Lymphocyte Protein Research Articles

MAL2 interacts with IQGAP1 to promote pancreatic cancer progression by increasing ERK1/2 phosphorylation

Pancreatic cancer is a digestive tract malignancy characterized by an occult onset and rapid progression. The genetic heterogeneity of pancreatic cancer is closely related to its highly malignant biological behavior. The myelin and lymphocyte protein 2 (MAL2) is upregulated in multiple cancers at the transcriptional level. However, the exact role of MAL2 in pancreatic cancer remains elusive. In this study, we demonstrated that MAL2 protein and mRNA levels were upregulated in pancreatic cancer. MAL2 overexpression was significantly associated with poor prognosis in patients with pancreatic cancer. We further showed that MAL2 interacted with IQGAP1 to increase ERK1/2 phosphorylation levels, which promoted pancreatic cancer progression. Therefore, these results suggest that MAL2 could be a novel therapeutic target for pancreatic cancer.

Targeted regulation of lymphocytic ER stress response with an overall immunosuppression to alleviate allograft rejection

Transplantation is the most effective, and sometimes the only resort for end-stage organ failure. However, allogeneic graft suffers greatly from lymphocyte-mediated immunorejection, which bears close relationship with a hyperactivation of endoplasmic reticulum (ER) stress response in host lymphocytes, especially in CD8+ T cells (T-8). Therefore, regulating lymphocytic ER unfolded protein response (UPR) might be a potential therapeutic breakthrough in alleviating graft rejection. Here, ER-targetable liposome is prepared via the surface modification of ER-targeting peptide (Pardaxin), which efficiently loads and directly delivers small molecule inhibitor of UPR sensor IRE1α into the ER of lymphocytes, inducing a systemic immunosuppression that facilitates tumorigenesis and metastasis in the tumor inoculation challenge in vivo. And in vitro, a stage-differential dependency of IRE1α in the phase transition of T-8 is identified. Specifically, inhibiting IRE1α at the early responding stages of T-8, especially at the activation phase, results in a shrunk proliferation, impaired effector function, and limited memory commitment, which might contribute centrally to the induced overall immunosuppression. Based on this, a classical acute rejection model, murine full-thickness trunk skin allograft that primary arises from the hyperactivity of T-lymphocyte, is used. Results suggest that lymphocytic IRE1α inactivation attenuates transplant rejection and prolongs graft survival, with a limited effector function and memory commitment of host T-8. Moreover, an even higher immunosuppressive effect is obtained when IRE1α inhibition is used in combination with immunosuppressant tacrolimus (FK506), which might owe to a synergistic regulation of inflammatory transcription factors. These findings provide a deeper insight into the biological polarization and stress response of lymphocytes, which might guide the future development of allogeneic transplantation.

Combination of lymphocyte count and albumin concentration as a new prognostic biomarker for rectal cancer

Although numerous studies have highlighted the prognostic values of various inflammation-related markers, clinical significance remains to be elucidated. The prognostic values of inflammation-related biomarkers for rectal cancer were investigated in this study. A total of 448 patients with stage II/III rectal cancer undergoing curative resection were enrolled from the discovery cohort (n = 240) and validation cohort (n = 208). We comprehensively compared the prognostic values of 11 inflammation-related markers-derived from neutrophil, lymphocyte, platelet, monocyte, albumin, and C-reactive protein for overall survival (OS) and recurrence-free survival (RFS). Among 11 inflammation-related markers, only “lymphocyte × albumin (LA)” was significantly associated with both OS and RFS in the discovery cohort (P = 0.007 and 0.015, respectively). Multivariate analysis indicated that low LA was significantly associated with poor OS (hazard ratio [HR] 2.19, 95% confidence interval [CI] 1.09–4.58, P = 0.025), and poor RFS (HR 1.61, 95% CI 1.01–2.80, P = 0.048). Furthermore, using the discovery cohort, we confirmed that low LA was significantly associated with poor OS (HR 2.89, 95% CI 1.42–6.00, P = 0.002), and poor RFS (HR 1.79, 95% CI 1.04–2.95, P = 0.034). LA can be a novel prognostic biomarker for stage II/III rectal cancer.

Runx3 Induces a Cell Shape Change and Suppresses Migration and Metastasis of Melanoma Cells by Altering a Transcriptional Profile.

