Intratumor Injection of Thermosensitive Polypeptide with Resveratrol Inhibits Glioblastoma Growth.

Local tumor treatment is a feasible measure for patients with glioblastoma (GBM) who are unsuitable for surgical resection. Interferon-elastin-like polypeptide [IFN-ELP(V)] is a slow-release, biodegradable, thermosensitive fusion protein with antitumor immunity, and resveratrol (Res) is a polyphenolic compound with an antitumor effect. In this study, we found that intratumor injection of IFN-ELP(V) combined with intraperitoneal injection of Res is more effective in delaying GBM growth in mice. Specifically, in an orthotopic GBM model, we found a significant improvement in the median survival with this strategy. Our results suggested that the combined use of IFN-ELP(V) and Res has a dramatic synergistic effect on GBM, thus providing a novel and effective therapeutic strategy for tumors. Impact statement We report a novel and effective strategy in which the combined use of interferon-elastin-like polypeptide [IFN-ELP(V)] and Res effectively inhibits glioblastoma growth. IFN-ELP(V) can create a reservoir in the tumor and continuously release IFN to produce a powerful in situ antitumor immune response; furthermore, the combination of IFN-ELP(V) and Res is more effective in inhibiting tumor growth.

Traumatic posterior atlantoaxial dislocation without fracture of the odontoid process: illustrative case.

BACKGROUND: Traumatic posterior atlantoaxial dislocation without fracture of the odontoid process is extremely rare. Only 24 cases have been documented since the first patient was reported by Haralson and Boyd in 1969. Although various treatment strategies are reported, no consensus has been yielded. OBSERVATIONS: A 58-year-old man experienced loss of consciousness and breathing difficulties after being struck by a car from behind. An immediate computed tomography scan showed subarachnoid hemorrhage, a posterior atlantoaxial dislocation without C1-2 fracture, and a right tibiofibular fracture. After the patient's respiration and hemodynamics were stabilized, closed reduction was attempted. However, this strategy failed due to unbearable neck pain and quadriplegia, resulting in surgical intervention with transoral odontoidectomy and posterior occipitocervical fusion. The patient developed postoperative central nervous system infection. After anti-infective and drainage treatment, the infection was controlled. At 1-year follow-up, the patient did not complain of special discomfort and was generally in good condition. LESSONS: The authors report their experience with transoral odontoidectomy and concomitant posterior occipitocervical fusion in a case of posterior atlantoaxial dislocation without related fracture. Although these procedures are highly feasible and effective, particular attention should be paid to their complications, such as postoperative infection.

The genomic distribution map of human papillomavirus in Western China.

Human papillomavirus (HPV) has been confirmed as the causative agent for cervical cancer. In this study, a total of 301 880 women were recruited from four different regions of Western China, with 301 880 exfoliated cervical cell samples collected from women for DNA isolation and purification. The HPV genotype was tested by polymerase chain reaction. The overall HPV prevalence rate, high-risk (HR) HPV infection rate, low-risk (LR) HPV infection rate and mixed HPV infection rate was 18.24%, 79.14%, 12.56% and 8.30%, respectively. The four most common HR HPV subtypes were HPV-52, 16, 58 and 53, which accounted for 20.49%, 19.93%, 14.54% and 10.01%, respectively. In LR HPV genotype, HPV-6 ranked the highest (28.17%), followed by HPV-81 (9.09%) and HPV-11 (3.78%). HPV genotype subgroup analysis also showed that single-type infection was the most common (77.26%) among HPV-positive individuals. Among multi-infection genotypes, double infection was the most common with frequencies of 76.04%. The overall prevalence of HPV is high in Western China, whose distribution demonstrates different patterns across different ages and regions. Viral genotypes HPV 53, 6 were frequently detected in this population, which is worth of significant clinical attention.

Comprehensive evaluation of risk factors for lymph node metastasis in patients with papillary thyroid carcinoma.

