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A bilayer electron system that is formed in a 60-nm-wide GaAs quantum well and has a large difference of the electron densities in the layers has been studied. It has been found that, when a magnetic field is tilted from the normal to the plane of the system, integer quantum Hall effect states at the filling factors of Landau levels of 1 and 2 disappear; instead, fractional quantum Hall effect states in the interval between these filling factors appear at the filling factors νF = 4/3, 10/7, and 6/5 with odd denominators and at the filling factor νF = 5/4. Several different states can be observed under the variation of the magnetic field. The detected fractional quantum Hall effect states are interpreted as combined states with the same filling factor 1 in the layer with the higher density and with the filling factors νF – 1 in the layer with the lower density. These states are formed because of the redistribution of electrons between the layers, which occurs under the variation of the magnetic field. The appearance of the state with the filling factor νF = 5/4 with the even denominator is presumably attributed to the dominance of the interlayer electron–electron interaction over the intralayer one for electrons in the layer with the lower density.

Summary ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour-initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti-ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5-reactive T cells are present in human melanoma patients and tested the applicability of ABCB5-derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)-γ and tumour necrosis factor (TNF)-α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co-cultured with ABCB5 antigen-loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5-reactive CD8+ T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART-1)-reactive CD8+ T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART-1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide-loaded autologous DC. As ABCB5-reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5-specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma.

ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour‐initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti‐ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5‐reactive T cells are present in human melanoma patients and tested the applicability of ABCB5‐derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co‐cultured with ABCB5 antigen‐loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5‐reactive CD8+ T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART‐1)‐reactive CD8+ T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART‐1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide‐loaded autologous DC. As ABCB5‐reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5‐specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma.

The convective radial transport effects of SOL turbulence have been estimated using recent turbulence data from the gas puff imaging (GPI) camera diagnostic on Alcator C-Mod. The average radial turbulence speed within the region 1-2 cm outside the separatrix near the outer was calculated by a 2-0 cross-correlation technique to be V-t similar to 0.2-0.3 km/s. Assuming this to be the local convective plasma velocity, the density SOL width lambda(n) was evaluated using a simple convective model to be lambda(n) similar to 4-7 cm, which is similar to 2-3 times higher than that measured using a Langmuir probe. This convective velocity was also similar to 2-3 times lower than the velocities estimated from analytic blob models, but showed a similar scaling with plasma current at constant q(95). The measured blob speeds were lower than both the convective speeds and the analytic blob model speeds.

Summary In recent years, percutaneous radiofrequency ablation (RFA) has been developed as a new tool in the treatment of non-small-cell lung cancer (NSCLC) in non-surgical patients. There is growing evidence that RFA-mediated necrosis can modulate host immune responses. Here we analysed serum inflammatory factors as well as immunosuppressive cells in the peripheral blood to discover possible prognostic indicators. Peripheral blood and serum samples were collected before RFA and within 3 months after the treatment in a total of 12 patients. Inflammatory cytokines and growth factors were measured in serum by the Bio-Plex assay. Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) were evaluated in the peripheral blood via flow cytometry. In patients developing local or lymphogenic tumour relapse (n = 4), we found an early significant increase in the concentration of tumour necrosis factor (TNF)-α as well as chemokine (C-C motif) ligand (CCL)-2 and CCL-4 compared to patients without relapse (n = 4) and healthy donors (n = 5). These changes were associated with an elevated activity of circulating MDSC indicated by an increased nitric oxide (NO) production in these cells. Elevated serum levels of TNF-α, CCL-2 and CCL-4 associated with an increased NO production in circulating MDSCs might be an early indicator of the incomplete RFA and subsequently a potential tumour relapse in NSCLC.

Problems with extremely high-transport anisotropy often arise in strongly magnetized plasmas. The numerical solution of the highly anisotropic transport equations becomes quite difficult when the computational grid is not aligned with the strong transport direction, since this can cause large numerical errors. Constructing a finite-difference scheme for a strongly anisotropic diffusion equation on a misaligned grid is discussed, and quantitative assessment of the numerical error is made for a set of example problems.