Runt-related transcription factor-3 (Runx3) is a tumor suppressor, and its contribution to melanoma progression remains unclear. We previously demonstrated that Runx3 re-expression in B16-F10 melanoma cells changed their shape and attenuated their migration. In this study, we found that Runx3 re-expression in B16-F10 cells also suppressed their pulmonary metastasis. We performed microarray analysis and uncovered an altered transcriptional profile underlying the cell shape change and the suppression of migration and metastasis. This altered transcriptional profile was rich in Gene Ontology/Kyoto Encyclopedia of Genes and Genomes (GO/KEGG) annotations relevant to adhesion and the actin cytoskeleton and included differentially expressed genes for some major extracellular matrix (ECM) proteins as well as genes that were inversely associated with the increase in the metastatic potential of B16-F10 cells compared to B16-F0 melanoma cells. Further, we found that this altered transcriptional profile could have prognostic value, as evidenced by myelin and lymphocyte protein (MAL) and vilin-like (VILL). Finally, Mal gene expression was correlated with metastatic potential among the cells and was targeted by histone deacetylase (HDAC) inhibitors in B16-F10 cells, and the knockdown of Mal gene expression in B16-F0 cells changed their shape and enhanced the migratory and invasive traits of their metastasis. Our study suggests that self-entrapping of metastatic Runx3-negative melanoma cells via adhesion and the actin cytoskeleton could be a powerful therapeutic strategy.

Effects of Tripterine on Adhesion Molecules and Cell Cycle in Human Acute Promyelocytic Leukemia Model Mice

To observe the effects of tripterine on adhesion molecules and cell biological characteristics in mice with acute promyelocytic leukemia (APL) tumor. Eighteen SCID beige mice were caudal vein injected with NB4 cell lines (5×106/only) to construct a human APL tumor-bearing model, then the mice were divided into tumor-bearing model group, arsenic trioxide group and tripterine group randomly, and another 6 mice which didn't construct model were set up as control group; after 3 weeks, the control group and the tumor-bearing model were intraperitoneally injected with normal saline as compared, arsenic trioxide was intraperitoneally injected according to 100 μg/kg, and tripterine was intraperitoneally injected according to 3 mg/kg. Four groups were all injected for 3 weeks. The biological characteristics of NB4 cells and the expression of adhesion molecules in venous blood of mice after treatment were observed. The neutrophil decrased and promyelocytes, NB4 cells, B lymphocytes and white blood cells increased in tumor-bearing group as compared with control group (P<0.05), and the expressions of serum P-selectin (P-selectin), soluble vascular adhesion molecule-1 (soluble vascular adhesion molecule-1, sVCAM-1) and soluble intercellular adhesion molecule-1 (soluble intercellular adhesion molecule-1, sICAM-1) all increased (P<0.05). The cell cycle showed that the proportion of G0 and G1 increased, but S, G2-M phases obviously decreased (P<0.05). Compared with the tumor-bearing group, the neutrophils increased in the tripterine group, and the expression of early promyelocyte, NB4 cell, B lymphocyte, leukocyte, P-selectin, sVCAM-1 and sICAM-1 protein decreased (P<0.05), moreover, the ratio of G0 and G1 decreased, the G2-M and S phases increased (P<0.05). Among which sVCAM-1 and sICAM-1 of leucocyte, NB4 cell, P-selectin and adhesion molecules in tripterine group showed significant difference as compared with arsenic trioxide group (P<0.05). Tripterine may not only inhibit the expression of sVCAM-1 and sICAM-1 proteins in APL tumor-bearing mice and reduce the adhesion of tumor cells, but also block tumor cells at G2-M and S phases, thereby exerting therapeutic effect on APL.

Differentiation and activation of human CD4 T cells is associated with a gradual loss of myelin and lymphocyte protein.

Upon generation of monoclonal antibodies to the T cell antigen receptor/CD3 (TCR/CD3) complex, we isolated mAb MT3, whose reactivity correlates inversely with the production of IFN‐γ by human peripheral blood T lymphocytes. Using eukaryotic expression cloning, we identified the MT3 antigen as myelin‐and‐lymphocyte (MAL) protein. Flow cytometry analysis demonstrates high surface expression of MAL on all naïve CD4+ T cells whereas MAL expression is diminished on central memory‐ and almost lost on effector memory T cells. MAL– T cells proliferate strongly in response to stimulation with CD3/CD28 antibodies, corroborating that MAL+ T cells are naïve and MAL– T cells memory subtypes. Further, resting MAL– T cells harbor a larger pool of Ser59‐ and Tyr394‐ double phosphorylated lymphocyte‐specific kinase (Lck), which is rapidly increased upon in vitro restimulation. Previously, lack of MAL was reported to prevent transport of Lck, the key protein tyrosine kinase of TCR/CD3 signaling to the cell membrane, and to result in strongly impaired human T cell activation. Here, we show that knocking out MAL did not significantly affect Lck membrane localization and immune synapse recruitment, or transcriptional T cell activation. Collectively, our results indicate that loss of MAL is associated with activation‐induced differentiation of human T cells but not with impaired membrane localization of Lck or TCR signaling capacity.

