Immunopotentiation effects of apigenin on NK cell proliferation and killing pancreatic cancer cells.

Apigenin is a kind of flavonoid with many beneficial biological effects. It not only has direct cytotoxicity to tumor cells, but also can boost the antitumor effect of immune cells by modulating immune system. The purpose of this study was to investigate the proliferation of NK cells treated with apigenin and its cytotoxicity to pancreatic cancer cells in vitro, and explore its potential molecular mechanism. In this study, the effect of apigenin on NK cell proliferation and killing pancreatic cancer cells were measured by CCK-8 assay. Perforin, granzyme B (Gran B), CD107a, and NKG2D expressions of NK cells induced with apigenin were detected by flow cytometry (FCM). The mRNA expression of Bcl-2, Bax and protein expression of Bcl-2, Bax, p-ERK, and p-JNK in NK cells were evaluated by qRT-PCR and western blotting analysis, respectively. The results showed that appropriate concentration of apigenin could significantly promote the proliferation of NK cells in vitro and enhance the killing activity of NK cells against pancreatic cancer cells. The expressions of surface antigen NKG2D and intracellular antigen perforin and Gran B of NK cells were upregulated after treating with apigenin. Bcl-2 mRNA expression was increased, while Bax mRNA expression was decreased. Similarly, the expression of Bcl-2, p-JNK, and p-ERK protein was upregulated, and the expression of Bax protein was downregulated. The molecular mechanism of the immunopotentiation effects of apigenin may be that it up-regulates Bcl-2 and down-regulates Bax expression at the gene and protein levels to facilitate NK cell proliferation, and up-regulates the expression of perforin, Gran B, and NKG2D through the activation of JNK and ERK pathways to enhance NK cell cytotoxicity.

Artesunate enhances γδ T-cell-mediated antitumor activity through augmenting γδ T-cell function and reversing immune escape of HepG2 cells.

OBJECTIVE: To explore the effect and mechanism of artesunate on γδ T cell-mediated antitumor immune responses against hepatoma carcinoma cells (HepG2) in vitro. METHODS: Human γδ T cells or HepG2 were respectively treated with artesunate, subjected to co-culture as appropriate, and the following assays were subsequently conducted: CCK8 to examine cell viability; LDH release assay to detect the killing effect of γδ T cells on HepG2 cells; flow cytometry to examine the expression of perforin (PFP) and granzyme B (GraB) of γδ T cells; ELISA to evaluate the levels of TGF-ß1 and IL-10 in the collected supernatant of HepG2 cells pretreated with artesunate; and Western blot analysis to examine Fas, FasL, STAT3, p-STAT3 expression of HepG2 cells induced by artesunate. Results: The results showed that the cytotoxicity effect of γδ T cells pretreated with artesunate on HepG2 cells was augmented via elevating the expression of GraB in γδ T cells. Furthermore, treatment with artesunate reversed the inhibition of HepG2 cells on γδ T cells by reducing the secretion of TGF-ß1 in HepG2 cells supernatant and enhanced the antitumor effect of γδ T cells against HepG2 cells through increasing the expression of Fas on HepG2 cells, which may be attributed to the inhibition of STAT3 signaling protein. CONCLUSION: Artesunate has several mechanisms for augmenting the antitumor immune responses mediated by γδ T cells. These results suggested artesunate may be an efficacious agent in the treatment of hepatocellular carcinoma.

Wnt pathway activator TWS119 enhances the proliferation and cytolytic activity of human γδT cells against colon cancer.