With the increasing incidence of papillary thyroid cancer (PTC), it is important to risk-stratify patients who may have a more aggressive tumor biology. The present study aimed to evaluate the risk factors for lymph node metastasis (LNM) in patients with PTC, which may provide a significant reference for clinical diagnosis and treatment. In total, 1,045 patients with PTC [313 with PT microcarcinoma (PTMC) and 732 with non-PTMC] between August 2016 and August 2019 were investigated. The B-type Raf kinase (BRAF) V600E mutation was tested in all samples. The clinical data (sex, age, tumor location, sample type and pathological features) were retrospectively analyzed. Logistic regression analysis was performed to evaluate independent risk factors for LNM. A total of 181/313 (57.8%) PTMC cases and 145/732 (19.8%) non-PTMC cases had a BRAF V600E mutation. In the PTMC cases, significant differences in sex and sample type were identified (BRAF V600E mutation vs. wild-type). In the non-PTMC cases, significant differences in sex and age were identified (BRAF V600E mutation vs. wild-type). Female sex and tumor diameter ≤1 cm were significant independent predictors of LNM in PTC. In PTMC, female sex was a significant independent predictor of LNM. A bilateral tumor was an independent protective factor for LNM in PTC, PTMC and non-PTMC. The BRAF V600E mutation rate of ultrasound-guided fine-needle aspiration cytology was higher compared with FFPE in PTMC (P=0.018). In contrast to previous studies, the results of the present study suggested that being female and having a tumor of diameter ≤1 cm were risk factors for LNM, and that the BRAF wild-type of PTMC may be more aggressive than other types. Notably, the position of the tumor in the bilateral thyroid was also an independent protective factor for LNM. Therefore, ultrasound-guided fine-needle aspiration should be recommended for gene analysis (BRAF V600E) in PTMC. In addition, clinicians should consider an individualized treatment according to gene mutations, sex, age, tumor size and the location of the tumor, in order to achieve an improved therapeutic efficacy.

A double mutation of BRAF L597Q and V600E in situ and solitary brain metastasis of occult papillary thyroid carcinoma: A case report.

RATIONALE: The rare BRAF L597Q (c.T1790A) point mutation has been previously reported in childhood acute lymphoblastic leukemia. We present the first rare case of occult papillary thyroid carcinoma with BRAF L597Q mutation in a Tibetan patient. PATIENT CONCERNS: A 57-year-old male patient presented with a protruding mass on the left forehead for 2 years and numbness in the right limb for 3 weeks. DIAGNOSES: The patient had a double mutation of BRAF L597Q and V600E in 2 separate lesions at thyroid and brain, the immunohistochemical staining showed that the cytokeratin (CK), thyroglobulin (Tg) and thyroid transforming factor-1 (TTF-1) were immunoreactive. All the findings supported the diagnosis of solitary brain metastasis of occult papillary thyroid carcinoma. INTERVENTIONS: The patient underwent left frontal lobe metastasis (thyroid cancer) resection that involved craniectomy and artificial skull repair. OUTCOMES: During the 24-month follow-up, no postoperative complications or recurrence and metastasis were found. LESSONS: This is the first case of solitary brain metastasis of occult papillary thyroid carcinoma with double mutation of BRAF L597Q and V600E in 2 separate lesions reported in the literature. Our study extends the disease spectrum of occult papillary thyroid carcinoma and suggests that the BRAF L597Q mutation might play a specific role in inducing the solitary brain metastasis of occult papillary thyroid carcinoma in a Chinese Tibetan patient, but the detailed molecular mechanism remains to be confirmed by a large number of functional experiments and clinical research.

Knockdown of nuclear factor erythroid 2-related factor 2 by lentivirus induces differentiation of glioma stem-like cells.

Glioma stem cells (GSCs) are key in the progression and recurrence of glioblastoma. Inducing the differentiation of GSCs is an important therapeutic target for glioblastoma. Nuclear factor erythroid 2-related factor 2 (Nrf2) has been reported to be important in maintaining the stem cell status of GSCs; however, its association with differentiation has not been studied. Herein, we knocked down Nrf2 from GSCs to investigate the role of Nrf2 in the differentiation of GSCs. First, Nrf2 expression was observed at different stages of differentiation; then, Nrf2 was knocked down and the association of Nrf2 with differentiation degree was observed in vitro. Finally, GSCs were planted in nude mice to study the association of Nrf2 with differentiation in vivo. The expression of Nrf2 decreased with the differentiation process. Following Nrf2 knockdown, the proportion of sphere-like colonies decreased and the dendritic cells in spheres increased; the expression of Nrf2 significantly decreased while the expression of differentiation marker glial fibrillary acidic protein (GFAP) and ßIII-tubulin increased both at the protein and the gene level. In the xenografts of nude mice, the differentiation of tumor cells was improved. These results suggest that Nrf2 is a key factor inhibiting the differentiation of GSCs, and knockdown of Nrf2 may promote the differentiation process, providing a therapy target for GSCs.

