The role of carbonic anhydrase III and autophagy in type 2 diabetes with cardio-cerebrovascular disease.

Type 2 diabetes mellitus (T2DM) is one of the most common chronic diseases among the elderly people. The T2DM increases the risk of cardio-cerebrovascular disease (CCD), and the main pathological change of the CCD is atherosclerosis (AS). Meanwhile, the carbonic anhydrases (CAs) are involved in the formation and progression of plaques in AS. However, the exact physiological mechanism of carbonic anhydrase III (CAIII) has not been clear yet, and there are also no correlation study between CAIII protein and T2DM with CCD. The 8-week old diabetic mice (db/db-/- mice) and wild-type mice (wt mice) were feed by a normal diet till 32 weeks, and detected the carotid artery vascular opening angle using the method of biomechanics; The changes of cerebral cortex and myocardium were watched by the ultrastructure, and the autophagy were observed by electron microscope; The tissue structure, inflammation and cell injury were observed by Hematoxylin and eosin (HE) staining; The apoptosis of cells were observed by TUNEL staining; The protein levels of CAIII, IL-17, p53 were detected by immunohistochemical and Western Blot, and the Beclin-1, LC3, NF-κB were detected by Western Blot. All statistical analysis is performed using PRISM software. Compared with wt mice, db/db-/- mice' carotid artery open angle increased significantly. Electron microscope results indicated that autophagy in db/db-/- mice cerebral cortex and heart tissue decreased and intracellular organelle ultrastructure were damaged. HE staining indicated that, db/db-/- mice' cerebral cortex and heart tissue stained lighter, inflammatory cells infiltration, cell edema were obvious, myocardial fibers were disorder, and myocardial cells showed different degrees of degeneration. Compared with wt mice, TUNEL staining showed that there was obviously increase in db/db-/- mice cortex and heart tissue cell apoptosis. The results of immunohistochemistry and Western Blot indicated that CAIII, Beclin-1 and LC3II/I expression levels conspicuously decreased in cortex and heart tissue of db/db-/- mice, and the expression level of IL-17, NF-κB and p53 obviously increased. The carotid artery' vascular stiffness was increased and which was probably related with formation of AS in diabetic mice. And the autophagy participated in the occurrence and development of diabetic CCD. CAIII protein might somehow be involved in the regulation of autophagy probably through affecting cell apoptosis and inflammation, but the underlying mechanism remains to be further studied.

EGb761 improves the cognitive function of elderly db/db-/- diabetic mice by regulating the beclin-1 and NF-κB signaling pathways.

To assess whether EGb761 could protect elderly diabetic mice with cognitive disorders and explore the role of beclin-1-mediated autophagy in these protective effects. Two-month-old male db/db-/- mice and wild-type C57/BL6 mice were randomly divided into six groups: db/db-/- control, db/db-/- 50 mg, db/db-/- 100 mg, wild-type (WT) control, WT 50 mg, and WT 100 mg. EGb761 (50 mg/kg or 100 mg/kg of bodyweight) was given by gavage once a day for 1 month from the age of 6 months. Y-maze and social choice tests were performed at 8th months. The blood pressure was measured. The imaging changes in the brain were measured using magnetic resonance imaging (MRI). The expression and distribution of beclin-1, LC3, and NF-κB were detected using immunohistochemistry staining and western blotting. Ultrastructure alterations in the hippocampus were observed using transmission electron microscopy. Compared with WT mice, the learning ability, memory and overall cognitive function of db/db-/- mice decreased (P < 0.05), and EGb761 could significantly improve the learning and memory function of db/db-/- mice (P < 0.05). EGb761 significantly improved systolic blood pressure in db/db-/- mice (P < 0.01). In addition, fMRI-bold showed a decline in the hippocampus of mice in the db/db-/- group compared with WT. EGb761 could improve these above changes. Immunohistochemistry staining and western blotting confirmed that EGb761 significantly increased beclin-1 and reduced LC3-II/I levels in the brains of db/db-/- mice (P < 0.05). NF-κB levels were obviously higher in the db/db-/- group than that in the WT group, and EGb761 significantly reduced NF-κB levels in db/db-/- mice (P < 0.05). There was a trend of increased autophagosomes in db/db-/- mice, but EGb761 did not change obviously the number of autophagosomes. Compared with normal aged WT mice, aging db/db-/- mice had more common complications of cerebral small vessel disease and cognitive dysfunction. EGb761 could significantly improve the cognitive function of aging db/db-/- mice via a mechanism that may involve the regulation of beclin-1, LC3, and NF-κB.

