B-positive Cells Research Articles

Human collagen type I‑based scaffold retains human‑derived fibroblasts in a patient‑derived tumor xenograft mouse model.

The present study aimed to investigate the role of a recombinant protein based on human collagen type I (RCPhC1) as a scaffold in maintaining the human tumor microenvironment within a patient-derived tumor xenograft (PDTX) model. RCPhC1, synthesized under animal component-free conditions, was explored for its potential to support the human-specific stroma associated with tumor growth. PDTX models were established using resected colorectal cancer liver metastasis specimens, and stromal cell populations from humans and mice were compared using three scaffolds: No scaffold (control), Matrigel and recombinant human collagen type I, across two passages. Specific antibodies for human Lamin B and mouse Lamin B were used for immunostaining to distinguish between human and mouse cells. Additionally, the impact of each scaffold on the invasive ability of mouse fibroblasts was assessed using an invasion assay. Patient-derived tumor tissues embedded with RCPhC1 hydrogels had significantly more human Lamin B-positive cells and fewer mouse Lamin B cells than those embedded with no scaffolds or Matrigel. The human Lamin B-positive cells in PDTX tumors with RCPhC1 hydrogels were recognized as fibroblasts. Additionally, these hydrogels significantly reduced the invasion of mouse fibroblast cell lines in vitro compared with Matrigel. The present study investigated RCPhC1 hydrogels as a new scaffold material for tumor engraftment in PDTX mouse models, and identified a promising experimental tool for maintaining the tumor microenvironment.

Long-Term Survival in Canine Hepatosplenic T-Cell Lymphoma Treated with Toceranib Phosphate Following Splenectomy: A Case of Atypical Lymphoma

Toceranib phosphate (toceranib) is approved for canine mast cell tumor treatment. However, no long-term response to toceranib in canine HSTCL has been reported. Here, we describe a case of a 10-year-old castrated mixed-breed dog that presented with a 3-month history of weight loss, polydipsia, and polyuria. The clinicopathological and imaging abnormalities included icterus, biliary obstruction, and splenomegaly with multiple diffuse splenic hypoechoic nodules. On day 21, a cholecystectomy was performed to remove the obstruction, followed by a liver biopsy and splenectomy. Cytology of the spleen and liver showed many small lymphocytes with intracytoplasmic granules (sGLs). Splenic and hepatic infiltration of neoplastic CD3/granzyme B-positive small cells and lymphocytic cholecystitis with granzyme B-negative small cells were noted. T-cell receptor gene clonal rearrangements were observed in the liver tissues. The dog was diagnosed with a hepatosplenic T-cell lymphoma (HSTCL) of sGLs concurrent with lymphocytic cholecystitis. The icterus resolved after surgery, but there was progressive elevation of liver enzyme levels. Toceranib was administered from day 39, resulting in decreased liver enzyme levels, and the dog remained in good condition. The dog stayed in remission after toceranib administration and survived for 460 days. Toceranib should be considered an effective treatment option for canine HSTCL.

Chlorogenic acid regulates the expression of protein phosphatase 2A subunit B in the cerebral cortex of a rat stroke model and glutamate-exposed neurons

BackgroundIschemic stroke is a serious neurological disorder caused by blockages in cerebral artery. Protein phosphatase 2A (PP2A) is a phosphatase that performs a critical role in cell signaling and growth. PP2A subunit B acts as a neuroprotective agent in the nerve system. Chlorogenic acid, which is mainly found in roasted coffee, has antioxidant, anti-inflammatory, and anti-apoptotic effects. We hypothesized that chlorogenic acid modulates PP2A subunit B expression in ischemic stroke models and glutamate-mediated neurons. Middle artery occlusion (MCAO) surgery was operated and chlorogenic acid (30 mg/kg) or phosphate buffer saline was treated 2 h after MCAO. The cerebral cortex was collected 24 h after surgery and the change of PP2A subunit B expression was analyzed. Glutamate and/or chlorogenic acid were treated in cultured neurons, further study was performed.ResultsA decrease in PP2A subunit B expression in MCAO animals was identified. Chlorogenic acid alleviated this decrease due to ischemic injury. Moreover, the number of PP2A subunit B-positive cells in the ischemic cerebral cortex was significantly decreased, chlorogenic acid alleviated this decrease. We also found protective effects of chlorogenic acid in neurons exposed to glutamate. Glutamate decreased the expression of PP2A subunit B and chlorogenic acid mitigated this decrease. Our results elucidated that chlorogenic acid performs neuroprotective functions and attenuates the reduction of PP2A subunit B by brain damage and glutamate-mediated excitotoxicity.ConclusionsWe showed that chlorogenic acid attenuated the decrease of PP2A subunit B in ischemic injury and neurons exposed to glutamate. Since PP2A subunit B contributes to the protection of brain tissue, we can suggest that chlorogenic acid preserves neurons by modulating PP2A subunit B during ischemic damage.