Differential Nrf2 expression between glioma stem cells and non-stem-like cells in glioblastoma.

Glioblastoma multiforme (GBM), the most commonly occurring primary intracranial tumor, is associated with a negative outcome, regardless of the availability of multimodal therapies. However, the identification of glioma stem cells (GSCs), which are small groups of cells within the GBM, has resulted in novel avenues for research. GSCs are resistant to numerous types of environmental stress, such as irradiation, antitumor drugs and hypoxia. Nuclear factor erythroid 2-related factor 2 (Nrf2) has a significant role the cellular response to oxidative stress and previous studies have supported the significance of Nrf2 in GBM; however, the role of Nrf2 in GSCs remains unclear. In the present study, Nrf2 in CD133- GBM cells and CD133+ GSCs from GBM were compared. GSCs from GBM, which express the surface marker CD133, were separated by magnetic cell sorting and analyzed by immunofluorescence in 24-well clusters and cell counting using flow cytometry. The expression of Nrf2 was detected at the transcriptional and translational levels in CD133+ and CD133- cells, and the result indicated that GSCs were successfully isolated from the GBM. The percentage of tumor stem cells in total cells was between 0.49 and 0.91%. Nrf2 was overexpressed in CD133+ GSCs when compared with CD133- GBM cells, which indicated that the expression of Nrf2 in GSCs was closely correlated with malignant proliferation and differentiation of the GBM. Therefore, it was concluded that Nrf2 may be a potential biomarker and rational therapeutic target in GBM.

Growth inhibitory in vitro effects of glycyrrhizic acid in U251 glioblastoma cell line.

Despite dramatic advances in cancer therapy, the overall prognosis of glioblastoma (GBM) remains dismal. Nuclear factor kappa-B (NF-κB) has been previously demonstrated to be constitutively activated in glioblastoma, and it was suggested as a potential therapeutic target. Glycyrrhizic acid (GA) has been proved to have cytotoxic effects in many cancer cell lines. However, its role in glioblastoma has not yet been addressed. Therefore, this study aimed to investigate the effects of GA on human glioblastoma U251 cell line. The effects of GA on proliferation of U251 cells were measured by CCK-8 assay and plate colony-forming test. Cellular apoptosis was detected by Hoechst 33258 fluorescent staining and flow cytometry with annexin V-FITC/PI dual staining. The expression of nuclear p65 protein, the active subunit of NF-κB, was determined by Western blot and immunofluorescence. Our results demonstrated that the survival rate and colony formation of U251 cells significantly decreased in a time- and dose-dependent manner after GA addition, and the apoptotic ratio of GA-treated groups was significantly higher than that of control groups. Furthermore, the expression of NF-κB-p65 in the nucleus was remarkably reduced after GA treatment. In conclusion, our findings suggest that GA treatment can confer inhibitory effects on human glioblastoma U251 cell line including inhibiting proliferation and inducing apoptosis, which is possibly related to the NF-κB mediated pathway.

Elevated cerebral cortical CD24 levels in patients and mice with traumatic brain injury: a potential negative role in nuclear factor κb/inflammatory factor pathway.

Increasing evidence indicates that sterile inflammatory response contributes to secondary brain injury following traumatic brain injury (TBI). However, the specific mechanisms remain largely unknown, as is whether CD24, known as an important regulator in the non-infectious inflammatory response, plays a role in secondary brain injury after TBI. Here, the expression of CD24 was detected in samples from patients with TBI by quantitative real-time polymerase chain reaction (PCR), western blotting, immunohistochemistry and immunofluorescence. RNA interference was used to investigate the effects of CD24 on inflammatory response in a mouse model of TBI. Nuclear factor kappa B (NF-κB) DNA-binding activity was measured by electrophoretic mobility shift assay, and the levels of downstream pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and Interleukin 1ß (IL-1ß) were detected by real-time PCR. The results indicated that both the mRNA and protein levels of CD24 were markedly elevated after TBI in humans and mice, showing a time-dependent expression. The expression of CD24 could be observed in neurons, astrocytes and microglia in both humans and mice. Meanwhile, downregulation of CD24 significantly induced an increase of NF-κB DNA-binding activity and mRNA levels of TNF-α and IL-1ß. These findings indicated that CD24 expression could negatively regulate the NF-κB/inflammatory factor pathway after experimental TBI in mice, thus providing a novel target for therapeutic intervention of TBI.

