[Effect of dNLR and LIPI scores on the prognosis of elderly patients with non-surgical treatment of non-small cell lung cancer].

Objective: To investigate the effects of derived neutrophil to lymphocyte ratio (dNLR) and lung immune prognostic index (LIPI) score on the overall survival (OS) of non-surgical elderly non-small cell lung cancer (NSCLC) patients. Methods: Clinical and pathological data of NSCLC patients in Hebei General Hospital from January 2014 to June 2018 were collected retrospectively. The dNLR value was calculated based on the results of blood routine before treatment, and the optimal cut-off value of dNLR was obtained by ROC curve. The patients were divided into low dNLR level group and high dNLR level group based on the optimal dNLR cut-off value. The groups were classified as good, intermediate and poor based on the LIPI score consisting of lactate dehydrogenase (LDH) and dNLR tested before treatment. The Kaplan-Meier method and Log rank test were used for survival analysis, and the Cox risk proportional regression model was used for analysis of prognostic influences. Results: The area under the ROC curve for dNLR predicting prognosis in non-surgical elderly NSCLC patients was 0.591 (95% CI: 0.491, 0.692; P=0.093). The optimal cut-off value for dNLR predicting prognosis in elderly NSCLC patients was 2.515, with a sensitivity of 45.5% and a specificity of 81.8%. The gender, BMI, pathological type and degree of tumor differentiation were associated with dNLR levels (P<0.05). The median survival times were 16 and 10 months for patients in the low dNLR level group (dNLR<2.51) and high dNLR level group (dNLR≥2.51), respectively (P<0.001), and 15, 10 and 6 months for patients with good, intermediate and poor LIPI scores, respectively (P<0.001). The age, gender, smoking, pathological type, tumor differentiation, clinical stage, BMI, dNLR level, LDH level and LIPI scores were all associated with patient prognosis (P<0.05), and age≥76 years, tumor differentiation and clinical stage Ⅲ and Ⅳ were independent factors influencing patient prognosis (P<0.05). Conclusion: No matter what treatment measures are taken, dNLR level and LIPI score are related to patients' prognosis, and non-surgical elderly NSCLC patients with high dNLR level and poor LIPI score before treatment have worse prognoses.

[Prognostic value of new TNM staging and serum CA125 in malignant peritoneal mesothelioma].

Objective: To explore the relationship between the new Tumor-Node-Metastasis (TNM) staging system and the serum CA125 level with the prognosis of malignant peritoneal mesothelioma (MPeM) . Methods: The clinical data of 74 patients with MPeM diagnosed by pathology and immunohistochemistry were collected from January 2005 to June 2016 in Cangzhou Central Hospital. According to the results of CT-peritoneal carcinoma index (PCI) , the tumor load was divided into T1 (PCI 1-10) , T2 (PCI 11-20) , T3 (PCI 21-30) and T4 (PCI 31-39) , combined with lymph node metastasis and extraperitoneal metastasis, a new TNM staging system was established. And serum CA125 level was measured in the same time. The median survival time of patients with MPeM, the effect of the new TNM staging system, and serum CA125 levels on their prognosis were retrospectively analyzed. Results: Among the 74 patients with MPeM, 25 (33.8%) cases were males and 49 (66.2%) cases were females. There were 8 cases with systemic chemotherapy, 8 cases with heated intraperitoneal chemotherapy, and 1 case with combination chemotherapy. 10 cases were T1, 22 cases were T2, 27 cases were T3, 15 cases were T4, 12 cases had lymph node metastasis and 10 cases had distant metastasis. The median survival time of T1, T2, T3 and T4 were 12, 10, 6 and 3 months respectively. There were 38 (77.6%) cases with high serum CA125 in all 49 cases who have been tested for CA125. The median survival time of positive group and negative group were 6 months and 11 months respectively. 68 (91.9%) patients had died by the end of collecting data. The median survival time was 8 months. Univariate analysis showed that there were significant differences in survival time between patients with different CT-PCI stages, serum CA125 levels, and with or without lymph node and extraperitoneal metastasis (P<0.05) . Multivariate analysis showed that CT-PCI was independent risk factors for the prognosis of MPeM (HR=2.203, 95%CI: 1.475-3.289) . Conclusion: The new TNM staging system and serum CA125 are important for the prognosis of patients with MPeM. Early detection, early diagnosis and comprehensive treatment can improve the survival time of patients with MPeM.