Early Onset of Rapid Lesion Growth in an Acute Subdural Hematoma Model in Rats

Acute subdural hematoma (ASDH) leads to the highest mortality rates of all head injuries with secondary brain damage playing a pivotal role in terms of morbidity and mortality. In patients with ASDH, a delay in surgery leads to disproportional mortality. The benefit of (very) early therapy is therefore, a target of ongoing research. As the process of delayed brain damage in ASDH has not yet been described, this study therefore aimed to examine secondary lesion growth in an experimental rat model of ASDH to define the ideal timing for testing potential neuroprotective therapies. Cerebral blood flow was monitored during ASDH induction with 300μl of autologous blood. Lesion growth was characterized using Hematoxylin-Eosin- , Cresyl-Violet-, and Fluoro-Jade B-staining for early signs of neuronal degeneration. Histological evaluations were performed between 15minutes and 24hours after ASDH. There was a significant reduction of cerebral blood flow after ASDH. Fluoro-Jade B-positive cells were visible 15minutes after ASDH in the lesioned hemisphere. Nonlinear growth of lesion volume from 3.7±0.4mm3 to 17.5±0.6mm3 was observed at 24hours in Hematoxylin-Eosin-staining. The most damage develops between 15minutes and 1hour and again between 2 and 6hours after ASDH. The time course of lesion growth supports the approach of early surgery for patients. It furthermore constitutes a basis for further ASDH research with more clearly defined time windows for therapy in animal models.

Anethole Pretreatment Modulates Cerebral Ischemia/Reperfusion: The Role of JNK, p38, MMP-2 and MMP-9 Pathways.

Anethole (AN) is one of the major constituents of several plant oils, demonstrating plentiful pharmacological actions. Ischemic stroke is the main cause of morbidity and death worldwide, particularly since ischemic stroke therapeutic choices are inadequate and limited; thus, the development of new therapeutic options is indispensable. This study was planned to explore the preventive actions of AN in ameliorating cerebral ischemia/reperfusion-induced brain damage and BBB permeability leakage, as well as to explore anethole's potential mechanisms of action. The proposed mechanisms included modulating JNK and p38 as well as MMP-2 and MMP-9 pathways. Sprague-Dawley male rats were randomly assigned into four groups: sham, middle cerebral artery occlusion (MCAO), AN125 + MCAO, and AN250 + MCAO. Animals in the third and fourth groups were pretreated with AN 125 or 250 mg/kg orally, respectively, for two weeks before performing middle cerebral artery occlusion (MCAO)-induced cerebral ischemic/reperfusion surgery. Animals that experienced cerebral ischemia/reperfusion exhibited amplified infarct volume, Evans blue intensity, brain water content, Fluoro-Jade B-positive cells, severe neurological deficits, and numerous histopathological alterations. MCAO animals exhibited elevated MMP-9 and MMP-2 gene expressions, enzyme activities, augmented JNK, and p38 phosphorylation. On the other hand, pretreatment with AN diminished the infarct volume, Evans blue dye intensity, brain water content, and Fluoro-Jade B-positive cells, improved the neurological score and enhanced histopathological examination. AN effectively lowered MMP-9 and MMP-2 gene expression and enzyme activities and diminished phosphorylated JNK, p38. AN decreased MDA content, amplified GSH/GSSG ratio, SOD, and CAT, decreased the serum and brain tissue homogenate inflammatory cytokines (TNF-α, IL-6, IL-1β), NF-κB, and deterred the apoptotic status. This study revealed the neuroprotective ability of AN against cerebral ischemia/reperfusion in rats. AN boosted blood-brain barrier integrity via modulating MMPs and diminished oxidative stress, inflammation, and apoptosis through the JNK/p38 pathway.

