Regulation of inflammation and immunity in sepsis by E3 ligases.

Sepsis is a life-threatening organ dysfunction caused by an abnormal infection-induced immune response. Despite significant advances in supportive care, sepsis remains a considerable therapeutic challenge and is the leading cause of death in the intensive care unit (ICU). Sepsis is characterized by initial hyper-inflammation and late immunosuppression. Therefore, immune-modulatory therapies have great potential for novel sepsis therapies. Ubiquitination is an essential post-translational protein modification, which has been known to be intimately involved in innate and adaptive immune responses. Several E3 ubiquitin ligases have been implicated in innate immune signaling and T-cell activation and differentiation. In this article, we review the current literature and discuss the role of E3 ligases in the regulation of immune response and their effects on the course of sepsis to provide insights into the prevention and therapy for sepsis.

Targeting UDP-α-d-glucose 6-dehydrogenase alters the CNS tumor immune microenvironment and inhibits glioblastoma growth.

Glioblastoma (GBM, WHO grade IV glioma) is the most common and lethal malignant brain tumor in adults with a dismal prognosis. The extracellular matrix (ECM) supports GBM progression by promoting tumor cell proliferation, migration, and immune escape. Uridine diphosphate (UDP)-glucose 6-dehydrogenase (UGDH) is the rate-limiting enzyme that catalyzes the biosynthesis of glycosaminoglycans that are the principal component of the CNS ECM. We investigated how targeting UGDH in GBM influences the GBM immune microenvironment, including tumor-associated microglia/macrophages (TAMs) and T cells. TAMs are the main immune effector cells in GBM and can directly target tumor cells if properly activated. In co-cultures of GBM cells and human primary macrophages, UGDH knockdown in GBM cells promoted macrophage phagocytosis and M1-like polarization. In orthotropic human GBM xenografts and syngeneic mouse glioma models, targeting UGDH decreased ECM deposition, increased TAM phagocytosis marker expression, reduced M2-like TAMs and inhibited tumor growth. UGDH knockdown in GBM cells also promoted cytotoxic T cell infiltration and activation in orthotopic syngeneic mouse glioma models. The potent and in-human-use small molecule GAG synthesis inhibitor 4-methylumbelliferone (4-MU) was found to inhibit GBM cell proliferation and migration in vitro, mimic the macrophage and T-cell responses to UGDH knockdown in vitro and in vivo and inhibit growth of orthotopic murine GBM. Our study shows that UGDH supports GBM growth through multiple mechanisms and supports the development of ECM-based therapeutic strategies to simultaneously target tumor cells and their microenvironment.

Monoallelic IDH1 R132H Mutation Mediates Glioma Cell Response to Anticancer Therapies via Induction of Senescence.

Heterozygous isocitrate dehydrogenase (IDH) R132H mutation (IDH1R132H/WT) is an early event during gliomagenesis. Clinically, patients with glioma carrying mutant IDH1 respond better to antitumor therapies. However, the mechanism by which IDH1 mutations contribute to gliomagenesis and therapeutic response remains elusive. Here we report that senescence is involved in the improved therapeutic responses of mutant IDH1 glioma cells. Knocking-in IDH1R132H/WT in glioma cells significantly enhanced gliomas cell senescence in response to temozolomide and radiation via a DNA-damage mediated mechanism. We further asked if senescence plays a role in IDH1R132H/WT-induced gliomagenesis. Together with ATRX knockout and p53/RB loss, IDH1R132H/WT transformed nonneoplastic human astroglial cells to form tumors in mouse brains. In-depth characterization revealed that a subset of these precancerous cells underwent senescence-like phenotypic changes, including flat and enlarged-cell morphology, increased senescence marker expression, decreased cell proliferation, and cell-cycle arrest at the G2-M phase. Mechanistic studies indicated that the combination of glioma driver genes (p53/RB/IDH1/ATRX) dramatically increased DNA damage and activated DNAdamage response (DDR) pathways ATR/ATR and Chk1/Chk2 in senescent cells. To determine how senescent cells drive tumor formation, we investigated non-cell-autonomous mechanisms such as senescence-associated secretory phenotype (SASP), a panel of proinflammatory and tissue-remodeling factors implicated in a tumor-permissive microenvironment. We found that astroglial cells carrying p53/RB/ATRX loss and IDH1R132H/WT upregulated key factors in SASP via an epigenetic-mediated mechanism. Our work suggests that drugs that specifically eliminate senescent cells could help kill precancerous cells and senescent tumor cells following antitumor therapies. IMPLICATIONS: The mechanisms by which IDH1 mutations contribute to gliomagenesis and therapeutic responses remain incompletely characterized; this work reveals senescence as a novel mechanism of IDH-mutant-mediated biological impact and describes new therapeutic opportunities concerning IDH1-mutant gliomas.

