Experimental study on regulation of immune effect of brain glioma cells by NKG2D mediated by miR-10b / 中国免疫学杂志

Objective:To observe the regulatory effect of microRNA-10b(miR-10b)on the immune effect of glioma cells and explore its mechanism.Methods:Human glioma cell U251 was cultured to obtain cells in logarithmic growth stage.The cell suspen-sion was prepared according to the concentration of 1.0×105 cells/ml,and the control group,overexpression group,low expression group and blank group were set up,with 6 wells in each group.The negative control,miR-10b mimics and miR-10b inhibitor were transfected by liposome transfection in control group,overexpression group and low expression group,respectively.The blank group was given the same amount of sterile normal saline.Natural killer(NK)cells from peripheral blood of a healthy volunteer was isolated and cultured.The killing activity of NK cells was detected by MTT method.The expression of NK cell activated receptor(NKG2D)on the surface of NK cells in each group were detected by flow cytometry,and the expression of major histocompatibility complex class Ⅰ chain-related gene A(MICA),UL16 binding protein 2(ULBP2)and UL16 binding protein 3(ULBP3)on the surface of U251 hu-man glioma cells in each group were detected.Results:The transfection efficiency of control group,overexpression group and low ex-pression group were(93.55±2.05)％,(95.67±3.14)％,(94.18±3.26)％,respectively.Compared with control group and blank group,the expression of miR-10b increased in overexpression group and decreased in low expression group,and the difference were statisti-cally significant(P<0.05).There was no significant difference in the expression of miR-10b between control group and blank group(P>0.05).Compared with control group and blank group,the killing activity of NK cells with different effect target ratios in overex-pression group decreased,the expression of NKG2D decreased,the killing activity of NK cells with different effect target ratios in low expression group increased,and the expression of NKG2D increased,and the difference were statistically significant(P<0.05).The killing activity of NK cells in each group increased with the increase of effect target ratio,and the difference were statistically signifi-cant(P<0.05),and there was no significant difference in NK cell killing activity and NKG2D expression between control group and blank group(P>0.05).Compared with control group and blank group,the expression of MICA,ULBP2 and ULBP3 on the surface of human glioma cell U251 in overexpression group decreased,and the expression of MICA,ULBP2 and ULBP3 on the surface of human glioma cell U251 in low expression group increased,the difference were statistically significant(P<0.05),and there was no signifi-cant difference in the expression of MICA,ULBP2 and ULBP3 on the surface of U251 glioma cells between control group and blank group(P>0.05).Conclusion:Inhibiting the expression of miR-10b can increase the expression of NKG2D on the surface of NK cells and MICA,ULBP2 and ULBP3 on the surface of human glioma cell U251,and enhance the killing activity of NK cells against human glioma cell U251.

Mutational analysis of ribosomal proteins in a cohort of pediatric patients with T-cell acute lymphoblastic leukemia reveals Q123R, a novel mutation in RPL10.

T-cell acute lymphoblastic leukemia (T-ALL) is a subtype of ALL involving the malignant expansion of T-cell progenitors. It is driven by a number of different possible genetic lesions, including mutations in genes encoding for ribosomal proteins (RPs). These are structural constituents of ribosomes, ubiquitous effectors of protein synthesis. Albeit the R98S mutation in RPL10, recurring with a higher frequency among RP mutations, has been extensively studied, less is known about the contribution of mutations occurring in other RPs. Alterations affecting translational machinery may not be well tolerated by cells, and there may be a selective pressure that determines the emergence of mutations with a compensatory effect. To explore this hypothesis, we sequenced the exomes of a cohort of 37 pediatric patients affected by T-ALL, and analyzed them to explore the co-occurrence of mutations in genes involved in ribosome biogenesis (including RPs) and translational control, and in known T-ALL driver genes. We found that some of the mutations in these sub-classes of genes tend to cluster together in different patients, indicating that their co-occurrence may confer some kind of advantage to leukemia cells. In addition, our sequencing highlighted the presence of a novel mutation in RPL10, namely the Q123R, which we found associated with a defect in protein synthesis. Our findings indicate that genetic alterations involving ribosome biogenesis and translational control should be carefully considered in the context of precision medicine in T-ALL.