[Role and mechanism of lipopolysaccharide induced exosome in the pathogenesis of acute lung injury].

Objective: To explore the effect and mechanism of exosome derived from lipopolysaccharide (LPS)-induced mouse macrophage (RAW264.7) on acute lung injury. Methods: RAW264.7 were cultured in vitro and divided into 2 groups: control group and LPS-induced group. The exosomes were extracted from the two groups of cell supernatant by ultracentrifugation and classified into 2 groups: C-EXO group and LPS-EXO group. In vivo, random allocation was used to averagely divide the eighteen male C57BL/6 mice into 3 groups: control group, EXO-control group and EXO-LPS group. All mice were sacrificed after 12 h. The lung tissue was used for HE staining to assess the degree of acute lung injury as well as immunohistochemical staining for interleukin (IL) -1ß and tumor necrosis factor (TNF)-α. The tissue protein expression levels of IL-1ß, TNF-α, ß-catenin, E-cadhein, ZO-1 and Occludin were measured by Western blot. In vitro, alveolar type Ⅱ epithelial cells (MLE-12) were cultured and divided into 3 groups: C-control group, EXO-control-induced group and EXO-LPS-induced group. The tissue protein expression levels of IL-1ß, TNF-α, and Occludin were measured by Western blot after 12 h. Results: The two samples of C-EXO group and LPS-EXO group was proved to be exosomes. Under a light microscope, the lung tissue of EXO-LPS group showed inflammatory cell infiltration, hemorrhage, interstitial and alveolar edema, and the thickness of alveolar septum. The tissue protein levels of IL-1ß and TNF-α in EXO-LPS group were obviously higher than the control group, EXO-control group (1.331±0.203 and 0.274±0.018, 0.892±0.074; 0.800±0.096 and 0.596±0.025, 0.441±0.061; all P<0.05). While the tissue protein levels of Occludin showed the opposite phenomenon (0.251±0.021 and 0.862±0.029, 0.453±0.013; all P<0.05). In vitro, Compared with the C-control group and the EXO-control-induced group, the expression levels of IL-1ß and TNF-α increased significantly in the EXO-LPS-induced group (0.900±0.033 and 0.320±0.030, 0.661±0.028; 0.739±0.045 and 0.151±0.024, 0.360±0.037; all P<0.05). whereas the protein levels of Occludin expression were reversed in MLE-12 (0.585±0.082 and 0.941±0.090, 0.732±0.083; all P<0.05). Conclusion: Exosomes derived from LPS-induced RAW264.7 can induced the acute lung injury by affecting barrier function, which probably is related to the low degree of Occludin in alveolar type Ⅱ epithelial cells.

[Role of Yes-associated protein 1 in angiotensinâ ¡-induced pulmonary fibrosis in rats].