The prognostic value of Foxp3+ tumor-infiltrating lymphocytes in patients with glioblastoma.

Forkhead box protein 3 (Foxp3) is known as a specific marker for regulatory T cells which contribute to immunosuppression in tumor microenvironment. However, existing studies regarding clinical significance of Foxp3+ tumor-infiltrating lymphocytes (TILs) in glioblastoma (GBM) remained discrepant. In this study, we aimed to explore whether this subtype of TILs correlated with prognosis in patients with GBM. Foxp3+ TILs as well as CD8+ ones were detected by immunohistochemistry on paraffin-embedded tumor samples from 62 patients. Staining for p53, MGMT and Ki-67 were also performed. The correlation of TIL subtypes with clinicopathologic features were analyzed. Progression-free survival (PFS) and overall survival (OS) were estimated by Kaplan-Meier method and compared using log-rank test. Independent prognostic factors for PFS and OS were determined through univariate and multivariate analysis. Significant correlation was found between Foxp3 and CD8 expression (P = 0.003), but not between TIL subtypes and clinicopathologic characteristics. Patients with higher density of Foxp3+ TILs showed relatively shorter PFS (P < 0.001) and OS (P = 0.003) whereas patients with higher density of CD8+ TILs obtained no significant differences in survival. Survival analysis based on molecular classifications further clarified these predictive values. Univariate and multivariate analysis revealed that frequency of Foxp3+ TILs was probably associated with both PFS (P = 0.002) and OS (P = 0.003). In conclusion, the results suggest that Foxp3 positive infiltrates could provide an independent predictive factor in GBM.

Comparison of USPIO-enhanced MRI and Gd-DTPA enhancement during the subacute stage of focal cerebral ischemia in rats.

BACKGROUND: Being one class of magnetic resonance imaging (MRI) contrast agents, ultrasmall superparamagnetic particles of iron oxides (USPIO) bear the potential to study neuroinflammation following stroke, but there is still debate over whether the iron oxides particles may enter the brain tissue passively over a damaged blood-brain barrier (BBB). PURPOSE: To compare the enhancement patterns of USPIO and gadopentate dimeglumine (Gd-DTPA) during the subacute stage of focal cerebral ischemia, to examine the relationship between USPIO enhancement and BBB disturbance, as well as with neuroinflammatory cell response. MATERIAL AND METHODS: Multiple MR sequences were obtained on days 3 and 6 after transient middle cerebral artery occlusion induced in rats with and without the application of USPIO and Gd-DTPA. The enhancement patterns of the two contrast agents were compared and correlated to histology, including IgG for BBB permeability, Prussian Blue staining for iron particle detection, and CD68 immunohistochemistry staining to identify macrophage/microglia. RESULTS: Gd-DTPA enhancement depicted BBB breakdown being in line with IgG leakage. The USPIO enhanced images demonstrated both diffuse and focal signal alteration in ischemic lesions. The diffuse enhanced pattern had a similar spatial and temporal profile as with Gd-DTPA enhancement. In addition, focal enhanced signal loss was visible on T1-, T2-, and T2*-weighted images, with a peak tendency of MR signal loss, macrophage/microglia concentration and iron particle accumulation at a later phase of the study. CONCLUSION: After focal cerebral ischemia, Gd-DTPA-enhanced MRI showed a higher sensitivity in detecting BBB alterations than did USPIO enhancement. USPIO provided complementary information regarding inflammatory cell activity in neuroinflammatory to cerebral ischemia that had not been visualized using conventional Gd-DTPA. The assessment using multiple MR parameters may identify intracellular and extracellular USPIO in vivo.

Density of tertiary lymphoid structures predicts clinical outcome in breast cancer brain metastasis.

