An effective cytokine adjuvant vaccine induces autologous T-cell response against colon cancer in an animal model.

BACKGROUND: Despite recent advances in early detection and improvements in chemotherapy for colon cancer, the patients still face poor prognosis of postoperative recurrence and metastasis, the median survival for patients with metastatic colorectal cancer is approximately 22-24 months. Some immunotherapeutic approaches had been attempted in colon cancer patients to significantly increase overall survival. A vaccine based approach has shown a novel direction for colon cancer prevention and therapy. METHODS: In this study, the experiments were designed including prevention and therapeutic stages in order to attain effect against tumor recurrence in clinical settings. The anti-tumor efficacy of a novel cytokine adjuvant vaccine that contained cytokines GM-CSF and IL-2 and inactivated colon CT26.WT whole cell antigen was evaluated in BALB/c mouse tumor models by measuring tumor growth post vaccination and the survival time of tumor-bearing mice, analyzing the expression and distribution of CD4, CD8, CD11c, CD80, CD86 and CD83 positive cells in control and treated mice by flow cytometry and immunochemistry. The tumor-specific cytotoxic T cells (CTL) were analyzed by tumor proliferation and the lactic dehydrogenates (LDH) release assays. IFN-γ, IL-2 and GM-CSF secretion in serum was assayed by ELISA. RESULTS: Our results suggested that cytokine adjuvant vaccine significantly inhibited tumor growth and extended the survival period at least 160d. It was found that the levels of CD8 + T and the tumor-specific cytotoxicity were significantly higher in prevention and treatment group vaccinated by cytokine adjuvant vaccine. CD8 + T cells play a key role in anti-tumor response. CONCLUSIONS: The novel GM-CSF and IL-2 based adjuvant vaccine effectively activated autologous T-cell response and represented a promising immunotherapeutic approach for patients with colon cancer.

Mass spectrometry in measurement of thyroid biomarkers.

In 2022, the number of patients with thyroid disease in China exceeded 200 million (10 million with hyperthyroidism, 90 million with hypothyroidism, and 100 million with other thyroid disease such as goiter, thyroid nodules, and thyroid cancer). Well-established markers include FT3, FT4, TT3, TT4, and TSH tested by a number of immunoassay methods. This approach is based on the primary binding of antigen with antibody and a subsequent secondary chemical reaction that provides an indirect measure. The use of traceable standards for quantitation remains an important factor to ensure inter-assay reliability and precision. Recently, mass spectrometry (MS) has received considerable attention as an analytic tool due to high resolution and quantitative accuracy. In addition, MS allows for sensitive determination of low-abundance markers making it ideal for development of traceable standards. Furthermore, this technology will allow for the development of highly accurate thyroid biomarker assays to facilitate diagnosis, enable early treatment and improve outcomes. Herein, we provide a systematic review and summary of MS in enhancing the analysis of thyroid biomarkers.

Unexpected serum phosphorus lost in an amniotic fluid embolism patient.

BACKGROUND: Serum phosphorus concentration reflects body energy equilibrium and functions of the kidney and the coagulation system. It is regulated by serum calcium concentration and parathyroid hormone (PTH). CASE REPORT: We present a case of constant low concentrations of serum phosphorus in a 34-year-old female who was diagnosed with amniotic fluid embolism (AFE) and was continuously treated with extracorporeal membrane oxygenation (ECMO), continuous renal replacement therapy (CRRT) and blood component transfusion during an observational period of 11 days. CONCLUSION: This case highlights that the release of inorganic phosphorus from platelets and plasma into the blood prompts PTH secretion. The administration of active vitamin D supplement and PTH antagonism should be considered to neutralize the negative regulatory effect of PTH on serum phosphorus and to benefit patients' recovery in the intensive care unit.

IGFBP3 induced by the TGF-ß/EGFRvIII transactivation contributes to the malignant phenotype of glioblastoma.