Levels of Soluble NKG2D Ligands and Cancer History in Patients Starting Hemodialysis.

Background: Immune dysfunction in hemodialysis patients is partially due to NK cell impairment. Ligands for NK activating receptors such as NKG2D expressed on cancer cells are involved in NK cell dysfunction and can lead to cancer development. Methods: A cohort with 370 patients who started hemodialysis (HD) was investigated. Serum levels of soluble NKG2D ligands were measured. Cancer history was defined as any cancer diagnosis at induction and hospitalization and death due to cancer during 2-year follow-up. Results: Sixty-two patients with and 308 patients without a cancer history showed mostly comparable biochemical parameters and uremic status at HD induction. Soluble MICB, ULBP-1, and ULBP-2 were detected in sera from most patients starting HD rather than MICA, the most representative NKG2D ligand. Measured NKG2D ligands, except for ULBP-1, were strongly correlated with each other. Correlations between NKG2D ligands and renal function were significant but modest in patients starting HD. Cancer history did not have any impact on levels of soluble NKG2D ligands. Discussion: Even though this investigation lacked a control cohort and serial measurement of parameters, expression patterns of NKG2D ligands were comprehensively described, and the significance of cancer in patients starting HD was elucidated for the first time. Elevated levels of soluble NKG2D ligands occurred potentially due to complex mechanisms of oxidative stress, with insufficient metabolism and excretion in a uremic milieu, but they might mask the significance of elevations in serum levels of soluble NKG2DLs in patients with a cancer history.

Complement Inactivation Strategy of Staphylococcus aureus Using Decay-Accelerating Factor and the Response of Infected HaCaT Cells.

Staphylococcus aureus is a species of Gram-positive staphylococcus. It can cause sinusitis, respiratory infections, skin infections, and food poisoning. Recently, it was discovered that S. aureus infects epithelial cells, but the interaction between S. aureus and the host is not well known. In this study, we confirmed S. aureus to be internalized by HaCaT cells using the ESAT-6-like protein EsxB and amplified within the host over time by escaping host immunity. S. aureus increases the expression of decay-accelerating factor (CD55) on the surfaces of host cells, which inhibits the activation of the complement system. This mechanism makes it possible for S. aureus to survive in host cells. S. aureus, sufficiently amplified within the host, is released through the initiation of cell death. On the other hand, the infected host cells increase their surface expression of UL16 binding protein 1 to inform immune cells that they are infected and try to be eliminated. These host defense systems seem to involve the alteration of tight junctions and the induction of ligand expression to activate immune cells. Taken together, our study elucidates a novel aspect of the mechanisms of infection and immune system evasion for S. aureus.

Immune evasion by cancer stem cells.

Tumor immunity represents a new avenue for cancer therapy. Immune checkpoint inhibitors have successfully improved outcomes in several tumor types. In addition, currently, immune cell-based therapy is also attracting significant attention. However, the clinical efficacy of these treatments requires further improvement. The mechanisms through which cancer cells escape the immune response must be identified and clarified. Cancer stem cells (CSCs) play a central role in multiple aspects of malignant tumors. CSCs can initiate tumors in partially immunocompromised mice, whereas non-CSCs fail to form tumors, suggesting that tumor initiation is a definitive function of CSCs. However, the fact that non-CSCs also initiate tumors in more highly immunocompromised mice suggests that the immune evasion property may be a more fundamental feature of CSCs rather than a tumor-initiating property. In this review, we summarize studies that have elucidated how CSCs evade tumor immunity and create an immunosuppressive milieu with a focus on CSC-specific characteristics and functions. These profound mechanisms provide important clues for the development of novel tumor immunotherapies.

A Time-Resolved Cryo-EM Study of Saccharomyces cerevisiae 80S Ribosome Protein Composition in Response to a Change in Carbon Source.