γδT cells are a distinct T-cell subset that display unique characteristics regarding T-cell receptor gene usage, tissue tropism and antigen recognition. Adoptive γδT cell transfer therapy has recently been gaining importance as an efficient approach in cancer immunotherapy. However, exploiting γδT cell response for tumour immunotherapy is a challenge due to cell numbers, activities and differentiation states that minimize the clinical therapeutic effects. Previous studies have indicated that the wnt/ß-catenin signalling pathway plays a crucial role in the differentiation, survival and enhancement of the immune response of T lymphocytes. In this study, we sought to evaluate whether the activation of the wnt/ß-catenin pathway through inhibition of glycogen synthase kinase-3ß (GSK-3ß) using 4,6-disubstituted pyrrolopyrimidine (TWS119) could be an efficient strategy to improve the proliferation, differentiation and cytolytic activity of γδT cells against colon cancer cells. Remarkably, we found that TWS119 significantly enhanced the proliferation and survival of γδT cells via activation of the mammalian target of rapamycin (mTOR) pathway, upregulation of the expression of the anti-apoptotic protein Bcl-2 and inhibition of cleaved caspase-3 in addition to the Wnt pathway. Our results also showed that enhancement of the cytolytic activity of γδT cells against human colon cancer cells by TWS119 was chiefly associated with upregulation of the expression of perforin and granzyme B in vitro and in vivo. Additionally, TWS119 can induce the expression of CD62L or CCR5 to generate a population of CD62L+γδT or CCR5+γδT cells in a dose-dependent manner. These findings suggested that TWS119 could be a useful complementary agent for improving γδT cell-based immunotherapy.

Ratiometric optical sensor for high-resolution imaging of pH with low cross-talk.

We synthesize lipophilic, highly efficient, and pH-insensitive oleic acid-modified quantum dots (QDs) with maximum emission at a wavelength of 628 nm. The pH sensing film is fabricated by encapsulating 5-hexadecanoylamino-fluorescein and QDs as the reference in D4-hydromed and plasticized polystyrene. Using a light-emitting diode with a central wavelength of 410 nm as an excitation source, it is shown that the emission wavelengths of the pH sensitive indicator and reference dye have no spectral overlap and match respectively the channels of a 3CCD (RGB) camera with low cross-talk. A series of validation experiments shows that this ratiometric pH optode has good properties of high sensitivity, long-term stability, and photostability. It had a fast response time of <20 s when going from pH 6.3 to pH 8.0. The pH images suggest that the proposed ratiometric pH-sensing approach has great advantage and promise for field applications.

Enhancement of CD3AK cell proliferation and killing ability by α-Thujone.

Thujone is a monoterpene ketone natural substance found mainly in wormwood and sage. Previous studies have shown that Thujone has various pharmacological effects, such as anti-tumor, analgesic, and insecticide. The effect of α-Thujone to human immune cells is still unknown. Our study focuses on investigating the effects and mechanism of α-Thujone to CD3AK (anti- CD3 antibody induced activated killer) cells proliferation and cytotoxicity to colon cancer cell lines. With cell proliferation and FCM assay, it is found that α-Thujone could significantly enhance CD3AK cell proliferation and expression of CD107a in a dose-dependent manner. The cytotoxicity to colon cancer cells detected by CCK-8 assay is also improved. The expressions of TNF-α and FasL detected with ELISA assay were not significantly changed. Mechanically, the study shows that α-Thujone could enhance the expression of p-ERK1/2 and p-Akt. In addition, α-Thujone has no cytotoxicity to HCT116 and SW620 cells proliferation. In a word, α-Thujone enhances CD3AK cell proliferation and cytotoxicity via the improvement of expression of CD107a, p-Akt and p-ERK1/2.

[Activation of Wnt/ß-catenin pathway in NK cells by glycogen synthase kinase-3ß inhibitor TWS119 promotes the expression of CD62L].

