Immunogenicity and protective efficacy of recombinant adenovirus expressing a novel genotype G2b PEDV spike protein in protecting newborn piglets against PEDV.

IMPORTANCE: Porcine epidemic diarrhea (PED) is a highly infectious and economically significant gastrointestinal disorder that affects pigs of all ages. Preventing and controlling PED is achieved by immunizing sows with vaccines, enabling passive piglet immunization via colostrum. The prevalence of G2b porcine epidemic diarrhea virus (PEDV) continues in China despite the use of commercial vaccines, raising questions regarding current vaccine efficacy and the need for novel vaccine development. Adenovirus serotype 5 (Ad5) has several advantages, including high transduction efficiency, a wide range of host cells, and the ability to infect cells at various stages. In this study, we expressed the immunogenic proteins of spike (S) using an Ad5 vector and generated a PED vaccine candidate by inducing significant humoral immunity. The rAd5-PEDV-S prevented PED-induced weight loss, diarrhea, and intestinal damage in piglets. This novel vaccine candidate strain possesses the potential for use in the pig breeding industry.

Immune modulation of Th1/Th2/Treg/Th17/Th9/Th21 cells in rabbits infected with Eimeria stiedai.

Introduction: Despite long-term integrated control programs for Eimeria stiedai infection in China, hepatic coccidiosis in rabbits persists. Th1, Th2, Th17, Treg, Th9, and Th21 cells are involved in immune responses during pathogen infection. It is unclear whether Th cell subsets are also involved in E. stiedai infection. Their roles in the immunopathology of this infection remain unknown. Therefore, monitoring these T-cell subsets' immune responses during primary infection of E. stiedai at both transcriptional (mRNA) and protein (cytokines) levels is essential. Methods: In experimentally infected New Zealand white rabbits, mRNA expression levels of their transcript-TBX2 (Th1), GATA3 (Th2), RORC (Th17), Foxp3 (Treg), SPI1 (Th9), and BCL6 (Th21)-were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), whereas Th1 (IFN-g and TNF-a), Th2 (IL4), Th17 (IL17A and IL6), Treg (IL10 and TGF-b1), Th9 (IL9), and Th21 (IL21) cytokines were measured using enzyme-linked immunosorbent assays (ELISAs). Results: We found that levels of TBX2, GATA3, RORC, SPI1, and BCL6 in the livers of infected rabbits were elevated on days 5 and 15 post-infection (PI). The concentrations of their distinctive cytokines IFN-g and TNF-a for Th1, IL4 for Th2, IL17A for Th17, IL9 for Th9, IL21 for Th21, and IL10 for Treg IL10 were also significantly increased on days 5 and 15 PI, respectively (p < 0.05). On day 23 PI, GATA3 with its cytokine IL4, RORC with IL17A, Foxp3 with IL10 and TGF-b1, and SPI1 with IL9 were significantly decreased, but TBX2 with IFN-g and IL6 remained elevated. Discussion: Our findings are the first evidence of Th1/Th2/Treg/Th17/Th9/Th21 changes in E. stiedai-infected rabbits and provide insights into immune regulation mechanisms and possible vaccine development.

Application of the Method of Melanin Bleaching After Immunohistochemical Staining of Melanin-containing Tissues.

OBJECTIVE: The purpose of this research was to investigate the method of melanin bleaching in immunohistochemical staining of melanin-containing tissues. MATERIALS AND METHODS: Forty melanin-containing tissue samples were selected, including 30 with malignant melanoma and 10 with blue naevi. The samples were divided into the following 3 groups: the prestaining bleaching group, nonbleaching group, and poststaining bleaching group. Each group was subjected to HMB45 and MelanA immunohistochemical staining. The detection system used was Roche ventana ultraview universal alkaline phosphatase red kit. RESULTS: In the prestaining bleaching group, melanin pigment was mostly removed. However, antigen expression was also affected, which resulted in significantly weaker positive staining and even represented false-negative expression. In the nonbleaching group, positive staining resulted in a red color that could easily be distinguished from the brown granules of melanin pigment. However, some melanin granules covered the tissue, impacting our observation of tissue structure. In the poststaining bleaching group, the results of positive staining were comparable to those in the nonbleaching group. The melanin pigment in these sections was almost completely removed, resulting in clearer staining and easier observation of tissue and cell structure. CONCLUSION: The method of melanin bleaching after immunohistochemical staining of melanin-containing tissue can almost completely bleach the melanin pigment and do not impact the expression of antigen. In our work, poststaining bleaching allowed for clearer immunohistochemical staining results and more obvious observation of tissue structure. This method also has the advantages of requiring simple reagents, utilizing simple operation procedures, and producing practical staining results, which makes it worthy of promotion!