Mutant IDH1 promotes phagocytic function of microglia/macrophages in gliomas by downregulating ICAM1.

Tumor-associated microglia/macrophages (TAMs) are the main innate immune effector cells in malignant gliomas and have both pro- and anti-tumor functions. The plasticity of TAMs is partially dictated by oncogenic mutations in tumor cells. Heterozygous IDH1 mutation is a cancer driver gene prevalent in grade II/III gliomas, and IDH1 mutant gliomas have relatively favorable clinical outcomes. It is largely unknown how IDH mutation alters TAM phenotypes to influence glioma growth. Here we established clinically relevant isogenic glioma models carrying monoallelic IDH1 R132H mutation (IDH1R132H/WT) and found that IDH1R132H/WT significantly downregulated immune response-related pathways in glioma cells, indicating an immunomodulation role of mutant IDH1. Co-culturing IDH1R132H/WT glioma cells with human macrophages promoted anti-tumor phenotypes of macrophages and increased macrophage migration and phagocytic capacity. In orthotopic xenografts, IDH1R132H/WT decreased tumor growth and prolonged animal survival, accompanied by increased TAM recruitment and upregulated phagocytosis markers, suggesting the induction of anti-tumor TAM functions. Using human cytokine arrays that query 36 proteins, we identified significant downregulation of ICAM-1/CD54 in IDH1R132H/WT gliomas, which was further confirmed by ELISA and immunoblotting analyses. ICAM1 gain-of-function studies revealed that ICAM1 downregulation in IDH1R132H/WT cells played a mechanistic role to mediate the immunomodulation function of IDH1R132H/WT. ICAM-1 silencing in IDH1 wild-type glioma cells decreased tumor growth and increased the anti-tumor function of TAMs. Together, our studies support a new TAM-mediated phagocytic function within IDH1 mutant gliomas, and improved understanding of this process may uncover novel approaches to targeting IDH1 wild type gliomas.

Traditional Chinese medicine shenhuang granule in patients with severe/critical COVID-19: A randomized controlled multicenter trial.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is still a pandemic, with a high mortality rate in severe/critical cases. Therapies based on the Shenghuang Granule have proved helpful in viral infection and septic shock. HYPOTHESIS/PURPOSE: The objective of the current study was to compare the efficacy and safety of the traditional Chinese medicine, Shenhuang Granule, with standard care in hospitalized patients with severe/critical COVID-19. STUDY DESIGN AND METHODS: This was an open-label, multicenter, randomized, controlled clinical trial. At 4 medical centers, a total of 111 severe/critical patients were randomly assigned to receive Shenhuang Granule (SHG group) twice a day for 14 days, in addition to standard care, or to receive standard care alone (Control group). The maximal follow up time was 75 days. The clinical endpoint was clinical improvement and mortality. RESULTS: 54 patients were assigned to the control group and 57 to the SHG group. The overall mortality was 75.9% (41/54) in the control group, and 38.6% (22/57) in the SHG group (p < 0.01 vs. control). The post hoc analysis showed that in the severe category, the mortality of the control group vs. the SHG group was 58.8% (10/17) vs. 5.3% (1/19) (p < 0.01); while in the critical category, it was 83.8% (31/37) vs. 55.3% (21/38) (p < 0.05). In the severe category, the mortality of patients who eventually received an invasive ventilator in the control vs. the SHG group was 58.8% (10/17) vs. 0 (0/19) (p < 0.01). Administration of SHG was associated with increased lymphocytes and decreased adverse events. CONCLUSION: Shenhuang Granule is a promising integrative therapy for severe and critical COVID-19.

Clinical Effect of Traditional Chinese Medicine Shenhuang Granule in Critically Ill Patients with COVID-19: A Single-Centered, Retrospective, Observational Study.

