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Slow light is generated via the gain mechanisms of nonlinear optical phenomena-- stimulated stimulated Brillouin scattering in optical fiber. Slow light is characterized in terms of Brillouin threshold, gain spectrum, Brillouin frequency shift by using optical heterodyne detection. According to the measured Brillouin frequency shift, we set our signal frequency in the gain region by manual matching method to get the maximum gain, and the gain bandwidth is broadened to 450MHz using a phrase-modulated pump. A delay of 32ns is achieved for the 500ns input signal when the gain is 15dB.

The mechanism of irritable bowel syndrome (IBS) is still incompletely understood in the world although large amount of investigations have been carried out on it. There are many studies on the pathophysiology of IBS in China, which has huge amount of population suffering from IBS with special ethnicity and culture, including Mainland China, Hong Kong and Taiwan. We collected the literatures to show the results and discuss whether there were any differences in the pathophysiologic findings between China and other countries, whether there were any differences among different subtypes and how the pathophysiology correlated with the manifestations of patients. Gene polymorphism, disturbances of gastrointestinal motility, visceral hypersensitivity, intestinal infection and inflammation, psychological disturbances, food hypersensitivity and intolerance, and altered gut microflora were reviewed in this paper. Some conflicting outcomes between China and other countries were noted although most of them were similar.

Hemopoietic precursors with the ability to differentiate into wide varieties of cell types are considered primitive, as are precursors with long-term repopulating ability. Here we study the populations of marrow precursors from which both myeloid and lymphoid lineages are descended shortly after transplantation. Surprisingly, few or none of these precursors show long-term repopulating ability. Equal portions of a mixture of marrow cells from C57BL/6J (B6) and congenic B6-Hbbd Gpi-1a mice are transplanted into a group of recipients. Three weeks later, highly significant correlations between percentages of B6 type T cells, B cells, granulocytes, and platelets in each recipient indicate that many lymphoid and myeloid cells are descended from common precursors. After 4-6 weeks, most correlations between lymphoid and myeloid cells improve, indicating that most or all differentiated cells are descended from common precursors. The more differentiated myeloid-specific precursors found in spleen colony-forming cell assays apparently fail to contribute significantly to the differentiated myeloid cell populations tested. By using the binomial model, in which variability of the data among the recipients is inversely related to the number of precursors in the mixture, donor precursor concentrations are estimated as approximately 21 per 10(5) marrow cells after 3 weeks, falling 3-fold to 6.6 per 10(5) after 4-6 weeks. This trend continues, with higher correlations, greater variabilities, and donor precursor concentrations of 1.9 per 10(5) marrow cells after 12-14 weeks and 1.4 per 10(5) after 24 weeks. Strong increases in variances between 3 and 12 weeks after transplantation suggest that most or all of the initially active multilineage precursors are exhausted during this time period. The fact that the ability of a hemopoietic stem cell to differentiate into widely disparate lineages is not associated with long-term repopulating ability requires a change in stem cell definitions, since primitive hemopoietic stem cells have traditionally been defined by both these abilities.

Catechins are a group of polyphenolic compounds that are antioxidants having beneficial biological activities. There are four main catechins in green tea, and each has its own biological features. In order to fully exploit prominent biological activities of specific catechins and to develop new medicine from catechins, it is necessary to obtain pure catechin preparations by isolation from natural sources, by chemical synthesis, or by biotransformation reactions with high yield and specificity. In this study epigallocatechin gallate (EGCG) can be hydrolyzed to epigallocatechin (EGC) by a hydrolase from Aspergillus oryzae after induction by addition of EGCG to the cultures. However, cultures without EGCG induction did not show any EGCG hydrolysis activity. The yield of EGC could reach at least 70%. Thin layer chromatography and high performance liquid chromatography were applied to separate and quantify EGCG and EGC.

A novel 4 x 1 scheme of transmitting two real orthogonal space-time block codes (O-STBC) on the I- and Q-channels with pulse-shaped offset QAM has recently been proposed. In order to achieve good spectral confinement, a pulse shape that introduces a small amount of controlled interference between I/Q- channels is used for the offset QAM signal. In this paper, two-real- symbol joint decoding is investigated for the scheme. This joint decoding can outperform the single-real symbol decoding proposed previously and the best-performing quasi-orthogonal space-time block code (QO-STBC) by more than 1 dB at bit error probability of 10-5 when the interference introduced by the pulse shape is relatively large.

