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The diurnal pattern of nitrous oxide (N2O) emissions is essential in understanding how weather and soil conditions influence the daily mean estimate of N2O fluxes. Incubation experiments were conducted to investigate the effects of vertical soil moisture distribution patterns on diurnal variation of N2O emissions. Clear diurnal patterns of N2O emissions on both surface watering (SW) and subsurface watering (SUW) treatments (SUW12, SUW15, and SUW18) were detected from soil sample (I), silty clay, and soil sample (II), sandy loam, where peak N2O fluxes usually occurred between 12:00 and 18:00 h. Different vertical watering patterns resulted in changes in the daily range of N2O fluxes and peak time. Mean fluxes from the SUW12, SUW15, and SUW18 treatments were 37.4%, 32.7%, and 43.3% lower than those from SW treatments from soil sample I, and 32.0%, 40.3%, and 41.1% from soil sample II. Moisture distribution patterns under SUW soils could be effective to mitigate N2O emissions. The N2O emissions from soil sample I ranged from178.3 to 2741.0 μg N2O m-2 h-1, which was more than in soil sample II with 7.0 to 83.7 μg N2O m-2 h-1. The different soil texture and N content level might account for the differences in magnitude of N2O fluxes from soils. The optimal soil moisture condition for peak N2O fluxes in the SW treatment had relatively narrower ranges than the SUW treatments with 46% to 60% water-filled pore space (WFPS) for soil sample I and 26% to 34% WFPS for soil sample II even though surface soil moisture for peak N2O fluxes were somewhat different from the previously reported optimal soil moisture range of 45% to 75% WFPS.

Nitrous oxide (N2O) emission from croplands in China is a serious environmental concern. Water management is an important factor in regulating N2O emissions from croplands. In China, controlled irrigation (CI) is one mode of the water-saving irrigation for rice and is widely used. This study aims to assess the lasting effects of CI on N2O emissions from winter wheat croplands in Southeast China, with traditional irrigation (TI) as the control. CI performed during the rice-growing season had obvious lasting effects on N2O emissions of the subsequent winter wheat-growing season. Compared with TI, CI significantly increased the cumulative N2O emission by 129.1 % during the rice-growing season (p 0.05). The results suggest that CI does not significantly increase the cumulative N2O emission from the rice–winter wheat rotation systems while insuring rice and wheat yields. This study focuses on the lasting effects of water-saving irrigation mode during rice-growing season on N2O emissions during the following wheat-growing season. Thus, it is a development and complement of the previous researches on the effects of water-saving irrigation on N2O emissions from rice–winter wheat rotation croplands.

To reveal the influence of non-flooding controlled irrigation (NFI) on gaseous nitrogen (N) losses in forms of ammonia volatilization (AV) and nitrous oxide (N2O) emissions from high N inputs rice paddies, lysimeter experiments were conducted with flooding irrigation (FI) as check. Compared with FI paddies, AV losses in NFI paddies decreased by 18.5–20.5 % and N2O emissions increased by 1.43–1.9 kg N ha−1. Weekly AV losses immediately after fertilization accounted for over 83 % of seasonal losses in both treatments. High N2O emissions from NFI paddies always occurred in drying process after N application, with peaks observed when water-filled pore space (WFPS) fell in 75–85 %. Water management immediately after N fertilization is crucial for mitigating gaseous N losses from rice paddies. Bringing N into shallow rhizosphere by irrigation and covering it with deep water will be helpful in preventing AV. Maintaining a flooding period and keeping WFPS higher than 85 % in the first drying process after fertilization might be effective to reduce N2O emissions peaks for NFI paddies.

Ammonia volatilization (AV) is one of the main pathways of nitrogen (N) loss from a rice paddy, which results in low N use efficiency and many other environmental problems. To reveal AV losses from rice paddies with different combined irrigation and N managements, field experiments were conducted using site-specific nutrient management (SSNM), controlled released nitrogen management (CRN), and non-flooding controlled irrigation (NFI). The interactive effect of N management and irrigation management on AV losses is significant. N management is the dominant management factor of AV losses from a rice paddy. Weekly AV losses followed by fertilization comprise the majority of seasonal AV losses. Ammonium contents in surface water or top soil solutions determine the AV loss rate for both flooding irrigation (FI) and NFI paddies. Moreover, AV losses are less sensitive to surface ammonium nitrogen contents for the NFI paddy than that for the FI paddy. Shallower water condition in the NFI paddy immediately after fertilization may result in higher AV losses than that in the FI paddy in a short term during the first wet–dry cycle after fertilization. However, the following wet–dry cycles result in lower AV losses in most of the rice growth stages. Seasonal AV losses from the NFI paddy using farmers’ fertilization practice (FFP), SSNM, and CRN treatments were 125.27, 37.63, and 23.73 kg N ha−1, which account for 31.1%, 23.2%, and 13.2% of the seasonal N inputs, respectively. These results were reduced by 14.0%, −17.1%, and 28.7% compared with those from the FI paddy with the same N management. A combination of NFI and CRN is the optimal treatment, with the lowest AV losses and high potential in reducing nutrient leaching risks. Increasing the water depth and the duration of flooding for the first wet–dry cycle after fertilization is a promising measure to reduce AV losses and improve N use efficiency in an NFI paddy.

Water-saving practices for rice production has become one of the major agricultural policies in China due to the severe water scarcity. However, greenhouse gases emissions from paddy field may be changed with water-saving practices. In the two-year field experiment at Thaihu Lake Basin of China, CH 4 and N 2 O fluxes from paddy field under controlled irrigation and continuous flooding irrigation were monitored using closed chambers. Total CH 4 emission from paddy field under controlled irrigation during the rice-growing period were 2.47 and 1.88 g m −2 in 2006 and 2007, respectively, reduced by 79.1% on average compared with continuous flooding irrigation. The CH 4 emission from paddy field was further concentrated in the initial and middle tillering stages of rice growth under controlled irrigation for 2 years. The N 2 O emission from paddy field under controlled irrigation was mostly larger than those from flooding irrigation during the rice-growing period. Total N 2 O emission from paddy field under controlled irrigation during the rice-growing period was 106 mg m −2 , increased by 10.6% compared with those from flooding irrigation. Carbon dioxide equivalents of CH 4 and N 2 O emissions from paddy field during the rice-growing period under controlled irrigation were 788 kg CO 2 ha −1 , reduced by 61.4% compared with those from flooding irrigation. It can be concluded that controlled irrigation is an effective technique for mitigating the carbon dioxide equivalents of CH 4 and N 2 O emissions from paddy field.

