Interleukin-2 reverses CD8(+) T cell exhaustion in clinical malignant pleural effusion of lung cancer.

Malignant pleural effusion (MPE) is a poor prognostic sign for cancer patients, whereas the functional condition of MPE CD8(+) T cells is unknown. Intracavitary immunotherapy with interleukin (IL)-2 has been proven effective in controlling MPE. To elucidate the underlying mechanism, 35 lung cancer (LC) patients with MPE and 12 healthy donors were included in this study. For the IL-2 therapy experiments, after draining partial MPE, we treated 14 patients by administrating IL-2 (3 or 5 × 10(6) U in 50 ml saline) into the thoracic cavity. Before and after IL-2 treatment (40-48 h), the MPE and peripheral blood (PB) were obtained from the subjects. PB from healthy volunteers was collected as control. The expression of programmed cell death 1 (PD-1), granzyme B (GzmB), interferon (IFN)-γ and the proliferation were analysed in CD8(+) T cells from MPE and PB. The CD8(+) T cells in the MPE of LC patients showed lowest GzmB, IFN-γ and proliferation but highest PD-1 expression, compared with that in PB of LC patients and healthy donors. IL-2 treatment reduced the expression of PD-1, increased the expression of GzmB and IFN-γ and enhanced the proliferation of CD8(+) T cells in MPE. In addition, IL-2 treatment reduced carcino-embryonic antigen (CEA) level in MPE. These results indicate that MPE CD8(+) T cells exhibit exhaustion phenotype which can be reversed by IL-2 therapy.

Cloning and polymorphism analysis of the 2-methyl-6-phytyl-1,4-benzoquinol methyltransferase gene (VTE3) in Arachis hypogaea, A. duranensis, and A. ipaënsis.

One of the critical enzymes involved in vitamin E biosynthesis in plants is 2-methyl-6-phytyl-1,4-benzoquinol methyltransferase (MPBQ MT). The full-length VTE3 cDNA (designated rVTE3-1 and -2) encoding MPBQ MT and the full-length DNA of VTE3 (designated gVTE3-1 and -2) were isolated from cultivated peanuts (Arachis hypogaea). The full-length DNA of VTE3 (designated gVTE3-A and -B) was isolated from the wild groundnut species A. duranensis (A-genome) and A. ipaënsis (B-genome), and polymorphism analysis of VTE3 was performed. The results demonstrated that rVTE3-1 and -2 both have a DNA sequence that is 1059 bp long and encodes 351 amino acids; the homology of the 2 amino acid sequences was 98.6%. The gVTE3-1 of cultivated Fenghua 2 peanut samples was 2710 bp long, with 3 introns located at 44-163, 772-1295, and 1603-2437 bp, and the Fenghua 2 gVTE3-2 was 2706 bp long, with 3 introns located at 44-169, 778-1291, and 1599-2433 bp. The homology for gVTE3-1 and -2 across 13 cultivated peanut samples was 99.9 and 100%, respectively. gVTE3-1 and -2 were from A- and B-genome, respectively, with 96.6% homology between the 2 sequences. The present study demonstrated that abundant polymorphisms were present in the VTE3 genes from different genomes. Additionally, polymorphisms were observed in the gVTE3-1 alleles of the 13 cultivars and wild A. duranensis species but not in the gVTE3-2 alleles of the 13 cultivars and wild A. ipaënsis species.

IFN-gamma enhances paclitaxel-induced apoptosis that is modulated by activation of caspases 8 and 3 with a concomitant down regulation of the AKT survival pathway in cultured human keratinocytes.

The aim of this study was to investigate the molecular effects of paclitaxel and IFN-gamma on cultured human keratinocyte cells (HaCaT) assessing the induction of both the apoptotic pathway and cell survival signals. Cellular cytotoxicity assays were performed by MTT dye assay. Caspases 8, 3 and AKT (Ser473 and Thr308 residues) were assessed by Western blot analysis. Morphological characteristics were examined by Wright stain analysis. Paclitaxel reduced keratinocyte growth in a 3-day bioassay with an effective ED(50) of 6-600 ng/ml. A large variation in ED(50) can be attributed to the asynchronous population of cells. Paclitaxel treatment induced activation of the AKT survival pathway in a time-dependent manner. The down-regulation of AKT signal was preceded by the subsequent activation of caspases 8 and 3 leading to apoptosis. These results indicate that paclitaxel activates both the PI3-K/AKT cell survival pathway followed by induction of apoptotic signals in cultured human keratinocytes. The induction of apoptosis in paclitaxel-treated cells is enhanced by coadministration of IFN-gamma. The synergistic effect of these two agents on HaCaT cells relies on a pathway involving caspases 8 and 3, with activity increasing by 48 h. Collectively, our data indicate that i) paclitaxel-induced apoptosis is enhanced by IFN-gamma; ii) the down-regulation of PI3-K/AKT survival pathway may help potentiate the apoptotic effects of paclitaxel and iii) the apoptotic signaling pathways are initiated with the activation of caspases 8 and 3 activities.