OBJECTIVE: To investigate the effect of Wnt/ß-catenin pathway activation by glycogen synthase kinase-3ß inhibitor 4,6-disubstituted pyrrolopyrimidine (TWS119) on proliferation and phenotypic characteristics of human nature killer (NK) cells. METHODS: The peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and added to the complete medium containing recombinant human interleukin-2 (rhIL-2) and human AB serum to isolate NK cells from PBMCs. After co-cultured with 0-8.0 µmol/L TWS119 for 72 hours, growth curve and Wnt/ß-catenin activation of NK cells in each group were determined by CCK-8 and Western blotting. The CD107a and CD62L (L-selectin) expressions in the NK cells were detected using flow cytometry. RESULTS: NK cells were amplified to (61.76 ± 3.74)% after human PBMCs were cultured for 10 days. The 0-2.0 µmol/L TWS119 could promote the growth of NK cells in a dose-dependent manner, and the proliferation rate gradually dropped when TWS119 was more than 2.0 µmol/L. 0-8.0 µmol/L TWS119 could activate Wnt/ß-catenin pathway and up-regulate the expression of CD62L in a dose-dependent manner, but it decreased the expression of CD107a. CONCLUSION: Human NK cells isolated from peripheral blood treated with TWS119 gave rise to early mature CD62L⁺ NK cells.

Decreased levels of perforin-positive lymphocytes are associated with posttraumatic complications in patients with major trauma.

OBJECTIVE: Posttraumatic immune disorder can cause complications including systemic inflammatory response syndrome (SIRS) and multiple-organ dysfunction syndrome (MODS). Cytotoxic granules containing perforin and granzyme-B (GrB) are released by cytotoxic CD8(+) T lymphocytes, NK and γδT cells after major trauma. This prospective clinical study was designed to analyze the association between these immune components and complications after major trauma. METHODS: We retrospectively studied 48 patients aged between 16 and 65 years admitted within 90min of major trauma (Injury Severity Score>16) and surviving beyond 7 days, and 20 healthy controls. Blood samples were drawn on admission and after 1, 3 and 7 days. CD8(+) T, NK and γδT cell counts in peripheral blood and the levels of perforin and GrB in these cells were analyzed by flow cytometry. Clinical aspects of MODS and SIRS were recorded daily. RESULTS: CD8(+) T cell counts were not significantly different in patients with SIRS or uncomplicated group, but were depressed in the MODS group after trauma. However, NK cell counts in patients with MODS were significantly depressed only at day 7 after injury, and γδT cell counts were significantly depressed after trauma. Perforin levels in CD8(+) T, NK and γδT cells in patients with MODS were depressed after trauma. GrB levels in NK, CD8(+) T and γδT cells in patients with MODS were significantly depressed at 3 and 7 days post trauma. CONCLUSION: Posttraumatic MODS is associated with early, sustained, and severe depression of lymphocytes.

Shikonin inhibits prostate cancer cells metastasis by reducing matrix metalloproteinase-2/-9 expression via AKT/mTOR and ROS/ERK1/2 pathways.

Metastasis is one of the most important factors related to prostate cancer therapeutic efficacy. In previous studies, shikonin, an active naphthoquinone isolated from the Chinese medicine Zi Cao, has various anticancer activities both in vivo and in vitro. However, the mechanisms underlying shikonin's anticancer activity are not fully elucidated on prostate cancer cells. In the present study, we aimed to investigate the potential effects of shikonin on prostate cancer cells and the underlying mechanisms by which shikonin exerted its actions. With cell proliferation, flow cytometric cell cycle, migration and invasion assays, we found that shikonin potently suppressed PC-3 and DU145 cell growth by cell cycle arrest at the G2 phase and metastasis in a dose-dependent manner. Mechanically, we presented that shikonin could suppress the metastasis of PC-3 and DU145 cells via inhibiting the matrix metalloproteinase-2 (MMP-2) and MMP-9 expression and activation. In addition, shikonin significantly decreased the phosphorylation of AKT and mTOR in a dose-dependent manner while it induced extracellular signal-regulated kinase (ERK), p38 mitogen activated protein kinase (MAPK) and c-Jun N terminal kinase (JNK) phosphorylation. Further investigation of the underlying mechanism revealed that shikonin also induced the production of reactive oxygen species (ROS) that was reversed by the ROS scavenger dithiothreitol (DTT). Additionally, DTT reversed the shikonin induced activation of ERK1/2, thereby maintaining MMP-2 and MMP-9 expression and restoring cell metastasis. Together, shikonin inhibits aggressive prostate cancer cell migration and invasion by reducing MMP-2/-9 expression via AKT/mTOR and ROS/ERK1/2 pathways and presents a potential novel alternative agent for the treatment of human prostate cancer.