In this study, we used remotely sensed data, GIS modeling, and statistical methods to evaluate the damage caused by the Wenchuan Earthquake (May 12, 2008) to the giant panda (Ailuropoda melanoleuca) habitat in the World Nature Heritage Sichuan Giant Panda Sanctuary (WHSGPS) in China. A landscape ecological analysis found increases of landscape heterogeneity, complexity, and fragmentation in the giant panda habitat after the earthquake. A terrain analysis found that slope and elevation are directly associated with the distribution of the damaged areas. As slope and elevation increase, the size of the damaged area keeps increase until to a peak, and then starts to drop. The total area of the damaged vegetation in our study area is 114.26 km(2), accounting for 3.78% of the study area; 30.46% of that 114.26 km(2) is located in the Core Zone of WHSGPS. There are 18.57km(2) of the damaged vegetation located in the identified suitable giant panda habitat, accounting for 1.75% of the total area of suitable giant panda habitats in the study area. Based on these findings, we conclude that the Wenchuan Earthquake does not have significant impact on the WHSGPS as a whole.

Greenhouse gas emissions, nitrogen and phosphorous losses through ammonia volatilization, leaching and surface drainage from rice paddy under efficient irrigation and drainage were analyzed based on field experimental data in order to reveal the eco-environmental impacts of efficient irrigation and drainage on rice paddy. The results showed that total methane emission from rice paddy under the controlled irrigation was reduced by more than 80% and total nitrous oxide emission increased by 15.9% compared with flooding irrigation. Seasonal comprehensive global warming potentials (GWP) of methane and nitrous oxide were 62.23 gCO2 m−2 for rice paddy under the controlled irrigation, reduced by 68.0% compared with flooding irrigation. Due to large reduction in seepage and surface drainages, nitrogen and phosphorous losses through leaching were reduced by 40.1% and 54.8%, nitrogen and phosphorous losses through surface drainage were reduced by 53.9% and 51.6% from rice paddy under efficient irrigation and drainage compared with traditional irrigation and drainage. Nitrogen loss through ammonia volatilization was reduced by 14.0%. Efficient irrigation and drainage management is helpful to mitigate greenhouse gases emission, nitrogen and phosphorus losses and their pollution on groundwater and surface water.

While many water-saving rice production techniques have been adopted in China, the environmental effects of these techniques require further investigation. This study aims to assess nitrogen (N) and phosphorus (P) leaching losses under real conditions in different water and N managements. Two water and three N treatments are conducted in the Taihu Lake region of China. Results show that the total N leaching losses during the rice season under flooding irrigation (FI) are 12.4, 9.31, and 7.17 kg ha−1 for farmers’ fertilization practices (FFP), site-specific N management (SSNM), and controlled-release nitrogen fertilizer management (CRN), respectively. Under controlled irrigation (CI), the respective losses were 7.40, 5.86, and 3.79 kg ha−1 for the same management methods. The total P leaching losses during the rice season under FI were 0.939, 0.927, and 0.353 kg ha−1 for FFP, SSNM, and CRN, respectively. Under CI, the losses were 0.424, 0.433, and 0.279 kg ha−1, respectively, for the same management methods. Ammonium and nitrate N accounted for 42.2–65.5% and 11.8–14.7% of the total nitrogen leaching losses under different water and N management methods, respectively. Due to significant decrease of volumes of percolation water and nitrogen and phosphorus concentrations in percolation water, N and P leaching losses were reduced in the CI treatment compared to the FI treatment under the same N management. The reduction of N input and application of controlled-release nitrogen fertilizer can reduce N and P leaching losses from paddy fields.

Biochar has been proposed as a new countermeasure to mitigate climate change because of its potential in inhibiting greenhouse gas emissions from farmlands. A field experiment was conducted in Taihu Lake region in China to assess the effects of rice-straw biochar amendment on methane (CH4) emissions from paddy fields under water-saving irrigation using three treatments, namely, control with no amendment (C0), 20 t ha−1 (C20), and 40 t ha−1 rice-straw biochar amendments (C40). Results showed that biochar application significantly decreased CH4 emissions by 29.7% and 15.6% at C20 and C40 biochar addition level, respectively. C20 significantly increased soil dissolved organic carbon, total nitrogen, and NH4+-N by 79.5, 24.5, and 47.7%, respectively, and decreased NO3−-N by 30.4% compared with C0. On the other hand, no significant difference was observed in soil pH and soil organic carbon in all treatments. C20 and C40 significantly increased and decreased soil oxidation-reduction potential, respectively. Compared with C0, rice yield and irrigation water productivity significantly increased by 24.0% and 33.4% and 36.3% and 42.5% for C20 and C40, respectively. Thus, rice-straw biochar amendment and water-saving irrigation technology can inhibit CH4 emissions while increasing rice yield and irrigation water productivity. The effects of increasing rice yield and irrigation water productivity were more remarkable for C40, but C20 was more effective in mitigating CH4 emission.

T cells contribute to host-tumor interactions in patients with monoclonal gammopathies. Expansions of CD8+CD57+ T-cell receptor Vβ–positive (TCRVβ+)–restricted cytotoxic T-cell (CTL) clones are found in 48% of patients with multiple myeloma and confer a favorable prognosis. We now report that CTL clones with varying TCRVβ repertoire are present in 70% of patients with Waldenström macroglobulinemia (WM; n = 20). Previous nucleoside analog (NA) therapy, associated with increased incidence of transformation to aggressive lymphoma, significantly influenced the presence of TCRVβ expansions (χ2 = 11.6; P < .001), as 83% of patients without (n = 6) and only 7% with (n = 14) TCRVβ expansions had received NA. Clonality of CD3+CD8+CD57+TCRVβ+-restricted CTLs was confirmed by TCRVβ CDR3 size analysis and direct sequencing. The differential expression of CD3+CD8+CD57+TCRVβ+ cells was profiled using DNA microarrays and validated at mRNA and protein level. By gene set enrichment analysis, CTL clones expressed not only genes from cytotoxic pathways (GZMB, PRF1, FGFBP2) but also genes that suppress apoptosis, inhibit proliferation, arrest cell-cycle G1/S transition, and activate T cells (RAS, CSK, and TOB pathways). Proliferation tracking after stimulation confirmed their anergic state. Our studies demonstrate the incidence, NA sensitivity, and nature of clonal CTLs in WM and highlight mechanisms that cause anergy in these cells.

There has been little information available on how the use of dextran in vivo influences plasma viscosity and yield stress of blood. Hence, the main purpose of this study is to provide such information when level of red blood cell aggregation in rats was raised to levels seen in normal human blood with administration of Dextran 500. Our results show that plasma viscosity (P < 0.003) and yield stress (P < 0.01) of rat blood increased significantly after dextran infusion. Whole blood viscosity also significantly increased with the dextran treatment over the entire range of shear rates we measured. The viscosity changes at high shear rates after dextran infusion would be due mainly to elevation of plasma viscosity, whereas the changes at low shear rates would be attributed to red blood cell aggregation induced by the dextran treatment.