Identification of proteins associated with treatment response of neoadjuvant chemoradiotherapy in rectal mucinous adenocarcinoma by co-expression network analysis based on proteomic analysis.

For rectal mucinous adenocarcinoma (MAC), identifying biomarkers of neoadjuvant chemoradiotherapy (NCRT) response has become imperative. This study applied label-free mass spectrometry and weighted gene co-expression network analysis to identify hub proteins in association with the NCRT response in 20 rectal MAC patients. We identified 131 differentially abundant proteins and 7 candidate proteins associated with the NCRT response. The immunostaining expressions of six proteins (ENOA, ILEU, MDHM, RM11, PTGDS, and RL3) were significantly associated with the NCRT response. Logistic regression analysis revealed that ENOA (OR = 6.275, P = 0.006) was independent risk hub protein for the NCRT response. Tow hub proteins (ENOA and PTGDS) were identified as significant risk factors by Cox regression analysis. A prognostic risk score system was constructed: risk score = (0.910 × EXPENOA) + (-1.519 × EXPPTGDS), and found to be an independent predictor of DFS in rectal MAC patients (HR = 10.308, P < 0.001). Our study suggested that ENOA may be a novel biomarker for the NCRT response and prognosis in rectal MAC patients. A two-hub-protein-based risk score system might be used for predicting tumor recurrence in rectal MAC patients. SIGNIFICANCE: NCRT resistance is a major problem in the treatment of rectal MAC patients. Identifying robust predictive biomarkers for NCRT resistance is beneficial to the stratified treatment of rectal MAC patients. In this study, label-free mass spectrometry and weighted gene co-expression network analysis identified ENOA as a potential novel biomarker for the NCRT response and prognosis. ENOA may be involved in the process of the NCRT resistance and tumor recurrence through the carbon metabolism pathway.

Combination of Neutrophil-to-Lymphocyte Ratio and Red Cell Distribution Width With Serum Tumor Markers for the Differential Diagnosis of Breast Cancer and its Association With Pathological Features and Molecular Types.

OBJECTIVE: To explore the neutrophil-to-lymphocyte ratio (NLR), red cell distribution width (RDW) and serum tumor markers as differential diagnostic markers of breast cancer (BC) and breast fibroadenoma (FA) and their associations with histopathological indicators and molecular typing in BC patients. METHODS: We collected pathological and routine clinical test data [NLR, RDW, serum tumor markers (CEA, CA15-3, CA125, CA19-9)] of 653 patients with BC and 100 patients with FA. After identifying indicators with significant inter-group differences, we used ROC curves to determine clinically significant cutoff values. Binary logistic regression analyses and ROC curves were used to analyze combined models for the differential diagnosis of BC and FA. Ordinal multinomial logistic regression analysis was used to explore correlations between routine clinical test indicators and pathological BC features. RESULTS: The BC and FA groups had significantly different CEA, NLR, and CA19-9 levels (P < .05), with respective areas under the curve (AUC) of 0.799, 0.747, and 0.711 for differentiating BC from FA and respective optimal cutoff values of 1.35 ng/mL, 1.58, and 8.55 U/mL. Binary logistic regression and ROC curve analysis showed that CEA was superior to the other 2 factors for the differential diagnosis of BC and FA. whereas the combined use of all three indicators was diagnostically most effective (AUC = 0.886; 95% confidence interval: 0.838-0.933, P = .000; sensitivity and/or specificity: 76.5%/88.9%). Ordered multinomial logistic regression analysis showed that NLR, CEA, and CA15-3 correlated positively with BC TNM staging; RDW was negatively correlated with BC histological grade; RDW, CA15-3 and CA125 were obviously associated with BC molecular typing. CONCLUSION: The combination of CEA, NLR, and CA19-9 may be used to screen and diagnose BC. NLR, CEA and CA15-3 are related to the TNM staging of BC. RDW is related to BC histological grading. RDW, CA15-3 and CA125 are related to BC molecular typing.