Dual or multi-targets therapy targeting epidermal growth factor receptor variant III (EGFRvIII) and other molecular may relax the constraint for glioblastoma (GBM), putting forward the urgent requirement of finding candidate molecules. Here, the insulin-like growth factor binding protein-3 (IGFBP3) was considered a candidate, whereas the mechanisms of IGFBP3 production remain unclear. We treated GBM cells with exogenous transforming growth factor ß (TGF-ß) to simulate the microenvironment. We found that TGF-ß and EGFRvIII transactivation induced the activation of transcription factor c-Jun, which specifically bound to the promoter region of IGFBP3 through Smad2/3 and ERK1/2 pathways and promoted the production and secretion of IGFBP3. IGFBP3 knockdown inhibited the activation of TGF-ß and EGFRvIII signals and the malignant behaviors triggered by them in vitro and in vivo. Collectively, our results indicated a positive feedback loop of p-EGFRvIII/IGFBP3 under administration of TGF-ß, blocking IGFBP3 may be an additional target in EGFRvIII-expressing GBM-selective therapeutic strategy.

Construction of Novel Prognostic Nomogram for Mucinous and Signet Ring Cell Colorectal Cancer Patients with a Survival Longer Than 5 Years.

Purpose: Mucinous adenocarcinoma (MA) and signet ring cell carcinoma (SRCC) are aggressive colorectal cancer histological subtypes with dismal prognosis. This study investigated prognostic factors and constructed novel nomograms for MA and SRCC patients who survived for over 5 years to optimize the follow-up regime, especially for early-onset patients. Patients and Methods: Data from the Surveillance, Epidemiology, and End Results (SEER) database registered between 2004 and 2018 were extracted. MA and SRCC patients were divided into two groups with survival time of 5 years as a cut-off point. Prognostic factors for overall survival (OS) and cancer-specific survival (CSS) were determined by Cox regression models, and survival curves were plotted by the Kaplan-Meier method. Results: We identified 8286 MA patients (45.73%) and 551 SRCC patients (20.32%) who survived for over 5 years. Multivariable Cox analyses identified age, tumor location, N stage, metastasis, CEA level, surgery, and lymph nodes dissection as independent risk factors for MACSS. SRCC was more aggressive and only N2 stage (P = 0.011) and metastasis (P = 0.043) were inversely associated with SRCCSS. Furthermore, we observed that small tumor size, well differentiation, and chemotherapy no longer provided survival benefit to ≥5-year survivors. Therefore, we constructed novel nomograms appropriate for MA patients who survived for over 5 years. The consistency indexes for predicting 10-year OS and CSS were respectively 0.717, 0.712 in the training cohort and 0.727, 0.735 in the validation cohort. Conclusion: Our well-calibrated nomograms represent the first clinical prognostic models developed especially for MA patients with a survival longer than 5 years. For both MA and SRCC patients, TNM stage was a stable prognostic factor, while the prognostic values of tumor size, differentiation grade, and chemotherapy changed over time. We are hopeful that our prognostic models will help define personalized follow-up managements to further prolong patient survival.

Single-cell RNA sequencing reveals the characteristics of cerebrospinal fluid tumour environment in breast cancer and lung cancer leptomeningeal metastases.

Leptomeningeal metastases (LM) occur in patients with breast cancer (BC) and lung cancer (LC) showing exceptionally poor prognosis. The cerebrospinal fluid (CSF) tumour microenvironment (TME) of LM patients is not well defined at a single-cell level. Based on the 10× genomics single-cell RNA sequencing (scRNA-seq) data from GEO database including five patient-derived CSF samples of BC-LM and LC-LM, and four patient-derived CSF samples of idiopathic intracranial hypertension (IIH) as controls, we analysed single-cell transcriptome characteristics of CSF TME in LM patients compared to controls simultaneously and comprehensively. In addition, we performed 10× genomics scRNA-seq on CSF cells derived from a BC-LM patient to help generate a solid conclusion. The CSF macrophages in LM patients showing M2-subtype signature and the emergence of regulatory T cells in LM confirmed the direction of tumour immunity toward immunosuppression. Then, the characteristics of CSF circulating tumour cells (CTCs) of breast cancer LM (BC-LM) patients were classified into five molecular subtypes by PAM50 model. The communication between macrophages and five subtype-specific CSF-CTCs showed largest number of ligand-receptor interactions. The five subtypes-specific CSF-CTCs showed great heterogeneities which were manifested in cell proliferation and cancer-testis antigens expression. Gene regulatory networks (GRNs) analysis revealed that transcription factor SREBF2 was universally activated in the five subtypes-specific CSF-CTCs. Our results will provide inspiration on new directions of the mechanism research, diagnosis and therapy of LM.