The expanded T cell clones are associated with a prolonged survival in patients with multiple myeloma. We sought to confirm this prognostic significance in a multicenter patient cohort and investigate the effect of thalidomide on clones and T regulatory cells (T(regs)). Blood was collected from 120 patients enrolled in a Phase III trial of maintenance therapy +/- thalidomide after autologous stem cell transplantation. TCR Vbeta repertoire analysis identified T cell expansions in 48% of patients pre-transplant and 68% after 8-month maintenance. T cell expansions, previously shown to be clonal, were predominantly CD8+ (93%) and all 24 TCR Vbeta families tested were represented. Thalidomide therapy was associated with a significant increase in the incidence of patients with multiple expansions (49% vs. 23%; chi2 = 6.8; p = 0.01). The presence of expansions regardless of therapy was associated with a significantly longer median progression free survival (PFS) (32.1 vs. 17.6 months; chi2 = 5.6; p = 0.02) and overall survival (OS) (chi2 = 3.9; p0.05). Median PFS in the thalidomide arm was 50.9 months for patients with expansions and 28.3 months for patients without expansions (chi2 = 19.4; p = 0.0002). Thalidomide did not appear to modulate T(reg) numbers. Expanded T cell clones are prognostically significant and have an impact on progression after thalidomide therapy in a proportion of patients.

Multiple myeloma (MM) is an incurable B cell malignancy characterised by the accumulation of monoclonal plasma cells in the bone marrow and the presence of high level of monoclonal immunoglobulin i...

Generalized synchronization in a drive-response Chua circuits is investigated. A cascade of transitions to GS is observed with increasing the interaction strength. The mechanism on the transitions to GS is given based on the asymptotic behaviors of response dynamics.

As new therapies increase the incidence of stringent complete remission (sCR) and overall survival (OS) of patients with multiple myeloma (MM), there is a growing demand for a sufficiently sensitiv...

This article investigates the relationship of local air pollution pattern with urban land use and with urban thermal landscape using a GIS approach. Ambient air quality measurements for sulfur dioxide, nitrogen oxide, carbon monoxide, total suspended particles, and dust level were obtained for Guangzhou City in South China between 1981 and 2000. Landsat TM images and aerial photo derived maps were used to examine city's land use and land cover at different times and changes. Landsat thermal infrared data were employed to compute land surface temperatures and to assess urban thermal patterns. Relationships among the spatial patterns of air pollution, land use, and thermal landscape were sought through GIS and correlation analyses. Results show that the spatial patterns of air pollutants probed were positively correlated with urban built-up density, and with satellite derived land surface temperature values, particularly with measurements taken during the summer. It is suggested that further studies investigate the mechanisms of this linkage, and that remote sensing of air pollution delves into how the energy interacts with the atmosphere and the environment and how sensors see pollutants. Thermal infrared imagery could play a unique role in monitoring and modeling atmospheric pollution.

Thalidomide and its immunomodulatory derivatives have provided the most significant advance in the therapy of myeloma since the introduction of high dose chemotherapy followed by stem cell transplantation nearly 20 years ago. The mechanism of action of thalidomide is complex and involves many aspects of malignant plasma cell growth and bone marrow stromal cell microenvironment interaction. Thalidomide was first used because of its anti-angiogenic properties, however it is the immunomodulatory actions that involve increasing host tumour-specific immunosurveillance by both T cell and natural killer cells which may be the most important mode of action.

SUMMARY Guangzhou has ambitions to build itself into a world class metropolis by 2010. Sustainable development is the only way to achieve this magnificent goal. Based on the ecological perspective of sustainable development and the principles of ecosystem integrity, this paper develops an approach for evaluation of sustainable development in Guangzhou between 1986 and 1995. A hierarchical evaluation system of four tiers of sustainability indicators was established. Using the method of fuzzy multistage synthetic evaluation, sustainability development level index, QIx, was calculated for the indicators at the B, C, D, and E tiers. Development stages were identified based on these index values. The coordination degree among the economic, social, and natural subsystems was also computed. Further, an overall sustainability index for each year was computed by combining the development level index and the coordination degree. It was found that the urban ecosystem in Guangzhou had generally become more sustainabl...

Idiotypic determinants are potential patient-specific tumor antigens in multiple myeloma (MM). In this study, we have determined the DNA sequence of the variable region of the tumor immunoglobulin (Ig) in 6 patients with MM. We then selected high affinity class I-restricted T-cell peptide epitopes in tumor Ig using two different internet-based epitope prediction programs. High affinity binding peptides were identified by at least one program in 4 out of 6 patients. Of these 35 peptides, only 3 scored high by both analyses. Given that all 6 patients had expanded T-cell clones with a cytotoxic (CD57+CD8+CD28-perforin+) phenotype, known to be associated with a longer survival and postulated to recognise tumor epitopes, this analysis indicates that such clones are unlikely to be exclusively directed towards tumor immuoglobulin, and suggests the need to expand the scope of the search for tumor epitopes with the ability to stimulate cytotoxic T cells in vivo.

This paper examines urban land development in Guangzhou in different periods of time between 1960 and 1997 using aerial photos and Landsat Thematic Mapper images. Results indicate that urban land development has been spatially and temporally uneven. Urban growth was sluggish and constrained in the period from the 1950s to 1970s. However, it has shown an accelerated rate after the 1980s, and eastward expansion replaced the traditional southern and western expansions. In recent years, northeastward expansion has increasingly become a favorite direction due to the need for more land and serious west-east-running traffic congestion. The patterns of urban land development can be better understood when relating them to the changing urban planning policies and the spatial structures of the city. The spatial structure has evolved over time in response to various State-approved Master Plans. However, decentralized development has always been a core planning policy. Both the 1985 and 1996 Master Plans call...

Trogocytosis, which results in the acquisition of myeloma cell-derived membrane proteins by T cells, and hence generates novel regulatory T cells, adds to the growing list of immune defects of multiple myeloma patients. The increasing complexity of the cancer-associated immune defects must be attentively considered for attempting to improve the so-far unsatisfactory rates of clinical responses to immunotherapy in patients affected by multiple myeloma and other malignancies.

© 2014 Informa UK, Ltd. Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells that have been implicated as inhibitors of lymphopoiesis in patients with malignancies. They have a consensus phenotype of CD33+/CD11b+/HLA-DRlo/-and can be further divided into CD15 + granulocytic (G-MDSC) and CD14 + monocytic (M-MDSC) subsets. We characterized MDSCs in patients with multiple myeloma (MM) and found a significant increase in G-MDSCs in the blood of patients with progressive MM. Flow-sorted MDSCs from patients with MM induced the generation of regulatory T cells (Treg). MDSCs from both patients with MM and aged-matched controls demonstrated a dose-dependent inhibition of lymphocyte proliferation in carboxyfluorescein succinimidyl ester (CFSE)-tracking experiments. Granulocyte colony stimulating factor (G-CSF) administered to induce stem cell mobilization caused an increase in the number of MDSCs in the peripheral blood of patients with MM and a concentration of these immune-suppressive cells in peripheral blood stem cell collections. MDSCs are likely to cause immune dysfunction in patients with MM.

