Tumor-associated macrophages promoting PD-L1 expression in infiltrating B cells through the CXCL12/CXCR4 axis in human hepatocellular carcinoma.

The inflammation-related tumor microenvironment (TME) is one of the major driving forces of hepatocarcinogenesis. We aimed to investigate cell-to-cell communication among Hepatocellular Carcinoma (HCC) through re-analyzing HCC single-cell RNA-seq data, and to confirm such cellular interaction through in vitro and in vivo study. We found a subset of Regulatory B cells with PD-L1 expression (PD-L1+ Bregs), mainly located in adjacent HCC tissues. In co-localization with PD-L1+ Bregs, a subset of Tumor Associated Macrophages with high expression of CXCL12 (CXCL12+ TAMs) was also mainly located in adjacent HCC tissues. Moreover, CXCL12+ TAMs can be stimulated in vitro using an HCC conditional medium. Using CellChat analysis and Multiplex Immunohistochemistry staining (mIHC), CXCL12+ TAMs were found to be first recruited by Cancer-Associated Fibroblasts (CAFs) through a CD74/macrophage migration inhibitory factor (MIF) pattern, and further differentiated into TGF-ß-enriched tissues. Furthermore, CXCL12+ TAMs recruited PD-L1+ Bregs via the CXCL12/CXCR4 axis, and CXCR4 expression was significantly positively correlated to PD-L1 expression in PD-L1+ Bregs. At last, we confirmed the communications among CAFs, Macrophages and B cells and their tumor-promoting effects by using an orthotopic mouse model of HCC. Immunosuppressive HCC TME involving cell-to-cell communications comprised MIF-secreting CAFs, CXCL12-secreting TAMs, and PD-L1-producing Bregs, and their regulation could be promising therapeutic targets in future immunotherapy for human HCC.

Prolyl hydroxylase 3 controls the intestine goblet cell generation through stabilizing ATOH1.

Intestinal epithelia self-renew constantly and generate differentiated cells such as secretary goblet cells. The intestine goblet cells secrete gel-forming mucins that form mucus to create a barrier of defense. We reported previously that loss of prolyl hydroxylase (PHD) 3 led to disruption of the intestinal epithelial barrier function. However, the underlying mechanism remains elusive. Here, we demonstrate that PHD3 controls the generation of intestine goblet cell. We found that genetic ablation of Phd3 in mice intestine epithelial cells reduced the amount of goblet cells. Mechanistically, PHD3 bounds the E3 ubiquitin ligase HUWE1 and prevented HUWE1 from mediating ubiquitination and degradation of ATOH1, an essential driver for goblet cell differentiation. The prolyl hydroxylase activity-deficient variant PHD3(H196A) also prevented ATOH1 destruction. A genetic intestine epithelial PHD3(H196A)-knockin had no effect on ATOH1 expression or goblet cell amount in mice, suggesting that the PHD3 prolyl hydroxylase activity is dispensable for its ability to control ATOH1 expression and goblet cell generation. In dextran sulfate sodium (DSS)-induced experimental colitis, PHD3-knockout rather than PHD3(H196A)-knockin sensitized the mice to DSS treatment. Our results reveal an additional critical mechanism underlying the regulation of ATOH1 expression and goblet cell generation and highlight that PHD3 plays a role in controlling intestine goblet cell generation in a hydroxylase-independent manner.

miR-519a enhances chemosensitivity and promotes autophagy in glioblastoma by targeting STAT3/Bcl2 signaling pathway.

