Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target

Expression of B7-H1, a costimulating glycoprotein in the B7 family, is normally restricted to macrophage-lineage cells, providing a potential costimulatory signal source for regulation of T cell activation. In contrast, aberrant expression of B7-H1 by tumor cells has been implicated in impairment of T cell function and survival, resulting in defective host antitumoral immunity. The relationship between tumor-associated B7-H1 and clinical cancer progression is unknown. Herein, we report B7-H1 expression by both renal cell carcinoma (RCC) tumors of the kidney and RCC tumor-infiltrating lymphocytes. In addition, our analysis of 196 clinical specimens reveals that patients harboring high intratumoral expression levels of B7-H1, contributed by tumor cells alone, lymphocytes alone, or tumor and/or lymphocytes combined, exhibit aggressive tumors and are at markedly increased risk of death from RCC. In fact, patients with high tumor and/or lymphocyte B7-H1 levels are 4.5 times more likely to die from their cancer than patients exhibiting low levels of B7-H1 expression (risk ratio 4.53; 95% confidence interval 1.94-10.56; P < 0.001.) Thus, our study suggests a previously undescribed mechanism whereby RCC may impair host immunity to foster tumor progression. B7-H1 may prove useful as a prognostic variable for RCC patients both pre- and posttreatment. In addition, B7-H1 may represent a promising target to facilitate more favorable responses in patients who require immunotherapy for treatment of advanced RCC.     

B7-H3 and B7x are highly expressed in human prostate cancer and associated with disease spread and poor outcome

B7-H3 and B7x are recently discovered members of the B7-CD28 family thought to dampen peripheral immune responses via negative costimulation. We evaluated their potential expression in human prostate cancer using a large cohort of patients with 7 years of follow-up. We identified 823 patients with tissue available treated with radical prostatectomy between 1985 and 2003. Immunohistochemistry was performed on tissue microarray sections using anti-B7-H3 and -B7x. The percentage and intensity of immunoreactivity by tumor cells were blindly evaluated by two urological pathologists, and outcome analyses were conducted. Both B7-H3 and B7x were highly expressed; 93% and 99% of tumors had aberrant expression, respectively. The median percentage of tumor cells staining positive was 80% for each molecule. Strong intensity for B7-H3 and B7x was noted in 212 (26%) and 120 (15%) patients, respectively. Patients with strong intensity for B7-H3 and B7x were significantly more likely to have disease spread at time of surgery (P < 0.001 and P = 0.005, respectively). Additionally, patients with strong intensity for B7-H3 and B7x were at significantly increased risk of clinical cancer recurrence (P < 0.001 and P = 0.005) and cancer-specific death (P = 0.004 and P = 0.04, respectively). To our knowledge, we present the largest investigation of B7 family molecules in a human malignancy and a previously undescribed evaluation of B7x in prostate cancer. B7-H3 and B7x are abundantly expressed in prostate cancer and associated with disease spread and poor outcome. Given the proposed immune-inhibitory mechanisms of B7-H3 and B7x, these molecules represent attractive targets for therapeutic manipulation in prostate cancer.     

Expression of B7-H4 and hepatitis B virus X in hepatitis B virus-related hepatocellular carcinoma

AIM: To investigate the expression and clinical significance of B7-H4 and hepatitis B virus X(HBx) protein in hepatitis B virus-related hepatocellular carcinoma(HBV-HCC).METHODS: The expression of B7-H4 in the human HCC cell lines Hep G2 and Hep G2.2.15 were detected by western blot, flow cytometry, and immunofluorescence. The expression of B7-H4 and HBx in 83 HBV-HCC was detected by immunohistochemistry, and the relationship with clinicopathological features was analyzed. Paraffin sections were generated from 83 HBV-HCC patients(22 females and 61 males) enrolled in this study. The age of these patients ranged from 35 to 77 years, with an average of 52.5 ± 11.3 years. All experiments were approved by the Ethics Committees of the Second Affiliated Hospital, Zhejiang University School of Medicine.RESULTS: B7-H4 was significantly upregulated in Hep G2.2.15 cells compared to Hep G2 cells. Specifically, the protein expression of B7-H4 in the lysates of Hep G2 cells was more than that in Hep G2.2.15 cells. In addition, HBx was expressed only in Hep G2.2.15 cells. Similar data were obtained by flow cytometry. The positive rates of B7-H4 and HBx in the tissues of 83 HBV-HCC patients were 68.67%(57/83) and 59.04%(49/83), respectively. The expression of HBx was correlated with tumor node metastases(TNM) stage, and the expression of B7-H4 was positively correlated with HBx(rs = 0.388; p 0.01). The expression level of B7-H4 in HBx-positive HBV-HCC tissues was substantially higher than that in HBx-negative HBV-HCC tissues. The expression level of B7H4 was negatively related to tumor TNM stage.CONCLUSION: Higher expression of HBx and B7-H4 was correlated with tumor progression of HBV-HCC, suggesting that B7-H4 may be involved in facilitating HBV-related hepatocarcinogenesis.     