The coronavirus disease 2019 (COVID-19) pandemic has become a public health emergency of global concern. In China, traditional Chinese medicine has been widely administered to COVID-19 patients without sufficient evidence. To evaluate the efficacy of Shenhuang Granule (SHG) for treating critically ill patients with COVID-19, we included in this study 118 patients who were admitted to the ICU of Tongji Hospital between January 28, 2020 and March 28, 2020. Among these patients, 33 (27.9%) received standard care plus SHG (treatment group) and 85 (72.1%) received standard care alone (control group). Enrolled patients had a median (IQR) age of 68 (57-75) years, and most (79 [67.1%]) were men. At end point of this study, 83 (70.3%) had died in ICU, 29 (24.5%) had been discharged from ICU, and 6 patients (5.2%) were still in ICU. Compared with control group, mortality was significantly lower in treatment group (45.4% vs. 80%, p < .001). Patients in treatment group were less likely to develop acute respiratory distress syndrome (ARDS) (12 [36.3%] vs. 54 [63.5%], p = 0.012) and cardiac injury (5 [15.1%] vs. 32 [37.6%], p = 0.026), and less likely to receive mechanical ventilation (22 [66.7%] vs. 72 [84.7%], p = 0.028) than those in control group. The median time from ICU admission to discharge was shorter in treatment group (32 [20-73] days vs. 76 [63-79] days, p = 0.0074). These findings suggest that SHG treatment as a complementary therapy might be effective for critically ill adults with COVID-19 and warrant further clinical trials.

Highly efficient magnetic labelling allows MRI tracking of the homing of stem cell-derived extracellular vesicles following systemic delivery.

Human stem-cell-derived extracellular vesicles (EVs) are currently being investigated for cell-free therapy in regenerative medicine applications, but the lack of noninvasive imaging methods to track EV homing and uptake in injured tissues has limited the refinement and optimization of the approach. Here, we developed a new labelling strategy to prepare magnetic EVs (magneto-EVs) allowing sensitive yet specific MRI tracking of systemically injected therapeutic EVs. This new labelling strategy relies on the use of 'sticky' magnetic particles, namely superparamagnetic iron oxide (SPIO) nanoparticles coated with polyhistidine tags, to efficiently separate magneto-EVs from unencapsulated SPIO particles. Using this method, we prepared pluripotent stem cell (iPSC)-derived magneto-EVs and subsequently used MRI to track their homing in different animal models of kidney injury and myocardial ischemia. Our results showed that iPSC-derived EVs preferentially accumulated in the injury sites and conferred substantial protection. Our study paves a new pathway for preparing highly purified magnetic EVs and tracking them using MRI towards optimized, systemically administered EV-based cell-free therapies.

Extracellular Matrix Protein Tenascin C Increases Phagocytosis Mediated by CD47 Loss of Function in Glioblastoma.

Glioblastomas (GBM) are highly infiltrated by myeloid-derived innate immune cells that contribute to the immunosuppressive nature of the brain tumor microenvironment (TME). CD47 has been shown to mediate immune evasion, as the CD47-SIRPα axis prevents phagocytosis of tumor cells by macrophages and other myeloid cells. In this study, we established CD47 homozygous deletion (CD47-/-) in human and mouse GBM cells and investigated the impact of eliminating the "don't eat me" signal on tumor growth and tumor-TME interactions. CD47 knockout (KO) did not significantly alter tumor cell proliferation in vitro but significantly increased phagocytosis of tumor cells by macrophages in cocultures. Compared with CD47 wild-type xenografts, orthotopic xenografts derived from CD47-/- tumor cells grew significantly slower with enhanced tumor cell phagocytosis and increased recruitment of M2-like tumor-associated microglia/macrophages (TAM). CD47 KO increased tumor-associated extracellular matrix protein tenascin C (TNC) in xenografts, which was further examined in vitro. CD47 loss of function upregulated TNC expression in tumor cells via a Notch pathway-mediated mechanism. Depletion of TNC in tumor cells enhanced the growth of CD47-/- xenografts in vivo and decreased the number of TAM. TNC knockdown also inhibited phagocytosis of CD47-/- tumor cells in cocultures. Furthermore, TNC stimulated release of proinflammatory factors including TNFα via a Toll-like receptor 4 and STAT3-dependent mechanism in human macrophage cells. These results reveal a vital role for TNC in immunomodulation in brain tumor biology and demonstrate the prominence of the TME extracellular matrix in affecting the antitumor function of brain innate immune cells. SIGNIFICANCE: These findings link TNC to CD47-driven phagocytosis and demonstrate that TNC affects the antitumor function of brain TAM, facilitating the development of novel innate immune system-based therapies for brain tumors.

Protective effect of tanshinone IIA against cardiac hypertrophy in spontaneously hypertensive rats through inhibiting the Cys-C/Wnt signaling pathway.

