Potentiated effects of lactate receptor GPR81 on immune microenvironment in breast cancer.

G protein-coupled receptor (GPR81), as lactate receptor, is an upstart in immune regulation, however, its mechanisms involved in tumor escape have not been fully elucidated. In this study, we explored the effects of GPR81 activation on triple-negative breast cancer (TNBC) cells and macrophages. The expression and relationship with immune infiltration of GPR81 were analyzed with TCGA database. Checkpoints and cytokines were evaluated with flow cytometry or ELISA. The TCGA-based data showed a marked decrease of GPR81 in breast cancer (BRCA) compared with normal breast, especially in the basal-like subtype. In normal mammary tissues, GPR81 had negative correlation with various immune checkpoints, nevertheless, this trend weakened accompanied with the reduction of GPR81. GPR81 stimulation had a significantly inhibitory influence on PD-L1 exposure in BT-549 and MDA-MB-231 cell lines, but not in MDA-MB-453 cell line. The pretreatment of siGPR81 to knockdown GPR81 expression resulted in a remitting of PD-L1 reduction when MDA-MB-231 cells were treated with GPR81 agonist 1. However, little effect of GPR81 activation was observed on the expression of PD-L1 on phorbol-12-myristate-13-acetate (PMA)-induced THP-1 cells. Furthermore, GPR81 agonist 1 exerted no significant impact on the secretion of cytokines in THP-1 cells. In general, it is suggested that GPR81 may facilitate immune monitoring via the reduction of PD-L1 in TNBC with glycolytic phenotype. Our results not only provide a novel insight into the effects of GPR81 on immune evasion but a potential therapy targeting GPR81 in BRCA.

Correlation Between IL-8 Gene Polymorphisms and Pathogenesis of Crohn's Disease.

Context: The risk of inflammatory bowel disease (IBD) is substantially heightened if patients' first-degree relatives have it. The genetic and immune factors related to the disease have attracted great attention, including patients innate genetic polymorphisms. Interleukin-8 (IL-8) plays a vital role in digestive-system diseases, especially in gastrointestinal diseases. Objective: The study intended to explore the expression of interleukin-8 (IL-8) in the colon tissues of patients with Crohn's disease and the correlation between its polymorphisms and the disease's occurrence. Design: The research team performed a prospective study. Setting: The study took place in the Department of Gastroenterology at Zhuji People's Hospital of Zhejiang Province in Zhuji, China. Participants: Participants were 100 patients with Crohn's disease at the hospital between November 2016 and June 2018 and 100 healthy individuals. The research team assigned participants with Crohn's disease to the Crohn's disease group and the healthy participants to the control group. Outcome Measures: The research team: (1) determined differences in the protein expression of the IL-8 between the groups; (2) examined the conformity of the data to that of the Hardy-Weinberg equilibrium; (3) analyzed the differences in the genotypes and alleles for the IL-8 single nucleotide polymorphisms (SNPs) rs102039, rs103284 and rs105432 between the groups; and (4) for the Crohn's disease group, examined the differences in the disease's location and behavior for the participants with different genotypes. Results: The protein expression level of IL-8 in the colon tissues in Crohn's disease group was significantly higher than that in control group (P < .05). The genetic association analysis showed significant correlations between the polymorphisms rs103284 and rs105432 and alleles of the IL-8 gene and the occurrence of Crohn's disease (P < .05), but no associations existed between the gene polymorphism rs102039 and alleles and Crohn's disease (P > .05). Significant correlations existed between the IL-8 gene polymorphisms rs103284 and rs105432 and the disease's location and behavior (P < .05). Conclusions: IL-8 had a significantly increased expression in the colon tissues of the participants with Crohn's disease, and some genotypes and alleles for the gene polymorphisms rs103284 and rs105432 were significantly higher in the Crohn's disease group than in the control group. In addition, the disease's location and behavior were significantly different for participants in the Crohn's disease group with different genotypes.

Radiation Therapy Changed the Second Malignancy Pattern in Rectal Cancer Survivors.