Several diaryl ethers were synthesized and tested in vitro against seven phytopathogenic fungi, namely Fusarium graminearum, Alternaria alternate, Helminthosporium sorokinianum, Pyricularia oryzae, Fusarium oxysporum f. sp. vasinfectum, Fusarium oxysporum f. sp. cucumarinum and Alternaria brassicae. Compared to a commercial agricultural fungicide, hymexazol, especially compounds a, b, e, g and k were found to be more effective at 50 mug/ml against F. graminearum, F. oxysporum f. sp. vasinfectum and F. oxysporum f. sp. cucumarinum. Meantime, some structure-activity relationships were also observed.

In a globally competitive environment, how corporations use strategic alliances to gain a competitive advantage has become an important topic. Based on data envelopment analysis (DEA) and the heuristic technique, this study proposes a model which is termed the candidate selection of strategic alliance (CSSA). The objective is to find the most proper partner for strategic alliance. Realistic data for the industry of department store are collected from Taiwan published stock market. The results are sound by review of many industrial managers and indicate that CSSA can effectively provide recommendations for enterprises to find the proper candidates of alliance.

Objective To develop a novel method of generating multiple autologous acute myeloid leukemia (AML) reactive T-cell lines as a step toward adoptive immunotherapy for AML. Materials and Methods AML peripheral blood mononuclear cells (MNC), including >90% AML blasts and 1% to 3% T cells, were seeded in limiting dilution culture in which AML blasts were induced to undergo dendritic cell (DC) differentiation. T cells were primed and activated with the addition of a cytokine combination. Results Highly reactive anti-AML T-cell lines (both CD4 + and CD8 + ) were generated, selected, and expanded. The estimated average frequency of AML-reactive T cells or precursors was 6 ± 3/1,000,000 AML peripheral blood mononuclear cells (n = 11). Robust intracellular interferon-γ (IFN-γ) release from T-cell lines was demonstrated by flow cytometry after stimulation by autologous AML cells, but not an autologous B-lymphoblastoid cell line (LCL). These T-cell lines caused specific lysis of autologous AML cells, but not autologous LCL or allogeneic AML cells, and they depleted autologous AML colony-forming cells (CFC), but not normal CFC. Most CD4 + T-cell lines exerted strong proapoptotic effects on AML cells. AML cell apoptosis by CD4 + T-cell lines correlated with IFN-γ secretion. Conclusion This study demonstrates a methodology for generating large numbers of AML-reactive cytotoxic T cell lines (either class I or II restricted) that may be useful clinically in adoptive immunotherapy. This study also provides estimates of AML-reactive T-cell frequency in patients with AML.

To investigate the effects of curcumin (Cur) on p210(bcr/abl) level in K562 cells, and the relationship between these effects and the molecular chaperone functions of heat shock protein 90 (Hsp90).Flow cytometry and Western blot were used to examine the abundance of p210(bcr/abl), Hsp90, p23, Hsp70, and p60(Hop) in K562 cells treated with Cur. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the bcr-abl mRNA level in K562 cells treated with Cur. After co-immunoprecipitation of p210(bcr/abl) and its molecular chaperones, the immunoprecipitate was then subjected to Western blot analysis with anti-Hsp90, anti-Hsp70, anti-p23, and anti- p60(Hop)mAb.An exposure of K562 cells to Cur produced time-dependent down-regulation of p210(bcr/abl), the inhibition rate of p210(bcr/abl) in K562 cells determined by flow cytometry after treatment with Cur 27.2 micromol/L for 1 h, 6 h, 12 h and 24 h was 31.2%, 63.7%, 81.3% and 94.5%, respectively. In contrast, Cur had almost no influence on bcr-abl mRNA level. Treatment with Cur for 24 h reduced the association of p210(bcr/abl) with Hsp90/p23 complex, while increasing the association of p210(bcr/abl) with Hsp70/ p60(Hop) complex; however, the total protein abundance of Hsp90, p23, and p60(Hop) in K562 cells had no apparent change, while Hsp70 increased greatly.Down-regulation of p210(bcr/abl) by Cur involves dissociating the binding of p210(bcr/abl) with Hsp90/p23 complex. In contrast, the association of p210(bcr/abl) with Hsp70/ p60(Hop) complex increased.