The study aimed to investigate the protective effect of tanshinone IIA against cardiac hypertrophy in spontaneously hypertensive rats (SHRs) through the Cys-C/Wnt signaling pathway. Thirty SHRs were randomly divided into cardiac hypertrophy, low- and high-dose tanshinone IIA groups. Ten Wistar-Kyoto rats were selected as control group. The systolic blood pressure (SBP), heart weight (HW), left ventricular weight (LVW) and body weight (BW) of all rats were recorded. HE staining and qRT-PCR were applied to observe the morphology of myocardial tissue and mRNA expressions of COL1A1 and COL3A1. ELISA and Western blotting were used to measure the serum asymmetric dimethylarginine (ADMA), nitric oxide (NO) and cardiac troponin I (cTnI) levels, and the expressions of the Cys-C/Wnt signaling pathway-related proteins, eNOS and Nox4. Compared with the cardiac hypertrophy group, the SBP, HW/BW, LVW/BW, swelling degree of myocardial cells, COL1A1 and COL3A1 mRNA expressions, serum cTnI and ADMA levels, and the Cys-C/Wnt signaling pathway-related proteins and Nox4 expressions in the low- and high-dose tanshinone IIA groups were decreased, but the endothelial NO synthase (eNOS), phosphorylated eNOS (Ser1177) and NO expressions were increased. No significant difference was found between the low- and high-dose tanshinone IIA groups. Our study indicated a protective effect of tanshinone IIA against cardiac hypertrophy in SHRs through inhibiting the Cys-C/Wnt signaling pathway.

Long-term outcomes of repeat hepatic resection in patients with recurrent hepatocellular carcinoma and analysis of recurrent types and their prognosis: a single-center experience in China.

BACKGROUND: Recurrent hepatocellular carcinoma (HCC) after curative resection usually originates from intrahepatic metastasis (IM) or multicentric occurrence (MO). The long-term outcomes of repeat hepatic resection in patients with different types of recurrence have not been evaluated in a large number of patients. The surgical indications for recurrent HCC remain controversial. The purpose of this study was to investigate long-term outcomes of repeat hepatic resection and clinicopathologic factors associated with different types of recurrent HCC, and to single out principle differentiating factors between IM and MO. METHODS: 82 patients who underwent repeat hepatic resection for recurrent HCC were retrospectively studied. The recurrent type was evaluated by histopathologic analysis of primary and recurrent HCC. The recurrence and survival rates as well as clinicopathologic factors associated with different types of recurrence were analyzed. RESULTS: 45 patients (54.9%) had confirmed with IM, and 37 patients (45.1%) had with MO. The recurrence rates in the MO patients after initial or repeat resection were significantly lower than those in the IM patients (p < 0.001). The overall survival rates in the MO patients after initial or repeat resection were significantly higher than those in the IM patients (p < 0.001). Recurrence-free time was identified as the most significant differentiating factor between IM and MO. A recurrence-free time of 18 months after initial resection was a significant cutoff time point for differentiating between IM and MO. A recurrence-free time of less than or equal to 18 months and microvascular invasion at repeat resection were independent adverse prognostic factors for overall survival after repeat hepatic resection. CONCLUSIONS: Repeat hepatic resection resulted in much higher survival rates in the MO patients than in the IM patients. Repeat hepatic resection could be recommended for those patients in whom the recurrent HCC occurs more than 18 months after initial resection.

Ku80 functions as a tumor suppressor in hepatocellular carcinoma by inducing S-phase arrest through a p53-dependent pathway.

Ku80 is a component of the protein complex called DNA-dependent protein kinase, which is involved in DNA double-strand break repair and multiple other functions. Previous studies revealed that Ku80 haplo-insufficient and poly (adenosine diphosphate-ribose) polymerase-null transgenic mice developed hepatocellular carcinoma (HCC) at a high frequency. The role of Ku80 has never been investigated in human HCC. Ku80 expressions in HCC and adjacent liver tissue were investigated by using immunohistochemical staining and western blot. Ku80 was transfected into a Ku80-deficient HCC cell line SMMC7721 cells, and the growth features of the Ku80-expressing cells and vector-transfected cells were studied both in vitro and in vivo. Cell cycle analysis and RNA interference were employed to investigate the mechanisms underlying the growth regulation associated with Ku80 expression. Ku80 was found frequently downregulated in HCC compared with adjacent liver tissue. Ku80 downregulation was significantly correlated with elevated hepatitis B virus-DNA load and severity of liver cirrhosis. Overexpression of Ku80 in SMMC7721 cells significantly suppressed cell proliferation in vitro and in vivo. Ku80 overexpression caused S-phase cell cycle arrest and was associated with upregulation of p53 and p21(CIP1/WAF1), and the inhibition of p53 or p21(CIP1/WAF1) expression by RNA interference overcame the growth suppression and S-phase arrest in the Ku80-expressing cells. A novel mechanism was revealed that Ku80 functions as a tumor suppressor in HCC by inducing S-phase arrest through a p53-dependent pathway.