Background and Objectives: Radiotherapy (RT) plays an important role in the treatment for locally advanced rectal cancer patients. It can bring radio exposure together with the survival benefit. Cancer survivors are generally at an increased risk for second malignancies, and survivors receiving RT may have higher risks than survivors not receiving RT. Whether the risk of an all-site second malignancy may increase after RT is still debated. This study aims to compare the second malignancy pattern in rectal cancer survivors after RT. Materials and Methods: The Surveillance, Epidemiology, and End Results (SEER) database was used for analysis. In total, 49,961 rectal cancer patients (20-84 years of age) were identified between 2000 and 2012 from 18 SEER registries. All patients underwent surgery. The occurrence of second malignancies diagnosed after rectal cancer diagnosis was compared in patients who received and did not receive RT. The standardized incidence ratios (SIRs) with 95% confidence intervals (CIs) were used. SEER*Stat was used to generate the 95% CIs for the SIR statistics using the exact method. Results: Of the total 49,961 patients, 5582 developed second malignancies. For all-site second primary malignancies, the age-adjusted SIRs were 1.14 (95% CI 1.1-1.18) and 1.00 (95% CI 0.96-1.04) in the no RT and RT groups, respectively. In 23,192 patients from the surgery-only group, 2604 had second malignancies, and in 26,769 patients who received RT, 2978 developed second malignancies. With respect to every site, the risk of secondary prostate cancer was significantly lower in the RT group (SIR = 0.39, 95% CI 0.33-0.46) than that in the surgery-only group (SIR = 1.04, 95% CI 0.96-1.12). Moreover, the risk of thyroid cancer was significantly higher in the RT group (SIR = 2.80, 95% CI 2.2-3.51) than that in the surgery-only group (SIR = 1.29, 95% CI 0.99-1.66). Conclusions: RT may change the second malignancy pattern in rectal cancer survivors; the risk of prostate cancer decreased, and the risk of thyroid cancer increased most significantly.

Sustained expression of HPV16 E7 oncoprotein promotes p-AKT(Ser473)/p-Src(Tyr527) signaling to drive precancerous lesions to invasive cervical cancer.

Human papillomavirus (HPV) E7 oncogene plays the most important role in cervical cancer. However, whether E7 oncoprotein is continuously expressed, associated with AKT(Ser473)/p-Src(Tyr527) signaling to trigger cervical carcinogenesis remains unclear. Here, we explored first if HPV16 E7 oncoprotein could be detected in clinical biopsies and is sustainedly expressed, and then investigated how this oncoprotein interacted with AKT(Ser473)/p-Src(Tyr527) signaling in cancer progression. We used ZHPV16E7384 affibody to detect E7 expression in HPV16-positive cervical cancer biopsies and animal tumors by immunohistochemistry (IHC). Results showed that ZHPV16E7384 affibody had intense and specific staining for E7 oncoprotein in the detected specimen. The E7 oncoprotein was continuously expressed to correspond with the development of precancerous lesions to invasive cervical cancer. IHC staining also revealed that AKT, p-AKT(Ser473), Src and p-Src(Tyr527) proteins were expressed in both patient biopsies and animal tumors, with the highest levels of p-AKT(Ser473)/p-Src(Tyr527) present in invasive cancer. Furthermore, siRNA experiments revealed that HPV16 E7 knockdown significantly impaired expression of p-AKT(Ser473)/p-Src(Tyr527) in both HPV16 E7-positive cancer cells and transformed cells. In addition, transient expression of HPV16 E7 protein promoted significantly expression of p-AKT(Ser473)/p-Src(Tyr527) in primary human keratinocytes. Finally, co-immunoprecipitation analysis proved that HPV 16 E7 protein interacted reciprocally with p-AKT(Ser473)/p-Src(Tyr527). In conclusion, we demonstrate that HPV16 E7 oncoprotein is continuously expressed to promote expression of p-AKT(Ser473)/p-Src(Tyr527) leading to drive the initiation and progression of cervical cancer. Our data provide a novel insight that HPV16 E7 activates p-AKT(Ser473)/p-Src(Tyr527) to establish a mechanistic link between the oncogene and the AKT/Src signaling to trigger cervical carcinogenesis.

A clinical prediction model for the presence of brain metastases from colorectal cancer.

