Assessment of natural and interleukin-2-induced production of interferon-gamma in patients with liver diseases

AIMS To clarify whether the lower interferon gamma (IFNγ) production by lymphocytes in patients with liver diseases is due to defects of lymphocytes themselves or of other cofactors such as interleukin-2(IL-2). METHODS Peripheral blood mononuclear cells (PBMCs) from patients with various liver diseases were cultured with or without PHA and IL-2. The cells were harvested and counted and the su- pernatants were tested for IFNγ by a sensitive and quantitative ABC-ELISA. RESULTS IFNγ was not round in serum samples from patients as well as normal individuals. However,in supernatants of non-in- duced and induced PBMCs,IFN7 was detected by ABC-ELISA. In non-induced PBMCs (group 1),the content of IFNγ in super- natants from control,CAH,CPH and HCC was 8.72 μg/L, 5.03 μg/L,6.02 μg/L and 4.91 μg/L respectively. The pro- duction of IFNγ in liver disease was significantly decreased,com- pared to control. In group 2 in which PBMCs were stimulated with PHA,the content of IFNγ was 22.71,17.12,14.54 and 17.63 μg/L respectively. In group 3 in which PBMCs were in- duced by IL-2,the amount of IFN7 in supernatant from control (60.67 μg/L) was much larger than those from CAH (21.70 μg/ L),CPH (24.00 μg/L) and HCC (19.15 μg/L) (P<0.01). Comparing the amount of IFNγ in group 3 (IL-2-induced) with that in group 1 (non-induced),we found that IFNγ production was en- hanced by nearly 4 folds in liver diseases and by over 7 folds in control,Whereas the number of PBMCs,whether from liver dis- eases or from control,was increased by only approximately 3 folds. CONCLUSIONS The decreased production of IFNγ in liver dis- eases including HCC is mainly due to endogenous defects of lym- phocytes though the defects of stimulating cofactors such as IL-2 may also be involved.

Determination of IL-2/IL-2R system in patients with liver cirrhosis.and carcinoma

AIMS To.determine the interleuking-2 and interleukin- 2 receptor (IL-2/IL-2R) system in patients with liver cirrhosis and carcinoma and compare their immune functions. The clinical significance is also discussed. METHODS Fifty patients with Liver cirrhosis (LC), 50 patients with hepatocellular carcinoma (HCC) and 30 normal controls were studied. The expression of mlL-2R was examined by immunofluorescence. IL-2's activity and serum level of soluble interleukin-2 recep- tor (sIL-2R) were measured by enzyme linked im- munosorbent assay. RESULTS IL-2's activity and the percentage of mIL- 2R expression in carcinoma were significantly lower than those in cirrhosis (P<0.01) and controls (P< 0.01),while the IL-2's activity and the expression of mlL-2R in cirrhosis were also lower than normal controls (P<0.05). The serum level of sIL-2R in carcinoma was significantly higher than that in cirrhosis (P<0.05) and controls (P<0.01),and the level in cirrhosis was higher than in controls (P<0.05). CONCLUSIONS Patients with liver cirhosis and car- cinoma share the decreased immune function of similar nature,but the latter has a more profound degree. Such resemblance in immune disturbances may be the important factor affecting the carcinogenesis of cirrhotic liver.

Serum Levels of Interleukin-2 and -10 in Patients with Early Syphilis

Objective: To investigate the roles of interleukin-2(IL-2) and interleukin-10 (IL-10) in pathogenesis ofearly syphilis. Methods: The serum levels of IL-2 and IL-10 in 48patients with early syphilis were detected by ABC-ELISA. Results: (1) The level of IL-2 in the patients withearly syphilis was significantly higher than that inhealthy controls, while that of IL-10 was lower(P<0.001 and P<0.001). (2) The levels of IL-2 and IL-10 were almost identical in patients with primary andsecondary syphilis (P>0.05), as well as between dif-ferent RPR titers (P>0.05). (3) After therapy, the levelof IL-2 decreased markedly (P<0.05), while that of IL-10 increase (p>0.05). (4) A significant correlation wasfound between the serum levels of IL-2 and IL-10 (r=0.5385 P<0.05). Conclusions: Th1 up-regulation occurs in patientswith early syphilis, and plays an active role in fightingagainst TP infection.