Regularity of distribution of immunoreactive pulmonary surfactant protein A in rat tissues.

Existing data has shown that SP-A-like protein or mRNA is widely distributed in lamellar bodies such as tissues and mucosal surfaces. Using immunohistochemistry method with a polyclonal antibody against human SP-A, in this study we investigated distribution of immunoreactive pulmonary surfactant protein A (IR-SP-A) in a number of rat tissues. The SP-A-like immunoreactivity was found in alveolar, parenchyma, pleura of lung; myelin sheath of brain; epithelia of Bowman's capsule, glomerulus and renal tubules of kidney; epithelia of colon, stomach, duct of salivary gland, pharynx; and blood vessel wall and connective tissue of extracellular matrix. The positive signal was blocked by pre-absorbed SP-A antigen from recombinant or bronchoalveolar lavage (BAL). SP-A has long been considered as an important frontier host defense molecule which participates in immune and inflammatory regulation of lung. With every inhalation, small particles, viruses, bacteria, and antigens from environment are continuously deposited onto the vast pulmonary epithelial surface. While a proper host defense is required to protect the lung, an over-exuberant response can disrupt the appropriate balance between pro- and anti-inflammatory. Traditional Chinese medicine believes that body is an open system relevant to the external environment. The physical, chemical and biological environmental factors constantly affect the open system, and the body properly reacts to maintain homeostasis of body machinery. The Chinese traditional medicine scholars have thus hypothesized that 'Qi' (meaning air) is the communication way between the body and external environment. What is 'Qi'? The results from our study suggest that IR-SP-A is a candidate of 'Qi'. It is compatible with the sites, theoretically containing collagenous and lectin domain molecules, also compatible with the primary injury sites of some autoimmune diseases. SP-A may be as one of 'Qi' molecules mentioned in traditional Chinese medicine that trigger some of autoimmune diseases.

Attenuation of cell migration and induction of cell death by aged garlic extract in rat sarcoma cells.

Previous studies have shown that aged garlic extract suppresses cancer growth and enhances immune system against cancer, and yet little is known about inhibition of the cancer cell migration. In this study we investigated whether the aged garlic extract inhibits growth and migration of rat sarcoma tumor cells. The suppression of tumor cell growth was demonstrated by counting the cell number in three groups (control, cultured with 10 mg/ml, 20 mg/ml of aged garlic extracts) after culturing for 3 days and 5 days. The results showed that aged garlic extract inhibited the growth of rat sarcoma cancer cells in a dose-dependent manner, compared to the numbers of the cells grown in control group. The inhibition of tumor cell migration was examined by measuring the distance of trails left behind by the tumor cells when they passed through the polybeads overnight in four groups (control, 5 mg/ml, 10 mg/ml, 20 mg/ml aged garlic extracts). The average distance of trails in control group was 7.44 mm, whereas the average distance of cell movement is only 2.48 mm when treated with the highest concentration (20 mg/ml) of the aged garlic extract. The results also showed that the inhibitory effect of aged garlic extracts on tumor cell migration was dose-dependent. This is the first report to show that the aged garlic extract inhibits rat sarcoma cell migration, a critical feature of tumor cell metastasis. It can be thus envisioned that if tumor cell metastasis could be attenuated if not completely stopped, it would be possible to stabilize the tumor in the local area for surgical removal. The results suggest that garlic, as a natural plant, unlike other cancer treatment methods, may play a role in fighting cancer without significant side effects.

Cytokine-induced p38 activation feedback regulates the prolonged activation of AKT cell survival pathway initiated by reactive oxygen species in response to UV irradiation in human keratinocytes.

A previous study has shown that UV activates the PI3K/AKT cell survival pathway while inducing cell death in human skin in vivo and cultured human keratinocytes in vitro, and yet the upstream pathway leading to the activation of AKT has not been thoroughly investigated. In this study we found that UV-induced phosphorylation of p38 and AKT in a time-dependent manner. The phosphorylation of p38 started at 5 min post UV irradiation, peaked at about 30 min, and remained elevated up to 2 h. The phosphorylation of AKT started at 15 min post UV treatment, peaked at about 1 h, and remained elevated up to 2 h. We also found that H2O2 induced phosphorylation of p38 and AKT in a time- dependent manner. Pretreatment with NAC abolished UV-induced AKT phosphorylation, suggesting the involvement of reactive oxygen species in AKT activation. Interestingly, SB203085, a known p38 inhibitor, had partially inhibited UV-induced AKT phosphorylation. Further studies showed that cytokines such as TNF-alpha and IL-1beta induced AKT phosphorylation in a time-dependent manner. Pretreatment with SB203085 inhibited IL-1beta-induced p38 and AKT phosphorylation. Collectively, our data suggest that UV activation of PI 3-kinase/AKT pathway is initiated by ROS and prolonged by feedback activation of p38 induced by released cytokines in response to UV irradiation in cultured human keratinocytes.