BACKGROUND: We conducted this study to explore clinicopathological profiles of brain metastases (BM) and establish a clinical prediction model that predicts the presence of BM in colorectal cancer (CRC) patients. METHODS: Patients with initially diagnosed CRC were reviewed between the year 2010 and 2015. Multiple imputations are used for handling missing values. Prognostic factors were identified by the univariate and multivariate Cox regression model. Univariate and multivariate logistic regression was used to identify the predictive factors for the presence of BM. A nomogram was constructed based on statistically significant risk factors of the presence of BM. The decision curve analysis (DCA) was used to assess the clinical usefulness and net benefits of the nomogram for the presence of BM. RESULTS: Four hundred ninety-five patients with brain metastasis at the initial diagnosis were identified, representing 0.24% of the whole cohort and 0.91% of the metastatic cohort. Multivariable logistic regression demonstrated that young age, positive CEA, adenocarcinoma, lower differentiated grade, presence of liver metastases, presence of lung metastases, and presence of bone metastases were significantly associated with higher risk of developing BM. The decision curve analysis inform clinical decisions were better than a scenario in which all patients or no patients are screened across a wide range of threshold at ≥ 0.027%. CONCLUSIONS: The risk estimates provided by the nomogram can be extremely useful for earlier diagnosis, especially when discussing screening strategy among high-risk patients.

Novel EBV LMP1 C-terminal domain binding affibody molecules as potential agents for in vivo molecular imaging diagnosis of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) latent infection and is common in Southern China and Southeast Asia. The viral latent membrane proteins LMP1 and LMP2 are persistently expressed in NPC tissues; the cytoplasmic domain of LMP1 (LMP1 C-terminal) and LMP2A (LMP2A N-terminal) proteins is essential for maintenance of latency and can alter host cell signaling to facilitate tumor growth and progression. Thus, targeting LMP1 or LMP2 oncoprotein has been an increasing interest for diagnosis and targeted therapy of NPC. Affibody molecules, a new class of small-affinity engineered scaffold proteins, have demonstrated high potential for therapeutics, diagnostics, and biotechnological applications. More recently, radiolabelled HER2-specific affibody molecules have demonstrated to be useful in imaging of HER2 expressing tumor. In this study, we report three novel EBV LMP1 C-terminal (EBV LMP1-C) domain affibody molecules (ZLMP1-C15, ZLMP1-C114, and ZLMP1-C277) were selected by biopanning from a random-peptide displayed phage library and used for molecular imaging in tumor-bearing nude mice. Surface plasmon resonance (SPR), indirect immunofluorescence, and immunohistochemistry (IHC) clearly showed that all three selected affibody molecules have high affinity and specificity in binding to EBV LMP1 protein. Moreover, in vivo tumor imaging revealed that Dylight-755-labeled affibody molecules accumulated rapidly in tumor site after injection (1 h) and then were continuously maintained for 24 h in EBV-positive NPC xenograft mice model. In conclusion, our findings highlight the potential use of ZLMP1-C affibody molecules as tumor-specific molecular imaging agents of EBV-associated NPC.Key points• We screened three novel affibody molecules (ZLMP1-C15, ZLMP1-C114, and ZLMP1-C277) targeting EBV LMP1-C terminal domain• ZLMP1-C recognize the recombinant and native LMP1-C with high affinity and specificity• ZLMP1-C can be used for molecular imaging.

The Undervalued Acute Leukopenia Induced By Radiotherapy In Cervical Cancer.