Hot cathodes are crucial components in a variety of plasma sources and applications, but they induce mode transitions and oscillations that are not fully understood. It is often assumed that negatively biased hot cathodes have a space-charge limited (SCL) sheath whenever the current is limited. Here, we show on theoretical grounds that a SCL sheath cannot persist. First, charge-exchange ions born within the virtual cathode (VC) region get trapped and build up. After the ion density reaches the electron density at a point in the VC, a new neutral region is formed and begins growing in space. In planar geometry, this 'new plasma' containing cold trapped ions and cold thermoelectrons grows towards the anode and fills the gap, leaving behind an inverse cathode sheath. This explains how transitions from temperature-limited mode to anode glow mode occur in thermionic discharge experiments with magnetic fields. If the hot cathode is a small filament in an unmagnetized plasma, the trapped ion region is predicted to grow radially in both directions, get expelled if it reaches the cathode, and reform periodically. Filament-induced current oscillations consistent with this prediction have been reported in experiments. Here, we set up planar geometry simulations of thermionic discharges and demonstrate several mode transition phenomena for the first time. Our continuum kinetic code lacks the noise of particle simulations, enabling a closer study of the temporal dynamics.

The effect of flowing metal walls on the resistive wall instabilities is analyzed for a general cylindrically symmetric diffusive pinch configuration. Two types of liquid metal flow are analyzed: a uniform flow which is poloidally symmetric, and a two-stream flow consisting of two opposite streams splitting at the top and merging at the bottom. It is found in both configurations that when the liquid wall flow velocity exceeds a critical value, the resistive wall mode is stabilized. However, for the two-stream flow the critical velocity is several times smaller than that for the uniform flow. Still in a realistic experiment one needs a flow velocity of a few tens m/s to stabilize the resistive wall mode.

From a cross between a tumor-susceptible mouse strain (DBA/2; D) and a tumor-resistant MHC-identical strain (B10.D2; D2) new recombinant inbred mouse strains were established over many generations of inbreeding and tumor resistance selection. Since resistance to the highly metastatic DBA/2 lymphoma variant ESb had an immunologic basis, and the two parental strains differed in endogenous viral superantigens (vSAGs), DNA of three D2×D recombinant inbred mouse lines was typed for endogenous mouse mammary tumor viruses using mouse mammary tumor virus long terminal repeat- and env gene-specific probes. The resistant D2×D mice were very similar to the susceptible parental strain D in their Mtv Southern blots, except for the lack of a single band corresponding to Mtv-7, the provirus coding for the strong DBA/2 superantigen Mls-1a. A backcross analysis revealed that Mtv-7-negative F2 mice were significantly more resistant than Mtv-7-positive F2 mice. When Mtv-7 was reintroduced into the resistant lines by crossing them with either CBA/J or BALB/D2.Mls-1a, the mice became again more tumor susceptible. Finally, we demonstrate the ability to transfer immunoresistance and graft-vs-leukemia reactivity from tumor-resistant to tumor-susceptible mice.

Detection of disseminated leukemia within organ is often very difficult and might lead to underestimation of the metastatic load. Therefore, we transduced the mouse ESb T lymphoma with the bacterial lacZ gene, which allowed us to follow metastasis at the single cell level. Intradermal primary tumor growth of lacZ transduced ESbL cells (L-CI.5s) comprised three phases: an initial expansion phase (day 0 to 9, increase from 0 to 8 mm, tumor diameter), a plateau phase (day 9 to 20, constant diameter of 8 mm and necrosis), and a second expansion phase (day 20 to 30, increase from 8 to 15 mm). Liver metastasis could already be detected at day 3 and maintained at that level until day 23, where exponential expansion started. A distinct mosaic-like metastasis pattern developed, with preferential localization of tumor cells to the periportal areas of the liver in immunocompetent animals. In contrast, in immunocompromised mice, primary tumor growth and metastasis were progressive and metastasis appeared as diffuse or focal/clustered. Healthy animals surviving a tumor cell inoculum of a variant cell ESbL- CI.5) with a reduced metastatic potential carried low levels of possibly dormant tumor cells in the bone marrow. Thus, this study showed that host immunocompetence determines to a large extent kinetics and load of spontaneous liver metastases and even influences the pattern and localization of disseminated lymphoma cells.

Thermal-spike evolution has been investigated using a method of numerical hydrodynamics. A pattern of origination of a shock-wave arising from a spike is presented. The fraction of energy taken away by the wave has been calculated as a function of the temperature of the spike. The fraction increases with the temperature but in the possible range of that the fraction is no more than about a half of the energy of the spike.