Epigallocatechin gallate restores the reduction of protein phosphatase 2 A subunit B caused by middle cerebral artery occlusion

BackgroundEpigallocatechin gallate (EGCG) is a flavonoid compound commonly found in green tea. It exhibits antioxidant, anti-inflammatory, and neuroprotective effects in cerebral ischemia. Protein phosphatase 2 A (PP2A) is an important serine/threonine phosphatase enzyme involved in various cellular activities. PP2A subunit B is present abundantly in the brain and plays an important role in the nervous system. We investigated the effect of EGCG on the expression level of PP2A subunit B in cerebral ischemia caused by middle cerebral artery occlusion (MCAO). EGCG (50 mg/kg) or vehicle was injected into the peritoneal cavity prior to MCAO surgery. Neurological behavior tests were performed 24 h after MCAO, and right cerebral cortex tissue was collected. Cerebral ischemia caused serious neurological abnormalities, which were alleviated by EGCG administration. We screened the expression of PP2A subunits containing A, B, and C using reverse-transcription PCR. We confirmed that PP2A subunit B exhibited significant changes in MCAO animals compared to subunits A and C. We continuously examined the expression of PP2A subunit B protein in MCAO animals using Western blot analysis.ResultsEGCG alleviated the reduction of PP2A subunit B protein by MCAO damage. In addition, immunohistochemistry demonstrated a decrease in the number of PP2A subunit B-positive cells in the cerebral cortex, and EGCG attenuated this decrease. Maintenance of PP2A subunit B is important for normal brain function.ConclusionTherefore, our findings suggest that EGCG exerts neuroprotective effects against cerebral ischemia through modulation of PP2A subunit B expression.

Proteasomal Processing Immune Escape Mechanisms in Platinum-Treated Advanced Bladder Cancer.

In recent years, the number and type of treatment options in advanced bladder cancer (BC) have been rapidly evolving. To select an effective therapy and spare unnecessary side effects, predictive biomarkers are urgently needed. As the host’s anti-cancer immune response is by far the most effective system to impede malignant tumor growth, immune system-based biomarkers are promising. We have recently described altered proteasomal epitope processing as an effective immune escape mechanism to impair cytotoxic T-cell activity. By altering the neoantigens’ characteristics through different proteasomal peptide cleavage induced by non-synonymous somatic mutations, the ability for T-cell activation was decreased (“processing escapes”). In the present study, we analyzed primary chemo-naïve tissue samples of 26 adjuvant platinum-treated urothelial BC patients using a targeted next-generation sequencing panel followed by the epitope determination of affected genes, a machine-learning based prediction of epitope processing and proteasomal cleavage and of HLA-affinity as well as immune activation. Immune infiltration (immunohistochemistries for CD8, granzyme B, CD45/LCA) was digitally quantified by a pathologist and clinico-pathological and survival data were collected. We detected 145 epitopes with characteristics of a processing escape associated with a higher number of CD8-positive but lower number of granzyme B-positive cells and no association with PD-L1-expression. In addition, a high prevalence of processing escapes was associated with unfavorable overall survival. Our data indicate the presence of processing escapes in advanced BC, potentially creating a tumor-promoting pro-inflammatory environment with lowered anti-cancerous activity and independence from PD-L1-expression. The data also need to be prospectively validated in BC treated with immune therapy.

Inhibition of cathepsin B protects pancreatic acinar cells against apoptosis in early pancreatic trauma in rats

Abstract Objectives: To observe the protective effect of cathepsin B inhibition against apoptosis of acinar cells in the early management of pancreatic contusion and laceration in rats, which would provide evidence of a potential early therapeutic for pancreatic contusion and laceration. Methods: Twenty-four rats were assigned to 2 groups: 1) Model (n = 12) with an induced pancreatic injury of severity I–II and 2) CA074-V (n = 12): an induced pancreatic injury, severity I–II treated with the cathepsin B inhibitor CA074-me (0.01 mg/g) by intravenous administration through the caudal vein at 5 minutes post model establishment. The mice in these two groups were further randomly divided into 4 subgroups containing 3 rats each that were sacrificed for quantitation of apoptosis, immunohistochemistry of cathepsin B, and serum amylase and lipase measurements at different time points after model establishment (0, 3, 6, and 12 hours). Results: The percentage of apoptotic pancreatic acinar cells collected from the injured tissues were much lower in the CA074-V group than the Model group at 3 hours [9.25 ± 3.94% vs. 64.76 ± 26.47%, P < 0.10] and 6 hours [14.71 ± 8.22% vs. 66.60 ± 13.54%, P < 0.10] post model establishment. The percentage of cathepsin B-positive pancreatic acinar cells were much lower in the CA074-V group than in the Model group at 3 hours [31.07 ± 12.02% vs. 69.16 ± 5.71%, P < 0.10], 6 hours [24.84 ± 0.93% vs. 47.06 ± 0.91%, P < 0.10], and 12 hours [28.33 ± 9.14% vs. 52.72 ± 1.25%, P < 0.10] post model establishment. Conclusions: Early cathepsin B inhibition effectively blocked acinar cell apoptosis in an experimental rat model of pancreatic contusion and laceration.