Nuclear factor-κB/Bcl-XL pathway is involved in the protective effect of hydrogen-rich saline on the brain following experimental subarachnoid hemorrhage in rabbits.

Early brain injury (EBI), a significant contributor to poor outcome after subarachnoid hemorrhage (SAH), is intimately associated with neuronal apoptosis. Recently, the protective role of hydrogen (H2 ) in the brain has been widely studied, but the underlying mechanism remains elusive. Numerous studies have shown nuclear factor-κB (NF-κB) as a crucial survival pathway in neurons. Here we investigated the role of H2 in EBI following SAH, focusing on the NF-κB pathway. A double blood injection model was used to produce experimental SAH, and H2 -rich saline was injected intraperitoneally. NF-κB activity within the occipital cortex was measured. Immunofluorescence was performed to demonstrate the activation of NF-κB; Bcl-xL and cleaved caspase-3 were determined via Western blot. Gene expression of Bcl-xL was detected by real-time PCR, and TUNEL and Nissl staining were performed to illustrate brain injury in the occipital cortex. SAH induced a significant increase of cleaved caspase-3. Correspondingly, TUNEL staining demonstrated obvious neuronal apoptosis following SAH. In contrast, H2 treatment markedly increased NF-κB activity and the expression of Bcl-xL and decreased the level of cleaved caspase-3. Additionally, H2 treatment significantly reduced post-SAH neuronal apoptosis. The current study shows that H2 treatment alleviates EBI in the rabbits following SAH and that NF-κB/Bcl-xL pathway is involved in the protective role of H2 .

Nrf2 is required to maintain the self-renewal of glioma stem cells.

BACKGROUND: Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioma stem cells (GSCs). Self-renewal is a complex biological process necessary for maintaining the glioma stem cells. Nuclear factor rythroid 2-related factor 2(Nrf2) plays a significant role in protecting cells from endogenous and exogenous stresses. Nrf2 is a key nuclear transcription factor that regulates antioxidant response element (ARE)-containing genes. Previous studies have demonstrated the significant role of Nrf2 in the proliferation of glioblastoma, and in their resistance to radioactive therapies. We examined the effect of knocking down Nrf2 in GSCs. METHODS: Nrf2 expression was down-regulated by shRNA transinfected with lentivirus. Expression levels of Nestin, Nrf2, BMI-1, Sox2 and Cyclin E were assessed by western blotting, quantitative polymerase chain reaction (qPCR) and immunohistochemistry analysis. The capacity for self-renewal in vitro was assessed by genesis of colonies. The capacity for self-renewal in vivo was analyzed by tumor genesis of xenografts in nude mice. RESULTS: Knockdown of Nrf2 inhibited the proliferation of GSCs, and significantly reduced the expression of BMI-1, Sox2 and CyclinE. Knocking down of Nrf2 changed the cell cycle distribution of GSCs by causing an uncharacteristic increase in the proportion of cells in the G2 phase and a decrease in the proportion of cells in the S phase of the cell cycle. CONCLUSIONS: Nrf2 is required to maintain the self-renewal of GSCs, and its down-regulation can attenuate the self-renewal of GSCs significantly.

Expression of the NLRP3 inflammasome in cerebral cortex after traumatic brain injury in a rat model.