Primary central nervous system Hodgkin's lymphoma: a case report.

Primary central nervous system Hodgkin's lymphoma (CNS-HL) is extremely rare. This current case report describes a 60-year-old male patient that presented with numbness of the left lower extremity and worsening headache. After a full range of investigations and a partial resection of the right cerebellum, external ventricular drainage reservoir placement and cranioplasty, he was diagnosed with primary CNS-HL. The patient was treated with 3 g/m2 methotrexate (intravenous [i.v.], once a day, day 1) and 1 g/m2 cytarabine (i.v., every 12 h, days 2 + 3), followed by anti-programmed cell death protein 1 antibodies (200 mg sintilimab, i.v., once a day, day 1, every 3 weeks). After six courses of treatment with intrathecal injections of 50 mg cytarabine (once a day, day 1) and 5 mg dexamethasone (once a day, day 1), there was no residual lesion on cranial magnetic resonance imaging. No significant drug-related adverse events were observed. The patient has been followed up every 3 months and no relapse has occurred.

Lymph Node Regression to Neoadjuvant Chemoradiotherapy in Patients with Locally Advanced Rectal Cancer: Prognostic Implication and a Predictive Model.

AIM: Currently, few studies have focused on the prognostic impact of lymph node regression to neoadjuvant chemoradiotherapy (NCRT) in rectal cancer. This study aimed to explore the prognostic impact of lymph node regression grade (LRG) in patients with locally advanced rectal cancer (LARC) following NCRT and radical surgery and develop a predictive nomogram for disease-free survival (DFS). METHODS: LARC patients undergoing NCRT and radical surgery between 2013 and 2014 were enrolled and divided into LRG low (≤ 2), middle (3-9), and high (≥ 10) groups. Clinicopathological characteristics and survival outcomes were compared. Predictors for DFS were identified by Cox regression analysis, and a nomogram was constructed. RESULTS: A total of 257 LARC patients were eligible, including LRG low (n = 149), middle (n = 59), and high (n = 49) groups. Higher LRG score was associated with higher TRG, more advanced ypT and ypN stages, and poorer OS and DFS (all P < 0.001). Cox regression analysis demonstrated that tumor differentiation (poor and anaplastic, HR = 2.048, P = 0.048), ypTNM stage (HR = 2.389, P = 0.015), and LRG-sum (HR = 1.020, P = 0.029) were independent prognostic determinants for DFS after NCRT. A nomogram for DFS was developed with a C-index of 0.68 (95%CI 0.64-0.72). CONCLUSION: LRG is an important prognostic indicator for DFS in LARC patients after NCRT. A predictive nomogram based on LRG was developed to guide more tailored adjuvant treatment and surveillance.

A Four Gene-Based Risk Score System Associated with Chemoradiotherapy Response and Tumor Recurrence in Rectal Cancer by Co-Expression Network Analysis.