Parameters of the gelatinase B activity in the integument epithelium and the lamina propria of the gastric mucosa in experimental acetate ulcer in rats

Objective: to determine the specifi c number and expression intensity of gelatinase B-positive cells in the gastric surface epithelium and lamina propria (cell component) in the area of the ulcerous defect in rats with experimental acetate ulcer of the stomach.Materials and methods: 18 white Wistar rats were divided into three groups: initial (intact), experimental (modeling of an acetate ulcer) and control (falsely operated) in the experiment. Tissues were obtained from the ulceration zone or the presumed ulceration zone (in the initial and control groups) to asses morphological changes and also indicators, characterizing the activity of gelatinase B 7 days after the simulation of the experiment.Results and Discussion. It was revealed some increase in the specifi c number of gelatinase B-positive cells against the background of moderate accumulation of lymphocytes in the integument epithelium of the gastric mucosa. A signifi cant increase in the specifi c number of gelatinase B-positive cells at the maximum intensity of its expression and a moderate accumulation of eff ector cells of tissue alterations was determined in the cellular component of the lamina propria of the gastric mucosa at the same time.

Characterization of metabolic activity induced by kainic acid in adult rat whole brain at the early stage: A 18FDG-PET study

ObjectiveBrain metabolic processes are not fully characterized in the kainic acid (KA)-induced Status Epilepticus (KASE). Thus, we evaluated the usefulness of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) as an experimental strategy to evaluate in vivo, in a non-invasive way, the glucose consumption in several brain regions, in a semi-quantitative study to compare and to correlate with data from electroencephalography and histology studies. Methods.Sixteen male Wistar rats underwent FDG-PET scans at basal state and after KA injection. FDG-PET images were normalized to an MRI-based atlas and segmented to locate regions. Standardized uptake values (SUV) were obtained at several time points. EEGs and cell viability by histological analysis, were also evaluated. ResultsFDG-PET data showed changes in regions such as: amygdala, hippocampus, accumbens, entorhinal cortex, motor cortex and hypothalamus. Remarkably, hippocampal hypermetabolism was found (mean SUV = 2.66 ± 0.057) 2 h after KA administration, while hypometabolism at 24 h (mean SUV = 1.83 ± 0.056) vs basal values (mean SUV = 2.19 ± 0.057). EEG showed increased spectral power values 2 h post-KA administration. Hippocampal viable-cell counting 24 h after KA was decreased, while Fluoro-Jade B-positive cells were increased, as compared to control rats, coinciding with the hypometabolism detected in the same region by semi-quantitative FDG-PET at 24 h after KASE. ConclusionsPET is suitable to measure metabolic brain changes in the rat model of status epilepticus induced by KA (KASE) at the first 24 h, compared to that of EEG; PET data may also be sensitive to cell viability.

Immunophenotypic analysis of the distribution of hepatic macrophages, lymphocytes and hepatic stellate cells in the adult rat liver.

The liver consists of parenchymal hepatocytes and non-parenchymal cells. Non-parenchymal cells, Kupffer cells, hepatic stellate cells and cholangiocytes have crucial roles in liver homeostasis and liver pathology. To establish baseline data, this study investigated immunohistochemically the distribution of non-parenchymal cells in perivenular areas (PV), periportal areas (PP) and Glisson's sheath (GS) of adult rat liver. Liver tissues were collected from the left lateral lobe of rats. CD163-positive macrophages were seen along the sinusoid of PV and PP areas, indicating Kupffer cells. Double immunofluorescence showed, Kupffer cells partly co-expressed CD68 and MHC class II antigens in the liver. The numbers of Kupffer cells were significantly high in PP areas as compared with PV or GS areas. CD68-positive exudative macrophages were highly localized in PP and GS areas and a comparatively low PV area. MHC class II-positive dendritic cells (activated macrophages) were localized mainly in GS. Granzyme B-positive NK cells were mainly localized in the Glisson's sheath. CD3-positive T cells and CD20-positive B cells were distributed along the sinusoids of the PP and PV areas of hepatic lobules. Vimentin and glial fibrillary acidic protein (GFAP)-positive hepatic stellate cells were localized along sinusoids in the hepatic lobules of the liver. Cholangiocytes reacting to cytokeratin 19 were seen on interlobular bile ducts in Glisson's sheath of the liver. This study shows that heterogeneous macrophage populations, liver-resident lymphocytes and hepatic stellate cells localized in PP and PV areas or GS areas of the liver with cells specific patterns.