BACKGROUND: Myelosuppression is common and threatening during tumor treatment. However, the effect of radiation on bone marrow activity, especially leukocyte count, has been underestimated in cervical cancer. The aim of this study was to evaluate the severity of radiotherapy- induced acute leukopenia and its relationship with intestinal toxicity. METHODS: The clinical data of 59 patients who underwent conventional radiation alone for cervical cancer were retrospectively analyzed. The patients had normal leukocyte count on admission, and the blood cell count, gross tumor volume (GTV) dose, and intestinal toxicity were evaluated. RESULTS: During radiotherapy (RT), 47 patients (79.7%) developed into leukopenia, with 38.3% mild and 61.7% moderate. The mean time for leukopenia was 9 days. Compared with leukopenianegative patients, leukopenia-positive ones had lower baseline leukocyte count, while neutrophil/ lymphocyte (NLR) and monocyte/lymphocyte (MLR) showed no significance. Logistic regression analysis indicated that excluding the factors for age, body mass index (BMI), TNM stage, surgery and GTV dose, baseline leukocyte count was an important independent predictor of leukopenia (OR=0.383). During RT, a significant reduction was found in leukocyte, neutrophil and lymphocyte count at week 2 while monocyte count after 2 weeks. Furthermore, NLR and MLR showed a significant and sustained upward trend. About 54.2% of patients had gastrointestinal symptoms. However, no significant relevance was noted between leukocyte count as well as NLR/MLR and intestinal toxicity, indicating leukopenia may not be the main factor causing and aggravating gastrointestinal reaction in cervical cancer. CONCLUSION: Our results suggest the underrated high prevalence and severity of leukopenia in cervical cancer patients receiving RT, and those with low baseline leukocyte count are more likely for leukopenia, for whom early prevention of infection may be needed during RT.

[Preparation and identification of rabbit anti-cyclin dependent kinase 6 (CDK6) antibodies].

Objective To prepare and identify rabbit anti-cyclin dependent kinase 6 (CDK6) antibody. Methods The recombinant pET21a (+)/CDK6 was successfully constructed, then the recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells and was induced by isopropyl-ß-D-thiogalactopyranoside (IPTG) for protein expression, which was detected by SDS-PAGE and Western blot analysis. The expressed protein was purified by nickel-chelating nitrilotriacetic acid (Ni-NTA) agarose and then analyzed by SDS-PAGE. Japanese white rabbits were immunized with purified CDK6 protein for many times every two weeks. The blood was collected at 0, 2, 4 and 6 weeks after immunization, and serum was separated from blood. The titer was detected by indirect ELISA. Western blot analysis, immunofluorescence assay and immunohistochemistry were employed to determine the specificity. Results High purity CDK6 protein and high specificity of rabbit anti-CDK6 antibody were successfully prepared. The titer of CDK6 rabbit serum antibody reached 1:30 000 after immunization, which could specifically recognize the CDK6 protein expressed in cervical cancer cell line and cervical cancer tissues. Conclusion The high titer and specificity of rabbit anti-CDK6 antibody is successfully prepared.

Acute small bowel obstruction: a rare initial presentation for the metastasis of the large-cell carcinoma of the lung.

We present one case with symptom of paroxysmal abdominal pain for over 20 days. Abdominal computerized tomography (CT) scan revealed intestinal obstruction and a mass of 6.0 cm × 6.0 cm in size located at the left adrenal. Chest CT scan showed a lobulated mass of 2.7 cm × 2.7 cm in size at the upper left lung. Core needle biopsy of the lung mass confirmed the diagnosis of large cell carcinoma. The patient underwent an emergency abdominal laparotomy and received a chemotherapy regimen that consisted of pemetrexed and cisplatin postoperatively. In addition, we made a review of the literature of the occurrence, diagnosis and outcome of this manifestation.

[K-ras gene mutation in colorectal cancer and its clinicopathologic significance].