BACKGROUND: Patients with breast cancer brain metastases (BCBM) experience a rapid decline in their quality of life. Recently, tertiary lymphoid structures (TLSs), analogs of secondary lymphoid organs, have attracted extensive attention. However, the potential clinical implications of TLSs in BCBMs are poorly understood. In this study, we evaluated the density and composition of TLSs in BCBMs and described their prognostic value. METHODS: Clinicopathological data were collected from 98 patients (2015-2021). TLSs were evaluated, and a TLS scoring system was constructed. Differences in progression-free survival (PFS) and overall survival (OS) between groups were calculated using the Kaplan-Meier method. Immunohistochemistry and multiplex immunofluorescence (mIF) were used to assess TLSs heterogeneity. RESULTS: TLSs were identified in 47 patients with BCBM. High TLSs density indicated favorable survival (OS, p=0.003; PFS, p<0.001). TLS was positively associated with OS (p=0.0172) and PFS (p=0.0161) in the human epidermal growth factor receptor type 2-positive subtype, and with prolonged OS (p=0.0482) in the triple-negative breast cancer subtype. The mIF results showed significant differences in the percentages of T follicular helper (Tfh) cells, M2 macrophages, cytotoxic T lymphocytes, and CD8+TIM-3+ T lymphocytes between the groups of TLS scores 0-3 (cytotoxic T lymphocytes, p=0.044; Tfh, p=0.021; M2 macrophages, p=0.033; CD8+TIM-3+ T lymphocytes, p=0.018). Furthermore, novel nomograms incorporating the TLS scores and other clinicopathological predictors demonstrated prominent predictability of the 1-year, 3-year, and 5-year outcomes of BCBMs (area under the curve >0.800). CONCLUSION: Our results highlight the impact of TLSs abundance on the OS and PFS of patients with BCBM. Additionally, we described the immune composition of TLSs and proposed novel nomograms to predict the prognosis of patients with BCBM.

In vivo USPIO-enhanced MR signal characteristics of secondary degeneration in the ipsilateral substantia nigra after middle cerebral artery occlusion at 3T.

BACKGROUND AND PURPOSE: Ultrasmall superparamagnetic iron oxide (USPIO) particles to enhance MRI have been used to study neuroinflammation in vivo. Our purpose was to observe the USPIO-enhanced MR signal alterations in the primary ischemic lesion and ipsilateral substantia nigra after middle cerebral artery occlusion (MCAO) to verify the subsequent sequelae of neuroinflammation seen in the primary ischemic focus and secondary degeneration region. MATERIALS AND METHODS: Sprague-Dawley rats were subjected to transient MCAO. In addition to conventional T2-, T1-weighted imaging, USPIO-enhanced MRI was performed in USPIO-injected stroke rats, while Gd-enhanced imaging was acquired in control stroke rats, on days 3, 6 using a 3-T MR scanner. The MR signal characteristics in the primary ischemic striatum, ipsilateral substantia nigra were noted, compared on histopathological H&E, Prussian blue (PB) staining. RESULTS: After MCAO, USPIO-induced T2 hypointensity changes were observed in the primary ischemic region with BBB impairment at both time points. In the substantia nigra ipsilateral to the primary ischemic lesion, there was no evidence of USPIO accumulation detected by MRI and PB staining, and no BBB leakage reflected by Gd-enhanced imaging on days 3 and 6. CONCLUSION: USPIO-enhanced MR signals have variable characteristics in both primary and remote sites after focal cerebral ischemia. This suggests that the neuroinflammatory response to brain ischemia in the primary ischemic focus and secondary degeneration region have different temporal patterns and pathophysiological mechanisms.

Corrigendum: H3K27M mutation doesn't mean worse prognosis in old patients.

[This corrects the article DOI: 10.3389/fonc.2022.912166.].

H3K27M Mutation Doesn't Mean Worse Prognosis in Old Patients.

Objective: Diffuse midline glioma (DMG), H3K27 altered is a new entity that has become widely recognized. However, studies concerning DMG in adult patients remain rare. We did a retrospective study covering the largest amount of patients to date to analyze the clinicopathological characteristics of diffuse glioma in midline structures of the adult. Methods: We reviewed 108 cases of adult DMG, collected their clinical data, and pathological results including H3K27 mutation. Summarized their features and the connection with overall survival in different age groups. Results: Among 108 cases, 79 tumors were located at the thalamus. 38 patients had H3K27M mutation, whose average age was 35.7 years. The median overall survival of H3K27M-mutant gliomas and the 80 H3K27M wild-type gliomas were both 12 months. For young patients (age ≤ 35), The median survival time of the H3K27M-mutant was 18 months, while that of the H3K27M wild-type was 37 months. For older patients (age>35), the median survival time of the H3K27M-mutant was 16 months, while that of the H3K27M wild-type was 13 months. Other clinicopathological factors including sex, tumor location, the approach of surgery, histological grade, ATRX, and P53 were statistically irrelevant to prognosis. Conclusion: The DMG in adults mainly occurred in the thalamus. H3K27M mutations tend to happen more frequently in young adults, and this genetic alteration results in a worse outcome only in young patients (≤35). For old patients, age is the only independent prognostic factor. Patients who underwent different surgical operations including biopsy, subtotal resection, and total resection had similar prognoses.

Organ-specific cholesterol metabolic aberration fuels liver metastasis of colorectal cancer.