Purpose: Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the bombesin peptide family and has significant homology to bombesin. We constructed a bispecific molecule, OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a bombesin/GRP antagonist (Antag2) and evaluated cytotoxicity against SCLC cells. Experimental Design: We tested binding of the bispecific molecule to SCLC cell lines and T cells by flow cytometry, antibody-dependent cellular cytotoxicity (ADCC) of SCLC cells in vitro and in a murine SCLC xenograft model. We studied SCLC apoptosis and necrosis during ADCC and the activity and cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). Results: The bispecific molecule functions as a cross-linker between T cells and SCLC cells, induces T cell activation, and mediates ADCC of SCLC cells; 40% to 80% growth inhibition of SCLC cells mediated by the bispecific molecule at low effector to target cell ratios was achieved. Activation of T cells by the bispecific molecule resulted in significant increases in IFNγ production and apoptosis and necrosis of SCLC cells associated with cleavage of PARP and caspase-3. Targeted immunotherapy with the bispecific molecule–armed human T cells significantly reduced SCLC tumor burdens in a mouse model. Conclusion: The bispecific molecule OKT3xAntag2 mediates growth inhibition and apoptosis of SCLC cells by activated T cells through activation and cleavage of caspase-3 and PARP in vitro and in vivo. Clinical trials of this bispecific molecule through adoptive transfer of ex vivo activated T cells in GRP receptor–positive tumors, such as SCLC, are warranted.

This paper is to investigate how we efficiently realize a real-time encryption and decryption system for data transmission and storage in the applications with different requirements. With the proposed new cryptographic system, we have designed the performance-driven reconfigurable signal encryption/decryption architecture and the corresponding parameterized IP core generator. The performance, hardware cost, and security of signal of the proposed IP core design can be configured by some parameters, including packet size, word-length of input data, and the number of processing elements to meet the requirement of application. This IP core has been qualified under RMM coding guidelines, and certified to reach near 100% code coverage using the provided test-benches. With the specified combinations of parameters, the proposed IP core design possesses the throughput rate ranges between 312.5 Mbps and 1.428 Gbps. Thus this design is useful for the embedded system in multimedia applications.

Stimulated Brillouin scattering (SBS) in optical fibers has long been used in frequency-selective optical signal processing, including in the realization of microwave-photonic (MWP) filters. In this work, we report a significant enhancement in the selectivity of SBS-based MWP filters. Filters having a single passband of 250 MHz–1 GHz bandwidth are demonstrated, with selectivity of up to 44 dB. The selectivity of the filters is better than that of the corresponding previous arrangements by about 15 dB. The shape factor of the filters, defined as the ratio between their −20 dB bandwidth and their −3 dB bandwidth, is between 1.35 and 1.5. The central transmission frequency, bandwidth, and spectral shape of the passband are all independently adjusted. Performance enhancement is based on two advances, compared with previous demonstrations of tunable SBS-based MWP filters: (a) the polarization attributes of SBS in standard, weakly birefringent fibers are used to discriminate between in-band and out-of-band components and (b) a sharp and uniform power spectral density of the SBS pump waves is synthesized through external modulation of an optical carrier by broadband, frequency-swept waveforms. The signal-to-noise ratio of filtered radio-frequency waveforms and the linear dynamic range of the filters are estimated analytically and quantified experimentally. Lastly, a figure of merit for the performance of the filters is proposed and discussed. The filters are applicable to radio-over-fiber transmission systems.

Based on the main physical processes of secondary electron emission, experimental results and the characteristics of backscattered electrons (BE), the formula was derived for describing the ratio (β angle ) of the number of secondary electrons excited by the larger average angle of emission BE to the number of secondary electrons excited by the primary electrons of normal incidence. This ratio was compared to the similar ratio β obtained in the case of high energy primary electrons. According to the derived formula for β angle and the two reasons why β > 1, the formula describing the ratio β energy of β to β angle , reflecting the effect that the mean energy of the BE W AV p0 is smaller than the energy of the primary electrons at the surface, was derived. β angle and β energy computed using the experimental results and the deduced formulae for β angle and β energy were analyzed. It is concluded that β angle is not dependent on atomic number z, and that β energy decreases slowly with z. On the basis of the two reasons why β > 1, the definitions of β and β energy and the number of secondary electrons released per primary electron, the formula for β E-energy (the estimated β energy ) was deduced. The β E-energy computed using W AV p0, energy exponent and the formula for β E-energy is in a good agreement with β energy computed using the experimental results and the deduced formula for β energy . Finally, it is concluded that the deduced formulae for β angle and β energy can be used to estimate β angle and β energy , and that the factor that W AV p0 increases slowly with atomic number z leads to the results that β energy decreases slowly with z and β decreases slowly with z.