Objective: To explore the mechanism of Yes-associated protein 1 (Yap1) in angiotensinⅡ(AngⅡ)-induced pulmonary fibrosis. Methods:In vivo, 18 male Wistar rats were randomly divided into three equal groups with 6 rats in each group, including control group, bleomycin-treated group (BLM), and BLM+ AngⅡ group. 28 days later, the lung tissues in all groups were harvested for the HE and Masson staining as well as the immunohistochemical (IHC) staining for Yap1. In vitro, the isolated fibroblasts were treated with 10(-7) mmol/L AngⅡor the AngⅡ-targeted inhibitor irbesartan for the scheduled time for mRNA and protein expressions of Yap1, PDZ-binding motif (TAZ), and collagen Ⅰusing PCR and Western blot, as well as the translocation test from the nucleus to the cytoplasm of Yap1 and TAZ. Subsequently, the fibroblasts were assigned into 4 groups: the empty plasmid (vector) group, the vector+ AngⅡ group, the Yap1 shRNA group, and the Yap1 shRNA+ AngⅡ group. Western blot was used to detect the relative expressions of Yap1, TAZ, Smad3 and collagen Ⅰ. The CCK-8 and EdU assays were performed to determine the proliferative capacity. Results:In vivo, severe lung fibrosis and increased Yap1 expression of IHC staining were found in BLM group. Additionally, more severe lung fibrosis and higher Yap1 expression were detected in the BLM+ AngⅡ group than the BLM group (both P<0.05). In vitro, both the mRNA and protein relative expressions of Yap1, TAZ and collagenⅠ were markedly higher in AngⅡ-treated groups than the control group (all P<0.05). Meanwhile, the relative expression of phosphorylated Yap1 reached its peak at 2 h after AngⅡ stimulation. In the protein translocation tests, after treated with AngⅡ for 24 h, the relative protein levels of Yap1 and TAZ in the nucleus of the AngⅡ group were significantly higher than those in the control group (0.382±0.007 vs 0.031±0.001, 1.097±0.030 vs 0.357±0.015). However, the relative protein expressions in the cytoplasm of the AngⅡ group were obviously less than that in the control group (0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015). Compared with the AngⅡ group, the expressions of Yap1 and TAZ in the AngⅡ+ irbesartan group were higher in cytoplasm (0.598±0.060 vs 0.323±0.058, 1.495±0.052 vs 0.858±0.059), while lower in the nucleus (0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015) (all P<0.05). Furthermore, the relative protein expressions of Yap1, TAZ, Smad3 and collagenⅠin Yap1 shRNA+ AngⅡ group were distinctly lower than the vector+ AngⅡ group (all P<0.05). In the cell proliferation tests, the absorbance and the percentage of EdU positive cells of vector+ AngⅡ group exceeded that of vector group (both P<0.05). However, the absorbance and the percentage of EdU positive cells in the Yap1 shRNA+ AngⅡgroup were less than the vector+ AngⅡ group (both P<0.05). Conclusion: AngiotensinⅡ promoted the collagen synthesis and cell proliferation in primary lung fibroblasts by increasing the Yap1 activity, leading to the progress of fibrosis.

[Mechanism of angiotensin-converting enzyme 2 overexpression improving collagen synthesis in lung].

Objective: To explore the mechanism of angiotensin-converting enzyme 2 (ACE2) overexpression improving collagen synthesis in lung. Methods: Lung fibroblasts of mice over-expressing ACE2 and the wild type (WT) were cultured in vitro and divided into 5 groups: WT-control, WT-AngiotensinⅡ (AngⅡ), ACE2(+ /+) -control, ACE2(+ /+) -AngⅡ and ACE2(+ /+) -AngⅡ+ A779. The protein relative expression levels of ACE2, collagen Ⅰ, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), autophagy-related protein (Beclin1), ubiquitin-binding protein p62 (P62), microtubule-associated proteins light chain 3-Ⅱ (LC3-Ⅱ) were measured by Western blot and triphosadenine (ATP) level was measured by ATP Assay Kit. Fibroblasts over-expressing ACE2 were pretreated with or without the autophagy inhibitor and were separated into 4 groups: ACE2(+ /+) -control, ACE2(+ /+) -AngⅡ, ACE2(+ /+) -AngⅡ+ 3-MA and ACE2(+ /+) -3-MA. In vivo, random allocation was used to averagely divide mice into four groups: WT-control, WT-Bleomycin (BLM), ACE2(+ /+) - control, ACE2(+ /+) -BLM. Wild type and ACE2 over-expressing mice were instilled with bleomycin endotracheally (3.5 mg/kg) or the same volume saline. All mice were sacrificed after 28 days and the lung tissue were used for HE and Masson staining as well as immunohistochemical staining for NOX4, P62 and LC3. Results: The vimentin in lung fibroblasts isolated from mice was proved to be positive by both immunohistochemical and immunofluorescence. The ACE2 protein level of lung fibroblasts over-expressing ACE2 was higher than the wild type (0.202±0.062 and 0.067±0.040, P<0.05). The protein levels of collagenⅠ, NOX4 and NLRP3 in WT-AngⅡ group were obviously higher than the WT-control group (0.861±0.129 and 0.417±0.076, 0.432±0.036 and 0.318±0.058, 0.367±0.125 and 0.045±0.012, all P<0.05). The difference of collagenⅠand NLRP3 between ACE2(+ /+) -AngⅡ group and ACE2(+ /+) -control group had no statistical significance (all P>0.05). CollagenⅠand NOX4 protein level in ACE2(+ /+) -AngⅡ+ A779 group were observably higher than ACE2(+ /+) - AngⅡ group (0.707±0.155 and 0.458±0.108, 0.299±0.038 and 0.149±0.090, all P<0.05). The autophagy related protein levels of Beclin1, P62 and LC3-Ⅱ in ACE2(+ /+) -control group were distinctly higher than WT-control group (0.834±0.051 and 0.274±0.018, 0.467±0.078 and 0.093±0.025, 0.494±0.065 and 0.150±0.054, all P<0.05). However, these protein levels in ACE2(+ /+) -AngⅡ+ A779 group were lower than ACE2(+ /+) -AngⅡ group (1.331±0.203 and 1.565±0.069, 0.298±0.096 and 0.438±0.077, 0.464±0.093 and 0.768±0.071, all P<0.05). ACE2(+ /+) -AngⅡ+ 3-MA group had higher collagenⅠ (0.383±0.125 and 0.032±0.013, P<0.05) and lower LC3-Ⅱ protein level (1.177±0.140 and 1.387±0.183, P<0.05) than AngⅡ group. In bleomycin induced lung fibrosis in mice, ACE2(+ /+) -BLM mice exhibited milder lung fibrosis and lower NOX4 protein level but higher LC3-Ⅱprotein level compared with WT-BLM mice. Conclusion: ACE2 over-expression ameliorated collagen synthesis through enhancing autophagy in lung.

