[Short term follow-up of femoral neck dynamic cross screw system and threaded cannulated screw in the treatment of vertically unstable femoral neck fractures].

OBJECTIVE: To analyze and compare the clinical effects of femoral neck dynamic cross screw system (FNS) and cannulated screws(CS) in the treatment of vertically unstable femoral neck fractures. METHODS: The clinical data and short-term follow-up results of 40 patients with vertically unstable femoral neck fractures admitted from July 2020 to August 2021 were retrospectively analyzed. According to different internal fixation methods, 40 patients were divided into two groups, 20 cases in FNS group included 11 males and 9 females with a median of 58.5(50.3, 62.5) years old, and 20 in CS group included 9 males and 11 females with a median of 52.0(40.5, 58.0) years old. The operation time, knife edge length, blood loss and treatment cost of two gruops were observed and compared. The postoperative fracture healing and internal fixation were evaluated with X-ray imaging data, and the femoral neck shortening of the affected side was measured. The incidence of thigh irritation, the time of partial weight bearing and full weight bearing, early necrosis of femoral head, reoperation revision and Harris scores were compared between two groups. RESULTS: FNS group was followed up for 18.0(15.0, 19.0) months, CS group for 17.0(15.0, 18.8) months. There was no significant difference in operation time, incision length and blood loss between two groups(P>0.05). The cost of diagnosis and treatment in FNS group was higher than that in CS group(P<0.001). In FNS group, there was no irritation sign of the affected side thigh, while in CS group, there were 6 cases with discomfort or irritation sign of the lateral thigh(P<0.05). The average time of partial weight bearing activity in CS group was later than that in FNS group(P<0.05); However, there was no significant difference in the activity time of complete weight bearing between two groups(P=0.011>0.05). At the last follow-up, the shortened length of the affected femoral neck in CS group was greater than that in FNS group(P<0.05). There was no early necrosis of femoral head and reoperation in both groups. There was no significant difference in Harris score between two groups 12 months after operation(P>0.05). CONCLUSION: FNS treatment of vertically unstable femoral neck fractures can significantly reduce the incidence of lateral thigh irritation sign, and effectively reduce the postoperative shortening rate of vertically unstable femoral neck fractures, which can provide a relatively stable anti rotation force and anti cutting force, so that patients can go to the ground relatively early, which is conducive to the recovery of the affected hip joint function after surgery. It is a new option for the surgical treatment of vertically unstable femoral neck fractures. However, due to the high cost of treatment, In clinical practice, appropriate surgical treatment is selected according to the actual situation.

Ameliorative Role of Mitochondrial Therapy in Cognitive Function of Vascular Dementia Mice.

BACKGROUND: Mitochondrial dysfunction plays a vital role in the progression of vascular dementia (VaD). We hypothesized that transfer of exogenous mitochondria might be a beneficial strategy for VaD treatment. OBJECTIVE: The study was aimed to investigate the role of mitochondrial therapy in cognitive function of VaD. METHODS: The activity and integrity of isolated mitochondria were detected using MitoTracker and Janus Green B staining assays. After VaD mice were intravenously injected with exogenous mitochondria, Morris water maze and passive avoidance tests were used to detect cognitive function of VaD mice. Haematoxylin and eosin, Nissl, TUNEL, and Golgi staining assays were utilized to measure neuronal and synaptic injury in the hippocampus of VaD mice. Detection kits were performed to detect mitochondrial membrane potential (ΔΨ), SOD activity and the levels of ATP, ROS, and MDA in the brains of VaD mice. RESULTS: The results showed that isolated mitochondria were intact and active. Mitochondrial therapy could ameliorate cognitive performance of VaD mice. Additionally, mitochondrial administration could attenuate hippocampal neuronal and synaptic injury, improve mitochondrial ΔΨ, ATP level and SOD activity, and reduce ROS and MDA levels in the brains of VaD mice. CONCLUSIONS: The study reports profitable effect of mitochondrial therapy against cognitive impairment of VaD, making mitochondrial treatment become a promising therapeutic strategy for VaD.

Gliclazide Ameliorates Neuronal Injury by Attenuating Oxidative Stress in D-gal-Induced Senescent Cells and Aging Mice.