Optical spectral analysis of closely spaced, subcarrier multiplexed fiber-optic transmission is performed, based on stimulated Brillouin scattering (SBS). The Brillouin gain window of a single, continuous-wave pump is scanned across the spectral extent of the signal under test. The polarization pulling effect associated with SBS is employed to improve the rejection ratio of the analysis by an order of magnitude. Ten tones, spaced by only 10 MHz and each carrying random-sequence on-off keying data, are clearly resolved. The measurement identifies the absence of a single subcarrier, directly in the optical domain. The results are applicable to the monitoring of optical orthogonal frequency domain multiplexing and radio over fiber transmission.

The therapeutic potential of the IgM complement-fixing murine monoclonal antibody (mAb) PM-81 (anti-CD15) against acute myeloid leukemia (AML was assessed in a SCID/hu leukemia model. Intraperitoneal (i.p.) injection of NB4 leukemia cells resulted in aggressive growth of leukemia cells in the peritoneal cavity of irradiated SCID/CB-17 mice. Flow cytometric analysis of human CD15, 33 and 45 expression, as well as cytologic examination, revealed that leukemia cells disseminated into the peripheral blood and multiple tissues of the mice. The approximately linear relationship between the injected leukemia cells and the subsequent leukemia cell proliferation provided a reliable model for monitoring the therapeutic effects of immunotherapy. Intraperitoneal injection of the mAb PM-81 markedly suppressed leukemia cell growth in this SCID/leukemia model. Most of the untreated mice died within 35–50 days of leukemia cell inoculation. Four weeks after inoculation of NB4 cells, five of nine mAb PM-81 treated mice had no solid tumor growth and six of nine had no detectable peritoneal exudate leukemia cells as determined by flow cytometry. In contrast, 100% of the mice in the untreated or control mAb groups were found to have both solid and peritoneal leukemia growth. In further experiments designed to evaluate the effects of therapy on survival, 50% ( 4 8 ) of PM-81 treated mice survived to 150 days, and had no detectable solid or suspension leukemia cells detectable at necropsy. In contrast, the median survival of untreated or negative control antibody-treated mice was 40 days (comparison to PM-81 treated; p = 0.006 and p = 0.03, respectively). The mechanism of leukemia cell suppression is not likely due to complement fixation since we could not demonstrate in vitro any cytotoxicity mediated by SCID mouse plasma. Further study is required to understand the mechanism of the antileukemia effect of PM-81 in this model.

Donor and host T cells were distinguished by T cell antigen marker Thy-1 isotype and cytoplasmic isozyme Gpi-1 in this study of bone marrow transplantation between congenic mice. During the first 3-6 weeks after irradiation and marrow transfer, percentages of cells bearing the donor Thy-1 isotype in the periphery are much lower than percentages of T cells bearing the donor Gpi-1 marker. Apparently a population of Thy-1- donor T cells exists for several weeks after bone marrow transplantation. Further study showed that this population of CD3+, Thy-1- donor T cells expressed CD4+ or CD8+ and was found in peripheral blood and spleen but not in the thymus. This finding suggests their extrathymic origin.