Role of transforming growth factor-alpha in von Hippel--Lindau (VHL)(-/-) clear cell renal carcinoma cell proliferation: a possible mechanism coupling VHL tumor suppressor inactivation and tumorigenesis

Mutations of the VHL tumor suppressor gene occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL(-/-) RCC). Loss of VHL protein function is associated with constitutive expression of mRNAs encoding hypoxia-inducible proteins, such as vascular endothelial growth factor. Overproduction of angiogenic factors might explain why VHL(-/-) RCC tumors are so highly vascularized, but whether this overproduction is sufficient for oncogenesis still remains unknown. In this report, we examined the activity of transforming growth factor-alpha (TGF-alpha), another VHL-regulated growth factor. We show that TGF-alpha mRNA and protein are hypoxia-inducible in VHL(-/-) RCC cells expressing reintroduced VHL. In addition to its overexpression by VHL(-/-) RCC cells, TGF-alpha can also act as a specific growth-stimulatory factor for VHL(-/-) RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of RCC. This role is in contrast to those of other growth factors overexpressed by VHL(-/-) RCC cells, such as vascular endothelial growth factor and TGF-beta1, which do not stimulate RCC cell proliferation. A TGF-alpha-specific antisense oligodeoxynucleotide blocked TGF-alpha production in VHL(-/-) RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture. Growth of VHL(-/-) RCC cells was also significantly reduced by a drug that specifically inhibits the epidermal growth factor receptor, the receptor through which TGF-alpha stimulates proliferation. These results suggest that the generation of a TGF-alpha autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells may provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.     

The expression and distribution of immunomodulatory proteins B7-H1, B7-DC, B7-H3, and B7-H4 in rheumatoid synovium

CD28/B7 signals have been shown to have the capacity to regulate T cell activation and participate in regulating the development of rheumatoid arthritis (RA). However, the expression and anatomical distribution of some members of the B7 superfamily including B7-H1, B7-DC, B7-H3 and B7-H4 in RA synovium is still unclear. We analyzed the expression of these molecules in synovial tissues from RA patients. Immunohistochemistry showed that all of these molecules were observed in synovium. On the cellular level, all of them were found on cell membrane and in cytoplasma. The expression of B7-DC and B7-H3 was major on capillaries, synovicytes and infiltrated inflammatory cells in the lining layer, while B7-H1 and B7-H4 were detected in some inflammatory cells residing in the sublining and lining layer. Fluorescent dual staining indicated that all these molecules were principally associated with CD31⁺ endothelial cells and CD68⁺ macrophages. In addition, B7-H1 and B7-H3 were also observed on CD3⁺ T cells (including CD4⁺ and CD8⁺ T cells). Interestingly, B7-H1/B7-H4, B7-H3/B7-DC were co-expressed on the same cells. The characteristic expression and distribution of these molecules in synovium indicated that they probably have different effects during the progress of RA, and a clear understanding of their functional roles may further elucidate the pathogenesis of this disease.     