The effect of controlled drainage (CD) on ammonia volatilization (AV) losses from paddy fields under controlled irrigation (CI) was investigated by managing water table control levels using a lysimeter. Three drainage treatments were implemented, namely, controlled water table depth 1 (CWT1), controlled water table depth 2 (CWT2), and controlled water table depth 3 (CWT3). As the water table control levels increased, irrigation water volumes in the CI paddy fields decreased. AV losses from paddy fields reduced due to the increases in water table control levels. Seasonal AV losses from CWT1, CWT2, and CWT3 were 59.8, 56.7, and 53.0 kg N ha−1, respectively. AV losses from CWT3 were 13.1% and 8.4% lower than those from CWT1 and CWT2, respectively. A significant difference in the seasonal AV losses was confirmed between CWT1 and CWT3. Less weekly AV losses followed by TF and PF were also observed as the water table control levels increased. The application of CD by increasing water table control levels to a suitable level could effectively reduce irrigation water volumes and AV losses from CI paddy fields. The combination of CI and CD may be a feasible water management method of reducing AV losses from paddy fields.

Many acute myeloid leukemia (AML) patients achieve a complete remission (CR) with chemotherapy but relapse is common. Removal of residual disease remains the greatest challenge. Allogeneic transplantation (alloHCT) addresses this through an immune-mediated graft versus leukemia effect (GVL), but has high morbidity and mortality. Therapeutic dendritic cell (DC) vaccination has the potential to provide immune control with limited toxicity. Previous trials using monocyte-derived DC (Mo-DC) have demonstrated modest clinical effects. This is understandable as Mo-DC have demonstrably poor migration in vivo and relatively inferior antigen processing and presentation compared to blood dendritic cells (BDC). We have developed a more practical, functionally superior vaccine composed of natural blood DC (BDC). This is achieved using the human-mouse chimeric monoclonal antibody CMRF-56 to enrich BDC from patient peripheral blood after a short incubation.We assessed the potential for preparing a CMRF-56+ BDC vaccine from AML patients in CR. We developed an extended flow cytometry panel to distinguish different BDC subsets from blasts in AML, sorted them to confirm morphology, then used TruCount methodology to enumerate them at diagnosis, post-chemotherapy (5-28 weeks) and post alloHCT. We correct previous reports that suggested BDC numbers are normal at AML diagnosis by demonstrating that the Lineage- HLA-DR+ CD11c+ cells commonly classified as myeloid DC contain myeloblasts. Exclusion of myeloblasts, revealed that CD1c and CD141 BDC are grossly depleted at AML diagnosis to 567/mL and 24/mL, 4% and 3% respectively of the the levels of healthy aged-matched controls (HC) (n=9; n=13), but recovered to 7323/mL and 294/mL, representing 57% and 39% HC levels (n=12) during CR1, and to 10282/mL and 299/mL, representing 80% and 40% of HC after alloHCT (n=6). In contrast, plasmacytoid dendritic cells (pDC) levels were 2229/mL and 27% of HC at diagnosis, but failed to recover further remaining at 1453/mL or 18% of HC at CR1 and at 1986/mL and 24% of HC post alloHCT. CD1c BDC from AML patients in CR upregulated the CMRF-56 antigen,. similarly to HC (n=5, p=0.4) but primary AML blasts did not, enabling myeloblast free, CMRF-56+ BDC purifications. CMRF-56+ BDC isolated from AML patients in CR expanded anti-viral and Wilms' Tumour 1-specific autologous CD8+ T cells in vitro. However, patients who failed standard induction chemotherapy and required fludarabine-containing salvage regimens produced good CMRF-56+ BDC preparations but did not expand functional T cells. These data support the feasabillity of preparing a functional BDC vaccine from AML patients in CR using CMRF-56 immune selection and highlight the potential detrimental effects of specific chemotherapeutics on cellular therapy. BDC vaccination may consolidate chemotherapy induced CR in AML, or enhance GVL post alloHCT, by stimulating specific immune responses to control residual disease. Disclosures Hsu: DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Bryant:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Fromm:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Papadimitrious:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Orellana:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Suen:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Yang:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Weatherburn:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Gasiorowski:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Iland:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Brown:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Joshua:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Ho:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Gibson:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Clark:DendroCyte BioTech Ltd: Equity Ownership, Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Hart:DendroCyte BioTech Ltd: Equity Ownership, Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd.

This article introduces the concepts of coordinated development of economy and environment and formulas of coordinated degree. Results obtained are that the coordinated degree in 1992 is over 0.87. The variation tendencies on coordinated development of urban environment and economy are forecasted upto the year 2010 in Guangzhou by adopting formula GM (1,1). It is considered that not only the index of environment quality in Guangzhou will come down to 0.31, but also coordinated degree which is equal to 0.64 will lower to middle level. Therefore, some effective countermeasures have to be taken to accomplish the coordination development of urban environment quality and economy.

The transfer of membrane proteins between cells during contact, known as trogocytosis, can create novel cells with a unique phenotype and altered function. We demonstrate that trogocytosis is more common in multiple myeloma (MM) than chronic lymphocytic leukemia and Waldenstrom macroglobulinaemia; that T cells are more probable to be recipients than B or natural killer cells; that trogocytosis occurs independently of either the T-cell receptor or HLA compatibility; and that after trogocytosis, T cells with acquired antigens can become novel regulators of T-cell proliferation. We screened 168 patients with MM and found that CD86 and human leukocyte antigen G (HLA-G) were antigens commonly acquired by T cells from malignant plasma cells. CD3+CD86acq+ and CD3+ HLA-Gacq+ cells were more prevalent in bone marrow than peripheral blood samples. The presence of either CD86 or HLA-G on malignant plasma cells was associated with a poor prognosis. CD38++ side population cells expressed HLA-G, suggesting that these putative myeloma stem cells could generate immune tolerance. HLA-G+ T cells had a regulatory potency similar to natural Tregs, thus providing another novel mechanism for MM to avoid effective immune surveillance.