Exploring the Emerging Role of the Gut Microbiota and Tumor Microenvironment in Cancer Immunotherapy.

The tumor microenvironment (TME) is a complex ecosystem, which includes many different types of cells, abnormal vascular systems, and immunosuppressive cytokines. TME serves an important function in tumor tolerance and escapes from immune surveillance leading to tumor progression. Indeed, there is increasing evidence that gut microbiome is associated with cancer in a variety of ways, as specific microbial signatures are known to promote cancer development and influence safety, tolerability, and efficacy of therapies. Studies over the past five years have shown that the composition of the intestinal microbiota has a significant impact on the efficacy of anticancer immunosurveillance, which contribute to the therapeutic activity of cancer immunotherapies based on targeting cytotoxic T lymphocyte protein 4 (CTLA-4) or programmed cell death protein 1 (PD-1)–programmed cell death 1 ligand 1 (PD-L1) axis. In this review, we mainly discuss the impact of TME on cancer and immunotherapy through immune-related mechanisms. We subsequently discuss the influence of gut microbiota and its metabolites on the host immune system and the formation of TME. In addition, this review also summarizes the latest research on the role of gut microbiota in cancer immunotherapy.

Immunohistochemical, histopathological, and biochemical studies of the NF-ҡB P65 marker in rat ovaries experimentally intoxicated by cadmium and the protective effect of the purslane plant extract.

The aim of this study is to describe the existence of the inflammatory marker nuclear factor kappa light chain B lymphocyte protein (NF-ҡB P65) in the tissue as a response to cadmium (CdCl2) toxicity. Next is to describe the disappearance of the NF-ҡB P65 in response to the purslane plant treatment to explore its anti-inflammatory effect, also describing the histopathological and biochemical changes that occurred from CdCl2 toxicity and the purslane plant tissue protections. There are four experimental groups, 32 rats (n = 8) intraperitoneally injected with CdCl2 and orally administered with purslane plant extract (according to groups) for 30days: group one (control), group two (purslane extract 2g/kg bw), group three (CdCl2 3.5mg/kg bw), group four (CdCl2 3.5mg/kg bw + purslane plant extract 2g/kg bw). The biochemical findings showed that ovaries and brain tissue homogenates in group three showed malondialdehyde increase and reduction in catalase, total antioxidant capacity, and acetylcholine esterase. A reduction in serum LH, FSH, and estradiol were also recorded. These parameters became normal in group four. The histopathological findings exhibited that group three showed ovarian and cerebral hemorrhage and lung pneumonia. Tissues of group four were protected and no pathological lesions were detected. The immunohistochemical results showed that the inflammatory marker NF-ҡB P65 in group three was strongly detected in the spleen and moderately detected in the ovaries, brain, and lung but negatively detected in the tissues of group four. In conclusion, CdCl2 induced ovarian toxicity and the NF-ҡB P65 existence was increased. Purslane plant protected rats from CdCl2 toxicity and decrease NF-ҡB P65.

Serum Levels of Interleukin-6, Ferritin, C-Reactive Protein, Lactate Dehydrogenase, D-Dimer, and Count of Lymphocytes and Neutrophils in COVID-19 Patients

Background: Since its first emergence in Wuhan city, China, severe acute respiratory syndrome coronavirus 2, which is responsible for the pandemic COVID-19, has become a significant health problem all over the world affecting over 2.1 million people globally. Methods: The current study aimed to investigate serum levels of interleukin 6 (IL-6), ferritin, C-reactive protein (CRP), lactate dehydrogenase (LDH), D-dimer as well as lymphocytes and neutrophils count in COVID-19 patients, and to clarify the correlation of these parameters with disease severity and progression. For these purposes, (100) patients with COVID-19 (confirmed by polymerase chain reaction) and (20) apparently healthy people (with matched age and sex) were included in the current study and considered as a control group. Results: All study population (patients and control) were subjected to the evaluation of serum levels of IL-6, ferritin, CRP, LDH, D-dimer, as well as lymphocytes and neutrophils, count. COVID-19 patients showed a significant elevation in the levels of all parameters included in this study when compared with healthy controls. We also found that all of IL-6, ferritin, CRP, LDH, D-dimer are significantly associated with the severity of the COVID-19 symptoms. Conclusion: Lymphopenia and increased neutrophils were also effectively correlated with disease progression. In line with these results, we concluded a proportional correlation between the aforementioned parameters and COVID-19 suggesting the uses of these tests to the diagnosis of critical cases.