Enhancement of oxidative stress and resultant neuronal injury play important roles in initiating cognitive impairment during the aging process. Thus, attenuating oxidative injury is regarded as a profitable therapeutic strategy for age-associated cognitive impairment. Previous studies showed that gliclazide (Gli) had a protective role in neuronal injury from cerebral ischemia/reperfusion (I/R) injury. However, whether Gli has a profitable effect on age-associated cognitive impairment remains largely unclear. The present study showed that Gli held the potential to attenuate neuronal apoptosis in D-gal-induced senescent cells and aging mice. Additionally, Gli could alleviate synaptic injury and cognitive function in D-gal-induced aging mice. Further study showed that Gli could attenuate oxidative stress in D-gal-induced senescent cells and aging mice. The p38 MAPK pathway was predicted as the downstream target of Gli retarding oxidative stress using in silico analysis. Further studies revealed that Gli attenuated D-gal-induced phosphorylation of p38 and facilitated Nrf2 nuclear expression, indicating that the anti-oxidative property of Gli may be associated with the p38 MAPK pathway. The study demonstrates that Gli has a beneficial effect on ameliorating D-gal-induced neuronal injury and cognitive impairment, making this compound a promising agent for the prevention and treatment of age-associated cognitive impairment.

Immunosuppressive effect of small extracellular vesicle PD-L1 is restricted by co-expression of CD80.

BACKGROUND: The PD-L1 on tumor cell-derived small extracellular vesicles (sEVs) can suppress the proliferation and cytokine production of T cells. However, PD-L1 can also be expressed by non-tumor cells. The present study is designed to test whether immunocytes release immunosuppressive PD-L1-positive sEVs. METHODS: sEVs were isolated from different clinical samples of head and neck squamous cell carcinoma (HNSCC) patients, the level and cellular origins of PD-L1-positive sEVs were assessed. Co-expression of CD80 on PD-L1-positive sEVs was examined to evaluate the immunosuppressive and tumor-promotive effects. RESULTS: PD-L1-positive sEVs in HNSCC patients had various cellular origins, including tumor cell, T cell, B cell, dendritic cell and monocyte/macrophage. However, PD-L1-positive sEVs derived from immune cells did not exert immunosuppressive functions due to the co-expression of CD80. It was verified that co-expression of CD80 disrupted the binding of sEV PD-L1 to its receptor PD-1 on T cells and attenuated the immunosuppression mediated by sEV PD-L1 both in vitro and in vivo. CONCLUSION: The study suggests that PD-L1-positive sEVs have the cellular origin and functional heterogeneity. Co-expression of CD80 could restrict the immunosuppressive effect of sEV PD-L1. A greater understanding of PD-L1-positive sEV subsets is required to further improve their clinical application.

Small extracellular vesicle PD-L1 in cancer: the knowns and unknowns.

According to the conventional wisdom, programmed death protein 1 ligand (PD-L1)-mediated immunosuppression was based on the physical contact between tumor cells and T cells in the tumor microenvironment. Recent studies demonstrated that PD-L1 was also highly expressed on the surface of tumor cell-derived small extracellular vesicles (sEVs). PD-L1 on sEVs, which could also directly bind to PD-1 on T cells, has a vital function in immunosuppression and immunotherapy resistance. Due to the heterogeneity and dynamic changes of PD-L1 expression on tumor cells, developing sEV PD-L1 as a predictive biomarker for the clinical responses to immunotherapy could be an attractive option. In this review, we summarized and discussed the latest researches and advancements on sEV PD-L1, including the biogenesis and secretion mechanisms, isolation and detection strategies, as well as the biological functions of sEV PD-L1. In the meantime, we highlighted the application potential of sEV PD-L1 as diagnostic and prognostic markers in tumor, especially for predicting the clinical responses to anti-PD-1/PD-L1 immunotherapies. In particular, with the gradual deepening of the studies, challenges and problems regarding the further understanding and application of sEV PD-L1 have begun to emerge. Based on the current research status, we summarized the potential challenges and possible solutions, and prospected several key directions for future studies of sEV PD-L1. Collectively, by highlighting the important knowns and unknowns of sEV PD-L1, our present review would help to light the way forward for the field of sEV PD-L1 and to avoid unnecessary blindness and detours.

A High-Quality Haplotype-Resolved Genome of Common Bermudagrass (Cynodon dactylon L.) Provides Insights Into Polyploid Genome Stability and Prostrate Growth.