Gut Microbiota Condition the Therapeutic Efficacy of Trastuzumab in HER2-Positive Breast Cancer.

Emerging evidence indicates that gut microbiota affect the response to anticancer therapies by modulating the host immune system. In this study, we investigated the impact of gut microbiota on immune-mediated trastuzumab antitumor efficacy in preclinical models of HER2-positive breast cancer and in 24 patients with primary HER2-positive breast cancer undergoing trastuzumab-containing neoadjuvant treatment. In mice, the antitumor activity of trastuzumab was impaired by antibiotic administration or fecal microbiota transplantation from antibiotic-treated donors. Modulation of the intestinal microbiota was reflected in tumors by impaired recruitment of CD4+ T cells and granzyme B-positive cells after trastuzumab treatment. Antibiotics caused reductions in dendritic cell (DC) activation and the release of IL12p70 upon trastuzumab treatment, a mechanism that was necessary for trastuzumab effectiveness in our model. In patients, lower α-diversity and lower abundance of Lachnospiraceae, Turicibacteraceae, Bifidobacteriaceae, and Prevotellaceae characterized nonresponsive patients (NR) compared with those who achieved pathologic complete response (R), similar to antibiotic-treated mice. The transfer of fecal microbiota from R and NR into mice bearing HER2-positive breast cancer recapitulated the response to trastuzumab observed in patients. Fecal microbiota β-diversity segregated patients according to response and positively correlated with immune signature related to interferon (IFN) and NO2-IL12 as well as activated CD4+ T cells and activated DCs in tumors. Overall, our data reveal the direct involvement of the gut microbiota in trastuzumab efficacy, suggesting that manipulation of the gut microbiota is an optimal future strategy to achieve a therapeutic effect or to exploit its potential as a biomarker for treatment response. SIGNIFICANCE: Evidence of gut microbiota involvement in trastuzumab efficacy represents the foundation for new therapeutic strategies aimed at manipulating commensal bacteria to improve response in trastuzumab-resistant patients.See related commentary by Sharma, p. 1937 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2195/F1.large.jpg.

Magnesium-containing composition influencing morphological parameters and gelatinase B activity in endometrial tissues in experimental chronic endometrial inflammation

Aim: to assess an effect of medicinal magnesium-containing composition on gelatinase B expression intensity and morphological parameters of chronic experimental endometrial inflammation.Materials and Methods. There were conducted experiments with 60 sexually mature female Wistar rats to determine an effect of medicinal magnesium-containing composition (contains magnesium chloride – a natural polymineral Bischofite) on gelatinase B activity in endometrial tissues (counting number of gelatinase-B-positive cells and intensity of gelatinase B expression), morphological parameters of cell infiltration, as well as amount of magnesium in red blood cell mass collected from the inferior vena cava and subclavian vein. All such parameters were assessed in experimental animals from 4 groups: group 1 – animals in baseline state, group 2 – control, group 3 – experimental chronic endometritis (CE), group 4 – experimental CE after treatment with medicinal magnesium-containing composition. Results. It was found that use of medicinal magnesium-containing composition increased amount of erythrocyte magnesium up to the baseline level and increased both number and expression intensity of stromal gelatinase B-positive cells. In addition, magnesium level in erythrocyte mass from the inferior vena cava was increased and accompanied with restored eosinophil-plasmacyte as well as significantly elevated macrophage-lymphocyte infiltration in endometrial tissues compared to CE animals lacking therapy. Conclusion. The results of this study allow us to conclude about importance of gelatinase B in pathogenesis of experimental chronic endometrial inflammation as well as an opportunity of regulating gelatinase B activity by applying medicinal magnesium containing drug in pathogenetic therapy of experimental CE.

The effect of piracetam on behavioral reactions of adult rats and morphological changes in the brain after whole body fractionated gamma irradiation: an exploratory study.

This research was aimed at examining the effect of piracetam on behavioral reactions and morphological changes in the brain of adult rats after fractionated gamma irradiation with a total dose of 5Gy. Fractionated gamma irradiation led to a decrease in freezing behavior in the Open Field and leukopenia. These behavioral and hematological disorders were accompanied by acell decrease in the cross-sectional area of granular layer of the dentate gyrus, an increase in the number of Fluoro Jade B-positive cells, and an increase in the number of irreversible changes in the cerebral cortex. The administration of piracetam immediately after irradiation for 14days maintained the freezing behavior at the level of intact animals and decreased in general motor activity. Also, an increase in morphometric parameters and a decrease of neurodegeneration were observed. We found a statistically significant decrease in the number of Fluoro Jade B-positive cells in comparison with the group of irradiated animals. The drug had no leukoprotective effect on laboratory animals, and led to the emergence of inconclusive trends in the alternation of the arms of the T-labyrinth. Piracetam application showed positive behavioral and morphological changes in rodents and might have a neuroprotective effect in brain tissue after gamma irradiation. Since it is the first experiment with piracetam we attempted, this exploratory study serves to provide more insight into the potential neuroprotection activity of piracetam, and following research is necessary.