The immunosuppressive microenvironment and immunotherapy in human glioblastoma.

Glioblastoma multiforme (GBM) is the most malignant intracranial tumor in adults, characterized by extensive infiltrative growth, high vascularization, and resistance to multiple therapeutic approaches. Among the many factors affecting the therapeutic effect, the immunosuppressive GBM microenvironment that is created by cells and associated molecules via complex mechanisms plays a particularly important role in facilitating evasion of the tumor from the immune response. Accumulating evidence is also revealing a close association of the gut microbiota with the challenges in the treatment of GBM. The gut microbiota establishes a connection with the central nervous system through bidirectional signals of the gut-brain axis, thus affecting the occurrence and development of GBM. In this review, we discuss the key immunosuppressive components in the tumor microenvironment, along with the regulatory mechanism of the gut microbiota involved in immunity and metabolism in the GBM microenvironment. Lastly, we concentrate on the immunotherapeutic strategies currently under investigation, which hold promise to overcome the hurdles of the immunosuppressive tumor microenvironment and improve the therapeutic outcome for patients with GBM.

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose.

Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.

MAb NJ001 inhibits lung adenocarcinoma invasiveness by directly regulating TIMP-3 promoter activity via FOXP1 binding sites.

BACKGROUND: Previously, we developed a monoclonal antibody (mAb) NJ001 that binds to the antigen SP70 in human non-small cell lung cancer (NSCLC) cells and showed it could inhibit lung adenocarcinoma (AD) growth. Here, we investigated the effect and mechanisms of NJ001 in lung AD metastasis. METHODS: Human lung AD cells (SPC-A1 and A549) were treated with different concentrations of mAb NJ001, and the effects of NJ001 on cell migration and invasive activity were investigated using wound-healing and Matrigel assays, respectively. The molecular mechanism of this inhibition was explored by microarrays, qRT-PCR, western blot, luciferase assays and electrophoretic mobility shift assays (EMSA). RESULTS: MAb NJ001 markedly suppressed lung AD cell migration; and the invasiveness of SPC-A1 and A549 cells treated with mAb NJ001 was diminished by 65%. Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) was highly expressed in SPC-A1 cells treated with mAb NJ001, whereas knockdown of TIMP-3 by shRNA significantly increased SPC-A1 and A549 invasiveness. MAb NJ001 affects lung AD by inhibiting TIMP-3 through direct transcriptional regulation of FOXP1 binding sites in the TIMP-3 promoter region, as shown in luciferase assays and EMSA. CONCLUSIONS: MAb NJ001 inhibits invasiveness and metastasis in lung AD through the FOXP1 binding sites in the TIMP-3 promoter region. It may have clinical applications in preventing and treating metastatic lung AD.

Long non-coding RNA Lnc-Tim3 exacerbates CD8 T cell exhaustion via binding to Tim-3 and inducing nuclear translocation of Bat3 in HCC.