Effects of Occupational Exposure to Waste Anesthetic Gas on Oxidative Stress and DNA Damage.

Objective The aim of the study was to investigate the potential effects of waste anesthetic gas (WAG) on oxidative stress, DNA damage, and vital organs. Methods A total of 150 members of the staff at a hospital were assigned to an exposure group or control group. The 68 operating room (OR) staff in the exposure group were exposed to WAG, and the 82 non-OR staff in the control group were not exposed to WAG. Air samples were collected in the OR, and the sevoflurane concentrations in the samples were determined. Superoxide dismutase (SOD), glutathione peroxidase (GSH-px), and malondialdehyde (MDA) in plasma from the participants were determined to assess oxidative stress. Western blot analysis was used to detect γH2AX in peripheral blood to assess DNA damage. Hematopoietic parameters, liver function, kidney function, and changes in electrophysiology were assessed to identify the effects on the vital organs. Results The mean (±standard deviation) sevoflurane concentration in 172 air samples from 22 operating rooms was 1.11 ± 0.65 ppm. The superoxide dismutase activity and vital organ parameters (lymphocyte, hemoglobin, and total protein concentrations and heart rate) were significantly lower (P < 0.05) in the exposed group than the control group. The malondialdehyde, total bilirubin, and creatinine concentrations and QT and QTc intervals were significantly higher (P < 0.05) in the exposed group than the control group. There were no significant differences between the glutathione peroxidase activities and γH2AX concentrations for the exposed and control groups. Conclusions Long-term occupational exposure to waste anesthetic gas may affect the antioxidant defense system and probably affects vital organ functions to some extent. No correlation between DNA damage and chronic exposure to WAG was observed.

P.832DRD1 protein and mRNA levels in peripheral blood lymphocytes: influence of SNPs of 5′-untranslated region and the schizophrenia disorder

Apoptosis is a powerful host cell defense to prevent viruses from completing replication. Poxviruses have evolved complex means to dampen cellular apoptotic responses. The poxvirus, Molluscum Contagiosum Virus (MCV), encodes numerous host interacting molecules predicted to antagonize immune responses. However, the function of the majority of these MCV products has not been characterized. Here, we show that the MCV MC163 protein localized to the mitochondria via an N-terminal mitochondrial localization sequence and transmembrane domain. Transient expression of the MC163 protein prevented mitochondrial membrane permeabilization (MMP), an event central to cellular apoptotic responses, induced by either Tumor Necrosis Factor alpha (TNF-α) or carbonyl cyanide 3-chlorophenylhydrazone (CCCP). MC163 expression prevented the release of a mitochondrial intermembrane space reporter protein when cells were challenged with TNF-α. Inhibition of MMP was also observed in cell lines stably expressing MC163. MC163 expression may contribute to the persistence of MCV lesions by dampening cellular apoptotic responses.

Cocaine Administration and Its Abstinence Conditions Modulate Neuroglia.

Cocaine induces neuronal changes as well as non-neuronal (astrocytes, microglia, oligodendroglia) mechanisms, but these changes can also be modulated by various types of drug abstinence. Due to the very complex and still incompletely understood nature of cocaine use disorder, understanding of the mechanisms involved in addictive behavior is necessary to further search for effective pharmacotherapy of this disease. The aim of this study was to investigate changes at the gene and protein levels associated with glial cell activity after cocaine exposure, as well as during early cocaine abstinence (3 days) with extinction training or in home cage isolation. Cocaine self-administration significantly decreased myelin regulatory factor (MYRF) and cyclic nucleotide phosphodiesterase (CNP) expression in the hippocampus as well as pleckstrin (PLEK) and T-lymphocyte activation antigen (CD86) in the rat striatum. Depending on cocaine abstinence conditions, microglial PLEK expression was increased through extinction training but did not change in the home cage isolation. In addition, downregulation of gene expression associated with oligodendrocytes (CNP, MYRF) and microglia regulator of G protein signaling 1 (RGS1) was observed in the hippocampus, regardless of the type of drug abstinence, while downregulation of myelin and lymphocyte protein (MAL) expression was found only in rats exposed to abstinence in the home cage. Taken together, the presented results strongly suggest that cocaine abstinence evokes significant changes in gene expression associated with the proper functioning of glial cells, suggesting their significant involvement in adaptive changes in the brain associated with cocaine exposure. Interestingly, drug abstinence conditions are important factors influencing observed changes at the transcript levels of selected genes, which may be of clinical interest.