mIL-2R,T cell subsets & hepatitis C

AIM: To study the levels of membrane interleukin-2 receptor (mIL-2R) and T cell subsets in peripheral blood mononuclear cells (PBMC) from patients with hepatitis C and their role in the pathogenesis of hepatitis C. METHODS: The levels of mIL-2R and T cells subsets in PBMC were detected by biotin- streptatividin (BSA) technique before and after stimulation with PHA in 203 patients with hepatitis C with HCV-RNA(+), anti-HCV(+), anti-HCV(-). RESULTS: The total expressive levels of mIL-2R before and after stimulation with PHA(0.03+/-0.01, 0.03+/-0.02, 0.04+/-0.02, 0.36+/-0.03), and T cell subsets in PBMC (0.62+/-0.06, 0.37+/-0.05, 0.35+/-0.07) were all lower in patients with hepatitis C than those in normal controls (0.66+/-0.07, 0.41+/-0.06, 0.31+/-0.05, P【0.01). Among the patients, the levels of mIL-2R were lower in silence than those in situation of PHA inducting (P【0.01). However, the levels of mIL-2R were similar in acute hepatitis C to that in chronic hepatitis C (P】0.05). The levels of CD(3)(+), CD(4)(+), CD(4)(+)/CD(8)(+) were lower and CD(8)(+) was higher in patients with acute and chronic hepatitis C with anti-HCV(+) than those in normal controls (0.62+/-0.06, 0.37+/-0.05, 0.35+/-0.07, 1.18+/-0.30, 0.61+/-0.07, 0.37+/-0.05, 1.39+/-0.33, 0.31+/-0.05, P【0.05-P【0.01). CONCLUSION: The cellular immunity is obviously changed in patients with hepatitis C. The levels of mIL-2R and activation of T cells are closely associated with chronicity of hepatitis C.

Helicobacter pyloripromote gastric cancer cells invasion through a NF-κB and COX-2-mediated pathway

AIM: To examine the effects of Helicobacter pylori(Hpylori) infection on the invasiveness of gastric cancer cells,and to elucidate its mechanism. METHODS: Gastric carcinoma cells, MKN-45, were incubated with CagA-positive H pylori, and cell invasion was determined by Matrigel analysis.The expression of matrix metallopr-oteinase-9 (MMP-9),vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2) were assessed by Western-blot analysis, and transcriptional activation of the COX-2 promoter was examined by measuring luciferase and β-galactosidase activities. Lastly,the proteinDNA interaction was confirmed by an electrophoretic mobility shift assay. RESULTS: The current studies showed that: (1) incubation of CagA-positive H pylori with MKN-45 cells significantly promotes gastric cancer cells invasion, and this effect is attenuated by pre-treatment with NS-398, a COX-2 inhibitor, or PDTC,a nuclear factor κB (NF-κB) inhibitor;(2) the induction of MKN-45 cells invasion by Hpylori is associated with increases in COX-2, MMP-9, and VEGF protein expression, and co-incubation of NS-398 or PDTC significantly reduces these effects;(3) H pylori infection transactivates COX-2 promoter activity and increases the binding of NF-κB to this promoter. CONCLUSION: Our data demonstrate that H pylori infection promotes gastric epithelial cells invasion by activating MMP-9 and VEGF expression. These effects appear to be mediated through a NF-κB and COX-2 mediated pathway, as COX-2 or NF-κB inhibitor significantly attenuate the invasiveness of gastric cancer cells and the expressions of MMP-9 and VEGF protein.

Serum soluble interleukin-2 receptor levels in patients with chronic hepatitis B virus infection and its relation with antiHBc