Abstract 2560 The mammalian orphan receptor tyrosine kinase-1 (ROR1) is expressed in a wide-variety of tissues during early embryonic development. By the late stages of embryogenesis the expression of this developmentally important protein is greatly diminished. Although not expressed in the tissues of post-partum animals, the ROR1 protein is expressed on neoplastic cells in chronic lymphocytic leukemia (CLL), some B-cell malignancies, and a variety of different carcinomas. We examined for expression of ROR1 in primary acute myeloid leukemia (AML) cells harvested from marrow aspirates and their normal counterparts by whole transcriptome paired-end RNA sequencing and by flow-cytometric analyses. These studies revealed selective expression of ROR1 in 62 (35%) of 179 AML samples examined. Many of these samples were found to have cells that co-expressed ROR1 and CD34, suggesting that ROR1 was present on the self-renewing leukemia stem-cell population, which resides in the marrow niche and potentially accounts for resistance to many cytotoxic drugs used in therapy. We investigated the activity of a chimeric anti-ROR1 mAb found effective in clearing CLL cells (UC99961) on AML expansion, growth, and renewal in a leukemia-stem-cell supportive niche assay. Mouse marrow cells lines SL/SL and M2–10B4 (transfected to produce hSCF,hIL3 and hIL3, hG-CSF respectively) were mixed 1:1 after mitomycin-C treatment, and used as a SLM2 stromal monolayer. CD34+ cells were selected from ROR1-positive (n=6) or negative (n=4) AML primary samples. As a normal control, CD34+ cells from cord blood (CB) were used (CB, n=3). In some experiments CD34+ cells were transfected with a GLP-lentivirus prior to co-culture. At the initiation of the co-culture, 10–50 μg/ml of the chimeric anti-ROR-1 mAb (UC99961) or control hIgG were added to the cultures. Two weeks after co-culture initiation, both stromal attached and floating cells were collected and their survival investigated by colony forming assay in methylcellulose. The UC99961 mAb was not cytotoxic to CB or ROR1-negative AML samples. In contrast, the UC99961 mAb provided a dose-dependent inhibition of colony formation for all ROR-1-positive AML samples examined. These results demonstrate the in vitro anti-leukemic specificity of this anti-ROR1 mAb in down-regulating AML stem and progenitor cell populations, without effecting normal CD34+ stem cells. To analyze the effect of ROR1 ligation on AML stem cell populations exclusively, AML self-renewal assays (2-ry colonies) were performed. In these studies, ROR1–positive AML samples were divided based on their response to mAb treatment. Half of the samples (n=3; 50%) demonstrated statistically significant (up to 90%) dose-dependent decreases in colony formation. However, another half was non-responsive and no correlation was found between ROR1 expression on leukemia CD34+ cells and response to anti-ROR1 mAb treatment in the self-renewal assays. Again UC99961 mAb treatment did not negatively impact CD34+ cells from CB or ROR1-negative AML, confirming the specificity and selective toxicity of the mAb for ROR1+ AML stem cells. These studies reveal selective expression of ROR1 on leukemia-stem-cells of large subset of AML patients. Furthermore, this study demonstrates that an anti-ROR1 mAb (UC99961) can inhibit survival and self-renewal in LSC supportive niche assays. Targeted ROR1 inhibition may represent a vital component of therapeutic strategies aimed at eradicating therapeutically recalcitrant malignant stem cells in AML and potentially other refractory cancer-stem-cell-driven malignancies. Disclosures: No relevant conflicts of interest to declare.

A general method has been developed to analyze all 2' hydroxyl groups involved in tertiary interactions in RNA in a single experiment. This method involves comparing the activity of populations of circularly permuted RNAs that contain or lack potential hydrogen-bond donors at each position. The 2' hydroxyls of the pre-tRNA substrate identified as potential hydrogen bond donors in intermolecular interactions with the ribozyme from eubacterial RNase P (P RNA) are located in the T stem and T loop, acceptor stem, and 3' CCA regions. To locate the hydrogen-bond acceptors for one of those 2' hydroxyls in the P RNA, a phylogenetically conserved adenosine was mutated to a guanosine. When this mutant P RNA was used, increased cleavage activity of a single circularly permuted substrate within the population was observed. The cleavage efficiency (kcat/Km) of a singly 2'-deoxy-substituted substrate at this position in the T stem was also determined. For the wild-type P RNA, the catalytic efficiency was significantly decreased compared with that of the all-ribo substrate, consistent with the notion that this 2' hydroxyl plays an important role. For the P RNA mutant, no additional effect was found upon 2'-deoxy substitution. We propose that this particular 2' hydroxyl in the pre-tRNA interacts specifically with this adenosine in the P RNA. This method should be useful in examining the role of 2' hydroxyl groups in other RNA-RNA and RNA-protein complexes.

Bispecific antibodies recognizing tumour-associated antigens and trigger molecules expressed on immune effector cells have been shown to redirect cytotoxicity of several types of peripheral blood cells against relevant tumour targets. Among various effector cells, natural killer (NK) cells appear to play a role in defence against leukaemia. Here we report the successful chemical conjugation of monoclonal antibodies to CD33 and CD16 to create a bispecific antibody (BsAb 251 x 3G8). This bispecific antibody is capable of augmenting the killing of otherwise resistant leukaemia cells by peripheral blood lymphocytes (PBL), purified resting NK (R-NK) cells, and activated NK (A-NK) cells. BsAb 251 x 3G8 may play a role in the therapy of acute myeloid leukaemia (AML) through redirecting the cytotoxic activity of endogenous or adoptively transferred NK cells.

