Dysregulation of blood lymphocyte subsets and natural killer cells in schistosomal liver cirrhosis and hepatocellular carcinoma

Abstract. Immunological factors are important in the pathogenesis of a wide spectrum of hepatobiliary diseases. Using flow cytometry, we determined the changes in lymphocyte subsets and natural killer cells in 123 individuals (81 patients with liver disease and 42 healthy volunteers). The liver diseases included periportal fibrosis (PPF, 10 patients), liver cirrhosis (LC, 31 patients), and hepatocellular carcinoma (HCC, 40 patients). Schistosomiasis and viral hepatitis B and C were the putative etiological agents of liver diseases. Immunophenotyping by indirect immunofluorescence was conducted using monoclonal antibodies to CD3 (T-lymphocytes), CD4 (helper/inducer T-cells), CD8 (suppressor/cytotoxic T-cells), and CD57 (natural killer cells) cell surface markers. Immunophenotyping of PPF patients showed no significant changes in all markers compared with the healthy controls. However, there was a significant decrease (P<0.01) in CD3 and CD4 T-cells, and a highly significant increase (P<0.001) in CD57 T-cells in patients with LC or HCC. In addition, LC and HCC patients showed no significant change in CD8 T-cells compared with controls. In conclusion, the progression of liver diseases is associated with a dysregulation of cellular immune responses. T-lymphocytes and natural killer cells may play a role in the immunopathogenesis of liver cirrhosis and HCC.     

Current approaches to the diagnosis and management of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, and incidence rates in Western countries are on the rise. Despite many options, no ideal treatment yet exists for this highly malignant tumour, and management strategies have varied accordingly. This review summarises current strategies for the diagnosis and management of HCC.     

Cryopreservation of human lymphocytes for assessment of lymphocyte subsets and natural killer cytotoxicity

Cryopreservation of lymphocytes has become increasingly important, especially when the cells are to be used in retrospective studies of selected and dwindling populations, such as A-bomb survivors. This report describes an efficient method for cryopreservation of human lymphocytes which does not significantly alter various immunological characteristics of these cells. The proportions of Leu-1⁺ cells (T cells), Leu-2a⁺ cells (suppressor-cytotoxic T cells), Leu-3a⁺ cells (helper-inducer T cells), HLA-DR⁺ cells, Mo2⁺ cells (monocytes), B1⁺ cells (B cells), and Leu-7⁺ cells (natural killer (NK) cells), as determined by monoclonal antibodies, were found to be stable following cryopreservation. NK cell activity against K-562 target cells showed a 40–60% decrease immediately after thawing, but recovered to approximate pre-freezing levels after preincubation for 18 h. Neither lymphocyte subsets nor cell viability significantly changed following preincubation after cryopreservation. However, the ratio of cells binding to K-562 cells increased after this preincubation and may account for the observed recovery of NK cell activity. NK cell activity remained relatively stable up to 14 months of storage which confirms that freezing damage depends on the freezing process rather than on the duration of cryopreservation.     

AFP-L3阳性肝细胞癌患者肿瘤分期和外周血淋巴细胞亚群的研究

目的: 分析小扁豆凝素结合型甲胎蛋白(AFP-L3)阳性的肝细胞癌患者肿瘤分期及外周血淋巴细胞的改变,探讨AFP-L3水平与肿瘤分期及免疫状态的关系。方法:采集60例肝细胞癌患者外周血,检测AFP及AFP-L3含量。根据外周血AFP-L3比例将60例原发性肝细胞癌患者分为2组:AFP-L3(+)组和AFP-L3(-)组。流式细胞术检测外周血中各淋巴细胞亚群(NK、CD4⁺、CD8⁺、CD4/CD8、Treg)的比例。结果:AFP-L3(+)组患者UICC分期Ⅲ+Ⅳ期为29例,AFP-L3(-)组为7例;AFP-L3(+)组患者BCLC分期C+D期为20例,AFP-L3(-)组为3例。AFP-L3(+)组患者发生血管侵犯为17例,远高于AFP-L3(-)组(1例)。AFP-L3(+)组患者外周血中NK细胞比例及CD4/CD8比值下降,CD8⁺T和Treg细胞显著比例上升。结论:外周血AFP-L3阳性的肝癌患者容易发生血管侵犯,且分期较差,可能与淋巴细胞亚群发生变化、免疫系统受到抑制有关。     