SLy2-deficiency promotes B-1 cell immunity and triggers enhanced production of IgM and IgG2 antibodies against pneumococcal vaccine.

BackgroundDespite the benefits of existing vaccines, Streptococcus pneumoniae is still responsible for the greatest proportion of respiratory tract infections around the globe, thereby substantially contributing to morbidity and mortality in humans. B‐1 cells are key players of bacterial clearance during pneumococcal infection and even provide long‐lasting immunity towards S. pneumoniae. Previous reports strongly suggest an essential role of the immunoinhibitory adapter Src homology domain 3 lymphocyte protein 2 (SLy2) for B‐1 cell‐mediated antibody production. The objective of this study is to evaluate S. pneumoniae‐directed B cell responses in the context of SLy2 deficiency.MethodsB‐1 cell populations were analyzed via flow cytometry before and after pneumococcal immunization of SLy2‐deficient and wild‐type control mice. Global and vaccine‐specific immunoglobulin M (IgM) and IgG antibody titers were assessed by enzyme‐linked immunosorbent assay. To investigate survival rates during acute pneumococcal lung infection, mice were intranasally challenged with S. pneumoniae (serotype 3). Complementary isolated splenic B cells were stimulated in vitro and their proliferative response was assessed by fluorescent staining. In vitro antibody secretion was quantified by LEGENDplex.ResultsWe demonstrate increased frequencies of B‐1 cells and elevated titers of preantigenic IgM in SLy2‐deficient mice. In addition, these mice produce significantly more amounts of IgM and IgG2 upon pneumococcal vaccination. Knocking out SLy2 did not induce survival advantages in our murine model of acute pneumonia, indicating the presence of compensatory mechanisms.ConclusionOur results reveal reinforced specific antibody responses towards pneumococcal polysaccharides and enhanced IgG2 secretion as a consequence of SLy2 deficiency, which could be relevant to the development of more efficient vaccines.

The involvement of TNF-α and TNF-β as proinflammatory cytokines in lymphocyte-mediated adaptive immunity of Nile tilapia by initiating apoptosis

Tumor necrosis factors (TNFs) are pleiotropic cytokines with important functions in homeostasis and disease pathogenesis. Recent advances have shown that TNFs are also involved in the regulation of adaptive immune responses. However, the knowledge about how TNF participates in and regulates adaptive immune response in early vertebrates is still limited. In present study, we identified two isoforms of TNF, TNF-α and TNF-β, from Nile tilapia Oreochromis niloticus (On-TNF-α and β). After analyzing the sequence characteristics, we investigated their regulatory roles in adaptive immune response of this fish species. On-TNF-α and β are evolutionarily conserved compare with their homologs from other vertebrates. Both TNFs were distributed in a wide range of tissues in O. niloticus, and with relative higher expression level in gill. After the animals were infected by Streptococcus agalactiae, mRNA levels of On-TNF-α and TNF-β in spleen lymphocytes were significantly upregulated during the primary response stage of adaptive immunity. Meanwhile, both TNF proteins in spleen lymphocytes were also dramatically elevated during the adaptive immune stage after bacterial infection. These results indicate the potential participation of On-TNF-α and TNF-β in adaptive immune response of Nile tilapia. Furthermore, On-TNF-α and β transcripts were obviously augmented, once spleen lymphocytes were activated by T cell-specific mitogen PHA. More importantly, both recombinant On-TNF-α and β could induce the apoptosis of head-kidney leukocytes of Nile tilapia. And On-TNF-β but not On-TNF-α promoted the apoptosis by activating caspase-8 in the target cells. Altogether, our study revealed that TNF-α and TNF-β participated in the lymphocyte-mediated adaptive immune response of Nile tilapia by initiating the apoptosis, and thus shed novel perspective for the regulatory mechanism of adaptive immunity in teleost.