Background IL-6 receptor/JAK/STAT is a major signalling pathway associated with pleiotropic functions including cell transformation and proliferation. STAT3, one of a family of 7 STAT members, sits at the apex of the signalling cascade and is a potential target for cancer therapy. Small molecular weight inhibitors either prevent STAT3 phosphorylation or target high constitutive levels of phosphorylated pSTAT3 (pSTAT3). Increased constitutive expression of pSTAT3 is a hallmark of solid tumors and is associated with increased morbidity. However recent studies suggest that this may not apply to hematological malignancies. Phospho-flow cytometry offers a novel approach for the quantitation of constitutive and cytokine induced pSTAT3 expression in subsets of hemopoietic cells. Constitutive pSTAT3 expression is not a prognostic factor for patients with either multiple myeloma (MM) or acute myeloid leukemia (AML), though we recently demonstrated that IL-6 induced pSTAT3 was associated with better prognosis in MM (χ2 =13.06; p We have determined constitutive and IL-6 induced pSTAT3 and 5 in malignant plasma cells of patients with MM (n=25) and for comparison, the blast cells in AML (n=8) and CD5+ B cells in chronic lymphocytic leukemia (CLL) (n=12) as well as the non-malignant B and T cells in each sample. Normal BM cells (n=9) served as a control. We have also determined pSTAT3 and 5 expression in "side population" cells (SP), the putative myeloma stem cells, and determined whether MM and AML tumor cells induce pSTAT3 on normal lymphocytes in vitro by co-culture experiments. IL-6 receptor expression was also determined on all cells. Results Constitutive pSTAT3 expression was increased (>5% of normal lymphocytes) in the malignant plasma cells of 44% of patients with MM but by comparison was not increased in AML or B-CLL. Constitutive pSTAT5 was increased (>5%) in 68% of MM plasma cells and 75% of AML cells and was either normal or decreased in CLL cells. Patients with increased pSTAT3 or 5 in MM cells also had high expression in autologous normal T cells. The level of constitutive pSTAT3 and 5 was not related to the disease status (relapse vs remission). In patients with MM, IL-6 induced an increased pSTAT3 expression (induced/constitutive ratio >1.5) in both MM cells and T cells in 88% of samples. IL-6 induced pSTAT3 expression in blasts of 89% of AML patients (median ratio = 79). There was no correlation between the level of pSTAT3 induced by IL-6 and IL-6 receptor expression. IL-6 did not increase pSTAT5 levels in MM, AML or CLL. SP cells in the MM cell line RPMI 8226 had lower constitutive pSTAT3 than the non-SP population and a lower IL-6 receptor expression, but had a greater response to cytokine stimulation, suggesting that SP cells may be more sensitive than non-SP cells to inhibitors of STAT phosphorylation. Addition of RPMI 8226 cells or supernatant to normal T cells caused an increase in pSTAT3, suggesting that the upregulation of pSTAT3 is at least partly due to a tumor-derived soluble agent in the microenvironment. Conclusions These results demonstrate the heterogeneity of constitutive and cytokine induced pSTAT3 and 5 expression in MM, AML and CLL. Elevated levels of constitutive pSTAT3 (approx 44% of MM) and pSTAT5 (88% of MM and AML) in the malignant clone were associated with high levels in autologous non-malignant T cells and are not of prognostic significance. Therefore elevated pSTAT3 and 5 may not be an exclusive hallmark of the malignant clone, as reported in solid tumors, but rather a result of the microenvironment and a high IL6-R expression. Phospho-flow cytometry enables precise quantitation of constitutive and cytokine-induced pSTAT3 and 5, and thus can identify the patients most likely to respond to direct STAT-3 or 5 inhibitor therapies. SP cells (putative MM stem cells) appear to be potentially targetable. Inhibitors targeting signalling pathways may be more efficacious if therapy is directed to patients with targetable checkpoints. Disclosures Hart: DendroCyte BioTech Pty Ltd: Equity Ownership.

Water management is one of the most important practices that affect methane (CH(4)) and nitrous oxide (N(2)O) emissions from paddy fields. A field experiment was designed to study the effects of controlled irrigation (CI) on CH(4) and N(2)O emissions from paddy fields, with traditional irrigation (TI) as the control. The effects of CI on CH(4) and N(2)O emissions from paddy fields were very clear. The peaks of CH(4) emissions from the CI paddies were observed 1-2d after the water layer disappeared. Afterward, the emissions reduced rapidly and remained low until the soil was re-flooded. A slight increase of CH(4) emission was observed in a short period after re-flooding. N(2)O emissions peaks from CI paddies were all observed 8-10d after the fertilization at the WFPS ranging from 78.1% to 85.3%. Soil drying caused substantial N(2)O emissions, whereas no substantial N(2)O emissions were observed when the soil was re-wetted after the dry phase. Compared with TI, the cumulative CH(4) emissions from the CI fields were reduced by 81.8% on the average, whereas the cumulative N(2)O emissions were increased by 135.4% on the average. The integrative global warming potential of CH(4) and N(2)O on a 100-year horizon decreased by 27.3% in the CI paddy fields, whereas no significant difference in the rice yield was observed between the CI and TI fields. These results suggest that CI can effectively mitigate the integrative greenhouse effect caused by CH(4) and N(2)O emissions from paddy fields while ensuring the rice yield.

Not all cameras based on the Camera Link interface can be displayed directly at present. Therefore, we designed a real-time display system based on the Xilinx Spartan3 FPGA XC3S1000-6FG456I. Our system could receive, store, read and display the Camera Link signal without the system computer. It contains three main modules. They are the input signal receiving module, the data storing module and the VGA signal bringing and exporting module. Two SDRAMs were used as the frame storage. Input Camera Link signal was converted to XGA signal with 1024×768 pixels/frame at 75 frame/s. The ADV7123JST was used as D/A converter. Its output signal was transmitted to the VGA interface and displayed on the screen of the VGA monitor. Our system could display the output signals of all cameras based on the Camera Link interface.

Dysfunctional T-cells associated with human tumors can be identified utilizing multi-parameter flow cytometry and studied for intracellular signalling checkpoints using phospho-flow or time-of-flight mass spectrometry. Whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent has not yet been determined. Anergic T-cells are hypo-responsive with low cytokine production and induce tolerance to protect the host from autoimmune disease. T-cell exhaustion is usually associated with chronic viral infection such as CMV but also with some cancers. Both exhausted and anergic T-cells express PD-1, LAG-3, Tim-3, CD160 and CD28. Hypo-responsive T-cells associated with melanoma have the phenotype of exhausted T-cells which can be reactivated using targeted immune check point blockade, resulting in clinical benefit. Senescent T-cells accumulate in an oligoclonal manner with ageing and chronic antigen exposure. Senescence can be characterised by telomere shortening, the expression of CD57, KLRG-1, CD160 and the absence of CD28. However, not all senescent cells have shortened telomeres as telomere independent senescence involving the p21-p53 and p16-pRb pathways and senescence associated secretory phenotype (SASP) T-cells have been described. The aim of this study was to determine whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent and to identify targetable checkpoints to restore T-cell function. Cytotoxic T-cell clones (CD57+ CD28- TCRVβ restricted) determined by BetaMark TCRVβ analysis were present in 51% of patients with myeloma (n=264), are protective (OS χ2=6.2; p Clonal T-cells lacked CD28, PD-1, CTLA-4, LAG-3, CD160 and Tim-3 expression and therefore are neither exhausted nor anergic. However, telomere assessment by qPCR demonstrated telomere T/S ratios were normal length for age and not significantly less than autologous CD57+TCR-Vβ- cells. T-cell clones also had normal p38 expression suggesting that the senescence was not due to TNF-α inhibiting the p38/MAPK pathway. The cytotoxic T-cell clones in patients with myeloma have the characteristics of telomere-independent senescence. The normal telomere length suggests that the hypo-responsiveness is induced independent of telomeres, and is therefore potentially reversible, though the pleiotropic nature of this dysfunction might require the targeting of multiple intracellular signalling pathways. The lack of PD-1 and CTLA-4 expression suggests that immune checkpoint blockade in myeloma would be unable to reverse the T cell dysfunction. Disclosures No relevant conflicts of interest to declare.