Although one of the first comprehensive examinations of long non-coding RNA (lncRNA) expression was performed in human CD8 T lymphocytes, little is known about their roles in CD8 T cells functions during the progression of hepatocellular carcinoma (HCC). Here, we show that Lnc-Tim3 is upregulated and negatively correlates with IFN-γ and IL-2 production in tumor-infiltrating CD8 T cells of HCC patients. Lnc-Tim3 plays a pivotal role in stimulating CD8 T exhaustion and the survival of the exhausted CD8 T cells. Mechanistically, Lnc-Tim3 specifically binds to Tim-3 and blocks its interaction with Bat3, thus suppressing downstream Lck/ NFAT1/AP-1 signaling, leading to nuclear localization of Bat3, and enhancing p300-dependent p53 and RelA transcriptional activation of anti-apoptosis genes including MDM2 and Bcl-2. In summary, Lnc-Tim3 promotes T cell exhaustion, a phenotype which is correlated with compromised anti-tumor immunity, suggesting that Lnc-Tim3 and its associated signaling pathways may influence the outcome of cancer therapies aimed at modulating the acquired immune system.

Xiaozhi decoction reduced posthemorrhoidectomy pain and analgesic medication consumption: a prospective study.

OBJECTIVE: The aim of this study was to investigate the effect of Xiaozhi decoction (XZD) on posthemorrhoidectomy pain and analgesic medication consumption. METHODS: From May 2013 to March 2015, 315 patients who underwent open hemorrhoidectomy in our hospital were enrolled in this study, of whom, 160 patients were randomly assigned to accept sitz bath with warm water after hemorrhoidectomy (control group) and 155 patients were randomly assigned to accept sitz bath with XZD (XZD group) after hemorrhoidectomy. Postoperative pain at 12 hours after surgery and on postoperative days (PODs) 1, 2, 7, 14 and 28 was evaluated by Visual Analog Scale (VAS). Pain on defecation on PODs 1, 2, 7, 14 and 28 was also recorded using the VAS. The consumption of analgesics was also analyzed. RESULTS: No significant difference was found in baseline characteristics between the two groups. Postoperative pain score of the XZD group was significantly lower on POD 2 (6.04±1.11 vs 6.33±1.14, P=0.0229), POD 7 (3.35±0.75 vs 4.22±0.87, P=0.0000) and POD 14 (2.87±0.64 vs 3.64±0.77, P=0.0000) than that of the control group. Similarly, patients in the XZD group experienced significantly less pain on defecation on POD 2 (5.02±1.34 vs 5.43±1.56, P=0.0130), POD 7 (3.08±1.17 vs 3.52±1.29, P=0.0017) and POD 14 (2.31±0.85 vs 2.68±0.99, P=0.0004). Patients in the XZD group consumed significantly less analgesic medication on POD 2 (P=0.0136), POD 7 (P=0.0074) and POD 14 (P=0.0046) than the control group. CONCLUSION: XZD could effectively relieve postoperative pain and reduce analgesic medication consumption after hemorrhoidectomy.

GFI1 downregulation promotes inflammation-linked metastasis of colorectal cancer.

Inflammation is frequently associated with initiation, progression, and metastasis of colorectal cancer (CRC). Here, we unveil a CRC-specific metastatic programme that is triggered via the transcriptional repressor, GFI1. Using data from a large cohort of clinical samples including inflammatory bowel disease and CRC, and a cellular model of CRC progression mediated by cross-talk between the cancer cell and the inflammatory microenvironment, we identified GFI1 as a gating regulator responsible for a constitutively activated signalling circuit that renders CRC cells competent for metastatic spread. Further analysis of mouse models with metastatic CRC and human clinical specimens reinforced the influence of GFI1 downregulation in promoting CRC metastatic spread. The novel role of GFI1 is uncovered for the first time in a human solid tumour such as CRC. Our results imply that GFI1 is a potential therapeutic target for interfering with inflammation-induced CRC progression and spread.

[Characterization and application of a monoclonal antibody against light chain of goose immunoglobulin].