Abstract 5167: Identification and validation of a novel immuno-oncology target and selection of a therapeutic antibody candidate with a pharmacologically beneficial activity profile

Abstract Identification of novel targets in cancer immunotherapy is needed to address the significant number of patients that either do not respond to current therapies or encounter unacceptable toxicities. The first two generations of immuno-oncology drugs have been antagonist antibodies against immune checkpoint proteins, such as cytotoxic T lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Moving forward, there has been progress in targeting co-stimulatory receptors like inducible T cell co-stimulator (ICOS), OX40 and CD137 with agonist antibodies. At Oxford BioTherapeutics, in-depth expression profiling of membrane proteins from intact tumors collected in the proprietary OGAP database revealed novel IO targets in primary tumor-derived lymphocytes (TILs). Proteomic and flow cytometry analysis of TILs and PBMCs establish that OX003R is a novel co-stimulatory IO target. It is expressed on naïve T and B cells; however, higher expression is observed in TILs and activated or exhausted T cells. OX003R expression is observed by immunohistochemistry in infiltrating lymphocytes in a variety of solid tumor types. A Fab phage display library was screened by FACS for binding to target on the cell surface. All the Fabs were also profiled by an interferon gamma release assay for T cell activation. Five best binders demonstrating T cell activation were reformatted into full-length chimeric mAbs and expressed in mammalian Expi 293 cells. Recombinant antibodies were extensively screened for T cell activation in an ex vivo 3D tumor culture system developed in-house using fresh non-small cell lung and colorectal carcinomas. Interferon gamma release was assessed by ELISpot assay and expression of the target was confirmed by immunohistochemistry on the corresponding tumor samples. Chimeric antibody 1B3 robustly activated T cells in most of the tumor samples in a dose-dependent manner as compared to isotype control and was chosen as the lead therapeutic antibody for humanization. The lead therapeutic antibody was tested for the propensity to facilitate the undesirable cytokine storm in whole blood and did not induce the release of dangerous levels of cytokines. Conclusion: OX003R is a validated immuno-oncology target and chimeric 1B3 is being developed as a promising therapeutic antibody with agonistic TIL activity, specifically in the tumor microenvironment. Citation Format: Arnima Bisht, Angelo Kaplan, Livija Deban, Chander Sekhar Peddaboina, Murray Cox, Lindsey Hudson, James Ackroyd, Nickolas Attanasio, San Lin Lou, Jason Allen, Martin Barnes, Robert Boyd, Eugene Zhukovsky, Abderrahim Fandi, Christian Rohlff. Identification and validation of a novel immuno-oncology target and selection of a therapeutic antibody candidate with a pharmacologically beneficial activity profile [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5167.

The Relationship Between Diabetes Mellitus and COVID-19 Prognosis: A Retrospective Cohort Study in Wuhan, China

BackgroundCoronavirus disease 2019 (COVID-19) is an emerging infectious disease that first appeared in Wuhan, China, and quickly spread throughout the world. We aimed to understand the relationship between diabetes mellitus and the prognosis of COVID-19. MethodsDemographic, clinical, laboratory, radiologic, treatments, complications, and clinical outcomes data were extracted from electronic medical records and compared between diabetes (n = 84) and nondiabetes (n = 500) groups. Kaplan-Meier method and multivariate Cox analysis were applied to determine the risk factors for the prognosis of COVID-19. ResultsCompared with nondiabetic patients, diabetic patients had higher levels of neutrophils (P = .014), C-reactive protein (P = .008), procalcitonin (P < .01), and D-dimer (P = .033), and lower levels of lymphocytes (P = .032) and albumin (P = .035). Furthermore, diabetic patients had a significantly higher incidence of bilateral pneumonia (86.9%, P = .020). In terms of complications and clinical outcomes, the incidence of respiratory failure (36.9% vs 24.2%, P = .022), acute cardiac injury (47.4% vs 21.2%, P < .01), and death (20.2% vs 8.0%, P = .001) in the diabetes group was significantly higher than that in the nondiabetes group. Kaplan-Meier survival curve showed that COVID-19 patients with diabetes had a shorter overall survival time. Multivariate Cox analysis indicated that diabetes (hazard ratio 2.180, P = .031) was an independent risk factor for COVID-19 prognosis. In subgroup analysis, we divided diabetic patients into insulin-required and non-insulin-required groups according to whether they needed insulin, and found that diabetic patients requiring insulin may have a higher risk of disease progression and worse prognosis after the infection of severe acute respiratory syndrome coronavirus 2. ConclusionsDiabetes is an independent risk factor for the prognosis of COVID-19. More attention should be paid to the prevention and treatment for diabetic patients, especially those who require insulin therapy.