Guangzhou city in South China has experienced an accelerated urban development since the 1980s. This paper examines the impact of the urban development on urban heat islands through a historical analysis of urban-rural air temperature differences. Remote sensing techniques were applied to derive information on land use/cover and land surface temperatures and to assess the thermal response patterns of land cover types. The results revealed an overriding importance of urban land cover expansion in the changes in heat island intensity and surface temperature patterns. Urban development was also related to a continual air temperature increase in the 1980s and 1990s. The combined use of satellite-derived vegetation and land cover distributions with land surface temperature maps provides a potential useful tool for many planning applications. The city's greening campaigns and landscaping designs should consider the different cooling effects of forest, shrubs and grassy lawns for temperature control and should plant more tall trees.

Background Identification of aberrant expression in signal transduction pathways is becoming increasingly relevant to the diagnosis, prognosis and pharmacodynamics of haematological malignancies. Aim To demonstrate the diagnostic and prognostic relevance of constitutive and IL-6 induced pSTAT3, pSTAT5, pAKT and pERK in patients with plasma cell disorders including multiple myeloma (MM), monocloncal gammopathy of uncertain significance (MGUS) and plasma cell leukaemia (PCL). Methods 65 frozen bone marrow samples collected from MM patients at diagnosis (2000-2010) were stained with anti CD 38,138, 45 and 130 (IL-6 receptor). Constitutive and IL-6 induced levels of pSTAT3, pSTAT5, pERK and pAKT were measured using phospho-flow. MGUS (n = 9) and PCL ( n = 6) samples were also analysed. Results Constitutive expression of pSTAT3, pSTAT5, pAKT, pERK or IL-6 receptor did not differentiate between plasma cell disorders nor have any prognostic significance. MM patients with upregulated levels of pSTAT3 following activation with IL-6 had a better overall survival, 55 versus 23 months, (x 2 =13.6, p p 0.001). Discussion IL-6 induced pSTAT3 is a novel and independent prognostic factor for MM. Constitutive expression of phosphory-lated proteins doesn't differentiate plasma cell disorders but could identify patients suitable for targeted inhibitor therapy.

Abstract 1818 There is clinical evidence for the presence of a limited degree of host-tumor control in patients with multiple myeloma (MM) but the exact mechanisms involved are not known. Patients who survive for more than ten years are likely to have the most active immunological host-tumour control and are an ideal cohort to study. We now add to our preliminary observations using a range of immunological biomarkers in the 29 of these patients who attend our clinic. 51% of MM patients (n=264) had expanded CD3+CD8+ TCRVβ+ CD57+T cell clones detected by TCRVβ analysis (Beckman, BetaMark). Clonality was confirmed by IgH CDR3 sequencing. These clones accounted for 14.3% (median) of the CD3 cells (range 4–49%). CFSE tracking demonstrated the anergic nature of these clonal T cells (median 6% proliferation) compared with other CD8 cells (70% proliferation) while Geneset analysis of mRNA microarrays (Affymetrix U133) identified that anergy was caused by upregulated RAS, CSK, TOB and suppressed ERK pathways. Unlike recent reports for T-LGL, microarray analysis suggested that there was no evidence of STAT3 upregulation in the MM T cell clones. Functional studies suggested that these non-proliferating clones have split anergy as interferon-γ production was normal. In contrast, all ten year survivors had expanded T cell clones and serial studies demonstrated a >8 year persistence of the same clone in 8 out of 12 patients studied. More importantly, unlike the other MM patients, the T cell clones in 19 out of 21 of the ten year survivors studied were not anergic. The Treg/Th17 ratio in the ten year survivors was significantly lower than other MM patients (median 1.9 vs 12.0; p In conclusion, MM patients have expanded clones of cytotoxic T cells that exhibit split anergy and these cells are potential candidates for restoring immunological control of MM and other cancers. The normalised Treg/Th17 ratio suggests that the induced tolerance associated with increased Treg cell numbers is also absent in these patients. Our observations that the ten year MM survivors do not have anergy in their clonal T cells and have less Treg suppression offers a unique cohort for future studies. Disclosures: No relevant conflicts of interest to declare.