[Expression of coxsackie-adenovirus receptor in keratinocytes of mouse skin after heat stimulation and the effect of coxsackie-adenovirus receptor on dendritic epidermal T lymphocytes].

OBJECTIVE: To study the influence of heat stimulation on expression of coxsackie-adenovirus receptor (CAR) in keratinocytes (KCs) of mouse skin and the effect of CAR on production of cell growth factors by dendritic epidermal T lymphocytes (DETCs). METHODS: (1) Twenty BALB/c mice were divided into heat stimulation group (HS) and control group (C) according to the random number table, with 10 mice in each group. Mice in group HS were inflicted with scald milder than superficial-thickness by dressing wet hot gauze, which had been soaked in 100°C hot water for 3 min, in the hair removed area on the back for 1 to 3 s, while mice in group C were sham injured by dressing a wet gauze which had been soaked in water of room temperature for 3 min in the hair removed area on the back for 1 to 3 s. Square full-thickness skin specimens measuring 2.0 cm × 2.0 cm in size were obtained from the center of the bare skin. The expression of CAR in skin tissue sections were detected by immunohistochemistry staining. The mRNA and protein expression levels of CAR in skin tissue sections were respectively determined by real-time fluorescent quantitation RT-PCR and Western blotting. (2) KCs were isolated and cultured from full-thickness skin obtained from the trunk of 2 fetal BALB/c mice, and they were divided into 2 groups according to the random number table, with 5 wells in each group. The cells in group HS and group C were respectively cultured in 42°C and 37°C, 5% CO2 incubator for 1 h, and then all the cells were cultured in 37 °, 5% CO2 incubator for 6 h. The apoptosis of the cells and their expression of CAR were detected by flow cytometer. (3) Five BALB/c mice were sacrificed, and full-thickness skin was obtained from the trunk. The DETCs were divided into 7 groups according to the random number table after being isolated and purified from the skin specimens. Cells in group C were cultured without any stimulation, and cells in the 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 mg/L CAR groups were respectively cultured with corresponding concentration of recombinant mice CAR nutrient solutions, with 5 wells in each group. The contents of insulin-like growth factor I (IGF-I) and keratinocyte growth factor (KGF) were determined with ELISA. Data were processed with independent samples t test and one-way analysis of variance. RESULTS: (1) The immunohistochemistry staining showed that there was mild positive staining in the skin tissue sections of mice in group C, while the positive staining was more obvious in group HS. The positive staining was mainly located in KCs, hair follicles, and sweat gland epithelial cells, while no positive staining was observed in fibroblasts. The mRNA expression levels of CAR in skin tissue sections in group C and group HS were respectively 0.157 ± 0.027 and 0.773 ± 0.029. There was statistically significant difference between them (t = 3.052, P < 0.01). The protein expression levels of CAR in skin tissue sections in group C and group HS were respectively 0.23 ± 0.09 and 0.89 ± 0.14. There was statistically significant difference between them (t = 2.556, P < 0.05). (2) The apoptosis rates of KCs in group C and group HS were respectively (5.7 ± 1.3)% and (7.4 ± 1.7)% (t = 0.464, P > 0.05). The expression rates of CAR in KCs in group C and group HS were respectively (48 ± 6)% and (80 ± 8)%. There was statistically significant difference between them (t = 2.585, P < 0.05). (3) The contents of IGF-Iin culture supernatants in group C and 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/L CAR groups were respectively (23.1 ± 1.8), (22.5 ± 2.1), (31.2 ± 2.5), (39.7 ± 2.3), (61.8 ± 3.5), (45.1 ± 2.8), and (29.0 ± 2.0) µg/L. There was statistically significant difference among 7 groups (F = 3.414, P < 0.05). The contents of KGF in culture supernatants in group C and 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/L CAR groups were respectively (131 ± 9), (217 ± 12), (355 ± 21), (563 ± 21), (535 ± 34), (292 ± 20), and (245 ± 10) ng/L. There was statistically significant difference among 7 groups (F = 5.063, P < 0.01). CONCLUSIONS: The expression of CAR in KCs would rise after HS. The optimum CAR concentration to increase IGF-I and KGF production in DETCs is low.

