33 deficiency resulted in less bodyweight loss at days 2 and 3 and decreased viral burden at day 4. Flow cytometry analyses showed that IL-33 −/−mice had reduced numbers of innate immune cells at day 2, including neutrophils, macrophages and NK cells. At day 4, IL-33 −/−mice displayed reduced activated T cells in the lungs as compared to WT mice. Lung tissue RNAseq transcriptomics revealed that IL-33 signals contributed to several inflammatory pathways, including type II interferon signaling, pathogen phagocytosis, macrophage activation, cytokine/chemokine and inflammatory responses and oxidative damage. Overall, these data uncover a detrimental role of the DAMP IL-33 in the lungs following SARS-CoV-2 infection and provide new insights to support the development of effective therapeutic strategies for COVID-19. Supported by grants from NIH (R21 AI153586)

THE IL-33/ST2 AXIS PROMOTES ACUTE RESPIRATORY DISTRESS SYNDROME BY NATURAL KILLER T CELLS.

Acute respiratory distress syndrome (ARDS) is characterized by uncontrolled inflammation, which manifests as leukocyte infiltration and lung injury. However, the molecules that initiate this infiltration remain incompletely understood. We evaluated the effect of the nuclear alarmin IL-33 on lung damage and the immune response in LPS-induced lung injury. We established a LPS-induced lung injury mouse model. We used genetically engineered mice to investigate the relationship among the IL-33/ST2 axis, NKT cells, and ARDS. We found that IL-33 was localized to the nucleus in alveolar epithelial cells, from which it was released 1 h after ARDS induction in wild-type (WT) mice. Mice lacking IL-33 (IL-33 - / - ) or ST2 (ST2 - / - ) exhibited reduced neutrophil infiltration, alveolar capillary leakage, and lung injury in ARDS compared with WT mice. This protection was associated with decreased lung recruitment and activation of invariant nature killer (iNKT) cells and activation of traditional T cells. Then, we validated that iNKT cells were deleterious in ARDS in CD1d - / - and Vα14Τg mice. Compared with WT mice, Vα14Τg mice exhibited increased lung injury in ARDS, and the CD1d - / - mice showed outcomes opposite those of the Vα14Τg mice. Furthermore, we administered a neutralizing anti-ST2 antibody to LPS-treated WT and Vα14Τg mice 1 h before LPS administration. We found that IL-33 promoted inflammation through NKT cells in ARDS. In summary, our results demonstrated that the IL-33/ST2 axis promotes the early uncontrolled inflammatory response in ARDS by activating and recruiting iNKT cells. Therefore, IL-33 and NKT cells may be therapeutic target molecules and immune cells, respectively, in early ARDS cytokine storms.

Abstract 777: Super-resolution imaging of heterochromatin structure detects early events in lung cancer

Abstract Early detection of lung cancer is essential to reduce lung cancer-related morbidity and mortality. Despite significant efforts, current approaches suffer from high false-positive rates or limited sensitivity. There is a critical need for non-invasive, cost-effective technologies that detect the early changes in lung carcinogenesis with high sensitivity and specificity. Abnormal chromatin structure is one of the hallmarks of cancer cells and is independent of molecular pathways making it an attractive target for early cancer detection. In this study, we assessed the use of super-resolution stochastic optical reconstruction microscopy (STORM) to detect abnormal heterochromatin disruption in lung carcinogenesis. First, utilizing a mouse model of tobacco carcinogen (NNK)-induced lung adenocarcinoma, we analyzed heterochromatin structure in the lungs of NNK-treated mice at 8, 15 and 20 weeks post-NNK exposure together with non-treated control mice at the same time points. Super-resolution images of NNK-induced lung lesions at all time points showed significant disruption of condensed heterochromatin nanodomains compared to normal alveolar epithelial cells from the control mice, consistent with our data from other tumor types. Importantly, the normal-appearing bronchial epithelial cell nuclei from NNK-treated mice also showed significant heterochromatin disruption in NNK-treated mice at 15 and 20 weeks (P< 0.0001), demonstrating that this method can detect early changes. Next, we sought to confirm this in humans. Super-resolution imaging showed that, compared to normal alveolar and bronchial epithelial cells from control subjects without cancer, the heterochromatin structure was more disrupted in tumor cells from patients with lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). We also observed significant disrupted heterochromatin structure in tumor-adjacent normal tissue at the tumor margin. Lastly, we performed a pilot study on bronchial epithelial cells isolated from sputum samples from 11 cigarette smokers with or without lung cancer. The heterochromatin structure in the bronchial epithelial cells collected from lung cancer patients at the time of diagnosis was significantly more disrupted than those from smokers without lung cancer (P= 0.004). Our results support disrupted heterochromatin structure as an early event in lung carcinogenesis and demonstrate the feasibility of utilizing super-resolution imaging of heterochromatin structure for early detection of lung cancer using sputum samples. Citation Format: Laura P. Stabile, Xuejiao Sun, Jianquan Xu, Beatriz Kanterewicz, David O. Wilson, Sanja Dacic, Lora H. Rigatti, Brenda Diergaarde, Yang Liu. Super-resolution imaging of heterochromatin structure detects early events in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 777.