Common bermudagrass (Cynodon dactylon L.) is an important perennial warm-season turfgrass species with great economic value. However, the reference genome is still deficient in C. dactylon, which severely impedes basic studies and breeding studies. In this study, a high-quality haplotype-resolved genome of C. dactylon cultivar Yangjiang was successfully assembled using a combination of multiple sequencing strategies. The assembled genome is approximately 1.01 Gb in size and is comprised of 36 pseudo chromosomes belonging to four haplotypes. In total, 76,879 protein-coding genes and 529,092 repeat sequences were annotated in the assembled genome. Evolution analysis indicated that C. dactylon underwent two rounds of whole-genome duplication events, whereas syntenic and transcriptome analysis revealed that global subgenome dominance was absent among the four haplotypes. Genome-wide gene family analyses further indicated that homologous recombination-regulating genes and tiller-angle-regulating genes all showed an adaptive evolution in C. dactylon, providing insights into genome-scale regulation of polyploid genome stability and prostrate growth. These results not only facilitate a better understanding of the complex genome composition and unique plant architectural characteristics of common bermudagrass, but also offer a valuable resource for comparative genome analyses of turfgrasses and other plant species.

Geraniin as a potential inhibitor of SARS-CoV-2 3CLpro.

Geraniin is a polyphenolic compound first isolated from Geranium thunbergii. The major protease (Mpro), namely 3 C-like protease (3CLpro), of coronaviruses is considered an attractive drug target as it is essential for the processing and maturation of viral polyproteins. Thus, our primary goal is to explore the efficiency of geraniin on 3CLpro of SARS-CoV-2 using the computational biology strategy. In this work, we studied the anti-coronavirus effect of geraniin in vitro and its potential inhibitory mode against the 3CLpro of SARS-CoV-2. We found that geraniin inhibited HCoV-OC43 coronavirus-infected cells during the attachment and penetration phases. Molecular docking and dynamics simulations exhibited that geraniin had a strong binding affinity and high stable binding to 3CLpro of SARS-CoV-2. Geraniin showed a strong inhibitory activity on coronavirus and may be a potential inhibitor of SARS-CoV-2 3CLpro.

MeCP2 prevents age-associated cognitive decline via restoring synaptic plasticity in a senescence-accelerated mouse model.

Age-related cognitive decline in neurodegenerative diseases, such as Alzheimer's disease (AD), is associated with the deficits of synaptic plasticity. Therefore, exploring promising targets to enhance synaptic plasticity in neurodegenerative disorders is crucial. It has been demonstrated that methyl-CpG binding protein 2 (MeCP2) plays a vital role in neuronal development and MeCP2 malfunction causes various neurodevelopmental disorders. However, the role of MeCP2 in neurodegenerative diseases has been less reported. In the study, we found that MeCP2 expression in the hippocampus was reduced in the hippocampus of senescence-accelerated mice P8 (SAMP8) mice. Overexpression of hippocampal MeCP2 could elevate synaptic plasticity and cognitive function in SAMP8 mice, while knockdown of MeCP2 impaired synaptic plasticity and cognitive function in senescence accelerated-resistant 1 (SAMR1) mice. MeCP2-mediated regulation of synaptic plasticity may be associated with CREB1 pathway. These results suggest that MeCP2 plays a vital role in age-related cognitive decline by regulating synaptic plasticity and indicate that MeCP2 may be promising targets for the treatment of age-related cognitive decline in neurodegenerative diseases.

Puerarin reduces impairment of intestinal and adipose immune responses to influenza virus infection in mice.

Influenza is an acute viral respiratory disease that can also cause gastroenteritis-like symptoms, such as abdominal pain, nausea, vomiting, and diarrhea. Immune dysfunction of adipose tissue is involved in the occurrence and prognosis of influenza viral pneumonia. In this study, we analyzed intestinal and adipose immune responses in mice infected with influenza virus and found that the impairment of intestinal and adipose immunity to influenza virus infection could be reversed by treatment with puerarin, a medicinal compound isolated from Pueraria lobata (called "gegen" in Chinese). We found that the lungs, small intestines (duodenum, ileum, jejunum) and large intestines (colon and rectum) of infected mice showed obvious inflammatory lesions, with significantly increased levels of virus, inflammatory cytokines (interleukin [IL]-6, IL-17, and tumor necrosis factor-α), Toll-like receptors 3, 4, and 9, and integrin αvß3 and α4, and a decreased level of secreted IgA compared to the normal control group (NC) (P < 0.05-0.001). Influenza virus infected mesenteric lymph nodes and adipose tissue, and adipokines (leptin, visfatin, "chemerin", and adiponectin) of lung and mesenteric adipose tissue were dysregulated. Puerarin treatment reversed the impairment of the intestinal and adipose immune responses in mice infected with influenza virus. Our findings suggest that influenza virus can infect adipose tissue and lead to intestinal adipose immune dysfunction in normal-weight mice and that the impairment of the intestinal and adipose immune response to influenza virus infection can be reversed by puerarin treatment.