Rationale: Metastasis, the development of secondary malignant growth at a distance from a primary tumor, is the main cause of cancer-associated death. However, little is known about how metastatic cancer cells adapt to and colonize in the new organ environment. Here we sought to investigate the functional mechanism of cholesterol metabolic aberration in colorectal carcinoma (CRC) liver metastasis. Methods: The expression of cholesterol metabolism-related genes in primary colorectal tumors (PT) and paired liver metastases (LM) were examined by RT-PCR. The role of SREBP2-dependent cholesterol biosynthesis pathway in cell growth and CRC liver metastasis were determined by SREBP2 silencing in CRC cell lines and experimental metastasis models including, intra-splenic injection models and liver orthotropic injection model. Growth factors treatment and co-culture experiment were performed to reveal the mechanism underlying the up-regulation of SREBP2 in CRC liver metastases. The in vivo efficacy of inhibition of cholesterol biosynthesis pathway by betulin or simvastatin were evaluated in experimental metastasis models. Results: In the present study, we identify a colorectal cancer (CRC) liver metastasis-specific cholesterol metabolic pathway involving the activation of SREBP2-dependent cholesterol biosynthesis, which is required for the colonization and growth of metastatic CRC cells in the liver. Inhibiting this cholesterol biosynthesis pathway suppresses CRC liver metastasis. Mechanically, hepatocyte growth factor (HGF) from liver environment activates SREBP2-dependent cholesterol biosynthesis pathway by activating c-Met/PI3K/AKT/mTOR axis in CRC cells. Conclusion: Our findings support the notion that CRC liver metastases show a specific cholesterol metabolic aberration. Targeting this cholesterol biosynthesis pathway could be a promising treatment for CRC liver metastasis.

[Relationship between breast cancer molecular subtypes with clinicopathological characteristics and prognosis].

OBJECTIVES: This study try to subclassify breast cancer into different prognostic subgroups according to immunohistochemical algorithm and discuss the relationship between subtypes and biological and clinical behavior and prognosis. METHODS: One hundred and twenty-eight cases of infiltrative ductal carcinoma were studied using immunohistochemical staining with an antibody panel of ER, PR, HER2 and CK5/6 and subclassified referring to previous reports, and the 9 cases of HER2 positive subtype were tested using FISH. RESULTS: The expression of ER, PR, HER2, and CK5/6 was detected in 67%, 45%, 27% and 27% cases, respectively. All cases were subclassified into five subgroups, with luminal A (55%), luminal B (20%), HER2 positive (7%), basal-like (10%) and unclassified cases (8%). Nine HER2 positive cases all showed amplification of HER2 gene. It was demonstrated that the luminal A group was associated with the best prognosis but the basal-like group worst by univariate analysis. Multivariate analysis demonstrated that both the clinical stage and immunohistochemical subtypes of tumor were related to overall survival. Menses status were different among these subtypes. CONCLUSION: According to the expression of ER, PR, HER2 and CK5/6, infiltrative ductal carcinoma could be subclassified into five subgroups with different biological features and outcome, having a role in evaluating the prognosis and guiding the clinical treatment.

Multi-centric solid-pseudopapillary neoplasm of the pancreas.

Multi-centric solid-pseudopapillary neoplasm (SPN) of the pancreas is a rare tumor: only 4 cases are reported in the literature. The clinical and pathological features have not yet been fully clarified. We report 3 cases of multi-centric SPN and discuss their clinical presentations and histological and immunohistochemical features, comparing with solitary SPN. Among the total of 7 cases, 6 were female and 1 was male. Patients had nonspecific symptoms at presentation. Tumors were often large and well demarcated with cystic degeneration and clear margin between lumps. Histologically, characteristic pseudopapilla was formed with uniform cells surrounding the delicate blood vessels. Tumor cells were positive for vimentin, synaptophysin, progesterone receptor, and CD10 and demonstrated nuclear localization of ß-catenin. The prognosis of patients was excellent after complete surgical resections. Multi-centric SPN shares similar clinical and pathological features to solitary SPN.

Retroperitoneal hemangiopericytoma: case report and literature review.

Retroperitoneal hemangiopericytoma is a kind of uncommon tumor. We report a case of 41-year-old man who was diagnosed retroperitoneal tumor without significant symptoms by abdominal ultrasonography. Abdominal CT and MRI scans show a 6 cm × 5 cm solid tumor delineated clearly from adjacent organs. Excision of the tumor was performed and the histopathological examination confirmed the diagnosis of hemangiopericytoma.