We studied nitric oxide (NO) production, adenosine deaminase (ADA) and 5'-nucleotidase (5-N) activity as a function of macrophage activation in the model of spontaneous metastasis of ESbL T lymphoma cells transduced with the lacZ gene. Liver and spleen macrophages were isolated and examined directly ex vivo without further experimental manipulation. Transient arrest of liver metastasis was accompanied by an increase of NO production and ADA activity and by a decrease of 5-N activity. An aggressive expansion of metastasis was correlated with a drop of NO production and ADA activity and with an increase of 5-N activity. To test the involvement of in situ activated Kupffer cells in an antimetastatic response, two immunotherapy protocols were used: i) active immunization with lymphoma cells and ii) adoptive transfer of antitumor immune spleen cells. Both treatments caused an upregulation of ADA activity and NO production in Kupffer cells, which correlated with host resistance against metastases.

The interaction between tumor and host cells determines to a large extent the outcome, namely tumor growth and progression toward metastases or tumor arrest, dormancy, or rejection. Most of the studies published so far on interactions of tumor cells and host cells were made in vitro and dealt with aspects such as cell adhesion, proliferation, invasiveness, cytotoxicity, or cytokine production. As the microenvironment in tissue culture differs in many respects from that in vivo, new approaches for in vivo studies of tumor-host cell interactions is of utmost importance in cancer research. To elucidate the metastatic phenotype, approaches have been made to relate, for instance, cell surface molecules expressed on the tumor cell lines from tissue culture to their propensity to generate metastases in vivo (1). Several authors have reported that certain steps of the metastatic cascade are rate limiting (2-6). To produce metastases, tumor cells must complete each of the sequential steps in the pathogenesis of cancer metastasis. Each discrete step appears to depend on the interaction between tumor cells and multiple host factors (i.e., the microenvironment of the tumor) and to be regulated by transient or permanent changes in multiple genes at the level of DNA, RNA, or protein. On this background, the need for comprehensive in vivo/ex vivo studies on tumor-host interactions and their kinetics in relevant model systems becomes obvious.

We report on the first experimental observation of neutral modes in particle‐hole conjugate fractional quantum Hall states using shot noise measurements. The presence of the neutral modes, in addition to producing excess noise in a quantum point contact (QPC), was seen to affect strongly the charge of the tunneling quasiparticles of the charge mode in the QPC and to increase their apparent temperature. The observation of an upstream neutral mode in the 5/2 state may constitute an added indication of its non‐abelian nature.

Charged excitations in the fractional quantum Hall effect are known to carry fractional charges, as theoretically predicted and experimentally verified. Here we report on the dependence of the tunneling quasiparticle charge, as determined via highly sensitive shot noise measurements, on system parameters, in the odd denominators states ν = 1/3 and ν = 7/3 and in the even denominator state ν = 5/2. In particular, for very weak backscattering probability and sufficiently small excitation energy (temperature and applied voltage), tunneling charges across a constriction were found to be significantly higher than the theoretically predicted fundamental quasiparticle charges.

We report on investigation of oscillations periodic in the magnetic field observed in magnetoresistence, photocur‐rent and photovoltage measurements in GaAs/AlGaAs high electron mobility field‐effect transistor subjected to sub‐terahertz radiation. The spectra show edge magnetoplasmons and cyclotron resonances which can be a basis of tunable resonant sub‐terahertz detectors.

The radiative decay of collective plasma oscillations as a new mechanism for THz emission is studied. This phenomenon is based on the attractive interaction of two intersubband plasmons. This interaction can be viewed as a collective e‐e scattering phenomenon. The emission results fit very well to the results of the current voltage measurements indicating that the conditions for a plasma instability are reached.

The ν = 5/2 quantum Hall state is conjectured to be a Moore‐Read state. According to that, quasiparticles carry a charge of e/4 and obey a “non‐abelian” statistics. Previous measurements of the tunneling quasiparticles charge (across a quantum point contact), via shot‐noise measurements [1], were indeed consistent with the charge of the quasiparticles being e/4, and were inconsistent with other potentially possible values, such as e/2 and e. The large scattering of the experimental data in these measurements made the precise determination of the quasi‐particle charge hard to achieve. Here we present new and improved shot noise data, which provide an even more conclusive evidence for the tunneling of e/4 quasiparticles at the ν = 5/2 state.