The role of the ribosome in the regulation of gene expression has come into increased focus. It is proposed that ribosomes are catalytic engines capable of changing their protein composition in response to environmental stimuli. Time-resolved cryo-electron microscopy (cryo-EM) techniques are employed to identify quantitative changes in the protein composition and structure of the Saccharomyces cerevisiae 80S ribosomes after shifting the carbon source from glucose to glycerol. Using cryo-EM combined with the computational classification approach, it is found that a fraction of the yeast cells' 80S ribosomes lack ribosomal proteins at the entrance and exit sites for tRNAs, including uL16(RPL10), eS1(RPS1), uS11(RPS14A/B), and eS26(RPS26A/B). This fraction increased after a change from glucose to glycerol medium. The quantitative structural analysis supports the hypothesis that ribosomes are dynamic complexes that alter their composition in response to changes in growth or environmental conditions.

Targeting hydrogen sulphide signaling in breast cancer.

INTRODUCTION: Hydrogen sulphide (H2S) has been established as a key member of the gasotransmitters family that recently showed a pivotal role in various pathological conditions including cancer. OBJECTIVES: This study investigated the role of H2S in breast cancer (BC) pathogenesis, on BC immune recognition capacity and the consequence of targeting H2S using non-coding RNAs. METHODS: Eighty BC patients have been recruited for the study. BC cell lines were cultured and transfected using validated oligonucleotide delivery system. Gene and protein expression analysis was performed using qRT-PCR, western blot and flow-cytometry. In-vitro analysis for BC hallmarks was performed using MTT, BrdU, Modified Boyden chamber, migration and colony forming assays. H2S and nitric oxide (NO) levels were measured spectrophotometrically. Primary natural killer cells (NK cells) and T cell isolation and chimeric antigen receptor transduction (CAR T cells) were performed using appropriate kits. NK and T cells cytotoxicity was measured. Finally, computational target prediction analysis and binding confirmation analyses were performed using different software and dual luciferase assay kit, respectively. RESULTS: The H2S synthesizing enzymes, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), exhibited elevated levels in the clinical samples that correlated with tumor proliferation index. Knock-down of CBS and CSE in the HER2+ BC and triple negative BC (TNBC) cells resulted in significant attenuation of BC malignancy. In addition to increased susceptibility of HER2+ BC and TNBC to the cytotoxic activity of HER2 targeting CAR T cells and NK cells, respectively. Transcriptomic and phosphoprotein analysis revealed that H2S signaling is mediated through Akt in MCF7, STAT3 in MDA-MB-231 and miR-155/ NOS2/NO signaling in both cell lines. Lastly, miR-4317 was found to function as an upstream regulator of CBS and CSE synergistically abrogates the malignancy of BC cells. CONCLUSION: These findings demonstrate the potential role of H2S signaling in BC pathogenesis and the potential of its targeting for disease mitigation.

Down-regulation of UL16-binding protein 3 mediated by interferon-gamma impairs immune killing in nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor characterized by a large number of tumor-infiltrating lymphocytes and high expression of programmed death ligand-1 (PD-L1). Interferon-gamma (IFN-γ) has proven to be the strongest inducers of PD-L1. This study aims at investigating the effect of IFN-γ on the expression of natural killer group 2, member D ligands (NKG2DLs), a series of immune-activating proteins, and their further effect on immune killing in NPC. METHODS: RNA-seq data from the Gene Expression Omnibus database was downloaded and analyzed for the correlation between IFN-γ and NKG2DLs. IHC staining of clinical biopsy samples was performed to support the correlation between IFN-γ and ULBP3. Different NPC cell lines were treated with IFN-γ (100 U/ml) and the expression of PD-L1 and ULBP3 were detected at different time points. The 5-8F cell lines with PD-L1 over-expression and ULBP3 knockout were established and the T-cell cytotoxicity assay was performed to investigate the effect of ULPB3 on cytotoxicity. RESULTS: Correlation analysis and IHC staining showed that the expression of ULBP3 had a significant negative correlation with IFN-γ in NPC patients. The vitro assays revealed that ULBP3 can be time-dependently down-regulated by IFN-γ. The cytotoxicity of CD8+ T-cells that were co-cultured with ULBP3 knockout 5-8F cells was significantly impaired compared to wild type 5-8F cells. CONCLUSIONS: IFN-γ can significantly down-regulate the expression of ULBP3 in NPC. And the down-regulation of ULBP3 and the up-regulation of PD-L1 are both mediated by IFN-γ and may collectively play a role in the inhibition of immune killing in NPC.