B7-H1 expression is associated with expansion of regulatory T cells in colorectal carcinoma

AIM: To investigate the expression of B7-H1 in human colorectal carcinoma (CRC) to define its regulating effects on T cells in tumor microenvironment.METHODS: One hundred and two paraffin blocks and 33 fresh samples of CRC tissues were subject to this study. Immunohistochemistry was performed for B7-H1 and CD3 staining in CRC tissues. Ficoll-Hypaque density gradient centrifugation was used to isolate peripheral blood mononuclear cells of fresh CRC tissues; flow cytometry and immunofluorescence staining were used for detection of regulatory T cells. Data was analyzed with statistical software.RESULTS: Costimulatory molecule B7-H1 was found strongly expressed in CRC tissues, localized in tumor cell membrane and cytoplasm, while weak or none expression of B7-H1 was detected in pared normal colorectal tissues. Meanwhile, CD3 positive T cells were found congregated in CRC tumor nest and stroma. Statistic analysis showed that B7-H1 expression level was negatively correlated to the total T cell density in tumor nest (P < 0.0001) and tumor stroma (P = 0.0200) of 102 cases of CRC tissues. Among the total T cells, a variable amount of regulatory T cells with a clear Foxp3⁺ (forkhead box P3) staining could be detected in CRC tissues and patients’ blood. Interestingly, in the 33 samples (15 cases of B7-H1^high CRC tissues and 18 cases of B7-H1^low CRC tissues) of freshly isolated mononuclear cells from CRC tissues, the percentages of CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ regulatory T cells were found remarkably higher in B7-H1^high CRC tissues than in B7-H1^low CRC tissues (P = 0.0024, P = 0.0182), indicating that B7-H1 expression was involved in proliferation of regulatory T cell. No significant difference was found in CRC peripheral blood (P = 0.0863, P = 0.0678). PD-1 is the specific ligand for B7-H1 pathway transferring inhibitory signal to T cell, which is expressed by activated T cell. Our further analysis of PD-1 expression on T cells in CRC tissues showed that conventional T cells (CD4⁺Foxp3^-/CD8⁺Foxp3^-), which was thought to contribute to the anti-tumor immune response, highly expressed PD-1; while regulatory T cells (CD4⁺Foxp3⁺/CD8⁺Foxp3^-) almost failed to express PD-1. The average percentage of PD-1 expression on regulatory T cells was significantly higher than the percentage of PD-1 on conventional T cells (CD4⁺Foxp3^- T cell, P < 0.0001; CD8⁺Foxp3^- T cell, P < 0.0001). The diverse expression of PD-1 might lead to different fate of T cell subsets in B7-H1 over-expression CRC tumor microenvironment.CONCLUSION: B7-H1 expression in tumor cells can inhibit the conventional T cell proliferation in tumor microenvironment through the PD-1 expression on conventional T cells.     

Investigation of the epithelial to mesenchymal transition markers S100A4, vimentin and Snail1 in gastroesophageal junction tumors

Epithelial to mesenchymal transition (EMT) promotes tumor progression and invasion. As no study has focused on gastroesophageal junction (GEJ) tumors, the expression of three EMT‐related proteins (S100A4, vimentin, and Snail1) was investigated with the aim of assessing their pathologic and prognostic significance. Resection specimens were obtained from 104 patients who underwent surgery for GEJ adenocarcinoma, without preoperative chemotherapy. Three tissue cores were obtained from each of the tumor body (TB), luminal surface (LS), and invasive edge (IE) to produce tissue microarrays, and immunohistochemical staining was performed. The microarrays were scored independently by two observers. The demographic and histopathologic details of the patients were collected. Overall positive expression was observed in 88 (S100A4, 85%), 16 (vimentin, 14%), and 92 (Snail1, 89%) tumors. Staining for S100 A4 was positive in 79 (76%) of TB, 69 (66%) of IE, and 69 (66%) of LS specimens. Staining for vimentin was positive in 7 (6%) of TB, 11 (11%) of IE, and 5 (5%) of LS specimens. Staining for Snail1 was positive in 83 (80%) of TB, 51 (49%) of IE, and 78 (75%) of LS specimens. Positive staining of TB for S100A4 (P = 0.04) and Snail1 at IE (P = 0.01) was associated with involvement of circumferential resection margins. Positive staining for S100A4 in the TB (P = 0.02) and LS (P = 0.01) was associated with poor 5‐year overall survival. Vimentin had no statistically significant relationships with pathologic factors or outcome. The acquisition of mesenchymal protein S100A4 is associated with a poor prognosis in patients with GEJ tumors who undergo potentially curative surgery, and LS samples can be used to obtain prognostic information. Increased EMT‐related protein expression (S100A4, Snail1) is associated with the involvement of circumferential resection margin.     