AIM: Resistance to neoadjuvant chemoradiotherapy (NCRT) and tumor recurrence presents a major clinical problem in locally advanced rectal cancer (LARC) patients. This study aimed to explore a genetic risk score related to NCRT response and tumor recurrence in rectal cancer after NCRT. MATERIALS AND METHODS: Weighted gene co-expression network analysis was employed to identify hub genes associated with NCRT response from the GSE93375 dataset. Prognostic hub genes were determined using Cox regression analysis and associated with disease-free survival (DFS). A risk score system was constructed and the prognostic significance of the risk score was validated in our patient cohort. A predictive nomogram for DFS was developed and validated internally. RESULTS: The Tan module had the highest correlations with NCRT response. Ten hub genes (COL15A1, THBS2, ITGB1, MMP2, CD34, SPARC, NOTCH3, PDGFRB, DCN, and SERPINH1) were associated with NCRT response. Immunostaining expression of four genes (NOTCH3, SPARC, DCN, and ITGB1) was found to be significantly associated with both NCRT response and DFS in our patient cohort and was selected to build a prognostic risk score for DFS as follows: risk score= (0.6188×Exp NOTCH3 ) + (0.6511×Exp SPARC ) + (-0.2976×Exp DCN ) + (1.0035×Exp ITGB1 ). Using this risk score, patients could be separated into high- and low-risk groups for tumor recurrence. A nomogram that incorporated the risk score, ypTNM stage, and tumor regression grade (TRG) was constructed and utilized to predict DFS in LARC patients. CONCLUSION: The four-gene expression-based risk score system presented here could be potentially used for predicting tumor recurrence in LARC patients after NCRT.

Pathological complete response may underestimate distant metastasis in locally advanced rectal cancer following neoadjuvant chemoradiotherapy and radical surgery: Incidence, metastatic pattern, and risk factors.

AIM: To evaluate the pattern of tumor relapse of pathological complete response (pCR) patients with locally advanced rectal cancer (LARC) following neoadjuvant chemoradiotherapy (nCRT) and total mesorectal excision (TME), and to identify predictive factors of distant metastasis in pCR patients after nCRT. METHOD: This was a retrospective analysis of 118 LARC patients who achieved a pCR following nCRT and TME from 2008 to 2015. Clinicopathological and therapeutic parameters were evaluated as possible predictors of distant metastasis-free survival (DMFS), and COX regression analysis was performed. RESULTS: After a median follow-up of 57 months, the 5-year overall and disease-free survival rates were 94.7% and 88.1%, respectively. Overall, 6 patients (5.1%) died, no local recurrence occurred, 13 patients (11%) developed distant metastases, including lung (n = 5), liver (n = 2), bone (n = 3), lung and brain (n = 1), peritoneal (n = 1), and spleen (n = 1) metastasis. On univariate analysis, tumor distance from the anal verge (HR = 0.706, P = 0.039), acellular mucin pools (HR = 6.687, P = 0.002), and MUC1 expression (HR = 8.280, P < 0.001) were independently associated with DMFS. COX regression demonstrated that MUC1 expression (HR = 3.812, P = 0.041) remained to be an independent predictor of DMFS in pCR patients. CONCLUSION: Distant metastasis still remained a major concern in pCR patients following nCRT and TME. Tumor distance from the anal verge, acellular mucin pools, and MUC1 expression were associated with distant metastasis in patients with pCR. MUC1 staining remained to be an independent risk factor for DMFS. Such information could facilitate treatment decision in these patients, such as adjuvant chemotherapy and follow-up.

Pirfenidone prevents radiation-induced intestinal fibrosis in rats by inhibiting fibroblast proliferation and differentiation and suppressing the TGF-ß1/Smad/CTGF signaling pathway.

Radiation-induced intestinal fibrosis (RIF) is a chronic toxicity following radiation, and can be very difficult to treat. Pirfenidone is a promising anti-fibrotic agent that inhibits fibrosis progression in various clinical and experimental studies. This study was aimed to explore whether pirfenidone could protect against RIF, and to evaluate the underlying mechanism. An animal model of RIF was induced by exposure of a single dose of 20 Gy to the pelvis. Rats were orally administered with pirfenidone (200, 400 md/kg/d) for 12 weeks. Primary rat intestinal fibroblasts were cultured to determine the effects of pirfenidone on TGF-ß1-induced (5 ng/ml) proliferation and transdifferentiation of fibroblasts. The expression of collagen I, α-SMA, and TGF-ß1/Smad/CTGF pathway proteins were analyzed by qRT-PCR and/or western blot analysis. The cell proliferation rate was determined by CCK-8 assay. The results indicated that pirfenidone significantly attenuated fibrotic lesion in irradiated intestines and reduced collagen deposition by inhibiting TGF-ß1/Smad/CTGF pathway in rat models. Moreover, in primary rat intestinal fibroblasts, pirfenidone decreased the up-regulation of TGF-ß1-induced collagen I and α-SMA by suppressing TGF-ß1/Smad/CTGF signaling pathway. Altogether, our findings suggested that pirfenidone attenuated RIF by inhibiting the proliferation and differentiation of intestinal fibroblasts and suppressing the TGF-ß1/Smad/CTGF signaling pathway.