Abstract 3950 Chemotherapy-resistant cancer stem cells are a major cause of relapse. It is considered that the small population of CD38++ cells which also exhibit “side population” (SP) characteristics after Hoescht 33342 staining contain chemotherapy resistant multiple myeloma (MM) stem cells. The hedgehog (Hh) signalling pathway is a key regulator of stem cell proliferation and differentiation. A range of Hh inhibitors are currently under development as therapeutics. Our aim was to determine the effect that NVP-LDE225, a novel Hh pathway inhibitor has on MM stem cells. NVP-LDE225 was kindly supplied by Novartis. Side population (SP) cells, considered to contain the MM stem cells, were detected after Hoescht 33342 staining using a BD ARIA II flow cytometer, Proliferation was monitored with CFSE tracking and viability with propidium iodide. MM cell lines RPMI8226, KMS-11, OPM2, U266 and primary MM cells were included. Expression of Hh pathway proteins PTCH1, Smo and Gli1 was determined by flow cytometry. Bone marrow samples were obtained from patients with myeloma after informed consent. CD38++ SP cells were identified in all 4 MM cell lines tested and 90% (26/29) of MM BM samples with the percentage of SP cells ranging from 0 to 9.5% of CD38++ cells. There was no correlation between %SP and %CD38++ and no significant difference in %SP during therapy. PTCH1 expression was higher on SP cells of KMS-11, OPM2 and U266 than non-SP cells (80.5% vs 51.7%) suggesting that the canonical Hh pathway was upregulated in CD38++ SP cells. PTCH1 expression varied from 0.1 – 87% on patient's CD38++ cells. In vitro dose response curves demonstrated increased killing of plasma cell lines above 1μM NVP-LDE225. The IC50 was 4.7uM for KMS-11 SP cells and 3.4uM for KMS-11 non-SP cells. During culture of flow-sorted CD38++ SP+ and non-SP cells (RPMI 8226 and KMS11), fluctuations in %SP suggest that SP cells differentiated to non-SP cells but also that the SP phenotype is unstable. This is supportive of the concept that the stem cell compartment is in a state of flux. The presence of NVP-LED225 caused a 90% inhibition of non-SP returning to SP and induced an additional 79% differentiation of SP to non-SP KMS11 cells on day 2. Proliferation studies using CFSE tracking of sorted SP cells demonstrated a 34% reduction in proliferation after exposure to NVP-LDE225. Studies with 9 primary human bone marrow samples from patients with MM confirmed the data from cell lines demonstrating that differentiation was significantly induced (t=2.78; p The studies confirm there is flux, to and from the SP and presumably MM stem cell compartment, that the canonical Hh pathway is upregulated in MM SP cells, and that the Hh pathway inhibitor NVP-LDE225 can induce SP differentiation, block de-differentiation, inhibit SP proliferation and at high concentrations is cytotoxic to all cells including MM stem cells. NVP-LDE25 could synergise with other maintenance therapy to reduce drug resistance by inducing the differentiation of chemo-resistant MM stem cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 3924 Prior to the introduction of novel therapies for patients with multiple myeloma (MM) few patients survived for more than 10 years. Several reports have suggested that 10 year survival is associated with a younger age at diagnosis and the duration of exposure to effective agents. Although the number of patients surviving 10 years is increasing, there have been no significant reports of immunological biomarkers in these patients. This is especially true in those for whom the prolonged survival has occurred without novel drugs. Previous studies have shown that the presence of expanded peripheral blood CD8 T-cell clones in MM patients is associated with a better prognosis; raising the possibility that these T-cells, confirmed as clones by TCR sequencing, mediate an anti-tumor effect. However microarray gene set enrichment analysis and proliferation tracking studies demonstrated that these cells are in an anergic state. In addition, there is evidence that Tregs inhibit and Th17 cells enhance autologous immune response in malignancy and that an imbalance in MM may impair disease control. Slan-DCs are a subset of myeloid dendritic cells and are of interest in MM because of their ability to stimulate cytotoxic T-cell responses and reverse anergy in tumor infiltrating lymphocytes. We have investigated the immune mechanisms of disease control which may contribute to long-term survival by analyzing Tregs, Th17 cells, Slan-DCs and the incidence and relative degree of anergy of T-cell clones in all current >10 year survivors. Peripheral blood samples were analyzed for the presence of CD3+ T-cell receptor Vβ restricted T-cell clones (BetaMark Kit), the number of CD3+CD4+CD25h+CD127- Tregs, CD4+IL-17+ Th17 cells and CD16+CD14low M-DC8+ Slan-DCs. Proliferation of T-cells was analyzed using CFSE tracked 4 day cultures stimulated with anti-CD3 and anti-CD28 beads. Results were compared with local data collected from 2 large MM cohorts and age-matched controls. Of 26 patients who had survived for >10 years, 22 were available for testing. At diagnosis the median age was 59y (median=57y in comparison cohort); 54% were female; median β2M was 2.2mg/L (range 1.1–7.3); 92% were ISS I; 73% of paraproteins were IgG, 23% IgA and 4% light chain only. 73% had end-organ effects with; 65% bone disease; 15% anemia and 8% renal impairment. 58% had received cytotoxic chemotherapy; 27% novel agents; 38% autologous transplants; 8% allogeneic transplants and 31% were untreated. Expanded T-cell clones were documented in all (100%) of the 10 year survivors, a significant increase compared with 54% (n=144) and 48% (n=120) in the previous MM cohorts (χ2=43.6;p Analysis of immunological biomarkers in >10 year survivors of MM was compared to a large MM cohort and demonstrated a statistically significant decrease in T-regs with an increase in Th17 cells, absolute Slan-DCs and incidence of T-cell clones. These T-cell clones are more responsive than those in an all-MM cohort suggesting they have acquired less MM-associated anergy. This demonstrates that immunological mechanisms are active in long term disease control in MM and suggests that overcoming anergy of cytotoxic T-cells should be a focus of future studies. Disclosures: No relevant conflicts of interest to declare.

Abstract 983 HLA-G is a “non-classical” HLA class I molecule which is considered to be responsible for the immune tolerance of the fetus, allografts or tumors by inhibiting the antigen-specific cytotoxic T lymphocyte response and decreasing NK cell function. CD86 is a costimulatory molecule which binds to a T cell counter receptor (eg CD28) during antigen stimulation and T cell receptor engagement. T cells do not express CD86mRNA but can acquire CD86 and HLA-G by trogocytosis. We have studied the incidence and prognostic significance of HLA-G and CD86 on malignant plasma cells, the incidence of HLA-G and CD86 on T cells and the immunosuppressive function of HLA-G on malignant plasma cells and T cells. HLA-G and CD86 expression were determined on T cells (CD3+) and plasma cells (CD38++) of patients with myeloma (MM). Flow-sorted CD3+ HLA-G- cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with anti-CD3/CD2/CD28 beads in the presence and absence of either HLA-G+ T cells or HLA-G+ plasma cells. Suppression of the proliferation of CFSE-tracked HLA-G- T cells by HLA-G+ T cells or plasma cells was determined by flow cytometry. CD86+ plasma cells are present in 54% of patients and are associated with a poor prognosis (χ2=4.6;p5% CD38++ cells and expressing >12% HLA-G; χ2=12.4; p Disclosures: No relevant conflicts of interest to declare.

Abstract 1820 Poster Board I-846 T cells contribute to the immunomodulatory control of the tumor in patients with monoclonal gammopathies. We previously found that CD3+CD8+CD57+TCRVβ+restricted cytotoxic T cell expansions were present in 48% of patients with multiple myeloma (n=221) and conferred a significant favorable prognosis. We now report the presence of these expansions in 70% of patients with Waldenstrom's Macroglobulinaemia (WM) (n=20) with a wide spectrum of the TCRVβ repertoire represented. Previous nucleoside analogue (NA) therapy, known to be associated with an increased incidence of transformation to aggressive lymphoma, significantly influenced the presence of TCRVβ expansions (χ2=11.6; p Disclosures No relevant conflicts of interest to declare.

The term trogocytosis is used to describe the fast, cell-to-cell contact-dependent transfer of membrane proteins between cells, with T and B-lymphocytes, natural killer (NK) cells, antigen presenting cells (APC) and tumour cells being the most widely studied. This study aimed to: (1) identify the extent of trogocytosis in patients with multiple myeloma (MM), a malignancy of bone marrow plasma cells, compared with the other B cell malignancies, (2) to identify some of the molecules involved with trogocytosis in these patients and then (3) determine if cells that had acquired molecules had altered function. An in vitro model of trogocytosis was established in which plasma cell lines and flowsorted bone marrow plasma cells (CD38++) of patients with MM (n=11) or the malignant B cells from patients with chronic lymphocytic leukaemia (CLL) (CD5+CD19+) (n=4) and Waldenstrom macroglobulinaemia (WM) (CD19+) were biotinylated and then cultured with either patient or normal mononuclear cells. The acquisition of biotinylated membrane proteins was determined by flow cytometry and confocal microscopy. Screening for potential molecules involved suggested that CD86 and HLA-G were likely candidates for trogocytosis. CD86 is a co-stimulatory molecule at the immune synapse and HLA-G is a non-classical MHC class I molecule which prevents antigen-specific cytolysis by cytotoxic T lymphocytes (CTLs), inhibits the function of circulating NK cells and prevents proliferation of allogeneic CD4+ T cells. These observations have lead to the hypothesis that expression of HLAG may aid in the escape of tumours from immune surveillance. T cells acquired significantly more biotinylated proteins (mean=13.55%) than B cells (mean=2.43%; t=2.80; p