Absence of gut microbiota impairs depletion of Paneth cells but not goblet cells in germ-free Atoh1lox/lox VilCreERT2 mice.

Mouse atonal homolog 1 (Math1/Atoh1) is a basic helix-loop-helix transcription factor important for the differentiation of secretory cells within the intestinal epithelium. The analysis of Paneth depletion efficiency on Math1lox/loxVilCreERT2 (Math1ΔIEC) mice treatment with tamoxifen in the presence or absence of intestinal microbiota showed a failure on Paneth cell depletion in germ-free mice as compared with specific pathogen-free (SPF) mice. However, goblet cells were efficiently depleted in Math1ΔIEC germ-free mice. The gene expression of Math1 was significantly reduced in the ileum of germ-free Math1ΔIEC mice 5 days after tamoxifen injection as compared with germ-free control, but its protein expression was still detectable in the nuclei of epithelial cells in the crypts. Germ-free mice showed low proliferative ileal crypts and apoptotic cells that were mainly detected in the tip of the villus, consistent with a slow turnover rate of epithelial cells. Although Paneth cells were not depleted in germ-free Math1ΔIEC mice for the first 7 wk after the last tamoxifen injection, far already from the 5 days time-laps observed in SPF conditions, an incomplete depletion of Paneth cells was observed 14 wk after the last tamoxifen injection. Colonization of germ-free mice restored the phenotype observed in SPF mice, highlighting the regulatory role of gut microbes in our model. We conclude that absence of intestinal microbiota in Math1ΔIEC mice is associated with reduced epithelial cell renewal and delays the depletion of preexisting Paneth cells.NEW & NOTEWORTHY Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1lox/loxVilCreERT2 mouse model.

Applicability of the histidine-tryptophan-ketoglutarate solution as a machine perfusion solution for marginal liver grafts.

There are very few reports comparing the use of the University of Wisconsin solution and histidine-tryptophan-ketoglutarate solution as machine perfusion solutions for marginal liver grafts. We aimed to clarify whether the use of the histidine-tryptophan-ketoglutarate solution in hypothermic machine perfusion improves the split-liver graft function in a large animal model. Porcine split-liver grafts were created by 75% liver resection. Hypothermic machine perfusion experimental groups were divided as follows: Group 1, perfusate, University of Wisconsin gluconate solution (UW group; n=5), and Group 2, perfusate, histidine-tryptophan-ketoglutarate solution (HTK group; n=4). After 4h of preservation, the liver function was evaluated using an isolated liver reperfusion model for 2h. In the HTK group, the portal vein and hepatic artery resistance during hypothermic machine perfusion and the portal vein resistance during isolated liver reperfusion were lower than those in the UW group. In addition, the total Suzuki score for hepatic ischemia-reperfusion injury in the HTK group was significantly better than that in the UW group. The number of anti-ETS-related genes staining-positive sinusoid epithelial cell nuclei in the HTK group was higher than that in the UW group (not significant). The histidine-tryptophan-ketoglutarate solution can be perfused with lower vascular resistance than the University of Wisconsin solution, reducing shear stress and preventing sinusoid epithelial cell injury in marginal grafts used as split-liver grafts.