Immunoglobulin (Ig) is considered a part of the innate immune system and cooperates with the complementary system as the first line of defense. In this study, a monoclonal antibody (MAb) direct against the light chain of goose Ig (GoIgCL) was generated, characterized and identified in various immunoassays to detect goose Ig. An immunoaffinity chromatography column prepared with this MAb was used to separate the goose Ig from sera. After being conjugated with horseradish peroxidase (HRP), this MAb was used as the secondary antibody to evaluate the goose-specific antibody. In addition, this MAb distinguished and localized the SIg+ lymphocytes from peripheral blood lymphocytes. MAb against GoIgCL may be good candidate to detect or purify goose Ig under various conditions and as a powerful tool for humoral immunity research on goose.

MicroRNA-206 attenuates the growth and angiogenesis in non-small cell lung cancer cells by blocking the 14-3-3ζ/STAT3/HIF-1α/VEGF signaling.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Angiogenesis is the major hallmark in NSCLC. So, further elucidation of molecular mechanisms underlying the angiogenesis of NSCLC is urgently needed. Here, we found that microRNA-206 (miR-206) decreased the angiogenic ability in NSCLC via inhibiting the 14-3-3ζ/STAT3/HIF-1α/VEGF pathway. Briefly, 14-3-3ζ bond with phosphorylated-STAT3, and in turn, elevated the expression of HIF-1α. Then, by enhancing the recruitment of HIF-1α to VEGF promoter, 14-3-3ζ increased the angiogenesis. However, miR-206 decreased the angiogenesis by targeting 14-3-3ζ, and inhibiting the STAT3/HIF-1α/VEGF pathway. In NSCLC cell xenograft model, either overexpression of miR-206 or inhibition of 14-3-3ζ inhibited the STAT3/HIF-1α/VEGF pathway and decreased the tumor growth and angiogenesis. Furthermore, there was a negative correlation between miR-206 and 14-3-3ζ in NSCLC specimens. NSCLC patients with low expressions of miR-206 but high expressions of 14-3-3ζ had the worst survival. Collectively, our findings provided the underlying mechanisms of miR-206/14-3-3ζ in tumor growth and angiogenesis, and implicated miR-206 and 14-3-3ζ as potential therapeutic targets for NSCLC.

EGFRvIII/integrin ß3 interaction in hypoxic and vitronectinenriching microenvironment promote GBM progression and metastasis.

Glioblastoma (GBM) is one of the most lethal brain tumors with a short survival time. EGFR amplification and mutation is the most significant genetic signature in GBM. About half of the GBMs with EGFR amplification express a constitutively autophosphorylated variant of EGFR, known as EGFRvIII. Our in vitro data demonstrated further enhanced EGFRvIII activity and tumor cell invasion in the tumor microenvironment of hypoxia plus extracellular matrix (ECM) vitronectin, in which EGFRvIII and integrin ß3 tended to form complexes. The treatment with ITGB3 siRNA or the integrin antagonist cilengetide preferentially interrupted the EGFRvIII/integrin ß3 complex, effectively reduced tumor cell invasion and activation of downstream signaling effectors. Cilengitide is recently failed in Phase III CENTRIC trial in unselected patients with GBM. However, we found that cilengitide demonstrated efficacious tumor regression via inhibition of tumor growth and angiogenesis in EGFRvIII orthotopic xenografts. Bioinformatics analysis emphasized key roles of integrin ß3, hypoxia and vitronectin and their strong correlations with EGFRvIII expression in malignant glioma patient samples in vivo. In conclusion, we demonstrate that EGFRvIII/integrin ß3 complexes promote GBM progression and metastasis in the environment of hypoxia and vitronectin-enrichment, and cilengitide may serve as a promising therapeutics for EGFRvIII-positive GBMs.

Grouping annotations on the subcellular layered interactome demonstrates enhanced autophagy activity in a recurrent experimental autoimmune uveitis T cell line.