A potential antiviral activity of Esculentoside A against binding interactions of SARS-COV-2 spike protein and angiotensin converting enzyme 2 (ACE2).

The recent emergence of the novel coronavirus (SARS-CoV-2) has resulted in a devastating pandemic with global concern. However, to date, there are no regimens to prevent and treat SARS-CoV-2 virus. There is an urgent need to identify novel leads with anti-viral properties that impede viral pathogenesis in the host system. Esculentoside A (EsA), a saponin isolated from the root of Phytolacca esculenta, is known to exhibit diverse pharmacological properties, especially anti-inflammatory activity. To our knowledge, SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) to enter host cells. This is mediated through the proteins of SARS-CoV-2, especially the spike glycoprotein receptor binding domain. Thus, our primary goal is to prevent virus replication and binding to the host, which allows us to explore the efficiency of EsA on key surface drug target proteins using the computational biology paradigm approach. Here, the anti-coronavirus activity of EsA in vitro and its potential mode of inhibitory action on the S-protein of SARS-CoV-2 were investigated. We found that EsA inhibited the HCoV-OC43 coronavirus during the attachment and penetration stage. Molecular docking results showed that EsA had a strong binding affinity with the spike glycoprotein from SARS-CoV-2. The results of the molecular dynamics simulation revealed that EsA had higher stable binding with the spike protein. These results demonstrated that Esculentoside A can act as a spike protein blocker to inhibit SARS-CoV-2. Considering the poor bioavailability and low toxicity of EsA, it is suitable as novel lead for the inhibitor against binding interactions of SARS-CoV-2 of S-protein and ACE2.

Overexpression of T-type calcium channel Cav3.1 in oral squamous cell carcinoma: association with proliferation and anti-apoptotic activity.

Cav3.1, a subfamily of T-type calcium channel, is overexpressed in various human cancers and exerts important functions in tumor progression. This study is to identify the expression pattern and clinical significance of Cav3.1 in oral squamous cell carcinoma (OSCC). Firstly, the expression levels of Cav3.1 in oral mucosa (OM), dysplasia and oral squamous cell carcinoma (OSCC) were determined and compared by real-time quantitative PCR and Western blot analysis. After that, human tissue microarrays, containing 29 OM, 23 dysplasia and 122 primary OSCC samples, were applied to investigate the expression levels of Cav3.1, proliferation markers [Ki-67, proliferating cell nuclear antigen (PCNA)] and cellular anti-apoptosis markers [B cell lymphoma 2 (Bcl-2)] by immunohistochemistry and digital pathology analysis. In addition, we determined the function of Cav3.1 using knockdown assays of Cav3.1 in vitro. The results demonstrated that the mRNA and protein expression of Cav3.1 were significantly higher in OSCC specimens, and Cav3.1 expression in primary OSCCs was correlated with tumor size and pathological grade. Statistical analysis of immunohistochemical staining showed that Cav3.1 was closely correlated with Ki-67, PCNA and Bcl-2. Functional studies showed that the knockdown of Cav3.1 in OSCC cell lines using RNA interference influenced cell proliferation and apoptosis in vitro. Taken together, these findings suggested that Cav3.1 is overexpressed in OSCC tissues, also associated with proliferative and anti-apoptotic activity in oral squamous cell carcinoma.

Development and validation of nomograms to intraoperatively predict metastatic patterns in regional lymph nodes in patients diagnosed with esophageal cancer.