Loss of heterozygosity of thenm23-H1 gene in human renal cell carcinomas

This study evaluates the potential contribution of thenm23-H1 gene to malignant transformation in patients with renal cell carcinoma. Using specific oligonucleotide primers for thenm23-H1 microsatellite repetitive sequence, gene instability was followed by polymerase chain reaction/loss of heterozygosity assay on 54 tumor specimens and the corresponding normal tissue samples. We also determined, immunohistochemically, the relative concentration and localization of thenm23-H1 protein product. From 77.7% informative cases, DNA from 6 tumors exhibited loss of heterozygosity, regardless of the tumor stage (TNM). Out of 39 samples analyzed, 30 were negative for Nm23-H1 protein, while the others were only slightly positive. No correlation with tumor stage was found. Normal renal tissue was also negative for this protein. Our results provide the evidence for loss of heterozygosity, followed by means of microsatellite tandem-repeat polymorphism, at thenm23-H1 locus in renal cell carcinoma. However, since no correlation was found between the tumor stage or metastatic potential on the one hand, and allelic loss and specific protein expression on the other, it seems thatnm23-H1 does not play a key role in the invasiveness of this tumor type.Abbreviations PCR polymerase chain reaction - LOH loss of heterozygosity     

Expression of survivin in gastric cancer and its relationship with tumor angiogenesis

BACKGROUND/AIMS: Survivin, a member of inhibitors of apoptosis, has been found in various human cancers. Its expression is associated with tumor progression and adverse outcome. Angiogenesis is an essential process for the primary tumor to grow and invade the adjacent normal structures. Angiogenic factors such as vascular endothelial growth factor induce survivin expression in endothelial cells. The current study was designed to investigate the possible role of survivin and vascular endothelial growth factor status for angiogenesis in human gastric cancer. METHODS: In this study, we conducted an immunohistochemical investigation of survivin and vascular endothelial growth factor expression in 106 tissue samples obtained from gastric cancer patients undergoing surgical treatment. To assess tumor angiogenesis, microvessel density was counted by staining endothelial cells immunohistochemically using anti-CD34 monoclonal antibody. RESULTS: The positive expression of survivin and vascular endothelial growth factor in gastric cancer tissues was demonstrated in 50.0 and 69.8% of cases, respectively. The expression of survivin did not associate with vascular endothelial growth factor expression. Expression of survivin was significantly associated with tumor size, depth of invasion, lymph node metastasis, tumor stage and poor survival (P=0.011, 0.004, 0.020, 0.002, 0.046, respectively). High microvessel density was significantly associated with lymph node metastasis and poor survival (P=0.006 and 0.017, respectively). The mean microvessel density value of survivin positive tumors was 87.4+/-34.4 and significantly higher than that of survivin negative tumors (P=0.016). The mean microvessel density value of vascular endothelial growth factor positive tumors was 98.7+/-37.0 and significantly higher than that of vascular endothelial growth factor negative tumors (P=0.001). A combined analysis of survivin and vascular endothelial growth factor status showed that the mean microvessel density value of both positive tumors was 103.7+/-33.1 and significantly higher than that of both negative tumors (P<0.001). CONCLUSION: These results suggest that survivin may play an important role in carcinogenesis by stimulating tumor angiogenesis in human gastric cancer.     

Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis

AIM:To investigate the expression of co-stimulatorymolecule B7-H3 in gastric carcinoma and adenomatissue as well as normal gastric tissue and to explore therelationship between B7-H3 expression and pathologicalfeatures and prognosis of gastric carcinoma.METHODS:B7-H3 expression was detected in 102samples of human gastric carcinoma and 10 samples ofgastric adenoma and 10 samples of normal gastric tissueby immunohistochemical assay.Correlation betweenthe expression of B7-H3 and the patients'age,sex,gastric carcinoma locus,tumor size,tissue type,tumorinfiltration depth,differentiation degree,lymph nodemetastasis,and survival time was analyzed.RESULTS:B7-H3 was expressed in all gastric adenomasamples and in 58.8% samples of gastric carcinoma.B7-H3 expression in gastric carcinoma samples wasnot related with the patients'age,sex,lymph nodemetastasis,and tumor size(P>0.05),but with thesurvival time,infiltration depth of tumor and tissue type.CONCLUSION:Detection of B7-H3 expression in gastriccarcinoma tissue is beneficial to the judgment of theprognosis of gastric carcinoma patients and the choice oftreatment.     