Primary extranodal nasal-type natural killer/T-cell lymphoma of the brain: a case report

Natural killer (NK)/T-cell lymphomas represent a rare type of lymphoma derived from either activated NK cells or, rarely, cytotoxic T cells. These lesions are most commonly extranodal and tend to present as destructive lesions within the midline facial structures. Other than the nasal cavity and paranasal sinuses, several other extranodal sites of involvement have been reported, including the pharynx, gastrointestinal tract, and testis. Although secondary involvement of the central nervous system has been reported, a convincing case of primary brain NK/T-cell lymphoma has not been previously reported. Here, we report a case of primary brain lymphoma of NK/T-cell type with a characteristic phenotype expressing CD3epsilon, CD56, granzyme B, Epstein-Barr virus-encoded small nuclear RNAs, with germline T-cell receptor gene configuration, and showing an unusual intravascular component. The patient underwent extensive imaging studies, revealing only the brain lesion. The lymphoma failed to respond to therapy and the patient eventually died after transfer to a hospice facility. This unusual case highlights an unusual presentation of a rare disease entity and highlights the need for a better understanding of the biology and treatment of T-cell lymphomas.     

Apelin/APJ信号在肝硬化及肝癌中的生物作用

Apelin/APJ信号在心脏、血管、脑、肺、胃肠道、肾脏和骨骼肌等多种组织中都有表达,可以参与多种细胞的信号转导过程,所以在调节人体生理和病理生理活动方面可发挥重要的调节作用。Apelin/APJ信号的作用主要包括调节心血管系统的稳态、维持水电解质的平衡、促进垂体激素释放、调节生物节律,还可以作为免疫调节因子对免疫系统进行调节。但是apelin/APJ信号在肝脏疾病中发挥什么样的作用还知之甚少。在肝脏疾病中,apelin/APJ信号可能参与到肝纤维化、肝硬化和肝癌的发生和发展,并且可抑制肝脏的再生。我们对apelin/APJ信号近年来在肝脏方面的研究做一综述。     

Relationship between impaired apoptosis of lymphocytes and distribution of dendritic cells in peripheral blood and synovial fluid of children with juvenile idiopathic arthritis

INTRODUCTION: The pathogenesis of juvenile idiopathic arthritis (JIA) is not fully understood. Recently the present authors described disturbed apoptosis of JIA lymphocytes in both peripheral blood (PB) and synovial fluid (SF) as well as an abnormal distribution of blood dendritic cells (BDCs) between the PB and SF in this disease. Possible relationships between these events during the development of JIA process are assessed here. MATERIALS AND METHODS: Lymphocyte apoptosis and BDC counts were assessed in the PB and SF of untreated JIA children. Lymphocyte apoptosis was analyzed by the Annexin-V/propydium iodide assay. Total DC (TDC) number was based on the sum of three BDC subpopulations determined using a panel of monoclonal antibodies against BDC antigens (BDCA): myeloid type 1 (mDC1, BDCA-1(+)/HLA-DR(+)/CD19(-)), myeloid type 2 (mDC2, BDCA-3(+)/HLA-DR(+)/CD14(-)), and plasmacytoid (pDC, BDCA-2(+)/HLA-DR(+)/CD123(+)). Cells were enumerated by the flow cytometric "single-platform" method. The concentration of tumor necrosis factor (TNF)-alpha and the distribution of particular lymphocyte subtypes in both PB and SF were also investigated. RESULTS: There was significant positive correlation between apoptosis of PB lymphocytes and SF TDC count (p=0.002) as well as SF TNF-alpha concentration (p=0.007). SF TNF-alpha levels also correlated with SF TDC count (p=0.003). Moreover, JIA SF was distinctly enriched with CD4+ and CD8+ T lymphocytes and included CD4(+)/CD25(high) cells as well. There was significant positive correlation between the number of CD4(+)/CD25(high) cells and SF JIA BDC count (p=0.015). CONCLUSIONS: These data suggest a possible link between impaired apoptosis of PB/SF lymphocytes and increased recruitment of PB BDCs to SF and other elements of the immune system in JIA, including regulatory CD4+/CD25high cells.     