Celecoxib upregulates ULBP-1 expression in lung cancer cells via the JNK/PI3K signaling pathway and increases susceptibility to natural killer cell cytotoxicity.

Lung cancer has the highest cancer mortality rate in the world, and effective therapies are still required. Cyclooxygenase-2 (COX-2) is highly expressed in numerous types of cancer, and is therefore considered a possible target of cancer treatment. Celecoxib, a selective COX-2 inhibitor, has binding pockets that interact with COX-2 and disrupt its enzymatic activities. In addition, celecoxib is able to affect cellular functions in a COX-2-independent manner. The present study aimed to investigate if celecoxib affected natural killer (NK) cell receptors and susceptibility to NK cell toxicity. For this purpose, PCR, immunoblotting, flow cytometry analysis and NK cell cytotoxicity assays were performed. The present study revealed that sublethal concentrations of celecoxib increased the expression levels of UL16-binding protein 1 (ULBP-1), a natural-killer group 2 member D (NKG2D) ligand, in lung cancer A549 and H460 cell lines. ULBP-1 mRNA and protein expression was induced in a dose- and time-dependent manner after celecoxib treatment. Expression levels of other NKG2D ligands, such as ULBP-2, ULBP-3, MHC class I-related chain A (MICA) and MICB did not change considerably compared to ULBP-1 in response to celecoxib treatment. Fluorescence microscopic images revealed abundant ULBP-1 in the cytoplasm after celecoxib treatment. Both JNK and PI3K may be involved in the induction of ULBP-1 expression after celecoxib treatment in A549 and H460 cells. In a NK cytotoxicity assay, celecoxib increased the sensitivity to NK cell-mediated cytotoxicity via interaction with ULBP-1 in lung cancer cells. Overall, the present results demonstrated that celecoxib treatment induced ULBP-1 expression in lung cancer cells, thereby increasing their susceptibility to NK cell cytotoxicity. These results suggest that the effects of conventional anticancer therapy may potentially be enhanced by using celecoxib, which targets COX-2, to enhance the sensitivity of lung cancer cells to NK cell-mediated cytotoxicity.

Staphylococcus aureus induces cell-surface expression of immune stimulatory NKG2D ligands on human monocytes.

Staphylococcus aureus is among the leading causes of bacterial infections worldwide. The pathogenicity and establishment of S. aureus infections are tightly linked to its ability to modulate host immunity. Persistent infections are often associated with mutant staphylococcal strains that have decreased susceptibility to antibiotics; however, little is known about how these mutations influence bacterial interaction with the host immune system. Here, we discovered that clinical S. aureus isolates activate human monocytes, leading to cell-surface expression of immune stimulatory natural killer group 2D (NKG2D) ligands on the monocytes. We found that expression of the NKG2D ligand ULBP2 (UL16-binding protein 2) is associated with bacterial degradability and phagolysosomal activity. Moreover, S. aureus-induced ULBP2 expression was linked to altered host cell metabolism, including increased cytoplasmic (iso)citrate levels, reduced glycolytic flux, and functional mitochondrial activity. Interestingly, we found that the ability of S. aureus to induce ULBP2 and proinflammatory cytokines in human monocytes depends on a functional ClpP protease in S. aureus These findings indicate that S. aureus activates ULBP2 in human monocytes through immunometabolic mechanisms and reveal that clpP inactivation may function as a potential immune evasion mechanism. Our results provide critical insight into the interplay between the host immune system and S. aureus that has evolved under the dual selective pressure of host immune responses and antibiotic treatment. Our discovery of an immune stimulatory pathway consisting of human monocyte-based defense against S. aureus suggests that targeting the NKG2D pathway holds potential for managing persistent staphylococcal infections.