Rubrofusarin Attenuates Chronic Restraint Stress-Induced Depressive Symptoms.

The aim of this study was to examine whether rubrofusarin, an active ingredient of the Cassia species, has an antidepressive effect in chronic restraint stress (CRS) mouse model. Although acute treatment using rubrofusarin failed, chronic treatment using rubrofusarin ameliorated CRS-induced depressive symptoms. Rubrofusarin treatment significantly reduced the number of Fluoro-Jade B-positive cells and caspase-3 activation within the hippocampus of CRS-treated mice. Moreover, rubrofusarin treatment significantly increased the number of newborn neurons in the hippocampus of CRS-treated mice. CRS induced activation of glycogen synthase kinase-3β and regulated development and DNA damage responses, and reductions in the extracellular-signal-regulated kinase pathway activity were also reversed by rubrofusarin treatment. Microglial activation and inflammasome markers, including nod-like receptor family pyrin domain containing 3 and adaptor protein apoptosis-associated speck-like protein containing CARD, which were induced by CRS, were ameliorated by rubrofusarin. Synaptic plasticity dysfunction within the hippocampus was also rescued by rubrofusarin treatment. Within in vitro experiments, rubrofusarin blocked corticosterone-induced long-term potentiation impairments. These were blocked by LY294002, which is an Akt inhibitor. Finally, we found that the antidepressant effects of rubrofusarin were blocked by an intracerebroventricular injection of LY294002. These results suggest that rubrofusarin ameliorated CRS-induced depressive symptoms through PI3K/Akt signaling.

Histological differentiation of mucus cell subtypes suggests functional compartmentation in the eel esophagus.

We investigated the morphological and histological changes in eel esophagus during the course of freshwater (FW) to seawater (SW) transfer and identified multiple types of mucus cells from tissues that were fixed using Carnoy's solution to retain the mucus structure. The FW esophageal epithelium is stratified and composed of superficial cells, mucus cells, club cells (exocrine cells with a large vacuole), and basal cells. Two types of periodic acid-Schiff (PAS)-positive mucus cells were identified, and they can be further distinguished by the periodic acid-thionin Schiff/KOH/PAS (PAT) method, indicating that C7/9- and C8-sialic acids were produced. Isolectin B4-positive mucus cells were found among the C8-sialic acid-producing cells and located at the tips of the villi at mid-posterior regions of the FW esophagus. The two different muci were immiscible and may form separate layers to protect the tissues from the high osmolality of imbibed SW during early SW acclimation. The densities of club cells and isolectin B4-positive cells decreased after SW acclimation, and cuboidal/columnar epithelial cells subsequently developed for active Na+ and Cl- absorption. Cuboidal/columnar epithelial cells proliferated in scattered array rather than at the bases of the villi, thereby retaining the characteristic of the stratified epithelium. Prominent leukocyte invasion was found at the base of the stratified epithelium at early SW transfer, indicating that the immune system was also activated in response to antigen exposure from imbibed SW. The mucus composition in FW is more complicated than that in SW, fueling further studies for their functions to form unstirred layers as osmoregulatory barriers.