Distribution of somatostatin in pancreatic ductal adenocarcinoma remodels the normal pattern of the protein during foetal pancreatic development: an immunohistochemical analysis

AIM: To determine the immunoreactivity of somatostatin during the development of the human fetal pancreas and pancreatic ductal adenocarcinoma, given that, somatostatin-positive cells were demonstrated either into its embryonic anlage or into pancreatic cancer. METHODS: Tissue sections from 15 pancreatic fetal specimens, and an equal number of ductal adenocarcinoma specimens were assessed. RESULTS: The density of positive cells in the primitive exocrine ductal epithelium and endocrine epithelium was significantly different from the relevant density in the neoplastic pancreatic tissue of mixed (ductal-endocrine) and pure ductal type (P1=0.021 P2=0.001, P3<0.0001, P4=0.003 respectively). The above values were estimated from the 8th to 10th week. There was no significant difference in the density of positive cells in the mantle zone of the islets from the 13th to the 24th week, and the neoplastic tissue of mixed (P5=0.16) and pure ductal type (P6=0.65). CONCLUSION: The immunostaining for somatostatin identifies a subgroup of pancreatic ductal adenocarcinomas with a neuroendocrine component, (initially considered as pure ductal tumors), and mixed ductal and neuroendocrine tumors. This pattern of expression in neoplasms recapitulates the normal pattern during the embryonal development of the organ, raising the question of therapeutic efficacy of somatostatin and analogues as monotherapy in pancreatic cancer management.     

Subjective and objective quality of sleep

Summary   Relatively few studies have tried to relate subjective sleep quality to objective sleep parameters and most have been carried out in laboratory settings and often with patients and usually only for a single night. The present study used a group of 33 subjects who had sleep polysomnographically recorded in their homes for three nights during a period of several weeks. First a multiple regression analysis was carried out for each night with a 4-item sleep quality index as the dependent variable and conventional sleep parameters as predictors. This yielded a significant beta value for percent Stage 0 for each of the three nights. Sleep efficiency showed a significant correlation with sleep quality for two nights but did not enter the regression. When the night with the best and poorest sleep were compared, the only significant variable became percent SWS. It was suggested that the differing results may have been due to the large age span confusing SWS/quality correlations.      

Frequency and phenotype of natural killer cells and natural killer cell subsets in bovine lymphoid compartments and blood

Natural killer (NK) cells are widely distributed in lymphoid and non‐lymphoid tissues, but little is known about the recirculation of NK cells between blood and tissues. This is relevant to understanding recirculation in the steady‐state and also for determining the roles for NK cells in vaccine‐induced immunity and responses to infection. Therefore, the percentage of NK cells and their phenotype across peripheral blood, afferent lymph and lymph nodes in steady‐state conditions was investigated in cattle using the pseudo‐afferent lymphatic cannulation model. CD2⁺ CD25^lo NK cells were the predominant subset of NK cells within the blood. In contrast, CD2⁻ CD25^hi NK cells were the main subset present within the skin‐draining afferent lymphatic vessels and lymph nodes, indicating that CD2⁻ NK cells are the principal NK cell subset trafficking to lymph nodes via the afferent lymphatic vessel. Furthermore, a low percentage of NK cells were present in efferent lymph, which were predominantly of the CD2⁻ subset, indicating that NK cells can egress from lymph nodes and return to circulation in steady‐state conditions. These compartmentalization data indicate that NK cells represent a population of recirculating lymphocytes in steady‐state conditions and therefore may be important during immune responses to vaccination or infection.     

Diagnosis, biology and treatment of hairy-cell leukaemia

Hairy-cell leukaemia (HCL) is usually readily diagnosed by seeing typical hairy cells (HCs) in the blood film. The diagnosis is then confirmed by tartrate-resistant acid phosphatase staining, marker analysis, and bone marrow examination. HCs are clonal mature memory B cells with specific features of activation. This HC activation is responsible for many of the pathological features of the disease, including its distinctive bone marrow fibrosis, splenic red pulp invasion, and pseudo-sinus formation. Chlorodeoxyadenasine is the treatment of first choice. Deoxycoformycin and rituximab are useful for the treatment of relapsed/refractory disease. The nature of the primary oncogenic event(s) remains unknown and is the major unresolved issue in HCL.     