BACKGROUND: Chemoresistance to temozolomide (TMZ) is a major challenge in the treatment of glioblastoma (GBM). We previously found that miR-519a functions as a tumor suppressor in glioma by targeting the signal transducer and activator of transcription 3 (STAT3)-mediated autophagy oncogenic pathway. Here, we investigated the effects of miR-519a on TMZ chemosensitivity and autophagy in GBM cells. Furthermore, the underlying molecular mechanisms and signaling pathways were explored. METHODS: In the present study, two stable TMZ-resistant GBM cell lines were successfully generated by exposure of parental cells to a gradually increasing TMZ concentration. After transfecting U87-MG/TMZ and U87-MG cells with miR-519a mimic or inhibitor, a series of biochemical assays such as MTT, apoptosis, and colony formation were performed to determine the chemosensitive response to TMZ. The autophagy levels in GBM cells were detected by transmission electron microscopy, LC3B protein immunofluorescence, and Western blotting analysis. Stable knockdown and overexpression of miR-519a in GBM cells were established using lentivirus. A xenograft nude mouse model and in situ brain model were used to examine the in vivo effects of miR-519a. Tumor tissue samples were collected from 48 patients with GBM and were used to assess the relationship between miR-519a and STAT3 expression. RESULTS: TMZ treatment significantly upregulated miR-519a in U87-MG cells but not in U87-MG/TMZ cells. Moreover, the expression of miR-519a and baseline autophagy levels was lower in U87-MG/TMZ cells as compared to U87-MG cells. miR-519a dramatically enhanced TMZ-induced autophagy and apoptotic cell death in U87-MG/TMZ cells, while inhibition of miR-519a promoted TMZ resistance and reduced TMZ-induced autophagy in U87-MG cells. Furthermore, miR-519a induced autophagy through modification of STAT3 expression. The in vivo results showed that miR-519a can enhance apoptosis and sensitized GBM to TMZ treatment by promoting autophagy and targeting the STAT3/Bcl-2/Beclin-1 pathway. In human GBM tissues, we found an inverse correlation between miR-519a and STAT3 expression. CONCLUSIONS: Our results suggested that miR-519a increased the sensitivity of GBM cells to TMZ therapy. The positive effects of miR-519a may be mediated through autophagy. In addition, miR-519a overexpression can induce autophagy by inhibiting STAT3/Bcl-2 pathway. Therefore, a combination of miR-519a and TMZ may represent an effective therapeutic strategy in GBM.

Contemporary Situation of Community-acquired Pneumonia in China: A Systematic Review.

The prevalence and microbial pattern reported for Community-acquired pneumonia (CAP) differ considerably and contemporary situation remains changing over time. We therefore searched both international and domestic databases for relevant references and pooled incidence of CAP and etiological distribution were estimated separately between children and adults groups. The results showed that CAP remained a major public health issue in China, with a relatively higher incidence than that reported in Western countries. Although pathogens were not detected in nearly half of patients, Mycoplasma pneumoniae remained to be the most frequently detected agent across age groups, the detection yield of which was lower than that reported from other countries. Notably, the incidence of influenza virus A in adults was almost four times higher than that in children while the prevalence of respiratory syncytial virus was much less common in adults than that in children. Despite some limitations, the value of this review, approaching to systematically review grey published data, is to sketch out the contemporary epidemiological and etiological situation of CAP in our country, which could be useful to help policymakers and clinicians make informed choices and to inspire future studies and surveillance.

[MACF1 knockdown in glioblastoma multiforme cells increases temozolomide-induced cytotoxicity].

OBJECTIVE: To investigate the role of microtubule-actin crosslinking factor 1 (MACF1) in the response of glioma cells to temozolomide (TMZ). METHODS: TMZ was applied to a human gliomablastoma cell line (U87) and changes in the protein expression and cellular localization were determined with Western blot, RT-PCR, and immunofluorescence. The responses of the cells with MACF1 expression knockdown by RNA interference to TMZ were assessed. TMZ-induced effects on MACF1 expression were also assessed by immunohistochemistry in a nude mouse model bearing human glioblastoma xenografts. RESULTS: TMZ resulted in significantly increased MACF1 expression (by about 2 folds) and changes in its localization in the gliomablastoma cells both in vitro and in vivo (P<0.01). Knockdown of MACF1 reduced the proliferation (by 45%) of human glioma cell lines treated with TMZ (P<0.01). TMZ-induced changes in MACF1 expression was accompanied by cytoskeletal rearrangement. CONCLUSION: MACF1 may be a potential therapeutic target for glioblastoma.

Origin of craniopharyngiomas: implications for growth pattern, clinical characteristics, and outcomes of tumor recurrence.

OBJECT Craniopharyngiomas are associated with a high rate of recurrence. The surgical management of recurrent lesions has been among the most challenging neurosurgical procedures because of the craniopharyngioma's complex topographical relationship with surrounding structures. The aim of this study was to define the determinative role of the site of origin on the growth pattern and clinical features of recurrent craniopharyngiomas. METHODS The authors performed a retrospective analysis of 52 patients who had undergone uniform treatment by a single surgeon. For each patient, data concerning symptoms and signs, imaging features, hypothalamic-pituitary function, and recurrence-free survival rate were collected. RESULTS For children, delayed puberty was more frequent in the group with Type I (infradiaphragmatic) craniopharyngioma than in the group with Type TS (tuberoinfundibular and suprasellar extraventricular) lesions (p < 0.05). For adults, blindness was more frequent in the Type I group than in the Type TS group (p < 0.05). Nausea or vomiting, delayed puberty, and growth retardation were more frequent in children than in adults (p < 0.05). Overall clinical outcome was good in 48.07% of the patients and poor in 51.92%. Patients with Type TS recurrent tumors had significantly worse functional outcomes and hypothalamic function than patients with the Type I recurrent tumors but better pituitary function especially in children. CONCLUSIONS The origin of recurrent craniopharyngiomas significantly affected the symptoms, signs, functional outcomes, and hypothalamic-pituitary functions of patients undergoing repeated surgery. Differences in tumor growth patterns and site of origin should be considered when one is comparing outcomes and survival across treatment paradigms in patients with recurrent craniopharyngiomas.