A study of RNA m6A demethylases in oral epithelial dysplasia and oral squamous cell carcinoma.

N6-Methyladenosine (m6A) modification is involved in the tumorigenesis of various cancers. However, the roles of RNA m6A demethylases, fat mass and obesity-associated protein (FTO), and AlkB homolog 5 (ALKBH5) in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC) remain unclear. This study focuses on FTO and ALKBH5 expression by using immunohistochemistry. Twenty specimens each of OED, OSCC, and normal oral mucosa (NOM) were included. The expression pattern, the number of positive cells, the cell-staining intensity, and the histochemical scoring (H-score) were examined and analyzed. In all the OED and OSCC specimens, FTO and ALKBH5 were mainly expressed with moderate to strong staining intensity in the nuclei of the abnormal epithelial cells, respectively. Regarding the NOM, both RNA demethylases showed mild cell staining intensity and was present in 50-60% of the specimens. Interestingly, the percentage of cell positivity, the cell-staining intensity, and the H-score of the FTO and ALKBH5 in NOM, OED, and OSCC were increased, respectively (p<0.001). There was also a positive correlation between the FTO and ALKBH5 expressions in OSCC (r=0.62, p=0.003), but not in the NOM and OED. These results suggest a possible prognostic role of FTO and ALKBH5 expression in the malignant transformation of OED and tumor progression. Further studies are needed to elucidate the mechanisms underlying the roles of FTO and ALKBH5 in carcinogenesis.

Abstract P3-03-27: Assessment of the proliferative activity of breast tissue after the administration of combined oral hormonal contraceptive, associated or not with estriol

Abstract The molecular mechanisms related to the breast cancer development are poorly understood, particularly those associated with estrogen. Evidence is mounting that developmental differences may influence the risk of cancer. However, understanding breast development, morphology and the biochemical factors that influence them is pertinent to study pre-malignant and malignant conditions that affect the breasts. Objective: To evaluate the proliferative activity of normal breast epithelium, through the c-myc expression, after the administration of combined oral hormonal contraceptive, associated or not with estriol. Retrospective double-blind study, in which the effect of combined oral contraceptives on the normal breast epithelium in women with fibroadenoma was evaluated. We analyzed 33 women In a cohort of 70 selected (37 excluded). The control group (1) received a compound containing levonorgestrel 0.15mg (LNG) and ethinylestradiol 0.03mg (EE), associated with 2mg of placebo (PLC) manufactured in the same capsule ingested daily for 21 days with an interval of 7 days, during four cycles. The study group (2) received a compound containing levonorgestrel 0.15 mg (LNG) and ethinylestradiol 0.03 mg (EE), associated with estriol 2 mg (E3) manufactured in the same capsule ingested daily for 21 days with an interval of 7 days, up to four cycles, when was proceeded the lumpectomy plus normal breast tissue sample. The UNIFESP-EPM Department of Pathology received the samples collected and fixed in 10% buffered formalin. We used an automation device to perform the Immunohistochemistry reactions, using c-myc antibodies, added DAB developer (diaminobenzidine) that provides a bright brown color that provides good contrast. We used conventional optical microscopy to read TMA slides. We considered positive the nuclei stained with dark brown in contrast to the blue negative to the c-myc reactions. We evaluated the epithelial areas at least five acinar units were present in the sample at 40X magnification. In the statistical analysis, the initial stages of consolidation as well as the analysis were performed in Software R (2020) and Rstudio (version 4.1), using the packages Tidyverse (2016), Psyco, SummaryTools, Janitor and DataExplorer. After verifying the assumptions of normality, independence and linearity, Student’s T test was performed for independent samples. An alpha of 0.05 was chosen for rejection of the null hypothesis, that is, a confidence interval of the test results of 95%. An effect size measure of the difference between the groups was also performed, called Cohen’s D; it ranges from -1 to 1, and the closer to 0, the smaller the effect size of the difference between the groups. Regarding the control group (30.30%) and the case group (69.70%). The table 2 presents the results for group 1 (COC + Placebo), while table 3 presents the results for group 2 (COC + Estriol). The T test was performed to verify a possible significant difference in the levels of cell proliferation in patients with or without Estriol. The normality was verified by the Shapiro-Wilk test, with data following a normal distribution (W = 0.901 and 0.96, p value &amp;gt; 0.3). The equitable variance was verified by the Levine test, which verified the equitable variance in both groups (F (1) = 0.24, p value = 0.62). A mean positive nucleus of 14 (SD = 10.65) was observed in the group of patients with contraceptive plus placebo and a mean of positive nuclei of 20.81 (SD = 9.88) in the group of patients with contraceptive plus Estriol. The ratio of positive nuclei to total was higher in the estriol group than in the control group. However, it was not possible to verify a significant difference in the two groups (T (14.57) = 1.3, p value = 0.215, d Cohen = 0.53). Cohen’s d pointed to a moderate difference effect (table 4). Table 2. Number of positive, negative and total normal epithelial cell nuclei in immunohistochemical investigation of c-myc antigens in Group 1 (COC + Placebo) (N = 10) Table 3. Number of positive, negative and total normal epithelial cell nuclei in immunohistochemical investigation of c-myc antigens in Group 2 (COC + Estriol) (N = 23) Table 4. Inferential results of the ratio of positive normal epithelial cell nuclei as a function of the control and case groups Citation Format: Osmar Pellegrini, Jr., Afonso Nazário, Angela Flavia L. Waitzberg. Assessment of the proliferative activity of breast tissue after the administration of combined oral hormonal contraceptive, associated or not with estriol [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-03-27.