Characterization of Human Cytomegalovirus UL16 and UL17 Transcripts.

Up to now, the UL16-17 region of human cytomegalovirus (HCMV) has not been well characterized at the level of mRNA and protein, especially for the Han strain, the first clinical HCMV strain in China. In previous studies, three transcripts were detected from the UL16-17 region by northern blot analysis for Merlin strain. Transcriptions of UL16 and UL17 were also studied by 5' rapid amplification of cDNA ends (5'RACE) and deep sequencing for AD169 and Towne strains, respectively. However, details of 3' end of UL16 and UL17 transcripts have never been confirmed by 3'RACE. The expressing phage of the UL16-17 region needs further research by northern blot, too. In the present study, cDNA library screening, northern blot and RACE were used to identify the transcription characteristics of the UL16-17 region. Mainly, 3 clusters of transcripts with the same 3' end were found to be expressed from the UL16-17 region in both Han and AD169 strains. The lengths of the core transcripts among the 3 clusters were 1,254nt, 718nt and 468nt, respectively. The corresponding 5' ends are at nt23119, nt23655, nt23905 in the HCMV Han genome. The consistent 3' end is located at nt24372 in the Han genome. The 1,254nt and 468nt transcripts are transcribed in early and late phases, and the 718nt transcript is transcribed only in the late phase.

Duck enteritis virus UL21 is a late gene encoding a protein that interacts with pUL16.

BACKGROUND: pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. RESULTS: We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293 T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. CONCLUSIONS: The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21.

Values of early diagnosis and prognosis evaluation of peripheral blood UL16 binding protein 2 expression in patient with colorectal cancer / 中国医师进修杂志

Objective@#To detect the level of peripheral blood UL16 binding protein 2 (ULBP2) in patients with colorectal cancer, and study its value on early diagnosis and prognosis evaluation.@*Methods@#Eighty patients with colorectal cancer (colorectal cancer group) and 60 healthy subjects (healthy control group) from May 2016 to May 2019 in Affiliated Dongfeng Hospital, Hubei University of Medicine were selected. Serum expression level of ULBP2 was detected by enzyme-linked immunosorbent assay. The diagnostic efficacy of serum ULBP2 in colorectal cancer was evaluated by receiver operating characteristic (ROC) curve. The influencing factors of survival in patients with colorectal cancer were analyzed by Cox regression model. Kaplan-Meier method was used for drawing the survival curve, and log-rank test method was used for comparison.@*Results@#The serum ULBP2 level in colorectal cancer group was significantly higher than that in healthy control group: (85.52 ± 12.18) ng/L vs. (66.20 ± 8.28) ng/L, and the serum ULBP2 level of stage Ⅰ to Ⅱ in colorectal cancer group was also significantly higher than that in healthy control group: (76.44 ± 7.56) ng/L vs. (66.20 ± 8.28) ng/L, and there were statistical differences (P<0.05). ROC curve analysis result showed that the optimal cut off value of serum ULBP2 for colorectal cancer diagnosis was 79.53 ng/L, and area under curve (AUC) was 0.869, with a sensitivity of 73.75% and a specificity of 91.67%; the optimal cut off value of serum ULBP2 for stage Ⅰ to Ⅱ colorectal cancer diagnosis was 71.86 ng/L, and AUC was 0.827, with a sensitivity of 78.57%, and a specificity of 78.33%. According to the median serum ULBP2 level, the patients were divided into ULBP2 high expression (ULBP2>85.52 ng/L, 38 cases) and ULBP2 low expression (42 cases). The serum expression level of ULBP2 was related to lymph node metastasis and tissue differentiation (P < 0.05). Univariate Cox regression analysis result showed that lymph node metastasis, distant metastasis, tissue differentiation and serum ULBP2 were risk factors of poor prognosis in patients with colorectal cancer (P<0.01 or <0.05); multivariate Cox regression analysis result showed that serum ULBP2 was the independent risk factor of poor prognosis in patients with colorectal cancer (HR = 0.194, 95% CI 0.077 to 0.490, P = 0.001). The median survival time in patients with serum ULBP2 high expression was significantly shorter than that in patients with serum ULBP2 low expression (28 months vs. 50 months), and there was statistical difference (P<0.05).@*Conclusions@#Serum ULBP2 can be used as an indicator for early diagnosis and prognostic evaluation in patients with colorectal cancer.