Abstract 3951: Apalutamide reworks the immune composition of prostate tumors

Abstract Prostate cancers depend on androgen receptor (AR) signaling for survival making it a major therapeutic target. Apalutamide is an oral nonsteroidal AR antagonist that is currently indicated for the treatment of patients with non-metastatic castration-resistant prostate cancer. Apalutamide hinders AR-mediated transcriptional activity by impeding AR nuclear translocation and binding to DNA in cancer cells. AR is also expressed by stromal and immune cells and can modulate innate and adaptive immune responses, moreover, androgen ablation can both hinder and enhance antitumor immunity. Thus far, success from immune checkpoint blockade monotherapy has been lacking in prostate cancer patients and novel strategies using of other modalities are being investigated. Here, we characterize the effect of apalutamide therapy on antitumor immunity in a preclinical mouse model of prostate cancer to assess its value as potential partner for combination with immunotherapy. Conditional prostate-specific Pten-knockout mice were treated with apalutamide (30 mg/kg/d, 5 days on/2 days off) or vehicle for a period of 4 or 8 weeks to coincide with androgen sensitivity and the shift towards castration resistance. Tumor reductions after 4 or 8 weeks of treatment with apalutamide were 38.2% (P<0.01) and 46.2% (P<0.001), respectively. Flow cytometric analysis of tumor tissues revealed 1.3 (P=0.033) and 1.9 (P<0.001) -fold increases of leukocyte (CD45+) infiltration in mice treated with apalutamide for 4 or 8 weeks, respectively. CD8+ T cell infiltration was 2.7-fold higher at 8 weeks, however, tumor reactive PD1+/CD8 T cells were 2-3-fold greater for apalutamide-treated mice at weeks 4 and 8. Quantitative immunohistochemical analysis confirmed increased intraepithelial CD8+ T and granzyme B-positive cells. Apalutamide treatment was also associated with increased peritumoral T regulatory cells and intraepithelial neutrophils. Immuno-profiling using a qRT-PCR-based panel of immune-relevant genes associated apalutamide therapy with an increased IFN-γ inflammatory signature, antigen presentation/dendritic cell, natural killer cell, T regulatory cell, tumor associated macrophage, and myeloid-derived suppressor cells (MDSC). These data also revealed a trend towards decreased CD8 T cell activation at week 8 accompanied by increased expression of the immunological checkpoints Ctla4, Tim3, and 2b4. In conclusion, our findings suggests that apalutamide-induced tumor cell death attracted phagocytes (macrophages and dendritic cells) that led to a innate and adaptive immune cell responses. While, cytotoxic T cell activity was improved, it was limited by the development of an immunosuppressive tumor microenvironment replete with MDSCs and T regulatory cells. Nevertheless, treatment with apalutamide turned immunologically “cold” tumors into more immunologically reactive tumors that may become more susceptible to targeted immunotherapy. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sato, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuko Sakai, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Apalutamide reworks the immune composition of prostate tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3951.

Neuroprotective effects of isoliquiritigenin against cognitive impairment via suppression of synaptic dysfunction, neuronal injury, and neuroinflammation in rats with kainic acid-induced seizures

Epileptogenesis is a dynamic process initiated by insults to brain and commonly accompanied by cognitive impairment. Isoliquiritigenin (ISL), a flavonoid in licorice, has a broad spectrum of biological effects including anti-inflammatory and antioxidant activities. However, the protective effects of ISL against cognitive impairment in epileptic processes and the underlying molecular mechanism are not well understood. To address these questions, we established an reproducible seizure model by intracerebroventricular injection of kainic acid (KA) in 21-day-old rats; ISL was intraperitoneally administered three times prior to KA injection, and changes in cognitive function; synaptic plasticity; neuronal injury; number of glial cells; and expression of pro-inflammatory cytokines and nuclear factor-like (NRF)2 signaling and NACHT, LRR, and PYD domains-containing protein (NLRP)3 inflammasome components in the hippocampus were examined. Rats with KA-induced seizures showed longer average escape latency and decreases in the number of platform crossings and average time spent in the target quadrant in the Morris water maze; ISL pretreatment reversed this decline in cognitive impairment and increased the protein levels of synaptophysin, postsynaptic density-95 and brain-derived neurotrophic factor while reducing the number of Fluoro Jade B-positive cells, microglia, and astrocytes; cleaved-Caspase-3 and -9 protein levels; and tumor necrosis factor-α, interleukin (IL)-1β, and IL-18 production. It also enhanced the nuclear localization of NRF2, hemeoxygenase-1, and NAD(P)H:quinone oxidoreductase (NQO) 1, and reversed the upregulation of NLRP3 inflammasome components NLRP3 and Caspase-1 induced by KA injection. Thus, ISL protects against cognitive impairment in KA-induced epileptic processes possibly through regulation of NRF2 signaling and the NLRP3 inflammasome pathway.

Inhibition of diacylglycerol kinase alpha to augment antitumor effector T cells in tumor-bearing host.