BACKGROUND: An accurate intraoperative prediction of lymph node metastatic risk can help surgeons in choosing precise surgical procedures. We aimed to develop and validate nomograms to intraoperatively predict patterns of regional lymph node (LN) metastasis in patients with esophageal cancer. METHODS: The prediction model was developed in a training cohort consisting of 487 patients diagnosed with esophageal cancer who underwent esophagectomy with complete LN dissection from January 2016 to December 2016. Univariate and multivariable logistic regression were used to identify independent risk factors that were incorporated into a prediction model and used to construct a nomogram. Contrast-enhanced computed tomography reported LN status and was an important comparative factor of clinical usefulness in a validation cohort. Nomogram performance was assessed in terms of calibration, discrimination, and clinical usefulness. An independent validation cohort comprised 206 consecutive patients from January 2017 to December 2017. RESULTS: Univariate analysis and multivariable logistic regression revealed three independent predictors of metastatic regional LNs, three independent predictors of continuous regional LNs, and two independent predictors of skipping regional LNs. Independent predictors were used to build three individualized prediction nomograms. The models showed good calibration and discrimination, with area under the curve (AUC) values of 0.737, 0.738, and 0.707. Application of the nomogram in the validation cohort yielded good calibration and discrimination, with AUC values of 0.728, 0.668, and 0.657. Decision curve analysis demonstrated that the three nomograms were clinically useful in the validation cohort. CONCLUSION: This study presents three nomograms that incorporate clinicopathologic factors, which can be used to facilitate the intraoperative prediction of metastatic regional LN patterns in patients with esophageal cancer.

Antiviral activity of puerarin as potent inhibitor of influenza virus neuraminidase.

Puerarin is a major isofiavone compound isolated from the root of Pueraria lobata. It was reported that puerarin had antioxidant, antiinflammatory, antitumor, cholesterol lowering, liver protective, and neuroprotective properties. However, few studies have explored the antiviral effect of puerarin and its target mechanism related to influenza virus. Here, the antiinfluenza activity of puerarin in vitro and in vivo and its mode of action on the potential inhibition of neuraminidase (NA) were investigated. Puerarin displayed an inhibitory effect on A/FM/1/1947(H1N1) (EC50 = 52.06 µM). An indirect immunofluorescence assay indicated that puerarin blocked the nuclear export of viral NP. The inhibition of NA activity confirmed that puerarin can block the release of newly formed virus particles from infected cells. Puerarin (100 and 200 mg/kg/d) exhibited effective antiviral activity in mice, conferring 50% and 70% protection from death against H1N1, reducing virus titers, and effectively alleviating inflammation in the lungs. The molecular docking results showed that puerarin had a strong binding affinity with NA from H1N1. The results of the molecular dynamics simulation revealed that puerarin had higher stable binding at the 150-loop region of the NA protein. These results demonstrated that puerarin acts as a NA blocker to inhibit influenza A virus both in cellular and animal models. Thus, puerarin has potential utility for the treatment of the influenza virus infection.

Mapping coastal circulations using moving vehicle acoustic tomography.

With the increased availability of highly maneuverable unmanned surface/underwater vehicles, abundant ocean data can now be collected. This study uses tomographic techniques to extend the survey area covered by moving vehicles. An acoustic reciprocal transmission experiment was conducted using three tomographic sensors installed on an autonomous underwater vehicle, a fishing ship, and a buoy. The distributed sensing method is applied for currents estimation. The estimated currents near the ship show consistent results with the direct measurements. In particular, an anticyclonic circulation was revealed. Further, a general least-squares method is employed to improve the estimate of this vortical structure.

Probucol, a "non-statin" cholesterol-lowering drug, ameliorates D-galactose induced cognitive deficits by alleviating oxidative stress via Keap1/Nrf2 signaling pathway in mice.

Oxidative stress plays a vital role in the initiation and progression of age-related neurodegenerative diseases. Ameliorating oxidative damage is therefore considered as a beneficial strategy for the treatment of age-related neurodegenerative disorders. Probucol (Prob), a lipid-lowering prototype agent, was reported to treat cardiovascular diseases, chronic kidney disease and diabetes mellitus. However, whether Prob has an effect on age-related neurodegenerative diseases remains unknown. In the study, it was found that Prob ameliorated D-galactose (D-gal) induced cognitive deficits and neuronal loss in the hippocampal CA1 region. Moreover, Prob alleviated ROS and MDA levels by elevating SOD, GSH-PX and HO-1 mRNA and protein expressions, and improving plasmic and cerebral SOD and GSH-PX activities in D-gal treated mice. Furthermore, Prob promoted the dissociation of Keap1/Nrf2 complex leading to the accumulation of Nrf2 in nucleus, implying that the improved anti-oxidant property of Prob is mediated by Keap1/Nrf2 pathway. The study firstly demonstrates the favorable effects of Prob against cognitive impairments in a senescent mouse model, rendering this compound a promising agent for the treatment or prevention of age-related neurodegenerative disease.