Knockdown of versican 1 blocks cigarette-induced loss of insoluble elastin in human lung fibroblasts.

COPD lung is characterized by loss of alveolar elastic fibers and an increase in the chondroitin sulfate (CS) matrix proteoglycan versican V1 (V1). V1 is a known inhibitor of elastic fiber deposition and this study investigates the effects of knockdown of V1, and add-back of CS, on CCL-210 lung fibroblasts treated with cigarette smoke extract (CSE) as a model for COPD. CSE inhibited fibroblast proliferation, viability, tropoelastin synthesis, and elastin deposition, and increased V1 synthesis and secretion. V1 siRNA decreased V1 and constituent CS, did not affect tropoelastin production, but blocked the CSE-induced loss in insoluble elastin. Exogenous CS reduced insoluble elastin, even in the presence of V1 siRNA. These findings confirm that V1 and CS impair the assembly of tropoelastin monomers into insoluble fibers, and further demonstrate that specific knockdown of V1 alleviates the impaired assembly of elastin seen in cultures of pulmonary fibroblasts exposed to CSE, indicating a regulatory role for this protein in the pathophysiology of COPD.

A membranous structure separating the adenohypophysis and neurohypophysis: an anatomical study and its clinical application for craniopharyngioma.

OBJECT This study aimed to identify the membranous septation between the adeno- and neurohypophysis. The clinical impact of this septation in the surgical removal of infradiaphragmatic craniopharyngioma (Id-CP) is also clarified. METHODS The sellar regions from 8 fetal and 6 adult cadavers were dissected. After staining first with H & E and then with picro-Sirius red, the membranous structures were observed and measured under normal light and polarization microscopy. The pre- and postsurgical images and intraoperative procedures in 28 cases of childhood Id-CP were reviewed and analyzed. RESULTS There is a significant membranous septation (termed the adenoneurohypophysis septation [ANHS]) lying behind the intermediate lobe to separate the adeno- and neurohypophysis. The average thicknesses are 21.9 ± 16.9 µm and 79.1 ± 43.2 µm in fetal and adult heads, respectively. The median segment of the septation is significantly thicker than the upper and lower segments. The ANHS extends from the suprasellar pars tuberalis to the sellar floor, where it is fused with the pituitary capsule. During Id-CP surgery performed via a transcranial approach, the ANHS can be identified to reserve the neurohypophysis. Moreover, by understanding the anatomy of this membrane, the pituitary stalk was preserved in 3 patients (10.7%). CONCLUSIONS There is a significant membrane separating the anterior and posterior lobes of the pituitary gland, which lies behind the intermediate lobe. Understanding the anatomy of this septation is important for identifying and preserving the neurohypophysis and pituitary stalk during Id-CP surgery.

IDH mutations occur frequently in Chinese glioma patients and predict longer survival but not response to concomitant chemoradiotherapy in anaplastic gliomas.

Mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1 and IDH2) appear to occur frequently and selectively in gliomas. Our aim was to assess whether IDH mutations are common in Chinese glioma patients and whether the mutations predict good response to concomitant chemoradiotherapy. In this study IDH1 and IDH2 mutations were detected in a series of 203 gliomas. IDH1 mutations were present in 75 of the 203 cases (36.9%) while IDH2 mutations in 5 of the 203 cases (2.5%). No tumor was mutated in both IDH1 and IDH2. IDH1/2 mutations were associated with prolonged overall survival in the whole series of patients exclusive of pilocytic astrocytoma (P<0.001), WHO grade Ⅱ patients who received no adjuvant therapy after surgery (P=0.014) and WHO grade Ⅲ patients who received concomitant chemoradiotherapy (standard schedule) after surgery (P=0.033). Furthermore, there was no correlation between IDH1/2 mutations and reponse to concomitant chemoradiotherapy in anaplastic gliomas. Our results suggest that IDH1 mutations also occur freuqently in Chinese glioma patients but the frequency of IDH1 mutations is below the findings reported by North American and European groups. Furthermore, we confirm the prognostic significance of IDH1/2 mutations in gliomas, but the mutations cannot predict a favorable response to concomitant chemoradiotherapy in anaplastic gliomas.