A novel ssDNA Bidnavirus in the giant freshwater prawn Macrobrachium rosenbergii

A purported parvovirus producing circular, eosinophilic inclusions in the nuclei of tubule epithelial cells of the hepatopancreas (HP) of the giant freshwater prawn Macrobrachium rosenbergii (Mr) was first reported from Malaysia in 1990. In 2009, similar inclusions in Mr from Thailand were confirmed to contain single-stranded DNA (ssDNA). In 2021, Mr samples showing similar HP inclusions were reported to give positive PCR test results for decapod hepanhamaparvovirus (DHPV) using universal DHPV primers designed from many viral isolates of 3 penaeid shrimp species. However, the matching DNA probe revealed positive in situ hybridization (ISH) results with host HP nuclei only, and not with the intranuclear inclusions. At the time, this anomaly could not be explained. Here we describe metagenomic analysis and de novo assembly from 2016 samples of Mr with similar intranuclear inclusions that showed negative PCR test results using the DHPV universal primers. Genomes identified through metagenomics revealed a novel ssDNA genome sequence of 6723 bases that was confirmed by a single PCR amplicon. ISH using specific probes derived from this genome sequence revealed that the ssDNA was present in both grossly normal nuclei and in the intranuclear inclusions. Tests with archived DNA from the samples used in the 2021 publication yielded positive PCR results for both DHPV and the novel sequence, suggesting that the shrimp had dual infections. Phylogenetic analysis of the novel sequence revealed no relationship to any known DHPV type. Instead, the sequence closely matched the polymerase B gene in the V1 fragment from Bombyx mori bidensovirus 2 (BmBDV2) that is characterized by 2 ssDNA genome fragments and has been classified in the family Bidnaviridae. The 2 BmBDV2 fragments V1 and V2 each produce their own independent capsid protein genes and virions. In contrast, the new Mr virus is independent with a single 6723 base ssDNA genome, and we propose the name Macrobrachium hepatopancreatic bidnavirus (MHBV).

Yield stress: A tipping point of cell unjamming.