[Anatomical characteristics of profunda artery perforator flap in the posteromedial femoral region and its application in the reconstruction of oral and maxillofacial defects].

Objective: To investigate the anatomical basis for the preparation of the profunda artery perforator flap (PAPF) in the posteromedial femoral region and its application in the reconstruction of oral and maxillofacial defects. Methods: Six lower limbs of Chinese adult cadavers were micro-surgically dissected. CT angiography (CTA) data of bilateral lower limbs of 6 patients was also collected retrospectively. The number, external diameter, pedicle length, and distribution of perforators in the posteromedial femoral region were recorded from the specimens and CTA data. Meanwhile, 10 patients with oral squamous cell carcinoma in the Department of Oral and Maxillofacial Surgery, Affiliated Hospital of Stomatology, Nanjing Medical University from August 2018 to June 2021 were treated with the PAPF. At each follow-up, contour and function of recipient and donor site, as well as swallowing and speech function were evaluated. Results: A total of 19 profunda artery perforator were identified in 6 lower limb specimens. The outer diameter at the beginning of the source artery was (2.34±0.25) mm and the total length of the pedicle was (11.12±1.06) cm. CTA data analysis of 12 legs identified 15 perforators of profunda artery in the posteromedial region. Eleven perforators were septocutaneous, including 2 perforators with a common trunk, while the remaining 4 perforators were musculocutaneous. As for different patterns of perforators (septocutaneous perforators, musculocutaneous perforators and perforators with a common trunk), the longitudinal distance to the pubic tubercle was (19.95±2.43), (21.84±2.54) and (19.48±0.55) cm respectively. The horizontal distance to the posterior edge of gracilis was (3.54±1.10), (3.72±0.30) and (3.85±1.48) cm, respectively. The initial diameters of perforators was (2.4±0.4), (2.6±0.6) and 1.9 mm respectively. Ten cases of the profunda artery perforator flaps survived successfully after operation. The flap sizes ranged from 8 cm×6 cm to 12 cm×7 cm. The patients were evaluated at 1, 3 and 6 months, and with 6 months interval ever since. During the follow-up, the shape of the recipient site was ideal, and the swallowing and language functions were not significantly affected. There was only linear scar in the donor area, and the function of the thigh was basically normal. Conclusions: PAPF possessed a good anatomic stability, suitable vascular pedicle length and diameter, minor influence to the donor area, sufficient amount tissue with good quality. It is an ideal choice for head and neck reconstruction.

BCL10 as a new candidate gene for immune response in pigs: cloning, expression and association analysis.