Clinical significance of skip lymph-node metastasis in pN1 gastric-cancer patients after curative surgery.

BACKGROUND: In addition to the stepwise manner of lymph-node metastasis from the primary tumour, the skip lymph-node metastasis (SLNM) was identified as a low-incidence metastasis of gastric cancer (GC). So far, both the mechanism and outcome of SLNM have not been elucidated completely. The purpose of this study was to analyse the clinical significance and the potential mechanism of SLNM in GC patients who had lymph-node metastasis. METHODS: Clinicopathological data and follow-up information of 505 GC patients who had lymph-node metastasis were analysed to demonstrate the significance of SLNM in evaluating the prognostic outcome. According to the pathological results, all GC patients who had lymph-node metastasis were categorized into three groups: patients with the perigastric lymph-node metastasis, patients with the perigastric and extragastric lymph-node metastasis and patients with SLNM.Results: Among the 505 GC patients who had lymph-node metastasis, 24 (4.8%) had pathologically identified SLNM. The location of lymph-node metastasis was not significantly associated with 5-year survival rate and overall survival (OS) (P = 0.194). The stratified survival analysis results showed that the status of SLNM was significantly associated with the OS in patients with pN1 GC (P = 0.001). The median OS was significantly shorter in 19 pN1 GC patients with SLNM than in 100 patients with perigastric lymph-node metastasis (P < 0.001). The case-control matched logistic regression analysis results showed that tumour size (P = 0.002) was the only clinicopathological factor that may predict SLNM in pN1 GC patients undergoing curative surgery. Among the 19 pN1 GC patients with SLNM, 17 (89.5%) had metastatic lymph nodes along the common hepatic artery, around the celiac artery or in the hepatoduodenal ligament. CONCLUSIONS: SLNM may be considered a potentially practicable indicator for prognosis among various subgroups of pN1 GC patients.

Application of fracture-sustaining reduction frame in closed reduction of femoral shaft fracture.

OBJECTIVES: This study aims to explore the clinical efficacy of applying a new reduction brace in the closed reduction of femoral shaft fracture. METHODS: A total of 18 patients with femoral shaft fracture, who were admitted to the Bone Trauma Surgery, Second Hospital of Shanxi Medical University, from September 2015 to January 2017, were chosen. A novel reduction brace combined with closed reduction intramedullary nail insertion on the traction table adopted for the orthopedic surgery was taken for the fixation. Then, surgical time, bleeding amount, and postoperational fracture healing time were recorded. RESULTS: All 18 patients with femoral shaft fracture successfully received closed reduction femoral nail with the application of the novel reduction brace. The follow-up period was 3-18 months, with an average of 12 months, and the femoral shaft fracture was well healed with good recovery of function. CONCLUSIONS: The design of the closed reduction brace of the femoral shaft fracture was reasonable, simple, and convenient to use and has a short learning curve. Furthermore, it led to little trauma to these patients and fully played the advantages of minimally invasive therapy for femoral fractures.

Lymphocyte­derived microparticles stimulate osteoclastogenesis by inducing RANKL in fibroblasts of odontogenic keratocysts.