Tissue mechanics is impacted not only by the properties of the composite cells, but also by the behavior of collective cells, such as cell unjamming and extended differential adhesion. In former studies, we used multicellular spheroids (MS) of healthy epithelial cells (jammed solid) and cancerous mesenchymal cells (unjammed fluid) as our model system to investigate the tissue mechanics of cell clusters. We realized even in a simple cell cluster system, the mechanical behavior differed between core/shell region, which we concluded due to the different domination of nuclei (bulk) and cortical rim formed by cell cytoskeleton (tissue surface tension). As we also observed in cancer spheroids, nuclei seem to dominate the whole mechanics of MTS (no reaction to chemical perturbation of the cell cytoskeleton). By specifically probing the nucleus region of a single cell under AFM, and combining its nucleus morphology aside as an indicator, we could measure how much yield stress a cell will need to become motile (unjammed). Based on our preliminary results, we see a different linear growth of the Young's modulus with elongated nuclei of healthy epithelial cells (high-slope) and cancerous mesenchymal cells (low-slope). Knowing the different behavior of such cell types under the constriction studies (mesenchymal cells do not pass through the confinement whereas epithelial cells do), the nuclei domination of the mesenchymal cells is unassailable. Therefore, to establish the connection between the nucleus-based yield stress to its motility are strongly required. To do so, we are able to derive an unjamming-based marker that detects the ability of cells to move in static histological images, which will be a big step to understand the role of the cancer cell unjamming in the metastatic cascade.

Pre-hyperglycemia immune cell trafficking underlies subclinical diabetic cataractogenesis

BackgroundThis work elucidates the first cellular and molecular causes of cataractogenesis. Current paradigm presupposes elevated blood glucose as a prerequisite in diabetic cataractogenesis. Novel evidence in our model of diabetic cataract challenges this notion and introduces immune cell migration to the lens and epithelial-mesenchymal transformation (EMT) of lens epithelial cells (LECs) as underlying causes.MethodsPaucity of suitable animal models has hampered mechanistic studies of diabetic cataract, as most studies were traditionally carried out in acutely induced hyperglycemic animals. We introduced diabetic cataract in the Nile grass rat (NGR) that spontaneously develops type 2 diabetes (T2D) and showed its closeness to the human condition. Specialized stereo microscopy with dual bright-field illumination revealed novel hyperreflective dot-like microlesions in the inner cortical regions of the lens. To study immune cell migration to the lens, we developed a unique in situ microscopy technique of the inner eye globe in combination with immunohistochemistry.ResultsContrary to the existing paradigm, in about half of the animals, the newly introduced hyper reflective dot-like microlesions preceded hyperglycemia. Even though the animals were normoglycemic, we found significant changes in their oral glucose tolerance test (OGTT), indicative of the prediabetic stage. The microlesions were accompanied with significant immune cell migration from the ciliary bodies to the lens, as revealed in our novel in situ microscopy technique. Immune cells adhered to the lens surface, some traversed the lens capsule, and colocalized with apoptotic nuclei of the lens epithelial cells (LECs). Extracellular degradations, amorphous material accumulations, and changes in E-cadherin expressions showed epithelial-mesenchymal transformation (EMT) in LECs. Subsequently, lens fiber disintegration and cataract progression extended into cortical, posterior, and anterior subcapsular cataracts.ConclusionsOur results establish a novel role for immune cells in LEC transformation and death. The fact that cataract formation precedes hyperglycemia challenges the prevailing paradigm that glucose initiates or is necessary for initiation of the pathogenesis. Novel evidence shows that molecular and cellular complications of diabetes start during the prediabetic state. These results have foreseeable ramifications for early diagnosis, prevention and development of new treatment strategies in patients with diabetes.

Transcripts of the Prostate Cancer-Associated Gene ANO7 Are Retained in the Nuclei of Prostatic Epithelial Cells.

Prostate cancer affects millions of men globally. The prostate cancer-associated gene ANO7 is downregulated in advanced prostate cancer, whereas benign tissue and low-grade cancer display varying expression levels. In this study, we assess the spatial correlation between ANO7 mRNA and protein using fluorescent in situ hybridization and immunohistochemistry for the detection of mRNA and protein in parallel sections of tissue microarrays prepared from radical prostatectomy samples. We show that ANO7 mRNA and protein expression correlate in prostate tissue. Furthermore, we show that ANO7 mRNA is enriched in the nuclei of the luminal cells at 89% in benign ducts and low-grade cancer, and at 78% in high-grade cancer. The nuclear enrichment of ANO7 mRNA was validated in prostate cancer cell lines 22Rv1 and MDA PCa 2b using droplet digital polymerase chain reaction (ddPCR) on RNA isolated from nuclear and cytoplasmic fractions of the cells. The nuclear enrichment of ANO7 mRNA was compared to the nuclearly-enriched lncRNA MALAT1, confirming the surprisingly high nuclear retention of ANO7 mRNA. ANO7 has been suggested to be used as a diagnostic marker and a target for immunotherapy, but a full comprehension of its role in prostate cancer progression is currently lacking. Our results contribute to a better understanding of the dynamics of ANO7 expression in prostatic tissue.