Effect of spironolactone combined with angiotensin-converting enzyme inhibitors and/or angiotensin II receptor blockers on chronic glomerular disease.

The aim of the present study was to observe the effects of spironolactone on urine protein level and kidney function in patients with chronic glomerular disease receiving angiotensin-converting enzyme inhibitors (ACEIs) and/or angiotensin II receptor blockers (ARBs). A total of 221 patients with chronic glomerular disease were divided into spironolactone and control groups. The spironolactone group was treated with spironolactone at a dose of 20 mg/day, in addition to the original treatment regime and doses of ACEIs and/or ARBs. The control group continuously received the original doses of ACEIs and/or ARBs alone. Twenty-four-hour urine protein levels, serum creatinine and potassium, plasma aldosterone (ALD) and blood pressure were monitored at 0, 4, 8, 12 and 16 weeks. The estimated glomerular filtration rates (eGFRs) were calculated based on the obtained serum creatinine results. Following treatment, the urine protein level in the spironolactone group was notably decreased compared with that prior to the treatment, whereas the urine protein level in the control group did not show a significant difference. No significant differences were observed with regard to the renal function, eGFR, serum potassium, plasma ALD and blood pressure in either group prior to and following treatment. In conclusion, spironolactone administration, when co-administered with ACEIs and/or ARBs, markedly decreases the urine protein levels in patients with chronic glomerular disease. The protective effect of spironolactone on renal function remains to be demonstrated.

Enhancement effect of dihydroartemisinin on human γδ T cell proliferation and killing pancreatic cancer cells.

γδ T cells play important roles in innate immunity against tumors and infections. Inhibitory effect of dihydroartemisinin on growth of cancer cells has been found in recent years. In this study, we investigated the effect of dihydroartemisinin on human γδ T cell proliferation by MTT assay and killing activity against pancreatic cancer cells SW1990, BxPC-3 and PANC-1 by LDH release assay in vitro. Intracellular molecule alterations were verified by flow cytometry. The results suggested that appropriate concentration of dihydroartemisinin favored the expansion of γδ T cells and enhanced γδ T cell mediated killing activity against pancreatic cancer cells. Up-regulation of intracellular perforin, granzyme B expression and IFN-γ production may be the important mechanism of dihydroartemisinin on increased antitumor activity of γδ T cells.

[Association of traumatic severity with change in lymphocyte subsets in the early stage after trauma].