Lymphocyte-C-reactive protein ratio as a novel prognostic index in intrahepatic cholangiocarcinoma: A multicentre cohort study.

The lymphocyte-C-reactive protein ratio (LCR) is a novel inflammatory-based score, based solely on the lymphocyte and C-reactive protein. We aimed to clarify the prognostic value of the LCR score in intrahepatic cholangiocarcinoma (ICC) patients after resection. We compared the prognostic accuracy of the LCR score with other inflammatory-based scores in this large, multicentre cohort study. The independent variables associated with overall survival (OS) were explored in both the primary (n=228) and validation cohorts (n=135). Harrell's concordance index (C-index) was used to compare the predictive ability of all the assessed inflammatory-based scores. The LCR score was differentiated two groups of ICC patients with distinct prognoses (1-, 3-, and 5-year OS rates: 94.4%, 66.3%, and 59.3%; and 66.6%, 45.6%, and 32.7%, respectively) (P<.001). Multivariate analysis showed that the LCR score, as well as the TNM stage and preoperative CA19-9 level, were independently associated with OS. The predictive accuracy of the LCR score (c score: 0.634) was superior to that of the other inflammatory-based scores (c scores: 0.508-0.615). These findings were supported by the external validation cohort. The LCR score is stable and consistently the best prognostic score and may offer as a simple, objective and discriminatory method in facilitating the risk stratification of ICC patients.

Lymphocyte activation gene-3 (LAG3) mRNA and protein expression on tumour infiltrating lymphocytes (TILs) in oesophageal adenocarcinoma

PurposeLymphocyte activation gene-3 (LAG3) is an immunosuppressive checkpoint molecule expressed on T cells. The frequency and distribution of LAG3 expression in oesophageal adenocarcinoma (EAC) is unknown. Aim of the study was the evaluation and distribution of LAG3 on tumour infiltrating lymphocytes (TILs) and correlation with clinico-pathological and molecular data.MethodsWe analysed tumor tissue samples using immunohistochemistry, multi-colour immunofluorescence and mRNA in-situ technology. The analyses were performed on a multi-spot tissue microarray (TMA) with 165 samples, followed by an evaluation on a single-spot TMA with 477 samples. These results were correlated with clinical and molecular tumour data.ResultsLAG3 expression on TILs was detectable in 10.5% on the multi-spot TMA and 11.4% on the single-spot TMA. There was a strong correlation between protein expression and mRNA expression (p < 0.001) in TILs. LAG 3 expression was correlated with CD4+ and CD8+ T-cells within the tumor (p < 0.001). LAG3 expression showed an improved overall survival (OS) compared to patients without LAG3 expression (median OS 70.2 vs. 26.9 months; p = 0.046). The effect was even clearer in the group of patients with tumour stages > pT2 (70.2 vs 25.0 months; p = 0.037).ConclusionThis is the first description of LAG3 expression on TILs in EAC, underscoring the importance of immunomodulation in EAC. Our data suggest an impact of LAG3 in a relevant subset of EAC. Therapeutic studies investigating the efficacy of LAG3 inhibition in EAC will also provide predictive evidence and relevance of the immunohistochemical determination of LAG3 expression.

A novel long noncoding RNA, ENSGALG00000021686, regulates the intracellular transport of fatty acids by targeting the FABP3 gene in chicken

Fatty acids (FAs) are essential for the vital movement of humans and animals. Their metabolism is, in part, regulated by FABP3. In our previous study, a novel lncRNA (ENSGALG00000021686, L21686) was identified, and FABP3 was predicted as its target gene. Here, using chicken myocytes, lymphocytes, and different tissues, L21686 target on the FABP3 gene, FABP3 mRNA expression, and their effect on FA metabolism are explored. The results show that the highest expression of L21686 is in muscle tissue, a significant energy-consuming tissue. L21686 expression is consistent with FABP3 mRNA expression. We also show that under the different treatments, the levels of FABP3 mRNA and protein in myocytes and lymphocytes change in tandem with L21686 expression. Moreover, the dual-luciferase reporter assay provided direct evidence that L21686 targets the FABP3 gene. Finally, it was found that the content of free FAs increases along with the up-regulation of L21686 and the FABP3 gene. Malonyl CoA content does not change under the different treatments, suggesting that L21686 regulates the intake of extracellular FAs in chicken. Further, the changes in lipoprotein lipase (LPL), sterol-regulatory element binding protein 1 (SREBP-1), fatty acid synthase (FASN), and acetyl-CoA carboxylase (ACC) mRNA levels support this view. In summary, our data show that the new lncRNA (L21686) regulates the intake of extracellular FAs in chicken cells in vitro by targeting the expression of the FABP3 gene. Our findings will help to establish the groundwork and provide a new clue for deciphering the regulation of FAs metabolism in chicken.