Prognostic significance of neoadjuvant rectal score in locally advanced rectal cancer after neoadjuvant chemoradiotherapy and construction of a prediction model.

AIM: To evaluate the prognostic significance of neoadjuvant rectal (NAR) score after neoadjuvant chemoradiotherapy (nCRT) for locally advanced rectal cancer (LARC), and to develop a nomogram predicting disease-free survival (DFS). METHOD: A total of 522 LARC patients undergoing nCRT and surgery were included. NAR scores were calculated using the equation [5pN-3(cT-pT) + 12]^2/9.61, and classified as low (<8), intermediate (8-16), and high (>16). Clinicopathological and survival outcomes were compared. Cox regression analysis was performed to identify risk factors of DFS. A predicting nomogram was developed and validated internally. RESULTS: For NAR score classification, 193 (37.0%) were low, 183 (35.0%) were intermediate, and 146 (28.0%) were high. Higher NAR score was associated with fewer pCR, lower tumor regression grade (TRG), and higher ypTNM stage. A total of 5-year DFS for low, intermediate, and high NAR groups was 85.6%, 71.9%, and 47.2%, respectively (P < 0.001). NAR score (HR = 2.488, P = 0.002), TRG (HR = 2.811, P = 0.047), CRM involvement (HR = 2.703, P = 0.002), and IMA nodal metastasis (HR = 2.441, P = 0.001) were independent prognostic factors of DFS. A predicting nomogram was developed with C-index of 0.701. CONCLUSION: NAR score could help in predicting DFS after nCRT. A nomogram was developed to identify subpopulations with aggressive tumors during clinical decision-making.

B7-H4 expression in bladder urothelial carcinoma and immune escape mechanisms.

B7-H4 is a recently identified member of the B7 family considered to negatively regulate the immune response, and has been associated with the occurrence and development of certain types of tumor. However, little is known regarding the importance of human B7-H4 expression in bladder urothelial carcinoma. In the present study, B7-H4 expression in the tissues and sera of patients with bladder urothelial carcinoma was investigated, along with the clinical significance. In addition, the effects of activated T-lymphocyte in vitro cytotoxicity in the BIU-87 bladder cancer cell line following the blockade of the B7-H4 signaling pathway were also analyzed. The results showed that in normal bladder tissues, B7-H4 was not detected, but in the bladder urothelial carcinoma tissue samples, B7-H4 was detected in 24/49 (49.0%) specimens. Additionally, positive B7-H4 expression was significantly associated with increased TNM stage and pathological grade (P<0.05). Compared with the healthy control group, the serum-B7-H4 (sB7-H4) concentrations in the patients were also significantly increased (P<0.05). The sB7-H4 concentrations in cases with high-grade histology were significantly higher than those in patients with low-grade histology (P<0.05). Following the blockade of the B7-H4 antigen in BIU-87 cells, the cytotoxic activity of activated T cells against such BIU-87 cells was significantly enhanced compared with that against the control BIU-87 cells. This occurred in a T cell density-dependent and blocking antibody dose-dependent manner. These observations suggest that B7-H4 is involved in tumor occurrence, and the development and immune escape of bladder urothelial carcinoma cells. Therefore, B7-H4 may be an important target in the diagnosis and/or treatment of bladder urothelial carcinoma.