Expression of Paired box 9 defines an aggressive subset of lung adenocarcinoma preferentially occurring in smokers.

A distinct subset of lung adenocarcinomas (LADs), arising from a series of peripheral lung cells defined as the terminal respiratory unit (TRU), is characterized by thyroid transcription factor 1 (TTF-1) expression. The clinical relevance of transcription factors (TFs) other than TTF-1 remains unknown in LAD and was explored in the present study. Seventy-one LAD samples were subjected to high-throughput transcriptome screening of LAD using cap analysis gene expression (CAGE) sequencing data; CAGE provides genome-wide expression levels of the transcription start sites (TSSs). In total, 1083 invasive LAD samples were subjected to immunohistochemical examination for paired box 9 (PAX9) and TTF-1 expression levels. PAX9 is an endoderm development-associated TF that most strongly and inversely correlates with the expression of TTF-1 TSS subsets. Immunohistochemically, PAX9 expression was restricted to the nuclei of ciliated epithelial and basal cells in the bronchi and bronchioles and the nuclei of epithelial cells of the bronchial glands; moreover, PAX9 expression was observed in 304 LADs (28%). PAX9-positive LADs were significantly associated with heavy smoking, non-lepidic subtype, EGFR wild-type tumors, and PD-L1 expression (all p < 0.0001). All these characteristics were opposite to those of TRU-type LADs with TTF-1 expression. PAX9 expression was an independent prognostic factor for decreased overall survival (p = 0.022). Our results revealed that PAX9 expression defines an aggressive subset of LADs preferentially occurring in smokers that may arise from bronchial or bronchiolar cells.

Features of changes in morphometric indicators of ovaries of laying hans during postnatal ontogenesis

The peculiarities of changes in the linear size and weight of ovaries of oviparous hens at different physiological stages of postnatal ontogenesis have been studied. The absolute indicator of the linear size and weight of the egg of hens is characterized by an intensive increase from the first 15 days of postnatal ontogenesis to 35 days, as well as the duration of this condition up to 120 days, physiological maturity, that the highest at 168 days, a decrease in this indicator to 570 days due to the stage of generative extinction of ovarian function at the stages after 168 days, morphometrically established that the coefficient of increase in indicators is higher in weight compared with linear dimensions in the period from 15 to 570 days of postnatal ontogenesis. It was also established that the cytoplasm of the oocyte is homogeneous, weakly basophilic, the nucleus is somewhat shifted to the periphery, the chromatin is in a dispersed state, and the nucleolus is clearly visible in the nucleus. Follicular epithelium is single-layer squamous. The nuclei of epithelial cells are hyperchromatic, flattened, the cytoplasm is weakly basophilic. Around the follicle lies loose connective tissue, the main cellular elements of which are fibroblasts.

Abstract B049: Comparison of keratin 17 and GATA6 as prognostic markers in pancreatic cancer