OBJECTIVE: To investigate the association of traumatic severity with changes in lymphocyte subsets in the early stage after trauma. METHODS: Sixty-three male patients admitted within 4 hours after trauma were enrolled. According to injury severity score (ISS), the patients were divided into two groups: mild trauma group (ISS<16, n=35) and severe trauma group (ISS≥16, n=28). At admission, the patients peripheral blood were extracted to detect T lymphocytes subsets, blood routine test, blood biochemical and arterial blood gas analysis which were used to calculate the acute physiology and chronic health evaluation II (APACHEII) scores. The correlation of lymphocyte subsets and ISS score, and the correlation of lymphocyte subsets and APACHEII score were both analyzed statistically. Another 20 cases of healthy male adults were enrolled as the control group. RESULTS: Compared with the healthy control group, CD3(+) T cell contents in blood were decreased obviously in mild trauma group and severe trauma group (0.648±0.112, 0.647±0.110 vs. 0.708±0.082, both P<0.05); CD4(+) T cells contents in severe group were decreased significantly (0.317±0.086 vs. 0.389±0.064, P<0.05), and natural killer (NK) cells were significantly increased (0.217±0.107 vs. 0.158±0.068, P<0.05). B cells content in severe group was decreased significantly than that of mild group (0.114±0.060 vs. 0.155±0.075, P<0.05). There were no significant difference in CD8(+) and CD4/CD8 ratio among the healthy control group, mild trauma group and severe trauma group (CD8(+): 0.260±0.074, 0.260±0.091, 0.271±0.105; CD4/CD8 ratio: 1.69±0.75, 1.56±0.83, 1.34±0.65, all P>0.05). Except that there were negative correlation between CD3(+) T cells and the ISS scores (r=-0.42, P=0.03), the other lymphocyte subsets showed no correlation with the ISS scores and the APACHEII scores (mild trauma group with ISS scores: CD3(+) r=-0.10, CD4(+) r=-0.31, CD8(+) r=0.18, B cells r=0.20, NK cells r=-0.04; mild trauma group with APACHEII scores: CD3(+) r=0.04, CD4(+) r=-0.07, CD8(+) r=0.06, B cells r=-0.10, NK cells r=0.05, severe trauma group with ISS scores: CD4(+) r=-0.12, CD8(+) r=-0.17, B cells r=0.02, NK cells r=0.31,all P>0.05;severe trauma group with APACHEII scores:CD3(+) r=-0.24, CD4(+) r=0.11, CD8(+) r=-0.26, B cells r=0.15, NK cells r=0.08, all P>0.05). CONCLUSIONS: CD3(+) and CD4(+) T cells decreased and NK cells increased significantly in blood in the early stage after severe trauma. CD3(+) T cells are independent indexes which reflect body injury. Therefore, it is necessary to monitor the changes of immune cells dynamically after severe trauma.

The enhanced effect of lupeol on the destruction of gastric cancer cells by NK cells.

Lupeol, a triterpene, was reported to possess beneficial effects as a therapeutic and preventive agent for a range of disorders. Many studies have confirmed that lupeol possesses strong activities such as antioxidative, antiinflammatory, antiarthritic, antimutagenic, and antimalarial, both in vitro and in vivo, and at its effective therapeutic doses exhibit no toxicity to normal cells and tissues. Lupeol was observed to inhibit the proliferation of gastric tumour cells in a dose-dependent manner, as assessed by MTT assay, and induce the proliferation of NK cells, as assessed by flow cytometry and Western blotting. The killing effect of NK cells on gastric tumour cells was assessed by LDH. Our experiment demonstrated that lupeol at appropriate concentrations could promote the proliferation of NK cells, inhibit the proliferation of gastric cancer cell lines BGC823, N87 and HGC27, and increase the killing effect of NK cells on gastric cancer cells. We speculated that lupeol might increase the expression of PFP, IFN-γ, and CD107a via the activation of PI3K/Akt and Wnt/ß-catenin signalling pathways. Lupeol could serve as a potential agent against gastric cancer; however, further in-depth in vivo studies are still required.

The effect of phloretin on human γδ T cells killing colon cancer SW-1116 cells.