[Microsurgical removal of huge tuberculum sellae meningiomas through bi-subfrontal anterior longitudinal fission approach].

OBJECTIVE: To summary the microsurgical techniques for removal of huge tuberculum sellae meningiomas through the bi-subfrontal anterior longitudinal fission approach. METHODS: Eleven patients with huge tuberculum sellae meningiomas underwent microsurgical removal of the meningiomas between January, 2005 and November, 2009. The microsurgical techniques were summarized, and the factors affecting the prognosis were analyzed. RESULTS: Among all the patients, 5 had Simpson grade I meningioma removal and the other patients had Simpson grade II removal. No death occurred in these patients. Nine patients showed vision improvement after the surgery, one had no significant improvement, and the other one experienced worsening of vision. Transient postoperative diabetes insipidus occurred in 5 cases. CONCLUSION: With satisfactory exposure of Dorsum sellae, bottom of the third ventricle and cavernous sinus, the bi-subfrontal anterior longitudinal fission approach is suggested for treatment of tuberculum sellae meningiomas. The key to improve the GTR and reduce the complication lies in the sequence of the operation, namely resection of the tumoral basement before dissection of the potential arachnoidal space and tuberculum.

[Microsurgical anatomy of the perforating arteries in the superior space of the internal carotid artery through a pterional approach].

OBJECTIVE: To study the microanatomy of the perforating arteries in the superior space of the internal carotid artery visualized through a pterional approach. METHODS: Twelve (24 sides) perfused cadaver heads were dissected via the pterional approach, and the perforating arteries in the superior space of the internal carotid artery were studied under microscope. The diameter, course and distribution in the anterior perforated substance of the perforating arteries were recorded. RESULTS: All the perforating arteries exposed lied on the side of the brain tissue. The carotid bifurcation on 8 sides had perforating arteries, and 11 sides showed medial lenticulostriate artery of the middle cerebral arteries, with short course and overlapped with another perforating arteries upon entry into the anterior perforated substance. On 4 sides, the medial lenticulostriate artery coincided with the perforating arteries in A1. All 24 sides showed middle lenticulostriate artery and lateral lenticulostriate artery of the middle cerebral arteries. Most of the lenticulostriate arteries originated from the anterior segment of the bifurcation of the middle cerebral arteries. The earlier bifurcation occurred in M1 of the middle cerebral arteries, the more perforating arteries originated. On 22 sides, the anterior cerebral arteries had perforating arteries with many branches, and fewer perforating arteries in a main artery were associated with greater diameter of them. CONCLUSION: The superior space of the internal carotid artery allows a space for operation, and in some cases, part of the medial leticulostriate arteries and A1 perforating arteries can be severed to obtain larger space for the operation.

[Anatomical study of venous spaces in transsphenoidal approach for cavernous sinus surgery].

OBJECTIVE: To study the anatomical and morphological characteristics of the venous spaces involved in surgery via transsphenoidal approach to the cavernous sinus (CS). METHODS: Ten fixed cadaver heads (six male, four female) with red and blue latex injected in the arteries and veins, respectively, were used to perform the transsphenoidal approach. The anterior wall of the sphenoidal sinus and the floor of sellar turcica were opened as much as possible to expose the dura mater at the sellar floor and the inferior wall of CS, and the location of the anterior and inferior intercavernous sinuses were observed carefully. All the spaces of CS were observed and measured. According to the observations, the venous spaces available for operation were identified and analyzed. RESULTS: In all the cadaver heads, 4 anterior and 5 inferior intercavernous sinuses were found, with the former locating below the optic protuberance, while the latter situated at the turn of the sellar protuberance at the clival indentation. CS was subdivided into medial space, inferolateral space, and dorsolateral space. CONCLUSIONS: In transsphenoidal approach, opening of anterior and inferior intercavernous sinus is liable to result in intra- and postoperative venous bleeding, and understanding of the location of the intercavernous sinus and appropriate utilization of these CS may help reduce intraoperative vascular and nerve injury.

[FK506 promotes apoptosis of macrophages activated by homogenate of allogenic nerve].