BCL10 is an apoptotic regulatory molecule identified through its direct involvement in t(1; 14)(p22; q32) of mucosa-associated lymphoid tissue lymphoma, and was implicated in the pathogenesis of this and several other tumour types. BCL10 was recognized as an antigen receptor-specific regulator of NF-kappaB, which showed close association with immune responses. In this study, we cloned and characterized BCL10 from the porcine spleen and analysed its genomic structure. BCL10 was mapped to SSC4q21-q23 by the IMpRH panels, it is closely linked to the marker S0161 and SW1461. This gene has three exons and two introns. Reverse transcriptase-polymerase chain reaction analyses showed that BCL10 was widely expressed in all the examined tissues. Transient transfection indicated that porcine BCL10 was located in cytoplasm in Pig Kidney Epithelial cells. BCL10 gene displays the opposite expression trend between the two treatments mimic virus and bacteria of polyriboinosinic-polyribocytidylic acid (Poly I:C) and lipopolysaccharide (LPS). The level of the BCL10 mRNA was up-regulated during 12-24 h and peaking at 48 h when treated with LPS, whereas it was down-regulated during 0-48 h and highest at 0 h (cells without treating with Poly I:C) when treated with Poly I:C. One single nucleotide polymorphism (SNP) site was identified in the 3'-untranslated region of porcine BCL10. Association analysis revealed that this SNP was significantly associated with intermediate cell mass (eosinophile granulocyte, basophile granulocyte and histoleucocyte) percentage, absolute intermediate cell mass count and mean red blood cell volume of 0-day-old pigs, and red blood cell count of 17-day-old pigs (P < 0.05), and also had significant associations with red blood cell count and haemoglobin concentration of 32-day-old pigs (P < 0.01).

MicroRNA-365 inhibits the progression of lung adenocarcinoma through targeting ETS1 and inactivating AKT/mTOR pathway.

OBJECTIVE: MicroRNAs (miRNAs) act as important regulators in human cancers by regulating the gene expression. The dysregulation of miR-365 has been investigated in many cancers. However, the function of miR-365 remains unknown in lung adenocarcinoma. Therefore, the regulatory mechanism of miR-365 was explored in lung adenocarcinoma. PATIENTS AND METHODS: The expression of miR-365 was detected in cell lines and 67 lung adenocarcinoma tissues using qRT-PCR. The Kaplan-Meier analysis was used to determine the association between miR-365 expressions and the survival rate in patients with lung adenocarcinoma. Transwell assay was then performed to investigate the effect of miR-365 on invasion and migration of lung adenocarcinoma cells. RESULTS: Downregulation of miR-365 and upregulation of ETS1 were identified in lung adenocarcinoma. Furthermore, miR-365 reversely regulated ETS1 expression in lung adenocarcinoma. Functionally, the overexpression of miR-365 inhibited proliferation, migration, and invasion of lung adenocarcinoma cells. However, the upregulation of ETS1 lessened the inhibitory effect of miR-365 in lung adenocarcinoma. In addition, miR-365 inhibited EMT and inactivated AKT/mTOR pathway in lung adenocarcinoma. CONCLUSIONS: MiR-365 inhibits the progression of lung adenocarcinoma by targeting ETS1 and inactivating the AKT/mTOR pathway.

A gain-of-function mutation in TNFRSF13B is a candidate for predisposition to familial or sporadic immune thrombocytopenia.