Slow light technology will play a key role in future all-optical communication. The slow-light technology based on stimulated Brillouin scattering has become a research highlight because of its additional advantages, such as compatibility of the devices with existing telecommunication systems, room-temperature operation, and tunable at arbitrary wavelengths. According to the propagation of a cw pulse through a Brillouin fiber amplifier, whose frequency is near the Stokes resonance, via three-wave coupling equations, both pump depletion and fiber losses taken into consideration, the principle of how slow-light effect based on stimulated Brillouin scattering produced and the mathematical expression of time delay are strictly deduced. A delay of 8 ns is obtained when the input Stokes pulse is 200ns and the SBS (stimulated Brillouin scattering) gain G is ~18 in our designed experiment of SBS slow-light system. Then the extent of transformation from pump waves to Stokes waves is measured using MATLAB numerical simulation according to the experiment dates, based on the relation between output pump light power and input pump light power and also the relation between output Stokes light power and input pump light power. And the relation between the input light power and propagation distance is discussed as well. Finally the relation between slow light pulse delay and SBS gain is also obtained.

2035 Poster Board II-12 Although hematologic remissions can be achieved in the majority of patients with adult acute lymphocytic leukemia (ALL) by chemotherapy, long term survival is only 30–40%. The inability of the immune system to recognize and eliminate residual malignant leukemia cells may be an important mechanism contributing to relapse. In this study, we investigated the possibility of using pre-B-ALL cells as antigen presenting cells in an in vitro culture to induce autologous ALL reactive T cells. After 7 day culture of Pre-B-ALL peripheral blood mononuclear cells (CD19+ 93±4%) in 96 well culture plates in culture medium supplemented with a cytokine combination of IL-2/IL-3/IL-4/IL-7/GM-CSF, CD80, CD86, CD83, CD54, HLA-Dr and CD40 expression was analyzed. Significant enhancement of CD80, CD86, CD83 and CD40 on ALL cells was observed (n=8, P≦0.001-0.02). Addition of lipopolysaccharide (LPS) to the cytokine combination further increased CD80, CD86 or CD40 expression by 3 of 8 ALL samples above the baseline enhancement by cytokines (Figure), while CD40 ligand (CD40L) enhanced expression of the co-stimulatory molecules in 4 of 8 cases. Autologous T cells remaining in the culture after day 7 were then expanded with high dose IL-2 and autologous ALL reactive T cell lines were identified by IFN-g release by T cells in response to autologous ALL cells. Autologous ALL reactive T cells were generated from 5 of 8 pre-B ALL samples studied. The data from 3 experiments demonstrated that although the cytokine combination plus LPS or CD40L could effectively induce ALL cell expression of co-stimulatory molecules, the presence of CD40L and LPS in the culture induced significantly greater activation of autologous ALL-reactive T cells than did the cytokine combination plus LPS alone, as assessed by average IFN-g release of 24-48 culture wells (P≦0.004). ALL-reactive T cell lines selected by high IFN-g release response showed effective elimination of autologous ALL cells in a 2 day co-culture assay with an E:T ratio of 1:1 by flow cytometry analysis. Average residual CD19+ cells was 2.0±1.4% for highly reactive T cell lines (n=17) vs 16.5±25.9% for the less reactive control T cell lines (n=9) (P Disclosures: No relevant conflicts of interest to declare.

SiOlsubg2l/subg thin films containing Silsubg1-xl/subgGelsubgxl/subg quantum dots (QDs) are prepared by ion implantation and annealing treatment. The photoluminescence (PL) and microstructural properties of thin films are investigated. The samples exhibit strong PL in the wavelength range of 400?470 nm and relatively weak PL peaks at 730 and 780 nm at room temperature. Blue shift is found for the 400-nm PL peak, and the intensity increases initially and then decreases with the increase of Ge-doping dose. We propose that the 400?470 nm PL band originates from multiple luminescence centers, and the 730- and 780-nm PL peaks are ascribed to the Si=O and GeO luminescence centers.