293 Background: Diacylglycerol kinases (DGKs), lipid kinases transforming diacylglycerol to phosphatidic acid, play important roles in intracellular signal transduction. Diacylglycerol kinase alpha (DGKa), an isozyme of DGKs, is well-known to promote proliferation of cancer cells by suppression of the apoptosis. Additionally, a previous report demonstrated that activation of DGKa induced anergy state of T lymphocytes in vivo. In this study, we investigated whether inhibition of DGKa not only suppress the tumorigenesis of cancer cells but also activate anti-tumor immunity. Methods: We first investigated the effect of DGKa inhibitor on in vitro proliferation of murine hepatoma cell lines (Hepa1-6) by cell proliferation assay. Cytokine and Granzyme B productions by CD8+ T cells from OT-1 mice after the OVA antigen stimulation were evaluated by ELISA and flowcytometry, respectively. Next, we established a tumor-bearing mice model by injection of mCherry-transfected Hepa1-6 cells into spleen. Tumorigenesis and tumor-infiltrating T cells in the liver were evaluated by in vivo imaging system, HE staining, and immunohistochemistry. CD8+ T cells were collected from the liver and stimulated with PMA and Ca2+ ionophore and the IFN-g production levels were evaluated by flowcytometry. Results: Proliferation of Hepa1-6 cells were suppressed in the presence of DGKa inhibitor in vitro. IL-2 production levels of OT-1 CD8 T cells in control group was augmented by the addition of DGKa inhibitor (246 vs 579 pg/ml, p < 0.05). Granzyme B-positive cells in OT-1 CD8+ T cells were increased by the treatment with DGKa inhibitor compared to the control group (4.4 vs 8.9 %, P < 0.05) after the antigen stimulation. In vivo administration of DGKa inhibitor significantly suppressed the tumor size (fluorescence (AU) 2.0x1010 vs 6.3x109, area (μm2) 1.5x107 vs 0.9x107, p < 0.05) in the liver of tumor bearing mice. Then, the number of tumor-infiltrating T cells (582 vs 1506, 5 HPF, p < 0.05) and the IFN-g-producing cells (9.2 vs 16.0 %) in CD8+ T cells were elevated by the DGKa treatment. Conclusions: Inhibition of DGKa not only suppressed the proliferation of hepatoma but also activated anti-tumor effector T cells in vivo.

Abstract 947: Influence of abiraterone therapy on anti-tumor immunity in genetically engineered mouse prostate cancer models

Abstract Recent evidence has suggested that androgen deprivation therapies (ADTs) can influence tumor immune responses via androgen receptor (AR) regulation. On one hand, reports have indicated that certain ADTs can compromise T cell immune responses and enhance PD-L1 immune suppression, while others indicate that ADT can enhance anti-tumor responses via modulation of the apoptotic pathway. Abiraterone is a steroidal CYP17 inhibitor approved for the treatment of late-stage metastatic castration-resistant prostate cancer. In this study we use genetically engineered mouse models of prostate cancer (GEMMs-PCa) to investigate the antitumor activity of abiraterone and its influence on tumor immunity. In a mouse Pten-deficient prostate cancer model, chronic treatment with abiraterone acetate (40 mg/kg/d, 5 days on, 2 days off) reduced prostate tumor burden by 13.1% ± 9.0 (P=0.499) after four weeks of dosing and 30.5% ± 8.1 (P=0.0275) after eight weeks. Downregulation of classical mouse Ar-responsive genes (Fkbp5, Nkx3.1, Msmb and Timp4) confirmed the inhibition AR transcriptional activity after abiraterone therapy. In a model of advanced prostate cancer, driven by the conditional inactivation of Pten and Trp53, treatment with abiraterone after surgical castration modestly improved median overall survival from 7 days to 16 days vs. castration alone (P=0.240, n=8 mice/group). qRT-PCR-based analysis of a panel of 54 immune-responsive genes, revealed distinct expression signatures in abiraterone-treated tumors compared to tumors from orchidectomized mice. Relative to orchidectomized mice, tumors from abiraterone treated mice consistently demonstrated reduced mRNA levels of the T regulatory cell gene markers Cd4, Foxp3, Cd4, Tgfb1 and Il10. Furthermore, mRNA expression levels of representative immune checkpoint genes Cd274, Pdcd1lg2, Pdcd1 and Ctla4 were also lower in abiraterone treated mice. Follow-up immunohistochemical analysis showed a 1.8-fold increase of tumor infiltrating granzyme B-positive cells in tumors of mice treated with abiraterone compared to surgical castration. Our results show that abiraterone suppressed AR transcriptional activity and reduced tumor growth and progression in GEMMs-PCa. Our data also suggests that abiraterone induces lesser immunosuppressive responses than surgical castration and supports further investigation into developing rational combinations of ADT and immunotherapy in order to enhance therapeutic responses for patients suffering with prostate cancer. Citation Format: Eri Banno, Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sato, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuko Sakai, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Influence of abiraterone therapy on anti-tumor immunity in genetically engineered mouse prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 947.