Inflammatory response plays an important role in the pathogenesis of secondary damage after traumatic brain injury (TBI). The inflammasome is a multiprotein complex involved in innate immunity and a number of studies have suggested that the inflammasome plays a critical role in a host inflammatory signaling. Nucleotide-binding domain, leucine-rich repeat, pyrin domain containing 3 (NLRP3) is a key component of the NLRP3-inflammasome, which also includes apoptotic speck-containing protein (ASC) with a cysteine protease (caspase)-activating recruitment domain and pro-caspase1. Activation of the NLRP3-inflammasome causes the processing and release of the interleukin 1 beta (IL-1ß) and interleukin 18 (IL-18). Based on this, we hypothesized that the NLRP3-inflammasome could participate in the inflammatory response following TBI. However, the expression of NLRP3-inflammasome in cerebral cortex after TBI is not well known. Rats were randomly divided into control, sham and TBI groups (including 6 h, 1 day, 3 day and 7 day sub-group). TBI model was induced, and animals were sacrificed at each time point respectively. The expression of NLRP3-inflammasome was measured by quantitative real-time polymerase chain reaction, western blot and immunohistochemistry respectively. Immunofluorescent double labeling was performed to identify the cell types of NLRP3-inflammasome's expression. Moreover, enzyme linked immunosorbent assay was used to detect the alterations of IL-1ß and IL-18 at each time point post-injury. The results showed that, TBI could induce assembly of NLRP3-inflammasome complex, increased expression of ASC, activation of caspase1, and processing of IL-1ß and IL-18. These results suggested that NLRP3-inflammasome might play an important role in the inflammation induced by TBI and could be a target for TBI therapy.

Enhanced cortical expression of myeloid differentiation primary response protein 88 (Myd88) in patients with traumatic brain injury.

BACKGROUND: Inflammatory response has been proven to play a crucial role in the pathophysilogical process after traumatic brain injury (TBI). Myeloid differentiation primary response protein 88 (Myd88) is considered as a vital factor for inflammation and immunity. Therefore, it is essential to know the detailed expression of Myd88 after TBI. However, the expression patterns of Myd88 in patients with TBI remain obscure. Hence, the aim of present study was to investigate the cortical expression of Myd88 in human contused brain. METHODS: Nineteen contused brain tissue biopsies were obtained from 19 patients undergoing surgery for brain contusions 3 h-17 d after trauma, and samples of control group were from three patients in the pathway during surgical removal of deep benign tumors. The expression of Myd88 was assessed by quantitative real-time polymerase chain reaction, Western blotting, immunohistochemistry and double immunofluorescent staining, and the messenger RNA (mRNA) levels of tumor necrosis factor-alpha (TNF-α) and interleukin 1beta (IL-1ß) were measured by quantitative real-time polymerase chain reaction. RESULTS: The progressively elevated mRNA and protein levels of Myd88 were detected after trauma, with the maximum after 72 h post-injury, and the distribution of Myd88 was found in neurons, astrocytes, and microglia. TNF-α and IL-1ß mRNA levels ascended significantly within 12 h, and then descended gradually until after 72 h post-injury. Interestingly, there was a positive relationship between the expression of Myd88 and the proinflammatory cytokine TNF-α. CONCLUSIONS: These findings indicated that Myd88 might play an important role in the inflammatory response after human TBI.

Increased expression of ferritin in cerebral cortex after human traumatic brain injury.

Despite numerous researches and improvements in the past few years, the precise mechanisms underlying secondary brain injury after trauma remain obscure. Iron is essential for almost all types of cells, including nerve cells. However, excess of iron has been proved to contribute to the brain injury following trauma in animal models. As a key iron-handling protein in the brain, ferritin might be involved in iron-induced pathophysiological process of various brain disorders. Therefore, the current study was aimed to investigate the expression of ferritin in the human contused brain. Nineteen contused brain samples were obtained from 19 patients undergoing surgery for brain contusions 3 h-17 d after trauma, and three normal temporal pole samples from 3 patients with petroclival meningioma were collected as controls. Expression of ferritin-H-chain was measured by quantitative real-time polymerase chain reaction (PCR), western blot and immunohistochemistry, respectively. Perl's reaction was taken for iron staining. The results showed that human traumatic brain injury (TBI) could up-regulate ferritin-H-chain in pericontusional cortex. A marked increase of ferritin was detected in the early group (≤12 h), and remained elevated for a long time till after 48 h post-injury. The location of ferritin-H-chain was found mainly at the neuron-like cells and seldom at glia-like cells. Perl's reaction showed that most of the iron-positive cells were glia-like cells. These findings suggested that iron and ferritin might be involved in the secondary brain injury and could be therapeutic targets for patients with TBI.