Upregulation of HLA class II in pancreatic beta cells from organ donors with type 1 diabetes.

AIMS/HYPOTHESIS: We aimed to characterise and quantify the expression of HLA class II (HLA-II) in human pancreatic tissue sections and to analyse its induction in human islets. METHODS: We immunostained human pancreatic tissue sections from non-diabetic (n = 5), autoantibody positive (Aab+; n = 5), and type 1 diabetic (n = 5) donors, obtained from the Network of Pancreatic Organ Donors (nPOD), with HLA-II, CD68 and insulin. Each tissue section was acquired with a widefield slide scanner and then analysed with QuPath software. In total, we analysed 7415 islets that contained 338,480 cells. Widefield microscopy was further complemented by high resolution imaging of 301 randomly selected islets, acquired using a Zeiss laser scanning confocal (LSM880) to confirm our findings. Selected beta cells were acquired in enhanced resolution using LSM880 with an Airyscan detector. Further, we cultured healthy isolated human islets and reaggregated human islet microtissues with varying concentrations of proinflammatory cytokines (IFN-γ, TNF-α and IL-1ß). After proinflammatory cytokine culture, islet function was measured by glucose-stimulated insulin secretion, and HLA-I and HLA-II expression was subsequently evaluated with immunostaining or RNA sequencing. RESULTS: Insulin-containing islets (ICIs) of donors with type 1 diabetes had a higher percentage of HLA-II positive area (24.31%) compared with type 1 diabetic insulin-deficient islets (IDIs, 0.67%), non-diabetic (3.80%), and Aab+ (2.31%) donors. In ICIs of type 1 diabetic donors, 45.89% of the total insulin signal co-localised with HLA-II, and 27.65% of the islet beta cells expressed both HLA-II and insulin, while in non-diabetic and Aab+ donors 0.96% and 0.59% of the islet beta cells, respectively, expressed both markers. In the beta cells of donors with type 1 diabetes, HLA-II was mostly present in the cell cytoplasm, co-localising with insulin. In the experiments with human isolated islets and reaggregated human islets, we observed changes in insulin secretion upon stimulation with proinflammatory cytokines, as well as higher expression of HLA-II and HLA-I when compared with controls cultured with media, and an upregulation of HLA-I and HLA-II RNA transcripts. CONCLUSIONS/INTERPRETATION: After a long-standing controversy, we provide definitive evidence that HLA-II can be expressed by pancreatic beta cells from patients with type 1 diabetes. Furthermore, this upregulation can be induced in vitro in healthy isolated human islets or reaggregated human islets by treatment with proinflammatory cytokines. Our findings support a role for HLA-II in type 1 diabetes pathogenesis since HLA-II expressing beta cells can potentially become a direct target of autoreactive CD4+ lymphocytes.

Mutation and methylation profiles of ectopic and eutopic endometrial tissues.

Adenomyosis and peritoneal endometriosis are common gynecologic lesions; they are characterized by aberrant locations of normal-appearing endometrium in myometrium and peritoneal surface, respectively. Both ectopic lesions are speculated to originate from uterine eutopic endometrium, which is composed of epithelium and stroma, but how these two different tissue types co-evolve in ectopic locations remains unclear. Here, we analyzed exome-wide mutations and global methylation in microdissected epithelium and stroma separately in paired adenomyosis, peritoneal endometriosis, and endometrium to investigate their relationship. Analyses of somatic mutations and their allele frequencies indicate monoclonal development not only in epithelium but also in the stroma of adenomyosis and peritoneal endometriosis. Our preliminary phylogenetic study suggests a plausible clonal derivation in epithelium and stroma of both ectopic and eutopic endometrium from the same founder epithelium-stroma progenitor cells. While a patient-specific methylation landscape is evident, adenomyosis epithelium and stroma can be distinguished from normal-appearing eutopic endometrium epigenetically. In summary, endometrial stroma, like its epithelial counterpart, could be clonal and both ectopic and eutopic endometrium following divergent evolutionary trajectories. Our data also warrant future investigations into the role of endometrial stroma in the pathobiology of endometrium-related disorders. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Stereodivergent Attached-Ring Synthesis via Non-Covalent Interactions: A Short Formal Synthesis of Merrilactone A.