Human uveitis is a type of T cell-mediated autoimmune disease that often shows relapse-remitting courses affecting multiple biological processes. As a cytoplasmic process, autophagy has been seen as an adaptive response to cell death and survival, yet the link between autophagy and T cell-mediated autoimmunity is not certain. In this study, based on the differentially expressed genes (GSE19652) between the recurrent versus monophasic T cell lines, whose adoptive transfer to susceptible animals may result in respective recurrent or monophasic uveitis, we proposed grouping annotations on a subcellular layered interactome framework to analyze the specific bioprocesses that are linked to the recurrence of T cell autoimmunity. That is, the subcellular layered interactome was established by the Cytoscape and Cerebral plugin based on differential expression, global interactome, and subcellular localization information. Then, the layered interactomes were grouping annotated by the ClueGO plugin based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. The analysis showed that significant bioprocesses with autophagy were orchestrated in the cytoplasmic layered interactome and that mTOR may have a regulatory role in it. Furthermore, by setting up recurrent and monophasic uveitis in Lewis rats, we confirmed by transmission electron microscopy that, in comparison to the monophasic disease, recurrent uveitis in vivo showed significantly increased autophagy activity and extended lymphocyte infiltration to the affected retina. In summary, our framework methodology is a useful tool to disclose specific bioprocesses and molecular targets that can be attributed to a certain disease. Our results indicated that targeted inhibition of autophagy pathways may perturb the recurrence of uveitis.

Development and evaluation of a competitive ELISA using a monoclonal antibody for antibody detection after goose parvovirus virus-like particles (VLPs) and vaccine immunization in goose sera.

An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based C-ELISA) for detecting antibodies against goose parvovirus (GPV) and its virus-like particles (VLPs) is described. The assay was developed using baculovirus-expressed recombinant VP2 virus-like particles (rVP2-VLPs) as antigens and a monoclonal antibody against GPV as the competitive antibody. Of the four anti-GPV MAbs that were screened, MAb 1G3 was selected as it was blocked by the GPV positive serum. Based on the distribution of percent inhibition (PI) of the known negative sera (n=225), a cut-off value was set at 36% inhibition. Using this cut-off value, the sensitivity of the assay was 93.3% and the specificity was 95.8%, as compared with the gold standard (virus neutralization assay). The rVP2-VLPs did not react with anti-sera to other goose pathogens, indicating that it is specific for the recognization of goose parvovirus antibodies. The assay was then validated with serum samples from goslings vaccinated with several VLPs (rVP1-VLPs, rVP2-VLPs, rVP3-VLPs, and rCGV-VLPs) and other vaccines (inactivated and attenuated). The C-ELISA described in this study is a sensitive and specific diagnostic test and should have wide applications for the sero-diagnosis and immunologic surveillance of GPV.

A network-based, integrative approach to identify genes with aberrant co-methylation in colorectal cancer.

Epigenetic changes, including aberrations in DNA methylation, are a common hallmark of many cancers. The identification and interpretation of epigenetic changes associated with cancers may benefit from integration with protein interactomes. Based on the assumption that genes implicated in a specific tumor phenotype will show high aberrant co-methylation patterns with their interacting partners, we propose an integrated approach to uncover cancer-associated genes by integrating a DNA methylome with an interactome. Aberrant co-methylated interactions were first identified in the specific cancer, and genes were then prioritized based on their enrichment in aberrant co-methylation. By applying this to a large-scale colorectal cancer (CRC) dataset, the proposed method increases the power to capture known genes. More importantly, genes possessing high aberrant co-methylation patterns, located at the topological center of the original protein-protein interaction network (PPIN), affect several cancer-associated pathways and form hotspots that are frequently hijacked in cancer. Additionally, the top-ranked candidate genes may also be useful as an indicator of CRC diagnosis and prognosis. Five fold cross-validation of the top-ranked genes in diagnosis reveals that it can achieve an area under the receiver operating characteristic (ROC) curve ranging from 82.2% to 98.4% in three independent datasets. Five of these genes form a core repressive module. CCNA1 and ESR1 in particular are evidently silenced by promoter hypermethylation in CRC cell lines and tissues, whose re-expression markedly suppresses tumor cell survival and clonogenicity. These results show that the network-centric method could identify novel disease biomarkers and model how oncogenic lesions mediate epigenetic changes, providing important insights into tumorigenesis.