P0686THE CIRCADIAN RHYTHM IN CKD PATIENTS

Abstract Background and Aims It is common to see that patients with chronic kidney disease CKD are complaint with sleep disorders. The data reveal that lymphocytes are under the regulation of circadian rhythm in the peripheral blood of humans. CKD patients have shown to stay up in a low-grade inflammatory status. And these patients are associated with systemic inflammation and acquired immune deficiency. In this condition, there are disorders of expression of chemokine receptors and function of lymphocytes in patients with CKD. Since the disorders of circadian rhythm have been reported, it is supposed that some proteins regulating circadian rhythm of lymphocytes may involve in sleep disorders. It is reported that circadian clock gene edcoded proteins, nuclear PER and CRY ptochrome (CRY), play an important role in the regulation of circadian rhythm. Therefore, we designed a questionnaire to patients with CKD stages 1-5 and investigated the levels of CRY mRNA and protein in PB lymphocytes. Method This research enrolled 101 patients (52 male and 49 female) with CKD stages 1-5, who were admitted to Xiangya Hospital, Changsha, China from August 2017 to August 2018. CKD was defined as the reduced level of eGFR when they received the Pittsburgh Sleep Quality-Index (PSOI) questionnaire. Patients were screened by strict exclusion criteria. The clinical files included general information of patients and blood biochemical test data. Subjective sleep quality was assessed in all patients using the Pittsburgh Sleep Quality-Index (PSQI). The expression of circadian clock genes PER1 and CRY1 in peripheral blood leukocytes using quantitative RT-PCR and Western blotting. Results Sleep questionnaires were obtained from the 101 patients from CKD stage 1 to 5. There were significant differences between the group of PSQI ≤ 5 and PSQI &amp;gt; 5 in aging, Hb, HCT, Alb, ALT and CRP (Table1). The six valuables were chosen with Scr, eGFR, Ca, P, Hb and Anxiety/Depression score with logistic anlaysis. It was found that the values of Aging and Scr were significantly increased in high PSQI (Table 2). It also found that the PSQI score was negative correlated with the stage of CKD (Table 3). The higher level of PER1 and CRY1 in PBMC of CKD stage 1 patients was confirmed by Western blot and qRT-PCR. It was also found that the expression of PER1 and CRY1 significantly down regulated in the PBMC of CKD stage 5 patients. However, the expression of PER1 and CRY1 was no obviously differences of in the patients with CKD stage 2 to CKD stage 4 (Figure 1 and Figure 2). Conclusion It was found that the progress of CKD was associated with the degree of sleep disorders in our research. The score of PSQI was negativly correlated with the lower eGFR. It indicated that there is association between sleep quality and eGFR. It also found that Hb, HCT, Alb and ALT were significantly better in the group whose PSQI score was lower than 5. CRP is a hallmark of inflammations a close relation with sleep disorders. There is correlation between decreased sleep quality and elevated CRP level. It also found that the more aging and CRP, the higher PSQI score with logistical analysis in our research. It suggested that there is a chronic inflammation are associated with the sleep of the patients with CKD. PBMCs are invloved in status of inflammation. It indicated that the PBMCs of patients with CKD may be affected by the sleep disorders. The circadian rhythm can be also shown in human’s PBMCs. The proteins of Per and CRY were two of period circadian clock proteins. In our research, we test that the expression of PER1 and CRY1 in PBMCs of patients with CKD. It found that PER1 and CRY1 were higher expressed in patients with CKD stage 1 and lowest in patients with CKD stage 5. It suggested that the circadian rhythm of PBMCs was associated with the stage of CKD. It was said that the function of PBMCs also are affected in CKD, such as the regulation of cellular calcium homeostasis, which can influence the circadian rhythm.