Values of early diagnosis and prognosis evaluation of peripheral blood UL16 binding protein 2 expression in patient with colorectal cancer / 中国医师进修杂志

Objective To detect the level of peripheral blood UL16 binding protein 2 (ULBP2) in patients with colorectal cancer,and study its value on early diagnosis and prognosis evaluation.Methods Eighty patients with colorectal cancer (colorectal cancer group) and 60 healthy subjects (healthy control group) from May 2016 to May 2019 in Affiliated Dongfeng Hospital,Hubei University of Medicine were selected.Serum expression level of ULBP2 was detected by enzyme-linked immunosorbent assay.The diagnostic efficacy of serum ULBP2 in colorectal cancer was evaluated by receiver operating characteristic (ROC) curve.The influencing factors of survival in patients with colorectal cancer were analyzed by Cox regression model.Kaplan-Meier method was used for drawing the survival curve,and log-rank test method was used for comparison.Results The serum ULBP2 level in colorectal cancer group was significantly higher than that in healthy control group:(85.52 ± 12.18) ng/L vs.(66.20 ± 8.28) ng/L,and the serum ULBP2 level of stage Ⅰ to Ⅱ in colorectal cancer group was also significantly higher than that in healthy control group:(76.44 ± 7.56) ng/L vs.(66.20 ± 8.28) ng/L,and there were statistical differences (P ＜ 0.05).ROC curve analysis result showed that the optimal cut off value of serum ULBP2 for colorectal cancer diagnosis was 79.53 ng/L,and area under curve (AUC) was 0.869,with a sensitivity of 73.75％ and a specificity of 91.67％;the optimal cut off value of serum ULBP2 for stage Ⅰ to Ⅱ colorectal cancer diagnosis was 71.86 ng/L,and AUC was 0.827,with a sensitivity of 78.57％,and a specificity of 78.33％.According to the median serum ULBP2 level,the patients were divided into ULBP2 high expression (ULBP2 ＞ 85.52 ng/L,38 cases) and ULBP2 low expression (42 cases).The serum expression level of ULBP2 was related to lymph node metastasis and tissue differentiation (P ＜ 0.05).Univariate Cox regression analysis result showed that lymph node metastasis,distant metastasis,tissue differentiation and serum ULBP2 were risk factors of poor prognosis in patients with colorectal cancer (P ＜ 0.01 or ＜ 0.05);multivariate Cox regression analysis result showed that serum ULBP2 was the independent risk factor of poor prognosis in patients with colorectal cancer (HR =0.194,95％ CI 0.077 to 0.490,P =0.001).The median survival time in patients with serum ULBP2 high expression was significantly shorter than that in patients with serum ULBP2 low expression (28 months vs.50 months),and there was statistical difference (P ＜ 0.05).Conclusions Serum ULBP2 can be used as an indicator for early diagnosis and prognostic evaluation in patients with colorectal cancer.

Clinicopathological relevance of tumor expression of NK group 2 member D ligands in resected non-small cell lung cancer.

UL16-binding protein (ULBP) 1-6 and MHC class I chain-related molecule A and B (MICA/B) are NK group 2, member D (NKG2D) ligands, which are specifically expressed in infected or transformed cells and are recognized by NK cells via NKG2D-NKG2D ligand interactions. We previously reported that MICA/B overexpression predicted improved clinical outcomes in patients with resected non-small cell lung cancer (NSCLC). However, the clinicopathological features and prognostic significance of ULBPs in NSCLC remain unclear. Here,ULBP1-6 expression was evaluated based on immunohistochemistry of 91 NSCLC samples from patients following radical surgery. ULBPs were expressed by the majority of NSCLC. Either ULBP1 or ULBP2/5/6 overexpression was associated with squamous-cell carcinoma histology, whereas ULBP4 overexpression was associated with younger age and adenocarcinoma histology. Although overexpression of ULBP1-6 did not impact clinical outcomes in NSCLC patients, integrative profiling with cluster analysis classified patients into 3 subgroups based on the expression pattern of NKG2D ligands. The subgroup characterized by ULBP1 or ULBP2/5/6 high expressing but ULBP4 low expressing tumors showed poor overall survival. Taken together with previous results, NSCLC histological subtype strongly correlates with NKG2D ligands expression pattern. NKG2D ligands expression levels assessed by multiple immune parameters could predict clinical outcomes of patients with NSCLC.