AIM： To investigate the relationship between serum soluble interleukin-2 receptor （sIL-2R） level and anti-HBc in patients with chronic hepatitis B virus （HBV） infection. METHODS： Sera from 100 patients with chronic HBV infection and 30 healthy controls were included in this study. The patients were divided into group A [HBsAg （＋）, HBeAg （＋） and anti-HBc （＋）, n = 50] and group B [HBsAg （＋）, HBeAg （＋） and anti-HBc （-）, n = 50]. sIL-2R levels were determined using ELISA. HBV DNA and alanine aminotransferase （ALT） were also detected. RESULTS： Serum sIL-2R levels were significantly higher in patients with chronic HBV infection than in healthy controls. Moreover, serum sIL-2R levels were significantly higher in patients with HBsAg （＋）, HBeAg （＋） and antiHBc （＋） （976.56±213.51×10^3 U/L） than in patients with HBsAg （＋）, HBeAg （＋） and anti-HBc （-） （393.41±189.54 ×10^3 U/L, P〈 0.01）. A significant relationship was found between serum sIL-2R and ALT levels （P〈 0.01） in patients with chronic HBV infection, but there was no correlation between sIL-2R and HBV DNA levels. The anti-HBc status was significantly related to the age of patients （P〈 0.01）. CONCLUSION： The high sIL-2R level is related to positive anti-HBc in chronic hepatitis B patients. Positive anti-HBc may be related to T-lymphocyte activation and negative anti-HBc may imply immune tolerance in these patients.

Interleukin-2 gene-encoded stromal cells inhibit the growth of metastatic cholangiocarcinomas

AIM： To demonstrate bone marrow stromal cells （BMSCs） can be used as an attractive target for genetic modification in the treatment of malignant diseases. METHODS： Using a hamster model of biliary cancer, we investigated the therapeutic effects of interleukin-2 （IL-2） gene-modified BHSCs. Syrian golden hamsters were injected via the femoral vein with 5×10^5 cells of the KIGB-5 biliary cancer cell line （n=20）. One week later, the hamsters were injected intraperitoneally with BMSCs containing Ad/hIL-2 and Ad/△E1, unmodified BHSCs, or RPHI only （control） and observed for 12 wk （n=5/each group）. RESULTS： All hamsters treated with BMSCs containing Ad/hIL-2 survived with no evidence of the disease during this period. In contrast, hamsters in the other three groups showed disseminated metastases involving the lungs as eady as 4 wk.CONCLUSION： Ad/IL-2 therapy is effective in the treatment of biliary cancer.

Effects of Warm Needling at Zusanli(ST 36)on NO and IL-2 Levels in the Middle-Aged and Old People

42 middle-aged and old people at the age between 55-70 years were selected and given the warm needling at Zusanli (ST 36), and their IL-2 and NO contents of peripheral blood before and after acupuncture were determined. The results showed that IL-2 and NO contents increased significantly after the warm needling (P<0.01).

A Clinical observation of the therapy for malignant hydrothorax by intra-thoracic injection using IL-2 combining with Cisplatin

Objective: The aim of our study was to explore the short-term effects and complication of interleukin-2 (IL-2) combining with Cisplatin in the treatment of malignant hydrothorax.Methods: Sixty-two cases patients with malignant hydrothorax were randomly divided into two groups.Observation group was 31 examples,thoracic cavity injection IL-2 and Cisplatin;31 cases in control group using Cisplatin alone intra-thoracic injection.The regime of every week for 1–4 weeks was used to observe short term effects and complications.Results: The total response rate in observe group was higher than that in control group (90.3% vs.68.1%),which had statistically significant difference (P < 0.05).The complications included gastrointestinal tract reaction,bone marrow inhibition,chest pain and fever.The incidence rates of chest pain and fever in observe group was slightly higher than that in control group,but there was no statistically significant difference (P > 0.05).Conclusion: The IL-2 combining with Cisplatin intra-thoracic injection for malignant hydrothorax has the features of good therapeutic effects and slight poisonous side effects,which is worth to be used in clinic.

TREATMENT OF 23 PATIENTS WITH ADVANCED GASTRIC CANCER BY INTRAVENOUSLY TRANSFER OF AUTOLOGOUS TUMOR-INFILTRATING LYMPHOCYTES COMBINED WITH rIL-2

Tumor-infiltrating lymphocytes (TIL)isolated from metastatic lymph nodes in patients with nonoperable advanced gastric cancer were induced to become LAK-like cytotoxic activrty of TIL after in vitro culture with rlL-2.Twenty-three patients with advanced gastric cancer were treated by intravenously transfer of autologous TIL combined with rlL-2. The tumor forus disappeared (complete remission, CR) in 3 patients (13. 0%) and significantly decreased (partial remission, PR) in 5 patients (21. 7%). Fifteen patients did not respond to the treatment. The amount of soluable IL-2 receptor in serum was significantly decreased after treatment, the cytotoxicity of NK cells and OT test were significantly increased. No significant difference in CD4/CD8 was found between before and after treatment. No serious side effect was obseved in the treatment.