Computerized 3D-reconstructions of the ligaments of the lateral aspect of ankle and subtalar joints.

3D-reconstruction images of the structures of lateral aspect of the ankle and subtalar joints were produced using plastination to make equidistant serial sections of 1.2 mm in thickness. A SGI workstation was employed to reconstruct the structures of the ligaments of the lateral aspect of ankle and subtalar joints in three dimensions. Reconstructed structures were displayed singly, in groups or as a whole, and these were rotated continuously at different velocities in 3D space. Different diameters and angles of the reconstructed structures could be measured easily. Improved results could be achieved with the use of a special sectional anatomical technique, i.e. contours + marching cubes algorithm.

EGF receptor crosstalks with cytokine receptors leading to the activation of c-Jun kinase in response to UV irradiation in human keratinocytes.

Ultraviolet (UV) irradiation causes photoageing through induction of matrix-degrading metalloproteinases (MMP), which are upregulated by activator protein-1 (AP-1) (Jun/Fos). The c-Jun kinase activity proves to be critically important in the regulation of AP-1 activity. Our previous studies showed that UV irradiation activates epidermal growth factor receptor (EGFR) and cytokine receptors leading to the activation of c-Jun kinase in cultured human skin keratinocytes in vitro and in human skin in vivo. However, the mechanism of UV-induced cell surface receptor activation and the crosstalk among growth factor receptor and cytokine receptors were not fully investigated. This study showed that UV (30 mJ/cm(2))-induced EGFR tyrosine phosphorylation in a manner similar to EGF (100 ng/ml), or IL-1beta (10 ng/ml) in cultured human keratinocytes. In all cases, EGFR tyrosine phosphorylation was completely inhibited by pretreatment of PD153035 (100 nM, 1 h). Also observed was that UV induced autophosphorylation of interleukin 1 receptor associated kinase (IRAK) in a manner analogous to IL-1beta or EGF. In both UV and EGF cases, the phosphorylation of IRAK was inhibited by pretreatment of PD153035. However, IL-1beta-induced IRAK activation was not affected by PD153035. In vitro kinase assay using GST-c-Jun as a substrate revealed that pretreatment of PD153035 completely inhibited UV- and IL-1-induced c-Jun kinase activity in cultured keratinocytes. Taken together, the above data suggest that EGFR plays dominant role in the crosstalk among growth factor receptor and cytokine receptors leading to the activation of c-Jun kinase upon UV irradiation, and that EGFR could be one of the targets for clinical and cosmetical prevention of UV-induced skin aging.

Ultraviolet irradiation activates PI 3-kinase/AKT survival pathway via EGF receptors in human skin in vivo.

Growth factors interact with their cell surface receptors and activate the enzyme PI 3-kinase (PI 3-K) resulting in the formation of 3-phosphorylated phosphatidylinositols, which in turn activate the serine/threonine kinase AKT/PKB. AKT functions, in part, to promote cell survival by phosphorylating the BCL-2 family member BAD and the cell death pathway enzyme, caspase-9. Although induction of apoptosis by ultraviolet (UV) irradiation is well documented, little is known about UV activation of cell survival pathways in human skin cells. We have investigated whether UV activates the PI 3-K/AKT pathway in human skin in vivo. UV irradiation (2MED from UVB source) stimulated PI 3-kinase activity within 15 min. PI 3-K activity was maximal (2.5-fold, n=6) 30 min post UV and remained elevated for 4 h. UV stimulated AKT activity within 30 min. Maximal activity (4-fold, n=11) was observed 1 h post UV. UV also stimulated phosphorylation of the downstream AKT effectors, S6 kinase and BAD. S6 kinase was maximally stimulated 4 h post UV (15-fold, n=6). Increased BAD phosphorylation was observed 1 h post UV and remained elevated for 4 h. Western blot analysis revealed that UV-induced phosphorylation of BAD at Ser112, a site known to be phosphorylated by AKT. Inhibitors of EGFR and PI 3-kinase blocked UV-induced phosphorylation of BAD, suggesting that EGFR mediates UV-activated cell survival pathway. Collectively, both positive and negative roles for UV activation of the PI 3-K/AKT pathway in human skin can be envisioned. The PI 3-K/AKT pathway likely plays a critical role in balancing UV-induced apoptotic signals, thereby preventing widespread skin cell death. Conversely UV activation of the PI 3-K/AKT pathway may enhance survival of mutated cells, thereby promoting skin cancer, as has been found in several other types of cancer.