Hepatic B7 homolog 1 expression is essential for controlling cold ischemia/reperfusion injury after mouse liver transplantation

Ischemia/reperfusion (I/R) injury remains a key risk factor significantly affecting morbidity and mortality after liver transplantation (LT). B7 homolog 1 (B7-H1), a recently identified member of the B7 family, is known to play important roles in regulating local immune responses. We hypothesized that B7-H1 plays crucial roles during innate immune responses induced by hepatic I/R injury, and using B7-H1 knockout (KO) liver grafts, we tested this hypothesis in the mouse LT model with 24 hours of cold storage. Cold I/R injury in wild type (WT)-to-WT LT enhanced constitutive B7-H1 expression on dendritic cells and sinusoidal endothelial cells and promptly induced B7-H1 on hepatocytes. When B7-H1 KO liver grafts were transplanted into WT recipients, serum alanine aminotransferase (ALT) and graft necrosis levels were significantly higher than those after WT-to-WT LT. Augmented tissue injury in B7-H1 KO grafts was associated with increased frequencies and absolute numbers of graft CD3(+) T cells (particularly CD8(+) T cells). B7-H1 KO grafts had significantly fewer annexin V(+) CD8(+) T cells, and this indicated a failure to delete infiltrating CD8(+) T cells. To evaluate the relative contributions of parenchymal cell and bone marrow-derived cell (BMDC) B7-H1 expression, we generated and transplanted into WT recipients chimeric liver grafts lacking B7-H1 on parenchymal cells or BMDCs. A selective B7-H1 deficiency on parenchymal cells or BMDCs resulted in similar levels of ALT and liver injury, and this suggested that parenchymal cell and BMDC B7-H1 expression was involved in liver damage control. Human livers up-regulated B7-H1 expression after LT. CONCLUSION: The study demonstrates that graft tissue expression of B7-H1 plays a critical role in regulating inflammatory responses during LT-induced hepatic I/R injury, and negative coregulatory signals may have an important function in hepatic innate immune responses.     

The role of leukemia-derived B7-H1 (PD-L1) in tumor-T-cell interactions in humans

OBJECTIVE: Expression of the B7 homolog B7-H1 (PD1-Ligand) has been proposed to enable tumor cells to evade immune surveillance. Recently, B7-H1 on murine leukemia cells was reported to mediate resistance to cytolytic T-cell destruction. We here investigate the expression and function of the B7 homolog B7-H1 in human leukemia. PATIENTS AND METHODS: Leukemia cells from 30 patients and 9 human leukemia cell lines were investigated for B7-H1 expression by flow cytometry. Functional relevance of B7-H1 for tumor-immune interactions was assessed by coculture experiments using purified, alloreactive CD4 and CD8 T cells in the presence of a neutralizing anti-B7-H1 antibody. RESULTS: Significant B7-H1 expression levels on leukemia cells were detected in 17 of 30 patients and in eight of nine cell lines. In contrast to various other tumor entities and the data reported from a murine leukemia system, no significant inhibitory effect of leukemia-derived B7-H1 on CD4 and CD8 cytokine production (IFN-gamma, IL-2), proliferation or expression of T-cell activation markers (ICOS, CD69) was observed. Furthermore, in the presence of neutralizing B7-H1 antibody (mAb 5H1) occurred no significant changes in T cell IFN-gamma or IL-2 production or proliferation. CONCLUSIONS: Our data demonstrate that leukemia-derived B7-H1 seems to have no direct influence on T-cell activation, proliferation, and cytokine production in humans. Further experiments are warranted to delineate factors and characterize yet-unidentified B7-H1 receptor(s) that determine inhibitory and stimulatory functions of B7-H1 in human leukemia.     