Abstract Background: Clinically and histologically identical pancreatic ductal adenocarcinomas (PDACs) differ in response to treatment, disease progression, and survival. Clinical trials are testing the use of either keratin 17 (K17) or GATA6 as surrogate prognostic and predictive biomarkers of PDAC. Still, there is a need to contrast their expression and prognostic value in the same cohort of cases. K17, a biomarker of the most aggressive subtype of PDAC, promotes tumor growth and chemoresistance. GATA6 is a transcription factor necessary for normal pancreatic development, and a surrogate marker of the classical subtype of PDAC, associated with longer-term survival. Our goal was to test the hypothesis that there is a reciprocal relationship between K17 and GATA6 expression and that their detection informs prognostic stratification. Methods: Stony Brook University Hospital PDAC patients were selected, and representative sections were processed for dual-staining by indirect immunoperoxidase methods using antibodies to K17 (KDx Diagnostics) and GATA6 (R&amp;D Systems AF1700). Manual scoring of the proportion of cells with strong (2+) nuclear and/or cytoplasmic staining was determined for K17 versus nuclear staining for GATA6 (PathSQ). Automated image analysis of GATA6 expression was further scored using QuPath v0.3.2 on annotated PDAC sections. Cut-off values for the best COX model for each marker were established to stratify for survival. Results: K17 expression was observed in cancer epithelial cells and in few benign proliferative ducts of chronic pancreatitis but no staining was detected in stromal or acinar cells. By contrast, GATA6 was detected in the nuclei of most cancer epithelial cells and was also consistently detected in benign ductal cells, acinar cells, and stromal cells. Defining high K17 cases as those with staining in ≥10% of tumor cells, high K17 correlated with shorter overall survival (p=0.03), supporting its use as a negative prognostic biomarker. Due to the high level of expression of GATA6 in PDAC cells (range 60-100% of tumor cells across all cases), however, no threshold was found to provide prognostic value. By dual-color IHC, K17 positive cells almost always had strong GATA6 nuclear staining but GATA6 was also detected in all K17-negative tumor cells. Computational analysis demonstrated 98% concordance with manual scoring for high GATA6 and 90% concordance with manual scoring for low GATA6. Conclusions: GATA6 was expressed in most PDAC tumor cells, as well as in benign stromal and epithelial cells. However, K17 expression was focal, relatively tumor cell-specific, and was not detected in most benign cellular components. Although high K17 status identified cases with relatively short survival, a survival threshold for GATA6 could not be detected. Contradiction in our observations of GATA6 compared to previous reports could result from differences in case selection or immunohistochemical protocol. Thus, further work is needed to explore the relationship between K17 and GATA6 as complementary biomarkers of PDAC patient survival. Citation Format: Michael Horowitz, Lyanne Oblein, Jaymie Oentoro, Saumya Kasliwal, Natalia D. Marchenko, Luisa F. Escobar-Hoyos, Kenneth R. Shroyer. Comparison of keratin 17 and GATA6 as prognostic markers in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B049.

Mildly dysplastic oral lesions with optically-detectable abnormalities share genetic similarities with severely dysplastic lesions

ObjectiveOptical imaging studies of oral premalignant lesions have shown that optical markers, including loss of autofluorescence and altered morphology of epithelial cell nuclei, are predictive of high-grade pathology. While these optical markers are consistently positive in lesions with moderate/severe dysplasia or cancer, they are positive only in a subset of lesions with mild dysplasia. This study compared the gene expression profiles of lesions with mild dysplasia (stratified by optical marker status) to lesions with severe dysplasia and without dysplasia. Materials and methodsForty oral lesions imaged in patients undergoing oral surgery were analyzed: nine without dysplasia, nine with severe dysplasia, and 22 with mild dysplasia. Samples were submitted for high throughput gene expression analysis. ResultsThe analysis revealed 116 genes differentially expressed among sites without dysplasia and sites with severe dysplasia; 50 were correlated with an optical marker quantifying altered nuclear morphology. Ten of 11 sites with mild dysplasia and positive optical markers (91%) had gene expression similar to sites with severe dysplasia. Nine of 11 sites with mild dysplasia and negative optical markers (82%) had similar gene expression as sites without dysplasia. ConclusionThis study suggests that optical imaging may help identify patients with mild dysplasia who require more intensive clinical follow-up. If validated, this would represent a significant advance in patient care for patients with oral premalignant lesions.