The efficacy of adoptive cell therapy of cancer and leukemia is often limited by the failure of cultured T cells, particularly cloned CD8+ T cells, to persist in vivo, and insight into the basis for the poor survival of the transferred cells is lacking. We previously reported a novel culture method that induces AML dendritic cell differentiation and primes in situ AML-reactive T cells (AMLDC culture) (Zhong et al. Exp Hematol2008, 36:486). Highly reactive anti-AML T-cell lines were generated. These T-cell lines caused specific lysis of autologous AML cells, but not autologous LCL or allogeneic AML cells, and they depleted autologous AML colony-forming cells (CFC), but not normal CFC. The culture procedure has been further optimized in this study. We found that by the addition of the TLR-4 agonist LPS,(1–100 ng/ml) in the last 24 hours of AMLDC culture (day 6), CD80, CD86, CD53, CD83 or HLA-Dr expression of AML cells pre-induced by cytokine combination GM-CSF/IL-4 or GM-CSF/IL4/IL2/IL7/IL12 could be significantly enhanced (n=6, P Conclusions: Timely exposure of AMLDC culture to TLR-4 agonists, followed by T cell expansion, may promote the generation of AML-reactive T cells and differentiation toward the central memory phenotype. Theoretically, this should promote long-term maintenance and potential of regulation of both humoral and cellular immune responses against AML upon infusion of AML reactive autologous T cells derived from such cultures and may therefore enhance the therapeutic efficacy of these cells.

We conducted a multi-center dose-escalation trial to assess whether CTLA-4 blockade is safe and efficacious as immunotherapy in patients with relapse of malignancy following allogeneic hematopoietic stem cell transplantation (allo-HCT). We hypothesized that since CTLA-4 is a negative regulator of effector T-cell activation, inhibition of CTLA-4 ligation with a blocking human monoclonal antibody (ipilimumab) will augment graft-versus-malignancy. Here we report on the effects of a single dose of ipilimumab on in vivo T cell subsets from 11 patients treated at the highest dose level (3 mg/kg) and compare these findings with those in normal volunteer donor lymphocytes. We analyzed the expression of intracellular CTLA-4 and FOXP3 on CD4+/CD25+ Treg cells, intracellular cytokines and surface markers for T-cell activation on peripheral T cells from 11 patients and 9 normal donors. Peripheral blood mononuclear cells (PBMC) from patients before (day 0) and after the treatment at day 1, 3, 7, 14 and 30 were stained with a panel of antibodies and analyzed by flow cytometry. Lymphocytes from normal donors at time zero and 3 days after culture in IL-2 (200u/ml) were used as controls. The pre-treatment expression of intracellular CTLA-4 was significantly higher in CD4+ T cells from patients than normal controls and was increased further after antibody treatment from 7.1±3.8% at day 0 to 18.2±7.1% at day 30 (p=0.02). Although the expression of FOXP3 in CD4+/CD25+ T cells was higher in patients than in normal donors (6.3±4.8% compared with 2.0±1.6%, p=0.02), there was no significant change in the levels following ipilimumab infusion. The expression of CD4+/CD25high in the patients was 7.7±2.8%, higher than the normal donors (2.3±1.1%). However, 6/11 cases had increased expression while others had decrease or no change, overall there was no statistically significant change. CD4+/CD25low activated T cells were elevated in 10/11 patients before ipilimumab (42.1±8.5%). Their levels were not affected by CTLA-4 blockade. CD8+/CD69+ activated T-cells were significantly increased in 8/11 patients within the 30 days after ipilimumab treatment but typically returned to baseline values on longer follow-up. CD4+/CD69+ and CD4+/HLA-DR+ T-cells were unchanged following ipilimumab treatment. These data show that a single dose of ipilimumab enhances levels of some subsets of activated T cells without a significant effect on cells with a T-regulatory phenotype.

Traditionally, the focus of anti-tumor immunity has been on CD8+ CTL responses. It is now realized that CD4+ T cells play a relevant role in protective anti-tumor responses. HLA Class II molecules are expressed on AML blasts, predicting that AML cells may stimulate CD4+ T cells. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we developed a novel method of generating multiple autologous AML reactive T cell lines by competitive limiting dilution (LD) AML MNC culture. Most autologous AML reactive T cell lines generated from this culture were CD4+. These CD4+ T cell lines with high IFN-gamma secretion in response to autologous AML cells showed low to moderate specific lysis of autologous AML cells in 4-hour 51Cr release assays. However, co-culture assays demonstrated that these CD4+ AML reactive T cell lines exerted intense cytotoxicity toward autologous AML cells, depleting more than 95% of autologous AML line cells in 2 days, and more than 99% in 7 days, with no or weak toxicity to allogeneic AML cells and cell lines, HL60, NB4, U937 KG1a and ak LCL cell line. Anti-HLA class II, Dr, Dp, Dq mAb (67±16% inhibition) and anti-HLA-Dr mAb (69±11% inhibition) but not anti-HLA class I mAb significantly inhibited IFN-gamma secretion of six CD4+ T cell lines stimulated by autologous AML cells confirmed the HLA class II restriction of reactivity of these CD4+ T cell lines. Flow cytometry analysis showed that these CD4+ T cell lines induced significant Annexin-V expression on autologous AML cells. The induction of Annexin-V+ on AML cells by CD4+ T cell lines correlated significantly with IFN-gamma secretion in response to autologous AML cells (n=11; r=0.84). This result suggested that the most important mechanism of autologous AML cell elimination by CD4+ AML reactive T cells generated from LD-AML-MNC cultures was apoptosis induction related to IFN-gamma secretion.