OBJECTIVE: To establish a simple, rapid and economical method in detecting mutations of oncogene K-ras and to investigate its mutations in colorectal cancer tissues and its relationship with clinicopathologic characteristics of colorectal carcinoma. METHODS: Forty colorectal cancer tissues were tested for K-ras mutations at codon 12 and codon 13 using polymerase chain reaction (PCR) followed by direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by sequence analysis. The other 113 colorectal cancer tissues were tested for K-ras mutations at codon 12 and codon 13 using PCR-RFLP followed by sequence analysis only. The mutation results were analyzed with the corresponding clinical pathological data. RESULTS: Among 40 colorectal cancer cases, none of K-ras mutations at codon 12 and codon 13 was detected by PCR followed by direct sequencing. However, K-ras mutations were found in 11 cases (11/40, 27.5%) by PCR-RFLP followed by sequence analysis, including 8 cases at codon 12 and 3 cases at codon 13 respectively. Among 153 colorectal cancer cases, point mutations were detected by PCR-RFLP followed by sequence analysis in 58 cases (37.9%). Point mutations at codon 12 were found in 46 cases and 12 cases at codon 13. Mutations with the highest frequency were G→A transitions (25/58, 43.1%) at codon 12. No significant correlation was observed between mutations of K-ras and gender, invasive depth, tumor differentiation, number of invaded lymph nodes, distant metastasis and clinical stage (P > 0.05). Mutation of oncogene K-ras at codon 12 and codon 13 was closely related with age and tumor location (P < 0.05). The incidence of K-ras mutation was significantly higher in younger patients and in patients with ascending colon cancer. CONCLUSIONS: PCR-RFLP followed by sequence analysis is a rapid, simple, sensitive and low-cost method. It is a suitable technology for detecting hot-spot mutations in the K-ras oncogene. Mutation of oncogene K-ras at codon 12 and codon 13 is a common molecular event in colorectal carcinogenesis, which might be related with age and tumor location.

Novel ELISA for serodiagnosis of nasopharyngeal carcinoma based on a B cell epitope of Epstein-Barr virus latent membrane protein 2.

Epstein-Barr virus (EBV) is widespread and is associated with nasopharyngeal carcinoma (NPC). Serological detection of EBV is commonly used for screening, diagnosis and epidemiological surveys of NPC. In the present study, a novel B cell multi-epitope peptide fusion protein (EBV-LMP2-3B), which is composed of three B cell linear epitopes (RIEDPPFNSLL, TLNLT and KSLSSTEFIPN) of EBV latent membrane protein 2 (LMP2), was expressed in a prokaryotic expression system and purified using Ni2+-nitrilotriacetate-Sepharose. The immunogenicity and binding specificity of EBV-LMP2-3B were evaluated on the basis of antibody responses in immunized BALB/c mice, western blotting and indirect immunofluorescence assay. Evaluation of EBV-LMP2-3B as a serological diagnostic reagent was performed using an indirect ELISA in 198 patients with NPC and 102 healthy adults. These results revealed that EBV-LMP2-3B was able to eliminate the high-titer serum antibody response in BALB/c mice. Western blot analysis and indirect immunofluorescence assay confirmed that the mouse immune sera recognized the native LMP2. Compared with healthy adults, patients with NPC demonstrated significantly greater reactivity to EBV-LMP2-3B (P<0.05). Furthermore, it was possible to effectively detect specific IgG in sera from patients with NPC, with a sensitivity of 91.91% and specificity of 93.14%, representing an improvement over the traditional viral capsid antigen-IgA-based detection method with 59.59% sensitivity and 75.49% specificity. In conclusion, the EBV-LMP2-3B protein may be used as a serological diagnostic reagent to screen for and diagnose NPC.

Evaluation of tandem Chlamydia trachomatis MOMP multi-epitopes vaccine in BALB/c mice model.

Chlamydia trachomatis (Ct), an obligate intracellular parasite, is the leading cause of bacterial sexually transmitted diseases worldwide. The best solution to control the spread of Ct is to develop safe and effective vaccines. However, an effective vaccine has not been developed due to some challenges such as selection of appropriate candidate antigens and an effective delivery system. In our previous study, we have developed a Ct vaccine that comprises a multi-epitope peptide of Ct major outer membrane protein (MOMP370-387) and Hepatitis B virus core antigen (HBcAg). The vaccine was evaluated in a murine model with chlamydial genital infection. The results indicated that Ct MOMP multi-epitope delivered by HBcAg could be an effective vaccine for the prevention of Ct. In this study, another two epitopes were selected from the MOMP protein and tandemly linked with MOMP370-387 to enhance the immunogenicity and the protective effect of the candidate vaccine. Our results revealed that both the immunogenicity and the protective effect of the tandem Ct MOMP multi-epitopes were much better than that of the single epitope. Therefore, vaccines based on the tandem Ct MOMP multi-epitopes could be more effective immune prophylactics to prevent Ct infection than the single epitope in murine model system.

Thyroid hormone level is associated with the frequency and severity of acute transverse myelitis.