A strategy to control the diastereoselectivity of bond formation at a prochiral attached-ring bridgehead is reported. An unusual stereodivergent Michael reaction relies on basic vs. Lewis acidic conditions and non-covalent interactions to control re- vs. si- facial selectivity en route to fully substituted attached-rings. This divergency reflects differential engagement of one rotational isomer of the attached-ring system. The successful synthesis of an erythro subtarget diastereomer ultimately leads to a short formal synthesis of merrilactone A.

Synthetic Studies on Selective, Proapoptotic Isomalabaricane Triterpenoids Aided by Computational Techniques.

The isomalabaricanes comprise a large family of marine triterpenoids with fascinating structures that have been shown to be selective and potent apoptosis inducers in certain cancer cell lines. In this article, we describe the successful total syntheses of the isomalabaricanes stelletin A, stelletin E, and rhabdastrellic acid A, as well as the development of a general strategy to access other natural products within this unique family. High-throughput experimentation and computational chemistry methods were used in this endeavor. A preliminary structure-activity relationship study of stelletin A revealed the trans-syn-trans core motif of the isomalabaricanes to be critical for their cytotoxic activity.

IL-17 is expressed on beta and alpha cells of donors with type 1 and type 2 diabetes.

PURPOSE: IL-17 is an important effector cytokine driving immune-mediated destruction in autoimmune diseases such as psoriasis. Blockade of the IL-17 pathway after the initiation of insulitis was effective in delaying or preventing the onset of type 1 diabetes (T1D) in rodent models. Expression of IL-17 transcripts in islets from a donor with recent-onset T1D has been reported, however, studies regarding IL-17 protein expression are lacking. We aimed to study whether IL-17 is being expressed in the islets of diabetic donors. METHODS: We stained human pancreatic tissues from non-diabetic (n = 5), auto-antibody positive (aab+) (n = 5), T1D (n = 6) and T2D (n = 5) donors for IL-17, Insulin, and Glucagon, and for CD45 in selected cases. High resolution images were acquired with Zeiss laser scanning confocal microscope LSM780 and analyzed with Zen blue 2.3 software. Cases stained for CD45 were also acquired with widefield slide scanner and analyzed with QuPath software. RESULTS: We observed a clear cytoplasmic staining for IL-17 in insulin-containing islets of donors with T1D and T2D, accounting for an average of 7.8 ± 8.4% and 14.9 ± 16.8% of total islet area, respectively. Both beta and alpha cells were sources of IL-17, but CD45+ cells were not a major source of IL-17 in those donors. Expression of IL-17 was reduced in islets of non-diabetic donors, aab+ donors and in insulin-deficient islets of donors with T1D. CONCLUSION: Our finding that IL-17 is expressed in islets of donors with T1D or T2D is quite intriguing and warrants further mechanistic studies in human islets to understand the role of IL-17 in the context of metabolic and immune stress in beta cells.

IL-6 is present in beta and alpha cells in human pancreatic islets: Expression is reduced in subjects with type 1 diabetes.

IL-6 is a pro-inflammatory cytokine upregulated in some autoimmune diseases. The role of IL-6 in the development of type 1 diabetes (T1D) is unclear. Clinical studies are investigating whether tocilizumab (anti-IL-6 receptor) can help preserve beta cell function in patients recently diagnosed with T1D. However, in some rodent models and isolated human islets, IL-6 has been found to have a protective role for beta cells by reducing oxidative stress. Hence, we systematically investigated local tissue expression of IL-6 in human pancreas from non-diabetic, auto-antibody positive donors and donors with T1D and T2D. IL-6 was constitutively expressed by beta and alpha cells regardless of the disease state. However, expression of IL-6 was highly reduced in insulin-deficient islets of donors with T1D, and the expression was then mostly restricted to alpha cells. Our findings suggest that the implication of IL-6 in T1D pathogenesis might be more complex than previously assumed.

Genomic landscape and evolutionary trajectories of ovarian cancer precursor lesions.