Expresión génica de ligandos mica, micb y ulbp (1-6) del receptor NKG2D de células natural killer y metaloproteinasas adam10, adam17 y mmp14 en lineas celulares de cáncer de cervical / Gene expression of mica, micb and ulbp (1-6) ligands of the NKG2D receptor of natural killer cells and metalloproteinases adam10, adam17 and mmp14 in cells lines of cervical cancer

RESUMEN El CCU es la segunda causa de muerte en mujeres de nuestro país. Dentro de los primeros mecanismos de defensa del hospedero se encuentra la respuesta inmune de las células NK y su función lítica a expensas de su receptor activador NKG2D, el cual posee como ligandos mica, micb y ulbp (1-6), los cuales se expresan en células transformadas y/o infectadas por virus. Uno de los mecanismos de evasión por parte de la célula tumoral es el clivaje de estas proteínas a través de metaloproteinasas como adam10, adam17 y mmp14. Se analizó la expresión de estos ligandos y metaloproteinasas mediante PCR tiempo real, en lineas celulares de referencia para cáncer cervical como HeLa (positiva para VPH-18) y C33A (negativa para VPH). Se obtuvieron valores representativos de expresion relativa genica con diferencias significativas asi: mmp14 en linea HeLa (p= 0.006); y mica y ulbp-3 en la linea C33A (p= 0.020 y p=0.003 respectivamente). Por lo tanto, se podría sugerir que la expresión de mmp14 se encuentra posiblemente involucrados con la presencia de VPH causante del cancer cervical y la respuesta inmunne innata desarrollada.

ABSTRACT Cervical cancer is the second leading cause of death in women in our country. Within the first host defense mechanisms is the immune response of NK cells and their lytic function at the expense of its NKG2D receptor activator which has as ligands mica, micb and ulbp (1-6), which are expressed in transformed cells and / or virally infected. One of the mechanisms of evasion by the tumor cell is the cleavage of these proteins through metalloproteinases as adam10, adam17 and mmp14. We analyzed the expression of these ligands and metalloproteinases by real time PCR, in reference to cell lines HeLa cervical cancer (positive for HPV-18) and C33A (negative for HPV). We obtained representing relative gene expression with significant differences from the other lines of study as follows: mmp14 in HeLa (p = 0.006); and mica and ulbp-3 in C33A (p = 0.020 and p = 0.003 respectively). Thus one might suggest that the expression of mmp14 is possible involved with HPV presence causing high risk of cervical cancer and innate inmunne response developed.

Differential Requirements for gE, gI, and UL16 among Herpes Simplex Virus 1 Syncytial Variants Suggest Unique Modes of Dysregulating the Mechanism of Cell-to-Cell Spread.