Acute transverse myelitis (ATM) is a progressive and autoimmune disease with inflammatory cell infiltrates into the spinal cord, and thyroid hormone (TH) level is associated with the oxidative and antioxidant status. Variations in oxidative stress and antioxidant levels are related to the pathogenesis of autoimmune and inflammatory diseases. Our study aimed to investigate the possible correlation between ATM and TH levels of thyroid-stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3), and FT4/FT3. We measured serum concentrations of TSH, FT4, and FT3 in 205 individuals, including 42 ATM patients, 49 multiple sclerosis patients, and 114 healthy controls. Our findings show that ATM patients had lower levels of TSH and FT3 and higher levels of FT4 and FT4/FT3 compared with healthy controls, whether male or female. Moreover, levels of TSH and FT3 in patients with ATM were inversely correlated with disease severity measured by the Expanded Disability Status Scale. Variations in TH level may represent the oxidative status and are surrogate biomarkers of the incidence and severity of ATM.

Solitary pulmonary mass in a patient with a history of lymphoma: a case report.

INTRODUCTION: With the progress made in treatments, the survival rate for patients with malignant lymphoma in the last 30 years has significantly improved. However, the risk of experiencing a second primary malignancy or other disease has increased significantly. CASE PRESENTATION: A 44-year-old Mongolian man with a large mass in his right lower abdomen was admitted to our hospital 15 years previously. The mass was removed, and confirmed via pathological examination to be a malignant B-cell lymphoma in the appendix and distal small bowel. Post-operative chemotherapy with standard cyclophosphamide, hydroxydaunomycin, vincristine (Oncovin®) and prednisolone regimen was given for six cycles. No obvious recurrence was detected over the following 12 years. Subsequently, a mass in the right lung was found on a regular X-ray follow-up; our patient did not report chills, fever or cough. Chest computed tomography and positron emission tomography scans confirmed the mass. A primary lung carcinoma was considered to be the most likely diagnosis. However, after an exploratory thoracotomy and right upper lobectomy was performed a pathological examination of tissue samples demonstrated a lung cryptococcal granuloma, with positive staining for periodic acid Schiff and periodic acid-silver metheramine. CONCLUSIONS: Compared to the normal population, second primary malignancy (in particular leukaemia and lung cancer) in patients with malignant lymphoma during their long-term survival has been seen occasionally. However, other diagnoses should also be considered such as pulmonary cryptococcosis. Other than computed-tomography-guided needle biopsy, surgery for some patients is a much more appropriate choice, which could also help attain correct diagnosis and treatment.

Clinical significance of K-ras and BRAF mutations in Chinese colorectal cancer patients.

AIM: To identify and assess mutations in the K-ras and BRAF genes in a cohort of Chinese patients with colorectal cancer (CRC) for their association with various clinicopathological parameters and prognosis. METHODS: Genomic DNA was isolated from frozen tissues. Pyrosequencing analysis was conducted to detect mutations in the K-ras (codons 12, 13, and 61) and BRAF genes (codon 600). Statistical analysis was carried out using SPSS-15.0 software. RESULTS: Among the 118 colorectal cancer patients, we detected 41 (34.7%) mutations in the K-ras gene. Mutation frequencies at codon 12 and codon 13 were 23.7% (28/118) and 10.2% (12/118), respectively. Only one patient harbored a point mutation at codon 61 (0.8%, 1/118). Gender was the only factor that showed an obvious relationship with K-ras gene mutation (female 44.7% vs male 28.2%, P = 0.037). Other clinicopathological features, such as age, location of the tumor, tumor differentiation, Tumor, Node and Metastases classification, and the Union for International Cancer Control staging, showed no positive relationship with K-ras gene mutations. No significant correlation was observed between the presence of K-ras mutations (codons 12, 13, and 61) and the survival of the patients. BRAF mutations were rare, and only two patients (1.7%) harbored a detectable mutation at codon 600. CONCLUSION: K-ras gene mutation is a common event in our 118 Chinese CRC patients, with an obvious relationship with gender. However, it seems not to be an independent prognostic factor in CRC patients. The BRAF gene is rarely mutated in Chinese CRC patients.