The clonal relationship between ovarian high-grade serous carcinoma (HGSC) and its presumed precursor lesion, serous tubal intraepithelial carcinoma (STIC), has been reported. However, when analyzing patients with concurrent ovarian carcinoma and tubal lesion, the extensive carcinoma tissues present at diagnosis may have effaced the natural habitat of precursor clone(s), obscuring tumor clonal evolutionary history, or may have disseminated to anatomically adjacent fimbriae ends, masquerading as precursor lesions. To circumvent these limitations, we analyzed the genomic landscape of incidental tubal precursor lesions including p53 signature, dormant STIC or serous tubal intraepithelial lesion (STIL) and proliferative STIC in women without ovarian carcinoma or any cancer diagnosis using whole-exome sequencing and amplicon sequencing. In three of the four cancer-free women with multiple discrete tubal lesions we observed non-identical TP53 mutations between precursor lesions from the same individual. In one of the four women with co-existing ovarian HGSC and tubal precursor lesion we found non-identical TP53 mutations and a lack of common mutations shared between her precursor lesion and carcinoma. Analyzing the evolutionary history of multiple tubal lesions in the same four patients with concurrent ovarian carcinoma indicated distinct evolution trajectories. Collectively, the results support diverse clonal origins of tubal precursor lesions at the very early stages of tumorigenesis. Mathematical modeling based on lesion-specific proliferation rates indicated that p53 signature and dormant STIC may take a prolonged time (two decades or more) to develop into STIC, whereas STIC may progress to carcinoma in a much shorter time (6 years). The above findings may have implications for future research aimed at prevention and early detection of ovarian cancer. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Phenotype onset in Huntington's disease knock-in mice is correlated with the incomplete splicing of the mutant huntingtin gene.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expanded CAG repeat within the huntingtin (HTT) gene. The Q140 and HdhQ150 knock-in HD mouse models were generated such that HdhQ150 mice have an expanded CAG repeat inserted into the mouse Htt gene, whereas in the Q140s, mouse exon 1 Htt was replaced with a mutated version of human exon 1. By standardizing mouse strain background, breeding to homozygosity and employing sensitive behavioral tests, we demonstrate that the onset of behavioral phenotypes occurs earlier in the Q140 than the HdhQ150 knock-in mouse models and that huntingtin (HTT) aggregation appears earlier in the striata of Q140 mice. We have previously found that the incomplete splicing of mutant HTT from exon 1 to exon 2 results in the production of a small polyadenylated transcript that encodes the highly pathogenic mutant HTT exon 1 protein. In this report, we have identified a functional consequence of the sequence differences between these two models at the RNA level, in that the level of incomplete splicing, and of the mutant exon 1 HTT protein, are greater in the brains of Q140 mice. While differences in the human and mouse exon 1 HTT proteins (e.g., proline rich sequences) could also contribute to the phenotypic differences, our data indicate that the incomplete splicing of HTT and approaches to lower the levels of the exon 1 HTT transcript should be pursued as therapeutic targets.

Effect of surgeon experience and bony pelvic dimensions on surgical performance and patient outcomes in robot-assisted radical prostatectomy.

OBJECTIVES: To evaluate the effects of surgeon experience, body habitus, and bony pelvic dimensions on surgeon performance and patient outcomes after robot-assisted radical prostatectomy (RARP). PATIENTS, SUBJECTS AND METHODS: The pelvic dimensions of 78 RARP patients were measured on preoperative magnetic resonance imaging and computed tomography by three radiologists. Surgeon automated performance metrics (APMs [instrument motion tracking and system events data, i.e., camera movement, third-arm swap, energy use]) were obtained by a systems data recorder (Intuitive Surgical, Sunnyvale, CA, USA) during RARP. Two analyses were performed: Analysis 1, examined effects of patient characteristics, pelvic dimensions and prior surgeon RARP caseload on APMs using linear regression; Analysis 2, the effects of patient body habitus, bony pelvic measurement, and surgeon experience on short- and long-term outcomes were analysed by multivariable regression. RESULTS: Analysis 1 showed that while surgeon experience affected the greatest number of APMs (P < 0.044), the patient's body mass index, bony pelvic dimensions, and prostate size also affected APMs during each surgical step (P < 0.043, P < 0.046, P < 0.034, respectively). Analysis 2 showed that RARP duration was significantly affected by pelvic depth (ß = 13.7, P = 0.039) and prostate volume (ß = 0.5, P = 0.024). A wider and shallower pelvis was less likely to result in a positive margin (odds ratio 0.25, 95% confidence interval [CI] 0.09-0.72). On multivariate analysis, urinary continence recovery was associated with surgeon's prior RARP experience (hazard ratio [HR] 2.38, 95% CI 1.18-4.81; P = 0.015), but not on pelvic dimensions (HR 1.44, 95% CI 0.95-2.17). CONCLUSION: Limited surgical workspace, due to a narrower and deeper pelvis, does affect surgeon performance and patient outcomes, most notably in longer surgery time and an increased positive margin rate.

Protease-activated receptor 4 activity promotes platelet granule release and platelet-leukocyte interactions.

Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.