Beneficial effect of an antibody against interleukin-2 receptor (daclizumab) in an experimental model of hepatocyte xenotransplantation

AIM: To investigate the use of Daclizumab (Dmab) as an immunosuppressive agent in an experimental model of hepatocyte xenotransplantation (XenoTx) in rats with fulminant hepatic failure (FHF). METHODS: Two white male New Zealand rabbits were used as donors and 68 Wistar rats as recipients. FHF was induced by intravenous application of dimethylnitrosamine (DMNA). The isolated hepatocytes of the rabbits were xenotransplanted into the spleen of the rats 24 h after FHF induction. Group A (n = 13): no treatment; Group B (n = 14): FHF and XenoTx; Group C (n = 14): FHF and XenoTx and cyclosporin (CsA); Group D (n = 14): FHF and XenoTx and Dmab; Group E (n = 13): FHF and XenoTx and CsA and Dmab. The rats were followed for 15 d. RESULTS: Statistical analysis showed better survival among groups D (92.86%) and E (76.92%) compared to group A (all rats died after 72 h), group B (28.57%) or group C (71.43%), although the differences were not statistically significant. Biochemical evaluation of the liver enzymes and histology confirmed satisfactory function and engraftment, respectively. CONCLUSION: This experimental model has shown the safe, effective and beneficial use of Dmab in a xenotransplantation model of rabbit hepatocytes in rats.

EFFECTS OF ACUMOXI ON IL2-IFN-NKC IMMUNOREGULATORY NETWORK AND ITS RELATED MACROPHAGE-IL1-Th NETWORK AND ON TUMOR——Observation on the Effect of Acumoxi on the Macrophage-IL1-Th Network and on the Metastasis of Hepatoma

Objective: To observe the effect of acumoxi (acupuncture and moxibustion) on macrophage (Mφ)-lL1-Th net-work and hydroperitoneum hepatoma (H 22) metastasis in mice. Methods: A total of 36 BALB/ c male mice bearing H 22 are randomly divided into control, acupuncture and acumoxi groups with 12 cases in each group. In the later 2 groups, Dazhui (GV 14) and Guanyuan (CV 4) are punctured once daily, continuously for 18 days, and in acumoxi group, the two acupoints were also moxibustioned alternatively with moxa stick once every day. After killing the mice, the tissue samples of the 3 groups are treated routinely step by step and analyzed by means of colorimetric analysis for determining the phagocytic function of the macrophages; and the content of IL1 of the Mφ supernatant is assayed with serum plate agglutination (SPA)-Ig floral hoop method of T helper cell (Th) monoclonal antibody; the weight of the reniportal lymph node, the kidney and the lung, and the number of the cancerous nodes on the pulmonary surface are calculated. Results: After acupuncture and moxibustion treatment, the immunoregulatory network indices of acumoxi group increase obviously compared with those of control group(P<0.01), showing an anti-metastasis effect of acumoxi on H 22. Conclusion: Results of the present study and those of our former research prove that acupuncture and moxibustion can suppress the tumor growth and H 22 metastasis by the enhancement of the immunoregulatory network.

THE ROLE OF RECOMBINANT HUMAN FUSION PROTEIN IL－6／IL－2（CH925） IN HEPADNA VIRUS INFECTION TREATMENT

The effects of CH925, a novel immune modulator, on hepadna virus infection was evaluated. Day-old ducklings were inoculated intravenously with LJ-76 DHBV containing serum. Infected ducklings were then treated with the CH925 and the mixture of IL-2 and IL-6 intravenously and the control ducklings received equivalent normal saline (NS). Blood and liver samples were taken and assayed for DHBV DNA and /or DHBsAg. At the completion of the experiment there was a inhibition of viremia with the CH925 and IL-2 + IL-6. Serum DHBV DNA was detected in 6 of 10 ducks in 100 000 unit/kg dosage group, 7 of 10 ducks in 50 000 unit/kg dosage group and 6 of 10 ducks in IL-2 + IL-6 dosage group, compared with 9 of 10 NS control, and it showed a similar result in circulating DHBsAg. When samples of liver DNA were processed for hybridization, a little difference in the DHBV DNA replication was noted between ducks receiving CH925, IL-2 + IL-6 or NS placebo. It is suggested that CH925 might be a potential remedy in HBV infection treatment.