Like all the herpesviruses, herpes simplex virus encodes machinery that enables it to move through cell junctions to avoid neutralizing antibodies. This cell-to-cell spread mechanism requires the viral fusion machinery (gD, gH/gL, and gB) and numerous accessory proteins. Of all of these, minor alterations to only four proteins (gB, gK, UL20, or UL24) will dysregulate the fusion machinery, allowing the formation of syncytia. In contrast, removal of individual accessory proteins will block cell-to-cell spread, forcing the virus to transmit in a cell-free manner. In the context of a Syn variant, removal of a required accessory protein will block cell fusion, again forcing cell-free spread. This has been investigated most thoroughly for gBsyn variants, which lose their syncytial phenotype in the absence of several accessory proteins, including gE, gI, UL16, and UL21, which are known to physically interact. Recently it was found that UL21 is not needed for gKsyn-, UL20syn-, or UL24syn-induced cell fusion, and hence it was of interest to ascertain whether gE, gI, and UL16 are required for Syn variants other than gBsyn. Null mutants of these were each combined with seven syncytial variants distributed among gK, UL20, and UL24. Surprisingly, very different patterns of accessory protein requirements were revealed. Indeed, for the three gKsyn variants tested, two different patterns were found. Also, three mutants were able to replicate without causing cytopathic effects. These findings show that mutations that produce Syn variants dysregulate the cell-to-cell-spread machinery in unique ways and provide clues for elucidating how this virus moves between cells.IMPORTANCE Approximately 2/3 of adults worldwide are latently infected with herpes simplex virus 1. Upon reactivation, the virus has the ability to evade neutralizing antibodies by moving through cell junctions, but the mechanism of direct cell-to-cell spread is poorly understood. The machinery that assembles between cells includes the viral fusion proteins and various accessory proteins that prevent cells from fusing. Alterations in four proteins will dysregulate the machinery, allowing neighboring cells to fuse to make syncytia, but this can be prevented by removing various individual accessory proteins to further disable the machinery. Previously, the accessory protein UL21 was found to be important for the activity of some syncytial variants but not others. In this study, we discovered that UL16, gE, and gI all act differently in how they control the fusion machinery. A better understanding of the mechanism of cell-to-cell spread may enable the development of drugs that block it.

Glycoprotein D of HSV-1 is dependent on tegument protein UL16 for packaging and contains a motif that is differentially required for syncytia formation.

Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) plays a key role in multiple events during infection including virus entry, cell-to-cell spread, and virus-induced syncytia formation. Here, we provide evidence that an arginine/lysine cluster located at the transmembrane-cytoplasm interface of gD critically contributes to viral spread and cell-cell fusion. Our studies began with the discovery that packaging of gD into virions is almost completely blocked in the absence of tegument protein UL16. We subsequently identified a novel, direct, and regulated interaction between UL16 and gD, but this was not important for syncytia formation. However, a mutational analysis of the membrane-proximal basic residues of gD revealed that they are needed for the gBsyn phenotype, salubrinal-induced fusion of HSV-infected cells, and cell-to-cell spread. Finally, we found that these same gD tail basic residues are not required for cell fusion induced by a gKsyn variant.

New Insights Into the Regulation of Natural-Killer Group 2 Member D (NKG2D) and NKG2D-Ligands: Endoplasmic Reticulum Stress and CEA-Related Cell Adhesion Molecule 1.

Natural-killer group 2 member D (NKG2D) is a well-characterized activating receptor expressed by natural killer (NK) cells, NKT cells, activated CD8+ T cells, subsets of γδ+ T cells, and innate-like T cells. NKG2D recognizes multiple ligands (NKG2D-ligands) to mount an innate immune response against stressed, transformed, or infected cells. NKG2D-ligand surface expression is tightly restricted on healthy cells through transcriptional and post-transcriptional mechanisms, while transformed or infected cells express the ligands as a danger signal. Recent studies have revealed that unfolded protein response pathways during endoplasmic reticulum (ER) stress result in upregulation of ULBP-related protein via the protein kinase RNA-like ER kinase-activating factor 4-C/EBP homologous protein (PERK-ATF4-CHOP) pathway, which can be linked to the pathogenesis of autoimmune diseases. Transformed cells, however, possess mechanisms to escape NKG2D-mediated immune surveillance, such as upregulation of carcinoembryonic antigen (CEA)-related cell adhesion molecule 1 (CEACAM1), a negative regulator of NKG2D-ligands. In this review, we discuss mechanisms of NKG2D-ligand regulation, with a focus on newly discovered mechanisms that promote NKG2D-ligand expression on epithelial cells, including ER stress, and mechanisms that suppress NKG2D-ligand-mediated killing of cancer cells, namely by co-expression of CEACAM1.