OBJECTIVE: To explore the effect and mechanism of Phloretin on human γδ T cells killing colon cancer SW-1116 cells. METHODS: γδ T cells were amplified in vitro from human peripheral blood mononuclear cells through isopentenyl pyrophosphate method (IPP). After cocultured different concentrations of Phloretin with γδ T cells or SW-1116 cells for 48h respectively, MTT assay was used to test the growth curve of these two cells; Flow cytometry to test the expression of Granzyme B (GraB), perforin (PFP), CD107a and IFN-γ of γδ T cells; Lactate dehydrogenase (LDH) release assay to test the cytotoxic activity of the γδ T cells on SW-1116 cells; and Western blot to test the Wnt3a expression of the γδ T cells. RESULTS: After cultured with IPP for ten days, the percentage of γδ T cells increased from 3.31±3.00% to 78.40±10.30%. Compared to the control group, when the concentration of Phloretin increased from 2.35µg/ml to 18.75µg/ml, it could significantly proliferate the γδ T cell growth (P<0.05) and inhibit the growth of SW-1116 cells in dose-response, and the expression of GraB, PFP, CD107a and Wnt3a significantly increased (P<0.05). Significant positive relationships were observed among CD107a and PFP, GraB, cytotoxicity (P<0.05). The percentage of IFN-γ producing γδ T cells treated with Phloretin was significantly higher than control group. CONCLUSION: Phloretin can enhance the killing effect of γδ T cells on SW-1116 cells; the mechanism may be that Phloretin could proliferate the γδ T cell growth, increase the expression of PFP and GraB, activate the Wnt signaling pathway, and produce higher level of IFN-γ. Indeed CD107a expression probably correlates quite well with antitumor activity.

[Inhibitory effect of toll-like receptor 7 agonist on K562 cells].

The aim of this study was to investigate the inhibitory effect of Toll-like receptor 7 (TLR7) agonist gardiquimod on K562 cells. Human γδT cells from peripheral blood cells were amplified by isopentenyl pyrophosphate. The proliferation capacity of γδT cells and K562 cells were measured with MTT assay after treatment with different concentrations of gardiquimod. Cytotoxicity of γδT cells on K562 cells was detected by CCK-8 kit, and the intracellular expression of TLR7, cell cycle and apoptosis of K562 cells before and after treatment with gardiquimod were measured by flow cytometry. The results demonstrated that gardiquimod could significantly stimulate the proliferation of γδT cells, and inhibit proliferation of K562 cells under the concentration of 11.0 µg/ml for 48 h. The expression of TLR7 increased after treatment with gardiquimod. No apoptosis was observed, but there were significant changes in cell cycle, moreover the K562 cells treated with gardiquimod were more killed by γδT cells. It is concluded that the gardiquimod can inhibit the proliferation of K562 cells and enhance their sensitivity to killing activity of human γδT cells.

[Co-stimulation of multiple activating factors on proliferation and phenotype of T lymphocytes in peripheral blood in vitro].

AIM: To observe the costimulation of multiple activating factors effects on the proliferation and phenotype of T lymphocytes in vitro. METHODS: Peripheral blood mononuclear cells (PBMCs) were separated by fractionation on Ficoll-Hypaque gradient. According to adding different cytokines (CD3 mAb, CD28 mAb, IFN-γ, IL-1α, IL-2 and IL-15), the experiments were divided into seven groups. Effects of different cytokines on the proliferation of PBMC were counted by automated hematology analyzer five categories. The phenotypes (CD3, CD4, CD8, CD28, CD16, CD56(+);CD16, CD3(+);CD8(+);, CD3(+);CD4(+);, CD3(+); CD56(+);, CD45RO) expressing on the surface of costimulatory cells were detected by flow cytometry, and the cytotoxicity of costimulatory cells on SGC-7901, SW-1990 and SW-116 cell lines was examined by lactate dehydrogenase release method. RESULTS: The proliferation has significant difference when adding different cytokines into PBMCs culture system, the highestest proliferation multiples group is the one contains cytokines CD3, CD28, IFN-γ, IL-2, IL-1α, IL-15 and IL-21, which proliferation multiple is 255.3±6.3 at the tenth day of cell culture, obviously higher than the other culture systems which only contains CD3, IFN-γ and IL-2 (166.6±5.5) (P<0.05). Part of cells'phenotype changed when adding different activating factors. Without IL-15, the proportion of CD16(+);CD56(+);(NK) cells and CD3(+);CD56(+); cells was higher than the other groups; CD45RO(+); memory cells is most evident when delayed adding IL-15 and IL-21 for three days. The cytotoxicity of PBMCs cultured for ten days with different activating factors had significant difference, the highest was the one which delayed adding IL-15 and IL-21 for three days (76.2%, 60.3% and 70.6%, respectively.), higher than the cell culture groups containing CD3, IFN-γ and IL-2 (54.9%, 44.6% and 50.4%, respectively) (P<0.05). The cultured cells had the strongest cytotoxicity on SGC-7901 gastric adenocarcinoma cells. CONCLUSION: The PBMCs' proliferation, phenotype and cytotoxicity had significant difference after being activated by different stimulating factors, adding matching stimulating factors into the culture system have great value on cell-directed culture.