CTLA-4 is expressed upon activation of T-cells,and serves as an important negative regulator of their effector function. It is also expressed constitutively on CD4+/CD25+ regulatory T cells (Treg), where its function is not clear. Following allogeneic hematopoietic stem cell transplantation (allo-HCT), CTLA-4 function may be involved in suppression of alloreactive T cells that mediate the graft-versus malignancy effect and GVHD. We have studied the administration of a single dose of Ipilimumab (MDX-010), a fully human monoclonal anti-CTLA-4 antibody, in a dose escalation trial in patients with relapse/progression of malignancy following allo-HCT. Here we report effects of ipilimumab on peripheral CD4+/CD25+ cell levels and FOXP3 mRNA expression in these patients. Seventeen patients with a variety of malignancies were enrolled in this study. Ipilimumab was given intravenously at a dose level of 0.1, 0.33, 0.66, 1, and 3mg/kg. The blood samples were obtained prior and after infusion at day 7, 14 and 30. The immunophynotyping of peripheral blood mononuclear cells (PBMC) was analyzed by flow cytometry. CD4+/CD25+ cells from nine patients at day 0, 7, and five normal donors were separated using a Dynal CD4+/CD25+ Treg kit. FOXP3 mRNA expression on CD4+/CD25+ cells were analyzed by a quantitative RT-PCR. Expression level of FOXP3 was normalized to 18S rRNA. Within CD4+ cell population, the percentage of CD4+/CD25high cells was significantly higher in patients at day 0 (11.6±6.7%, n=17), compared with normal donors (3.8±1.6%, n=12; P

Naturally occurring cytotoxic T cells directed against various leukemia associated antigens (LAA) expressed by acute myeloid leukemia (AML) cells have been described. However, these LAA-specific T cells are rare and obviously unable to initiate effective anti-leukemia responses. The challenge is how to investigate, select, activate and expand the rare LAA-specific T cells from the vast population of blood cells in patients with AML for immunotherapy. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we have developed a novel method of generating multiple autologous AML reactive T cell lines by limiting dilution AMLDC (LD-AMLDC) culture. The principle of LD-AMLDC is based on the assumption that autologous AML-reactive T cells or precursors are randomly distributed in the AML PBMC suspension, and that each one has an equal opportunity to respond to AML cells in the 96-well plates under optimized culture condition. By culturing AML PBMC (>90% blasts) in culture medium supplemented with GM-CSF/IL4/IL2/IL7/IL12 to induce AML DC differentiation and activate in situ autologous T cells, highly reactive anti-AML T cell lines (both CD4+ and CD8+ lines) were selected and expanded from LD-AMLDC culture using the appropriate numbers of AML PBMC in each culture well by the criterion of release of IFN-gamma in response to autologous AML blasts. By maximum likelihood solution, the estimated average frequency of AML reactive T cells or precursors is 6±3/1,000,000 AML PBMC (n=8). Strong intracellular IFN-gamma release of T cell lines obtained in LD-AMLDC was demonstrated by flow cytometry analysis after stimulation by autologous AML cells but not autologous B-lymphoblastoid cell line (LCL) (Figure). Effective specific lysis (up to 70% at E:T=20:1) of autologous AML cells but not autologous LCL or allogeneic AML cells by these T cell lines was observed. Two PR1 specific T cell lines were obtained by screening 39 AML reactive HLA-A2+ CD8+ T cell lines generated from 5 LD-AMLDC cultures, suggesting that other unidentified CD4 or CD8 lines with strong autologous AML responses may be reactive to known or unknown LAAs. These results encourage continued efforts to induce, activate and select T cells lines with high autologous AML reactivity using LD-AMLDC culture and to expand multi-LAA reactive T cell lines acquired from limiting dilution AML-DC culture for AML immunotherapy. Figure Figure