Leukocyte­derived microparticles (LMPs) include neutrophil­, lymphocyte­ and monocyte­derived MPs. LMPs act as proinflammatory mediators in autoimmune diseases, infectious diseases and vascular diseases. The present study examined the hypothesis that the percentage of LMPs was increased in patients with inflamed odontogenic keratocysts (OKCs), and investigated the biological effects of Jurkat cell­derived MPs on the fibroblasts of OKCs in vitro. Cyst fluid MPs, obtained by centrifugation of samples from 20 patients with inflamed OKCs, 3 patients with uninflamed OKCs, 15 patients with radicular cysts (RCs) and 12 patients with inflamed dentigerous cysts (DCs), were analyzed by transmission electron microscopy, dynamic light scattering and immunofluorescence staining. The percentages and concentrations of cyst fluid LMPs were further determined by flow cytometry. The cytokine levels of apoptotic Jurkat cell­derived MPs and Jurkat cell supernatants were compared by cytokine antibody arrays. Fibroblasts were isolated from 3 patients with OKC and co­cultured with apoptotic Jurkat cell­derived MPs with or without interleukin (IL)­15Rα to detect the levels of matrix metallopeptidase 9 (MMP­9) and receptor activator of nuclear factor­κB ligand (RANKL) by reverse transcription­quantitative polymerase chain reaction and enzyme­linked immunosorbent assay. The supernatant from Jurkat MPs­treated fibroblasts was collected to make conditioned medium in which the osteoclastogenesis of Raw264.7 cells was determined. Antibodies against human soluble (s)RANKL were added to the conditioned medium to investigate the inhibitory effects. Mean percentages of lymphocyte­ and neutrophil­derived MPs were significantly higher in inflamed OKCs than in DCs. Significant elevations in IL­15 were detected in apoptotic Jurkat cell­derived MPs compared with that in Jurkat cell supernatant. Furthermore, higher levels of MMP­9 and RANKL were detected in Jurkat cell MP­treated OKC fibroblasts, and this was partially blocked by IL­15Rα. Increased osteoclast­like cell formation was observed in the Jurkat MPs­treated fibroblast supernatant and Raw264.7 co­culture groups. The anti­human sRANKL antibody in the Jurkat MPs­treated fibroblast supernatant group decreased the osteoclastogenesis of the Raw264.7 cells. These results indicate that LMPs serve as novel communication tools that contribute toward the bone resorption of inflamed OKCs by inducing RANKL of OKC fibroblasts via IL­15.

Overexpression of Fra-1, c-Jun and c-Fos in odontogenic keratocysts: potential correlation with proliferative and anti-apoptotic activity.

AIMS: The purpose of this study was to explore the potential involvement of Fra-1, c-Jun and c-Fos, three vital members of the AP-1 complex, in the pathogenesis of odontogenic keratocysts (OKCs). METHODS AND RESULTS: Tissue samples, containing 10 normal oral mucosa (OM), 10 dentigerous cysts (DC) and 32 OKC specimens, were applied to investigate the expression levels of Fra-1, c-Jun and c-Fos by immunohistochemistry and real-time-quantitative polymerase chain reaction (RT-qPCR). The association between Fra-1, c-Jun and c-Fos expression levels and markers of proliferation [Ki-67, proliferating cell nuclear antigen (PCNA)], anti-apoptosis (Bcl-2) was then investigated in the OKC serial tissue sections. The results showed that Fra-1, c-Jun and c-Fos expression levels were increased significantly in OKCs compared to these in OM and DC tissue samples. Meanwhile, the expression levels of Fra-1, c-Jun and c-Fos were associated positively with the expression levels of Ki-67, PCNA and Bcl-2, as confirmed further by double-labelling immunofluorescence analysis and hierarchical analysis. CONCLUSIONS: This study revealed for the first time that Fra-1, c-Jun and c-Fos were overexpressed in OKCs and had a close correlation with proliferation and anti-apoptosis potential of OKCs.

Increased level of cell-derived microparticles in the cyst fluids of odontogenic keratocysts.

The aim of this study was to examine the level and basic characteristics of cell­derived microparticles (MPs) in the cyst fluids of odontogenic keratocysts (OKCs). For this purpose, MPs from the cyst fluids (CFMPs) of OKCs were purified by a classic differential centrifugation method and characterized by a transmission electron microscope and fluorescence microscope. Flow cytometric analysis was used to determine the size, concentration and cellular origins of the CFMPs. Moreover, the expression level of receptor activator for nuclear factor­κB ligand in the OKCs was evaluated by immunohistochemical staining and then analyzed for its correlation with the concentration of CFMPs by Spearman's rank correlation test. In addition, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and tartaric­resistant acid phosphatase (TRAP) staining were performed to examine the osteoclastogenesis of mouse bone marrow­derived macrophages (BMMs) in response to CFMPs. The results revealed that the levels of total CFMPs were significantly elevated in OKCs compared with dentigerous cysts (DCs) and radicular cysts (RCs). In addition, in vitro experiments further revealed that CFMPs derived from the OKCs of patients could be taken up by BMMs, leading to a significant increase in the mRNA expression levels of nuclear factor of activated T­cells 1 (NFATc1) and TRAP. Moreover, TRAP­positive multinucleated osteoclasts were successfully cultured in the presence of macrophage colony­stimulating factor (M­CSF) and CFMPs with BMMs. On the whole, our findings indicate that patients with OKCs have higher levels of CFMPs compared with patients with DCs and RCs, which may be associated with the bone resorption of OKCs.