Multiple Myeloma (MM) is a B cell neoplasm in which malignant plasma cells accumulate in the bone marrow and produce excessive amounts of a monoclonal immunoglobulin. Among the large number of antigens expressed by plasma cells, CD38 is one of the highest expressed surface molecules, suggesting an alternative public tumour target. For instance, the myeloma cell line 8226 has a high surface expression of CD38. We plan to introduce genetically recombinant single-chain variable fragments (scFv) of anti-CD38 antibody, along with CD3 chain, into NK-92 a natural killer cell line which is highly cytotoxic against a variety of malignant cells (the only natural killer cell line being used in phase I/II clinical trials). It is likely that the retargeted NK-92 cells may specifically and efficiently kill CD38-expressing myeloma cells. By using a series of degenerate primer sets for mouse immunoglobulin variable region genes, VH and VL regions were isolated through reverse transcription PCR from OKT10 cell line. Further sequencing analysis provided novel information of the OKT10 immunoglobulin heavy and light chains. A PCR-based assembling strategy was employed to construct the scFv with FLAG sequence integrated as a tag. VH and VL domains were joined together with a hinge region of (Gly4Ser)3. The phagemid construct has been formed by subcloning the scFv fragment into the Sfi I/Not I cloning sites of the expression vector pHEN2, and subsequently transformed into the E. Coli HB2151. Five out of 7 transformed bacterial colonies screened did not contain the scFv fragment. For the remaining two clones with the correct-sized insert, we tested the specificity of the scFv fragments that were prepared through intracellular antibody capture as periplasmic proteins. We performed a competitive flow assay using periplasmic proteins with commercially available anti-CD38 PE conjugated antibodies on 8226 myeloma cell line. We found that one of these two clones (clone 6) showed a reduction in CD38 staining and presented as double peaks; while this pattern was not seen in the clone 7. Subsequent DNA sequencing revealed that there was a 100-bp truncation in the heavy chain region in clone 7; while the clone 6 has a complete productive heavy chain and light chain spanned by the linker region. We are going to transfect NK-92 cells with retrovial vector pL-scFv(clone 6) construct as the basis of an efficient approach for anti-myelome immunotherapy. (Dr. Daniel Sze is supported by the International Myeloma Foundation Senior Research Grant).

Different stages of differentiation of CD8 T cells based on the expression of costimulatory receptors - CD28 and CD27 has been previously described (Nature Medicine8:379, 2002). Specifically, CD8 T cells that are CD28−CD27− are designated as the “Late” stage, while those that are CD27+ CD28− belong to the “Intermediate” stage. We have previously demonstrated that in a total of 39 total patients with multiple myeloma that had expanded T cell receptor Vβ (TCRVβ) clones, 78% of patients had clonally expanded CD8+CD57+TCRVβ+ T cells of the “Late” subset while the remaining 22% were of the “Intermediate” subset. It was also demonstrated that patients whose expanded T cells expressed a “Late” T cell phenotype had a significantly improved survival over those with an “Intermediate” T cell phenotype (Blood102:927a, 2003). The survival of patients with an “Intermediate” phenotype was not significantly better than those who had no detectable T cell clones. It was suggested that the expanded CD8+CD57+ cells clones with late memory/effector phenotype are associated with good prognosis in patients with myeloma. Hence gene expression profiles of these clinically significant late stage T cells will be very useful for the understanding of the anti-tumor mechanisms of these cells. Our laboratory has previously demonstrated that only the CD57+ CD8+ cells in patients with myeloma are monoclonal or biclonal, while the CD57− counterparts are polyclonal. Based on this finding, we performed an Affymetrix array analysis on these “Late” expanded T cell clones and “Intermediate” T cell clones from each of 2 patients. Eventually we aim to select for individual TCRVβ clone rather than from total CD8+CD57+. We have optimised the procedure for RNA extraction of small number of sorted cells (n=1000), RNA amplification and subsequent labelling for Affymetrix array analysis using U133Plus 2.0 GeneChips. Patient’s peripheral blood mononuclear cells were sorted using 6-color flow sorting with the phenotypes of DAPI−CD3+CD4−CD57+CD28−CD27+ (Intermediate) and DAPI−CD3+CD4−CD57+CD28−CD27− (Late). The RNA extracted from these two populations were then individually amplified and labeled for the array analysis. We implemented an algorithm called Rank Product (RP) to calculate the most significantly up-regulated genes in “Late” stage T cells in both patients’ samples. The top 2 of the Rank Product value list were: i.) KLCRs, killer cell lectin like receptor members that were associated with increased cell lysis and ii.) IGFBP7, insulin-like growth factor binding protein 7 that were associated with tumor suppression activity. Granulysin, a protein present in cytotoxic granules of cytotoxic T lymphocytes, was shown to be highly up-regulated in one of the patients but not in the other. Furthermore, we used Gene Spring software (Silicon Genetics) as an alternative data analytical approach. KLCRs were also found to be significantly up-regulated by more than 2 fold in both patients’ samples. To further validate our observation, more patients will be included in the future study and the up-regulation of these genes will be verified by real time PCR. (This work is supported by the Cancer Institute NSW)

Thalidomide is one of the current therapies for multiple myeloma (MM) which has multiple mechanisms on myeloma cells, bone marrow microenvironment, T cells and NK cells. Chinese herbal medicines have been used in the treatment of cancer and also have multiple pharmacological activities; however, there is a strong need for evidence-based scientific data to understand their anti-cancer mechanisms. The aim of this study is to integrate thalidomide and Chinese herbs to enhance the beneficial effects and minimize the side-effects of thalidomide. The melanins from an anti-cancer Chinese herb Ligustrum lucidum were extracted and tested. The dual roles of the herbal extracts on T/NK immunomodulatory and anti-tumor properties were investigated in the following three aspects: Proliferation measured by both [3H] thymidine assays and CFSE assays; Cell survival measured by propidium iodide exclusion and trypan blue staining; and NK cytotoxicity using myeloma cell line 8226 as target cell labeled with CalceinAM, and peripheral blood mononuclear cells (PBMC) from healthy subjects as the source of effector cells. It was found that in the [3H] thymidine assays, Ligustrum lucidum melanin (LLM) extracts exhibited both stimulatory effect on PBMC proliferation and inhibitory effects on myeloma cell line 8226 proliferation at the concentration of 0.02 to 20 μg/ml. The stimulatory effect on PBMC was also observed with CFSE assays when PBMC was incubated with LLM (20 μg/ml) for 2 days with a proliferated population of 33%, compared with thalidomide (5 μg/ml) 57%, and control, 12%. In the cell survival assay tumor cell line NK92-MI was completely inhibited by LLM (20 μg/ml). NK cytotoxicity assay showed the LLM (0.02 to 20 μg/ml) showed dose response increase of cytotoxic effects of NK cells from 50% to 70%, higher than 42% achieved by thalidomide (5 μg/ml). These results indicate that herbal medicine Ligustrum lucidum posses both T cell / NK cell immunomodulatory effects and anti-tumor effects to myeloma cells. Further works will be to determine the structure of the plant constituent and to assess the synergistic combinations of the active extracts and constituents with thalidomide in the cellular assays.