Serum levels of interleukin-18 and nitrite+nitrate in renal cell carcinoma patients with different tumor stage and grade

Interleukin (IL-18) and nitric oxide (NO) are immunoregulatory cytokines that have been suggested to participitate in the multistep process of carcinogenesis. In this study, serum IL-18 and nitrite+nitrate levels (as an index of NO generation) were measured in 26 patients with histologically confirmed renal cell carcinoma (RCC) before surgical procedure and in 8 healthy controls. RCC patients showed significantly higher serum IL-18 levels when compared to the control subjects (p<0.001). Serum IL-18 levels were found to be similar in patients with T1 and T2, T3, T4 tumors (p>0.05). Patients with grade 3 and 4 tumors showed significantly higher serum IL-18 levels when compared to the patients with grade 1 and 2 tumors (p<0.05). No significant difference was found in serum nitrite+nitrate levels between RCC patients and control subjects (p>0.05). Serum nitrite+nitrate levels were lower in patients with grade 3 and 4 tumors than those with grade 1 and 2 tumors, but this was not statistically significant (p=0.063). In conclusion, elevated serum level of IL-18 in RCC patients than in controls and extra-increase of this level in those with high grade tumors may reflect the degree of human defence mechanisms against tumor cells in RCC.     

核转录因子κB在非小细胞肺癌中的表达及意义

目的研究核转录因子κB（nucleus factor-кB,NF-кB）P65亚单位在非小细胞肺癌（non-small cell lung cancer,NSCLC）中的表达及与非小细胞肺癌临床病理特征的关系及意义。方法采用免疫组织化学染色方法（PV-6000通用型二步法）检测62例NSCLC组织和20例癌旁肺组织中NF-кBP65蛋白。结果P65蛋白在NSCLC组织中阳性表达率为79%（49/62）,明显高于癌旁肺组织50%（10/20）,差异有统计学意义（P〈0.05）。P65蛋白与NCSCLC细胞分化程度、肺癌分期和淋巴结转移有关,差异有统计学意义（P〈0.05）。结论NF-кBP65蛋白高表达与NSCLC的发生、细胞增殖及浸润转移相关,可作为预后观测的指标和分子治疗的新靶点。     

宫颈癌组织中B7-H3蛋白的表达变化及其与HPV感染的关系

目的观察宫颈癌组织中的B7家族同族3（B7-H3）蛋白的表达变化,探讨其与人乳头瘤病毒（HPV）感染的关系。方法采用免疫组化SP法检测50例宫颈癌组织肿瘤细胞和血管内皮细胞（宫颈癌组）中及15例子宫肌瘤组织（对照组）中的B7-H3蛋白。采用人乳头状瘤病毒核酸扩增分型检测试剂盒检测两组HPV感染情况。结果宫颈癌组肿瘤细胞B7-H3蛋白阳性41例,内皮细胞B7-H3蛋白阳性为24例;对照组宫颈组织细胞B7-H3蛋白阳性为3例,内皮细胞中无B7-H3表达,两组相比,P均〈0.05。宫颈癌组中HPV感染25例;对照组中无HPV感染者。宫颈癌组肿瘤细胞及血管内皮细胞中B7-H3蛋白表达与HPV感染呈正相关（r分别为0.364 4、0.400 3,P均〈0.05）。结论宫颈癌肿瘤细胞及血管内皮细胞中B7-H3呈高表达,且与HPV感染有关。     