MiR-615-5p depresses natural killer cells cytotoxicity through repressing IGF-1R in hepatocellular carcinoma patients

miR-615-5p was characterized by our group as a tumour suppressor. IGF-1?R activates a downstream signalling pathway, well characterized in liver cells, however, its role in immunity especially Natural Killer cells (NKs) remains vague. This study aimed at investigating the regulatory role of miR-615-5p on IGF signalling and its impact on NKs cytotoxicity in HCC. Our results showed an upregulation in miR-615-5p and IGF-1?R in NKs of 130 HCC patients compared to 35 controls. Forcing the expression of miR-615-5p, repressed IGF-IR, attenuated NKs cytotoxicity, decreased CD56^dim, increased CD56^bright NK subsets and reduced the cytotoxic markers NKG2D, TNF-α and perforins. It repressed NKG2D ligand (ULBP2) in Huh-7 cells. In conclusion, miR-615-5p represses IGF-1?R in NKs and their target hepatocytes; however, it has a contradicting impact on HCC progression on both cell types. These findings might pave the way for better understanding the role of microRNAs in NKs function and HCC immune-pathogenesis.     

Abnormal cytokine production by bone marrow stromal cells of multiple myeloma patients in response to RPMI8226 myeloma cells

INTRODUCTION: Recent studies indicate that bone marrow stromal cells (BMSCs) derived from patients with multiple myeloma (MM) differ from those of healthy donors in their expression of extracellular matrix compounds and in cytokine production. It is not known whether these abnormalities are primary or are acquired by BMSCs on contact with MM cells. MATERIALS AND METHODS: Interleukin (IL)-6, IL-11, IL-10, and tumor necrosis factor (TNF)-alpha production by CD166+ mesenchymal BMSCs and the CD38+/CD138+ RPMI8226 myeloma cell line cultivated in vitro in monocultures or co-cultivated under cell-to-cell contact or non-contact conditions in the presence of a tissue culture insert were measured. Intracellular cytokines were measured by flow cytometry analysis as the percentage of cytokine-producing cells or by mean fluorescence intensity as the level of cytokine expression in cells. Additionally, ELISA was used to measure IL-6, soluble IL-6 receptor (sIL-6R), IL-11, IL-10, TNF-alpha, B-cell-activating factor of the TNF family (BAFF), hepatocyte growth factor (HGF), and osteopontin (OPN) production in the supernatants of the cultures and co-cultures. RESULTS: A higher ability of the BMSCs of MM patients than in controls was detected to produce IL-6, IL-10, TNF-alpha, OPN, and especially HGF and BAFF in response to the RPMI8226 cells. Moreover, the BMSCs of the MM patients significantly enhanced the production of sIL-6R by the RPMI8226 cells. DISCUSSION: Cytokines over-expressed by BMSCs of MM patients can function as growth factors for myeloma cells (IL-6, IL-10, HGF), migration stimulatory factors for tumor plasma cells (TNF-alpha, HGF), adhesion stimulatory factors (HGF, BAFF and OPN), stimulators of osteoclastogenesis (IL-6, TNF-alpha), and angiogenic factors (TNF-alpha). The results of this experiment strongly suggest that the BMSCs from MM patients differed in spontaneous and myeloma cell-induced production of cytokines, especially of HGF and BAFF, and these abnormalities were both primary and acquired by the BMSCs on contact with the MM cells. This in turn suggests the presence of an undefined, autocrine stimulation pathway resulting in a prolonged production of cytokines even in long-term cultures in vitro and in vivo. These abnormalities might provide optimal conditions for the proliferation and differentiation of residual tumor cells or their precursors in the affected bone marrow.     

Lipid peroxidation levels in patients with acute brucellosis

The purpose of this study was to investigate levels of lipid peroxidation, indicated by plasma malondialdehyde (MDA), with consideration of clinical status and treatment outcomes in patients with acute brucellosis. Plasma MDA levels were measured in patients with acute brucellosis and healthy subjects. Significantly higher MDA levels were detected in plasma of patients with acute brucellosis compared to controls (P<0.01). Plasma levels of MDA were significantly decreased after the brucellosis treatment (P<0.01). The results of the present study indicate for the first time that a considerable level of lipid peroxidation is involved in acute brucellosis cases and this may be of importance with respect to the understanding of disease pathogenesis and may serve as a target for treatment regime.     