Effects of pituitary adenylate cyclase activating polypeptide on CD4(+)/CD8(+) T cell levels after traumatic brain injury in a rat model.

BACKGROUND: The effect of pituitary adenylate cyclase activating polypeptide (PACAP) during traumatic brain injury (TBI) and whether it can modulate secondary injury has not been reported previously. The present study evaluated the potential protective effects of ventricular infusion of PACAP in a rat model of TBI. METHODS: Male Sprague Dawley rats were randomly divided into 3 treatment groups (n=6, each): sham-operated, vehicle (normal saline)+TBI, and PACAP+TBI. Normal saline or PACAP (1 µg/5 µL) was administered intracerebroventricularly 20 minutes before TBI. Right parietal cortical contusion was produced via a weight-dropping method. Brains were extracted 24 hours after trauma. Histological changes in brains were examined by HE staining. The numbers of CD4(+) and CD8(+) T cells in blood and the spleen were detected via flow cytometry. RESULTS: In injured brain regions, edema, hemorrhage, inflammatory cell infiltration, and swollen and degenerated neurons were observed under a light microscope, and the neurons were disorderly arrayed in the hippocampi. Compared to the sham group, average CD4(+) CD8(-) lymphocyte counts in blood and the spleen were significantly decreased in rats that received TBI+vehicle, and CD4(-) CD8(+) were increased. In rats administered PACAP prior to TBI, damage was attenuated as evidenced by significantly increased CD4(+), and decreased CD8(+), T lymphocytes in blood and the spleen. CONCLUSION: Pretreatment with PACAP may protect against TBI by influencing periphery T cellular immune function.

[Clinical observation on adoptive immunotherapy with autologous cytokine-induced killer cells for advanced malignant tumor].

BACKGROUND & OBJECTIVE: Cytokine-induced killer (CIK) cells have the characteristics of rapid proliferation, high efficiency, and broad spectrum in killing of tumor cells. However, there was few report about its clinical application on treatment for cancer patients. The current study was designed to evaluate the effect of adoptive transfer of autologous CIK cells on the patients with advanced malignant tumor. METHODS: Peripheral blood mononuclear cells of the patients with advanced malignant tumor were separated by fractionation on Ficoll-Hypaque gradient, then cultured in the medium containing IFN-gamma, IL-2, and CD3McAb for 7 days in vitro, and than the cultured auto-CIK cells were transfused back to the patients. The numbers of transferred CIK cells per patient were 5-15 x 10(9) in one course of treatment. Among these patients, 47 cases received chemotherapy, 3 cases received radiotherapy before CIK cells transfusion. The interval between chemoradiotherapy and immunotherapy was over 2 to 4 weeks. RESULTS: Among 63 patients receiving CIK cells immunotherapy, the total effective rate (PR + MR) was 44.46% (28). In the patients with increasing of CEA level in serum, 14 cases showed reduction of serum CEA and 1 cases remained increasing after the treatment with CIK cell. In the patients with increasing of AFP level in serum, similarly, 9 cases showed reduction of serum AFD and 1 case remained increasing. The absolute members of CD3, CD4, and CD8T cells increased to over 45% after being treated with CIK cells. Among treated patients, the appetite of 51 cases and performance and sleep of 32 cases got improved. Among 18 cases, 13 cases showed the pain relief. CONCLUSION: Adoptive immunotherapy of auto-CIK cells can significantly enhance cellular immune functions and improve subjective symptoms, but without side effects, so this is a safe and effective treatment for the patients with malignant tumor.