CLONING AND EXPRESSION OF THE GENE CODING FOR IL－2（60）－PE40，A MOLECULAR TARGETED PROTEIN

It has recently been shown that chimeric toxin composed of IL2 fused tp PE40, a mutant form of Pseudomonas Exotoxin A devoid of its native cell recognition and binding domain was cytotoxic to IL-2 receptor bearing cells. We here amplified the gene IL-2(60), which cedes for the N-terminal 1-60 amino acids of human IL-2 by PCR. After that, we fused it to PE40 and the new chimeric protein IL-2(60)-PE40 was expressed in E. coli. SDS-PAGE revealed that IL-2(60)-PE40 chimeric protein accounts for more than 18% of total cell proteins. As the region IL-2 binds with its receptor was defined in the N-terminal residues 8-54 of IL-2, such fusion proteins will have the same activity with IL-2-PE40. Following primany purification, IL-2 (60)-PE40 was shown to be very toxic to IL-2 receptor-positive cells but non measurable effect on the cells lacking IL-2 receptors. Such a structure has not been reported by now. The fusion protein is useful for suppressing the immune response in cases of rejection of allografts and organ transplants and as therapeutic agents for the treatment of IL-2 receptor related diseases scuh as autoimmune disease, ATL(adult T-cell leukemia), et al.

EFFECT OF ACUPUNCTURE ON IL 2-IFN-NKC IMMUNOREGULATORY NETWORK IN MICE WITH TRANSPLANTED HEPATOCARCINOMA

: To study the effect of acupuncture on interleukin 2 (IL 2)-interferon(IFN)-natural killer cells (NKC) immunoregulatory network of mice with transplanted hepatocarcinoma(HAC) in order to provide new evidence for acupuncture treatment of hypofunction of immune system. Methods:The 28 HAC-vaccinated BALB/C mice are randomly divided into control group (n=14) and acupunctrue group (n=14). In the latter group bilateral'Dazhui' (BL 11) and 'Zusanili' (ST 36) which are located according to the same positions indicated in Comparative Anatomy of Macro-animals, are needled once every day, twelve sessions altogether. Twenty-four hours after the needling treatment the mice are killed and the spleen is taken out to be made into cell suspension for assaying concentrations of IL2 (MTT method) and NKC (colorimetric method) respectively. Obital Serum IFN is determined by using (CPE microplate staining), and the tumor mass is taken out and balanced with an analytical balance (1/10000) to calculate the tumor inhibition rate according to the formula. Results: The tumor weight of the mice of aupuncture group is obviously decreased (inhibition rate 43.06%) while the activity of the IL2 and NKC, and the IFN tiler are increased greatly, which have significant differences compared with those of control group (P>0.01). Conclusion:The results of the study show that acupuncture can strengthen the positive immunoregulatory function of the IL2 -IFN-NKC network in immune hypofunction mice bearing HAC.

Production of Interleukin 2 like Growth Factor from the Mitogen Activated Peripheral Blood Lymphocytes of Indian Major Carp, Labeo rohita

Optimum conditions for in vitro production of interleukin 2 （IL 2） like activity from the Peripheral blood lymphocytes （PBL） of an Indian major carp, Labeo rohita were studied. Culture supernatants were generated by culturing the PBL in RPMI-1640 media supplemented with Glutamine and 10% fetal bovine serum （FBS） and stimulating with two different mitogens： concanavalin A （Con A） and Phytohaemagglutinin （PHA） at different concentrations separately. Significantly （P 〈 0.01） higher proliferation response was obtained from the culture supematant stimulated with concanavalin A （Con A） at a concentration of 10 lag mLl. The effect of phorbol myristate acetate （PMA） was also studied by co-stimulating PBL with Con A and PHA separately and it was found to synergistically enhancing the stimulation index with Con A whereas the stimulation index remain unchanged with PHA. The Con A （10 μg mLl） stimulated PBL were also cultured at different cell density, incubation period and incubation temperature in order to optimize the in vitro L. rohita IL2 production. The IL2 like activity was studied by lymphocyte proliferation assay on 72 h Con A blasts using WST based assay technique. Significantly （P 〈 0.01） higher stimulation indices were obtained when the PBL were cultured at a cell density of 1 × 10^6 cells mL^-1 for 30-36 h at an incubation temperature of 30 ℃. The IL2 like activity was purified by DEAE-Sepharose anion exchange chromatography and recorded between 70-130 mM NaCI with peak activity at 110 Mm NaCI. The molecular weight of the factor responsible for IL2 like activity was found to be 15-17 KD.