Reduced N-cadherin expression is associated with metastatic potential and poor surgical outcomes of hepatocellular carcinoma.

BACKGROUND AND AIM: N-cadherin (N-cad), one of the classic cadherins, has been reported to be involved in tumor metastasis in some types of tumors. This study aims to investigate the expression status of N-cad in hepatocellular carcinoma (HCC) and the correlation between N-cad expression and metastatic potential, as well as the surgical outcomes of HCC. METHODS: N-cad expression in HCC and adjacent liver tissues, as well as normal liver tissues, was studied by immunohistochemistry and Western blot, and the relationship between N-cad expression and the clinicopathological features of HCC was evaluated. By using RNA interference technique, the correlation of N-cad expression and metastatic potential was investigated by downregulating N-cad expression in HCCLM3 cells, and the effects of N-cad downregulation on cell aggregation, migration, and invasion were then analyzed. Furthermore, the correlation between N-cad expression and the surgical outcomes of a cohort of HCC patients was analyzed. RESULTS: In liver tissues, N-cad was strongly expressed on cell-cell boundaries, whereas various reduced-expression patterns were observed in tumors. Of 64 HCC, 34 (53%) tumors showed reduced N-cad expression, compared with their adjacent liver tissues. The decreased expression of N-cad was significantly correlated with poorer tumor differentiation (P = 0.001) and vascular invasion (P = 0.003). N-cad knockdown in HCCLM3 cells resulted in decreased cell aggregation and increased cell migration and invasion. The decreased expression of N-cad in HCC was significantly associated with shorter postoperative disease-free survival (P = 0.039). CONCLUSIONS: N-cad expression is decreased in HCC, and the downregulation of N-cad is associated with the metastatic potential of HCC and poorer surgical prognosis.

Are surgical indications of Barcelona Clinic Liver Cancer staging classification justified?

Liver resection is the most effective treatment for hepatocellular carcinoma (HCC). The Barcelona Clinic Liver Cancer (BCLC) staging system is commonly accepted as a guideline for HCC treatment, but it only recommends liver resection for the patients with HCC at stage 0 to A1. The surgical indications of the BCLC staging system need to be re-evaluated. 120 HCC patients undergoing curative liver resection were retrospectively stratified to the BCLC staging system, and the survival of the patients at stages A, B and C was analyzed. The justification of the BCLC staging system was re-evaluated. Fifty-two patients were classified at stage A, 51 at stage B and 17 at stage C respectively. The hospital mortality of this cohort was zero and the morbidity was 24.1%. The 1-, 2-, 3-year overall survival rate of this cohort was 81.6%, 68.3%, and 57.5% respectively. There was no significant difference in the survival rate between the patients at stage A and B (P>0.05). If the treatment guidelines of BCLC staging system were followed, the majority of the patients at stages A and B (77.7%, 80/103) would not have been treated surgically. Our data suggest that the surgical indications of the BCLC staging system are not justified for HCC treatment. More studies may be needed as for how to further broaden the surgical indications of the BCLC staging system in the future.

Are Surgical Indications of Barcelona Clinic Liver Cancer Staging Classification justified? / 华中科技大学学报（医学）（英德文版）

Liver resection is the most effective treatment for hepatocellular carcinoma (HCC).The Barcelona Clinic Liver Cancer (BCLC) staging system is commonly accepted as a guideline for HCC treatment,but it only recommends liver resection for the patients with HCC at stage 0 to A1.The surgical indications of the BCLC staging system need to be re-evaluated.120 HCC patients undergoing curative liver resection were retrospectively stratified to the BCLC staging system,and the survival of the patients at stages A,B and C was analyzed.The justification of the BCLC staging system was re-evaluated.Fifty-two patients were classified at stage A,51 at stage B and 17 at stage C respectively.The hospital mortality of this cohort was zero and the morbidity was 24.1％.The 1-,2-,3-year overall survival rate of this cohort was 81.6％,68.3％,and 57.5％ respectively.There was no significant difference in the survival rate between the patients at stage A and B (P＞0.05).If the treatment guidelines of BCLC staging system were followed,the majority of the patients at stages A and B (77.7％,80/103) would not have been treated surgically.Our data suggest that the surgical indications of the BCLC staging system are not justified for HCC treatment.More studies may be needed as for how to further broaden the surgical indications of the BCLC staging system in the future.