Helicobacter pylori induces the release of α-defensin by human granulocytes

Objectives:  Little information is available on the potential role of α-defensins derived from neutrophils during H. pylori infection, or the effect of H. pylori on the α-defensin release. The effects of H. pylori on human granulocytes were investigated in vitro by flow cytometry and ELISA. Additionally we sought to identify by immunohistochemistry the α-defensins within the gastric mucosa of patients infected with H. pylori. Materials and Methods:  The intracellular expression of α-defensin in human granulocytes and in mononuclear cells was determined by flow cytometry. Induction of α-defensin release from granulocytes, mononuclear cells, or from whole blood cultures by H. pylori was detected by measuring the HNP1-3 (α-defensin) concentrations in the supernatants by ELISA. Immunohistochemistry was used to identify HNP1-3 in infiltrating neutrophils in the gastric mucosa of eight patients. Results:  A considerable intracellular α-defensin staining was observed in granulocytes. Stimulation of granulocytes with H. pylori resulted in a decrease in intracellular staining which was due to the extracellular release of α-defensin. In whole blood cultures H. pylori infection resulted in significantly high α-defensin concentrations (131623 ± 13986 pg/ml), which were mainly due to the activity of the granulocytes with only a minor amount furnished by the mononuclear cells. In H. pylori-infected mucosa, infiltrating neutrophils showed intense immunostaining with anti-HNP1-3. The intensity of α-defensin staining varied parallel with the density of H. pylori in the biopsy samples. Conclusions:  H. pylori induce α-defensin release from granulocytes which may well be important in local host response to H. pylori infection in gastroduodenal diseases. Received 19 May 2008; returned for revision 20 October 2008; received from final revision 28 October 2008; accepted by I. Ahnfelt-R?nne 23 November 2008     

Regulatory CD4+CD25+ T cells and macrophages: communication between two regulators of effector T cells

Regulatory T cells (Tregs) play an essential role in the induction and maintenance of peripheral tolerance as well as prevention of autoimmunity by limiting the strength of the immune response of effector T cells. Macrophages, a heterogeneous population of phagocytes and professional antigen presenting cells (APCs), can also exert suppressive effects on effector T cells to keep the peripheral balance of immunity. The bi-directional interactions of dendritic cells (DCs) and Tregs have been cell studied. However, much less is known about the reciprocal interaction between macrophages and Tregs. In this review, we will discuss recent observations regarding the interplay of these two regulators of immunity. Received 8 December 2006; returned for revision 17 January 2008; received from final revision 9 June 2008; accepted by G. Wallace 10 July 2008     

NK细胞对不同人肝癌细胞株的杀伤作用

目的: 观察自然杀伤(NK)细胞对不同肝癌细胞株的体内外抑瘤作用,并检测肝癌细胞MHC-I类链相关蛋白(MIC蛋白)的表达。方法: 抽取志愿者外周血50 mL,分离单个核细胞,置入NK细胞试剂盒行孵化及逐级扩增。计算NK细胞对人白血病细胞株K562及人肝癌细胞株BEL7402、HepG2、SMMC7721的杀伤率。接种建立人肝癌细胞株裸鼠移植瘤,进行NK细胞瘤内及瘤周注射;计算各组裸鼠移植瘤的体积,绘制各组肿瘤生长曲线;处死裸鼠,称瘤重,计算NK细胞对各组肿瘤抑制率。检测人肝癌细胞表面 MIC蛋白表达。结果: NK细胞对K562细胞杀伤最强,BEL-7402次之,SMMC-7721细胞株杀伤敏感性最低。裸鼠抑瘤实验结果显示NK细胞对于BEL-7402细胞株的抑瘤效果最好,而对于SMMC-7721细胞株的抑瘤效果较差。BEL-7402及HepG2细胞株表面表达MIC,而SMMC-7721则很少表达。结论: NK细胞对于不同人肝癌细胞株体内外抑瘤作用不同,其差异可能和不同肝癌细胞株MIC的表达有关。