CHANGES ON RECEPTOR EXPRESSIONAND PRODUCTION OF INTERLEUKIN-2 IN CIRCULATING LYMPHOCYTE POPULATION AFTER OPEN HEART SURGERY

To evaluate the change of perioperative cell mediated immunity after cardiac operation with cardiopul-monary bypass (CPB), so as to provide some information for timely prevention and treatment against post-operative immunological disorder, 40 patients were studied. By searching for the effects of CPB and anes-thesia, interleukln-2 receptor (IL-2R) expression upon the surface of peripheral blood mononuclear cells(PBMC), as well as interleukin-2 (IL-2) production in vitro was traced 55 min after anesthesia, at end ofCPB, on postoperative 1, 7, and 14 day versus preanesthesia control. Our data demonstrated that expres-sion of IL-2R on PBMC was significantly suppressed in all comparing with the baseline value, meanwhile,IL-2 production in vitro also statistically dropped. However,no statistical difference was found on perioper-ative IL 2R expression and IL-2 synthesis in the cholecystectomy group. We conclude that postoperativeimmunological disorder seems to be the main factor, which could be denoted as reduced IL 2R expressionon PBMC and lL-2 synthesis in vitro for sepsis, even multiple system organ failure developed after cardiacsurgery.

Interleukin-1 receptor-associated kinase-2 is involved in IL-18-induced NF-κB activation

objective: To investigate whether interleukin-1 receptor-associated kinase-2 (IRAK-2) is involved in interleukin-18 (IL-18)-induced nuclear factor- κ B (NF-κ B) activation. Methods: Phosphorothioate oligodeoxynucleotide (ODN) was designed antisense to sequences of IRAK-2. Antisense IRAK-2 ODN was delivered by lipofectin encapsulation into cultured HepG2 cells. IRAK-2 mRNA expression was assayed by semiquantitative reverse transcription-PCR. The levels of NF- K B were measured by sandwich ELISA. Results: Antisense IRAK-2 ODN blocked IRAK -2expression. IL-18 activated NT- K B and the A value increased from a basal level of 0.115±0.004 to 2.141 ±0.038. Antisense IRAK-2 ODN inhibited IL-18-induced NT- K B activation in a dose (1-8μg )-dependent fashion. When the cells were treated with 4μg antisense IRAK-2 ODN for 8 h, a maximum inhibition of 45.4% was induced as shown by the reduction of the OD value from a control level of 2.141±0.038 down to 1.168±0.026. Conclusion: IRAK-2 can regulate IL-18-stimulated NF- K B activation.

Cellular immune function and liver damage in post hepatitic cirrhosis

AIM To study the cellular immune function in patients with post hepatitic cirrhosis (PHC) and its relation with different liver damages.

CD4+ T cell responses in hepatitis C virus infection

Hepatitis C virus (HCV) infection is a major cause of liver damage, with virus-induced end-stage disease such as liver cirrhosis and hepatocellular carcinoma resulting in a high rate of morbidity and mortality worldwide. Evidence that CD4+ T cell responses to HCV play an important role in the outcome of acute infection has been shown in several studies. However, the mechanisms behind viral persistence and the failure of CD4+ T cell responses to contain virus are poorly understood. During chronic HCV infection, HCV-specific CD4+ T cell responses are rela- tively weak or absent whereas in resolved infection these responses are vigorous and multispecific. Persons with a T-helper type I profile, which promotes cellular effec- tor mechanisms are thought to be more likely to experi- ence viral clearance, but the overall role of these cells in the immunopathogenesis of chronic liver disease is not known. To define this, much more data is required on the function and specificity of virus-specific CD4+ T cells, especially in the early phases of acute disease and in the liver during chronic infection. The role and possible mechanisms of action of CD4+ T cell responses in deter- mining the outcome of acute and chronic HCV infection will be discussed in this review.