OBJECTIVE: To study the effect of FK506 in promoting apoptosis of peripheral blood-derived macrophages activated by homogenate of allogenic nerve tissues. METHODS: Homogenate of the allogenic nerve tissues was prepared using the sciatic nerve and injected in one-month-old SD rats, from which the macrophages activated by the homogenate were collected from the abdominal cavity and cultured in vitro. The cells were divided into 4 groups according to different concentrations of FK506 for treatment, namely 0 (group A, control group), 0.25 ng/ml (group B), 0.5 ng/ml (group C), and 1.0 ng/ml (group D). The cells of the 4 groups were inoculated into 96-well plate respectively for detecting the viability of the macrophages by MTT assay and for morphological evaluation of the cell apoptosis by transmission electron microscopy and fluorescence microscopy. RESULTS: The cells in groups B and C exhibited reduced viability and signs of apoptosis, and necrosis was observed in group D. Transmission electron microscopy and fluorescence microscopy identified early apoptotic changes and the presence of apoptotic body in the macrophages. The apoptotic rates of groups B and C were much higher than that in group A found by flow cytometry. CONCLUSION: FK506 can promote the apoptosis of macrophage activated by allogenic nerve homogenate and reduce macrophage-mediated immunological rejection of peripheral nerve allograft.

[In vitro study of formalin-fixed bone tissues for allogeneic bone grafting].

OBJECTIVE: To conduct an in vitro study to assess the preliminary possibility of using formalin-fixed, instead of fresh, human bone tissues for allografting. METHODS: Fresh cadaveric bone tissues were fixed by formalin for more than 6 months and dissected into 5 mmx5 mmx5 mm pieces and 5 mmx5 mmx40 mm sticks, followed by chemical treatments to prepare the allograft bone materials. When alls treatments were completed, the bone grafts were centrifuged and their properties and cellular compatibility assessed in comparison with the currently used bone grafts clinically. RESULTS: The residual formaldehyde of the fixed allograft bone material was much below the controlled level and no significant differences were noted between the bone graft materials tested in regard to the chemical and mechanical properties and biocompatibility. CONCLUSION: This material we have prepared may meet the clinical demands for bone grafting, with good biocompatibility and less chance for infection by pathological agents.

Construction of dataset for Virtual Chinese Male No.1.

OBJECTIVE: To establish digitized Virtual Chinese Human Male No.1 (VCH-M1) image dataset with a 0.2-mm equal interval. METHODS: The body of a 24-year-old male was used for this study. Perfusion with phenol and vermilion of the arteries was performed, followed by body shape adjustment by cold saline and pre-embedding with broken ices in an upside-down position, which was completed in a stepwise procedure to minimize body shape deformation. Section milling was conducted subsequently with the section thickness of 2 mm and the section images were captured by digital camera, which were immediately transferred to a computer for storage and processing. RESULTS: A total of 9 232 sections were obtained for the whole body, and the resolution of each of the image in TIF format was 3 024x2 016 pixels, resulting in the size of approximately 18 M for each image and about 161 G for the whole dataset. CONCLUSIONS: Compared with VCH-F1, the image quality in VCH-M1 dataset is significantly improved, demonstrated by much clearer tissue boundary in the images and minimized body shape deformation during the embedding process.

[Registration of the image data of the Virtual Chinese Female No.1].

In this study, the authors attempt to find a solution for the image registration before three-dimensional reconstruction of digitized Virtual Chinese Female No.1 (VCH-F1) image data can be completed. On the basis of a thorough understanding of the features of the image data, the parameters were determined based on the marker outside the specimen and the least-square procedure performed on the outline offset distance between two images, prior to the completion of the image registration by means of rigid body transformation with the parameters. In the trial application of these two methods in image registration for these image data, good results were obtained, which approved of the application of the two methods for accomplishing the image registration with simpler calculation processes and easier computer processing.

[Research report of experimental database establishment of digitized virtual Chinese No.1 female].

OBJECTIVE: To establish digitized virtual Chinese No.1 female (VCH-F1) image database. METHODS: A 19 years old female cadaver was scanned by CT, MRI, and perfused with red filling material through formal artery before freezing and em- bedding. The whole body was cut by JZ1500A vertical milling machine with a 0.2 mm inter-spacing. All the images was produced by Fuji FinePix S2 Pro camera. RESULTS: The body index of VCH-F1 was 94%. We cut 8 556 sections of the whole body, and each image was 17.5 MB in size and the whole database reached 149.7 GB. We have totally 6 versions of the database for different applications. CONCLUSIONS: Compared with other databases, VCH-F1 has good representation of the Chinese body shape, colorful filling material in blood vessels providing enough information for future registration and segmentation. Vertical embedding and cutting helped to retain normal human physiological posture, and the image quality and operation efficiency were improved by using various techniques such as one-time freezing and fixation, double-temperature icehouse, large-diameter milling disc and whole body cutting.