人蛋白酶体β亚基4型在肝细胞癌中的表达及与预后的关系

目的 探讨人蛋白酶体β亚基4型(PSMB4)在肝细胞癌(以下简称肝癌)组织及正常肝组织中的表达及其在临床预后中的意义。方法 收集2017年1月-10月在南通大学附属医院行手术治疗的8例肝癌组织及癌旁组织标本,应用Western Blot法检测肝癌组织及相应癌旁组织中PSMB4蛋白的表达情况。另收集2009年1月-2012年10月南通大学附属医院留存的105例肝癌组织和25例正常肝组织的石蜡包埋标本,应用免疫组化法进一步检测PSMB4的表达情况。根据PSMB4免疫组化结果差异将105例肝癌患者分为PSMB4高表达组(n=57)和PSMB4低表达组(n=48)。计量资料2组间比较采用独立样本t检验;计数资料2组间比较采用χ2检验。Cox风险回归模型分析2组患者临床病理特征及预后的关系,Kaplan-Meier生存曲线分析不同PSMB4水平患者的生存情况。结果 2组间HBV感染、肿瘤最大直径、肿瘤分化程度、TNM分期比较差异均有统计学意义(χ^2值分别为22.482、8.219、14.964、6.587,P值均<0.05)。105例肝癌患者中57例(54.29%)为PSMB4 高表达,48例(45.71%)为PSMB4低表达;而25例正常肝组织中8例(32%)为PSMB4高表达,17例(68%)为PSMB4低表达或不表达,2组表达情况比较差异有统计学意义(χ^2=4.011, P<0.05)。PSMB4染色主要分布于细胞浆和细胞核内,染色呈棕色及棕黄色颗粒样。8例新鲜肝癌及癌旁组织样本中有7例肝癌组织的PSMB4蛋白表达水平明显高于癌旁组织(P值均<0.05)。单因素分析显示患者的5年生存状态与肿瘤大小(P=0.01)、转移情况(P<0.001)、分化程度(P=0.01)、TNM分期(P=0.003)以及PSMB4表达水平(P<0.001)有关;多因素结果显示肿瘤转移[风险比(HR)=11.375,95%可信区间(95%CI):4.911~26.348)]和PSMB4高表达(HR=6.834,95%CI: 2.939~15.889)是肝癌患者的独立危险因素(P值均<0.001)。PSMB4高表达组患者5年生存率明显低于PSMB4低表达组(27.6% vs 79.2%, χ^2=22.96,P<0.05)。结论 PSMB4在肝癌组织中的表达增高,并且其高表达是肝癌患者预后的独立危险因素,PSMB4有可能作为肝癌预后的潜在标志及治疗的潜在靶点。     

Study of human B7 homolog 1 expression in patients with hepatitis B virus infection

AIM: To further investigate the role of human B7 homolog 1 (B7-H1) in the mechanism of persistent hepatitis B virus (HBV) infection. METHODS: Peripheral and intra-hepatic B7-H1 expression were compared by flow cytometry and immunochemical staining between two 2 distinct groups, one being chronic HBV tolerance patients (CHB-T) and the other being acute hepatitis B patients (AHB). B7-H1 mRNA expression level was also compared by real time polymerase chain reaction between CHB-T and AHB patients. The location of intra-hepatic B7-H1 and CD40 expression were analyzed by immunofluorescence. The levels of B7-H1 and CD40 expression on cultured myeloid dendritic cells (mDCs) with or without hepatitis B surface antigen (HBsAg) treatment were analyzed dynamically by flow cytometry. Intracellular interferon-γ (IFN-γ) staining and the stimulatory capacity of mDC of cultured mDC with or without HBsAg treatment were also compared by flow cytometry. RESULTS: Peripheral B7-H1 expression on mDCs was increased significantly in AHB compared to CHB-T patients (P 0.05). In the liver tissues from CHB-T patients, B7-H1 positive cells were almost absent despite a persistently elevated serum HBsAg load. In contrast, there were indeed increased B7-H1-positive cells in situ in the liver tissue from AHB. In vitro analysis showed the parallel upregulation of B7-H1 and CD40 on CD11c+ mDCs after the onset of stimulation. Addition of recombinant hepatitis B surface antigen (rHBsAg) significantly decreased CD40 expression (P 0.05 at 16 h, 20 h and 24 h time points). B7-H1 expression was also inhibited by rHBsAg, and the inhibition rate of CD40 was greater than that of B7-H1. This preferential inhibition of CD40 expression on mDCs by rHBsAg resulted in the dysfunction of mDCs and T cells in the mixed leucocyte reaction (MLR) system. With rHBsAg pretreatment, in a carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled MLR system at a ratio of 1:5 responder cell-stimulator cell (R/S), the CFSE dim percentage of T cells decreased from 85.1% to 25.4% and decreased from 30.3% to 12.0% at 1:10 R/S. IFN-γ production by CD8+ T cells, in the MLR system, was reduced significantly by HBsAg pretreatment. At ratios of 1:5 R/S, the percentage of IFN-γ and CD8 dual positive T cells decreased from 55.2% ± 5.3% to 15.1% ±3.1% (P 0.001), and decreased from 35.0% ± 5.1% to 7.3% ± 2.7% at ratios of 1:10 R/S (P 0.001). CONCLUSION: B7-H1 is not a signature of immune dysfunction, but an inflammation marker. HBsAg regulate immune response by tipping the balance between B7-H1 and CD40.