Tacrolimus loaded lipid-polymer hybrid nanoparticles incorporated thermosensitive gel as intravesical drug delivery system

A drug that is administered through a catheter into the bladder is referred to as an intravesical drug delivery system. The aim of this work is to improvise intravesical drug delivery of Tacrolimus loaded lipid polymer hybrid nanoparticles thermosensitive gel for bladder wall to provide control release, increase intravesical residence time and avoid fast removal by urination. The Tacrolimus loaded lipid polymer hybrid nanoparticles mimic cell membrane and enhance cellular uptakes. Our data reveals nanocarrier formation with a mean particle size, zeta potential and entrapment efficiency of optimal formula was found to be 124±0.01 nm,-27.5±0.102mv and 72% ± 0.15% respectively. In situ gelling formulations containing TAC-CS-LPHNs can adhere to the mucosal layer of the bladder and help in the diffusion of therapeutic agent across the bladder wall. To impart mechanical strength and mucoadhesive properties, the chitosan concentration was adjusted to 0.5% and 0.25%.The image of field emission scanning electron microscope shows an irregular surface with filled pores with nanoparticles. The gel was syringeable and had reasonable viscosity within room temperature. A Tissue uptake study reveals that Tacrolimus loaded lipid polymer hybrid nanoparticles have been penetrating bladder tissues and accumulated inside the cytoplasm and nucleus of urothelium epithelial cells. In addition, the bioimaging study shows that the gel resided inside the bladder for up to 2 hrs and completely urinated out of the bladder with negligible distribution to other tissues.

Predicted Structure and Functions of the Prototypic Alphaherpesvirus Herpes Simplex Virus Type-1 UL37 Tegument Protein.

The alphaherpesvirus UL37 tegument protein is a highly conserved, multi-functional protein. Mutagenesis analysis delineated the UL37 domains necessary for retrograde transport and viral replication. Specifically, the amino-terminal 480 amino acids are dispensable for virus replication in epithelial cell culture, but it is unknown whether this amino-terminal deletion affects UL37 structure and intracellular transport in epithelial cells and neurons. To investigate the structure and function of UL37, we utilized multiple computational approaches to predict and characterize the secondary and tertiary structure and other functional features. The structure of HSV-1 UL37 and Δ481N were deduced using publicly available predictive algorithms. The predicted model of HSV-1 UL37 is a stable, multi-functional, globular monomer, rich in alpha helices, with unfolded regions within the linker and the C-tail domains. The highly flexible C-tail contains predicted binding sites to the dynein intermediate chain, as well as DNA and RNA. Predicted interactions with the cytoplasmic surface of the lipid membrane suggest UL37 is a peripheral membrane protein. The Δ481N truncation did not alter the predicted structure of the UL37 C-terminus protein and its predicted interaction with dynein. We validated these models by examining the replication kinetics and transport of the Δ481N virus toward the nuclei of infected epithelial and neuronal cells. The Δ481N virus had substantial defects in virus spread; however, it exhibited no apparent defects in virus entry and intracellular transport. Using computational analyses, we identified several key features of UL37, particularly the flexible unstructured tail; we then demonstrated that the UL37 C-terminus alone is sufficient to effectively transport the virus towards the nucleus of infected epithelial and neuronal cells.

Shoseiryuto Ameliorated TDI-Induced Allergic Rhinitis by Suppressing IL-33 Release from Nasal Epithelial Cells.

Toluene diisocyanate (TDI) is a major cause of occupational asthma and rhinitis. Shoseiryuto (SST) is one of the traditional herbal medicines (Kampo medicine) and has long been used as a natural medicine for allergic diseases such as allergic rhinitis (AR) and asthma. Recent studies have shown that the expression and release of IL-33, which regulates the TH2 cytokine response in epithelial cells, is an important step in developing the inflammatory response of the nasal mucosa. In this study, we investigated whether SST may ameliorate the TDI-induced AR-related symptoms in rats and inhibit IL-33 release from nasal epithelial cells. An AR rat model was generated by sensitization and induction with TDI. SST was administered during the sensitization period. AR-related symptoms in rats were evaluated, and IL-33 release was measured both in vivo and in vitro. SST suppressed symptoms appearing in TDI-induced AR model rats, such as elevated serum histamine and IL-33 levels in nasal lavage fluid (NLF)/serum, which were suppressed by SST administration. TDI-induced IL-33 release from the nasal epithelial cell nuclei was also observed and suppressed in SST-treated rats and cultured nasal epithelial cells. These results suggest that SST ameliorates the symptoms of TDI-induced AR at least partially by inhibiting IL-33 release from nasal epithelial cells.