Essentials Positive family histories suggest the existence of hereditary immune thrombocytopenia (ITP). The predisposing gene for familial ITP was screened in two familial ITP patients. The G76S mutation on TNFRSF13B led to immune dysfunction and induced megakaryocyte apoptosis. The G76S mutation on TNFRSF13B is a gain-of-function mutation and a predisposing variant for ITP. SUMMARY: Background Most immune thrombocytopenia (ITP) is sporadic but a positive family history of ITP in some patients suggests that hereditary forms exist. Because of the rarity of familial ITP families available for study and the heterogeneity of sporadic ITP, family linkage analysis or genome-wide association studies are limited. Objectives Based on one ITP pedigree, we try to identify the predisposing gene in familial or sporadic ITP and reveal the way in which it causes thrombocytopenia. Methods Gene expression profiling analysis and whole-exome sequencing were performed on samples from family members with ITP, sporadic ITP cases and healthy individuals. We also evaluated the influence of potential pathogenic mutation on immune function and megakaryocyte apoptosis. Results Whole-exome sequencing identified a potential pathologic p.G76S heterozygous mutation on the TNFRSF13B gene in familial ITP patients. ITP patients harboring the G76S mutation displayed an upregulated 'cytokine-cytokine receptor interaction' signal, increased serum TNFα, IL-17α, IFNγ and BAFF levels, and enhanced binding capacity of APRIL ligand to B cells. Additionally, Epstein-Barr virus (EBV)-transformed B cells with the G76S mutation could induce human megakaryocyte line (Meg-01) apoptosis significantly. Conclusion p.G76S mutation on the TNFRSF13B gene is responsible for ITP, and is a genetic predisposing factor for familial or sporadic ITP.

Molecular cloning, chromosomal organization, and functional characterization of a sodium-dicarboxylate cotransporter from mouse kidney.

The sodium-dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs filtered Krebs cycle intermediates and plays an important role in the regulation of urinary citrate concentrations. (1) Low urinary citrate is a risk factor for the development of kidney stones. As an initial step in the characterization of NaDC-1 regulation, the genomic structure and functional properties of the mouse Na(+)-dicarboxylate cotransporter (mNaDC-1) were determined. The gene coding for mNaDC-1, Slc13a2, is found on chromosome 11. The gene is approximately 24.9 kb in length and contains 12 exons. The mRNA coding for mNaDC-1 is found in kidney and small intestine. Expression of mNaDC-1 in Xenopus laevis oocytes results in increased transport of di- and tricarboxylates. The Michaelis-Menten constant (K(m)) for succinate was 0.35 mM, and the K(m) for citrate was 0.6 mM. The transport of citrate was stimulated by acidic pH, whereas the transport of succinate was insensitive to pH changes. Transport by mNaDC-1 is electrogenic, and substrates produced inward currents in the presence of sodium. The sodium affinity was relatively high in mNaDC-1, with half-saturation constants for sodium of 10 mM (radiotracer experiments) and 28 mM at -50 mV (2-electrode voltage clamp experiments). Lithium acts as a potent inhibitor of transport, but it can also partially substitute for sodium. In conclusion, the mNaDC-1 is related in sequence and function to the other NaDC-1 orthologs. However, its function more closely resembles the rabbit and human orthologs rather than the rat NaDC-1, with which it shares higher sequence similarity.

Drotrecogin alfa (activated) prevents smoke-induced increases in pulmonary microvascular permeability and proinflammatory cytokine IL-1beta in rats.

In an established rat model of smoke inhalation injury, we conducted a dose-response study to examine the protective effects of Xigris [drotrecogin alfa (activated) (DrotAA)], a recombinant form of human activated protein C (APC). DrotAA is a serine protease (approximately 55 kD molecular weight) with the same amino acid sequence and the glycosylation site as human plasma-derived APC. A total of 120 F344/NH rats (half each gender, approximately 175 g body weight) were randomly divided into five groups and exposed nose-only to air or diesel fuel smoke for 20 min. These rats were then i.v. administered with DrotAA in 0, 5, 10, and 20 mg/kg body weight, respectively, immediately following smoke exposure. Treatment with DrotAA significantly attenuated smoke inhalation injury in a dose-dependent manner at 2 hours after insult, as indicated by preserving microvascular permeability and proinflammatory cytokine IL-1beta (but not TNF-alpha and neuropeptide substance P) in bronchoalveolar lavage fluid (BALF). Moreover, the rats treated with 20 mg/ kg of DrotAA had an improvement of the expiration phase of pulmonary dynamic compliance. At all dosages, however, DrotAA also significantly increased all phases of pulmonary resistance compared with either the controls or to smoke inhalation alone. Generally, these data suggest that DrotAA may exert an anti-inflammatory effect by inhibiting cytokine-mediated inflammatory amplification. However, additional studies following a clinical course are needed to confirm the maximum efficiency and possible side effects of this recombined human activated protein C.