We examine the phase and the period of the radiation-induced oscillatory magnetoresistance in GaAs/AlGaAs devices utilizing in situ magnetic field calibration by electron spin resonance of diphenyl-picryl-hydrazal. The results confirm a f-independent 1/4-cycle phase shift with respect to the hf=j variant Planck's over 2pi omega(c) condition for j/=1, and they also suggest a small ( approximately 2%) reduction in the effective mass ratio, m(*)/m, with respect to the standard value for GaAs/AlGaAs devices.

Variations in the width of a quantum well (QW) are known to be a source of broadening of the exciton line. Using low temperature near-field optical microscopy, we have exploited the dependence of exciton energy on well width to show that in GaAs QWs, these seemingly random well-width fluctuations actually exhibit well-defined order-strong long-range correlations appearing laterally, in the plane of the QW, as well as vertically, between QWs grown one on top of the other. We show that these fluctuations are correlated with the commonly found mound structure on the surface. This is an intrinsic property of molecular beam epitaxial growth.

We employ shot noise measurements to characterize the effective charge of quasiparticles, at filling factor nu=1/3 of the fractional quantum Hall regime, as they scatter from an array of identical weak backscatterers. Upon scattering, quasiparticles are known to bunch, e.g., only three e/3 charges, or 'electrons' are found to traverse a rather opaque potential barrier. We find here that the effective charge scattered by an array of scatterers is determined by the scattering strength of an individual scatterer and not by the combined scattering strength of the array, which can be very small. Moreover, we also rule out intra-edge equilibration of e/3 quasiparticles over length scale of hundreds microns., 4 pages, 4 figures

Nitric oxide (NO), an important molecule involved in neurotransmission, vascular homeostasis, immune regulation, and host defense, is generated from a guanido nitrogen of L-arginine by the family of NO synthase enzymes. Large amounts of NO produced for relatively long periods of time (days to weeks) by inducible NO synthase in macrophages and vascular endothelial cells after challenge with lipopolysaccharide or cytokines (such as interferons, tumor necrosis factor-alpha, and interleukin-1), are cytotoxic for various pathogens and tumor cells. This cytotoxic effect against tumor cells was found to be associated with apoptosis (programmed cell death). The mechanism of NO-mediated apoptosis involves accumulation of the tumor suppressor protein p53, damage of different mitochondrial functions, alterations in the expression of members of the Bcl-2 family, activation of the caspase cascade, and DNA fragmentation. Depending on the amount, duration, and the site of NO production, this molecule may not only mediate apoptosis in target cells but also protect cells from apoptosis induced by other apoptotic stimuli. In this review, we will concentrate on the current knowledge about the role of NO as an effector of apoptosis in tumor cells and discuss the mechanisms of NO-mediated apoptosis.

In a murine tumor model, complete tumor remission is achievable at even advanced metastasized stages by transfer of immune T cells from donor B10.D2 (H-2d, Mls(b)) into tumor-bearing DBA/2 (H-2d, Mls(a)) mice. We showed previously that this graft-versus-leukemia (GvL) effect is dependent on synergistic interactions of transferred CD4+ and CD8+ T cells with host sialoadhesin (SER)-positive macrophages. We now show that the CD40-CD40L (CD154) interaction is involved in the induction of inducible nitric oxide synthase (iNOS) expression during adoptive immunotherapy (ADI). We demonstrate that during ADI, the level of CD40 expression in the liver becomes significantly augmented in comparison to livers of tumor-bearing, untreated animals. CD40 expression is found mostly on SER+ macrophages and to a lesser extent on dendritic cells (DCs). In GvL animals, more SER+ macrophages express iNOS than untreated animals. iNOS expressing cells are found in close proximity to apoptotic cells, at early time points of the therapy in areas of metastasis, and at late stages around portal veins, where CD4+ and CD8+ T lymphocytes form clusters with SER+ macrophages. Blocking of CD40L in vivo at days 5 and 20, when all iNOS+ cells express CD40, leads to significantly reduced CD40 and iNOS expression as well as to a marked inhibition of the therapeutic effect. These data provide functional and in situ evidence that the increased CD40 and iNOS expression observed during ADI contribute to the eradication of liver metastases and to the clearance of donor lymphocytes from the liver.

Newcastle Disease Virus (NDV), an agent with interesting immune stimulatory and anti-tumor activity, was investigated for its capacity to activate anti-tumor activity in murine macrophages in vitro and in vivo. Direct macrophage activation was seen under a variety of experimental conditions using two different strains of NDV, different sources of macrophages (spleen and peritoneum) and different strains of mice (DBA/2, C57BL/6, 615). Various macrophage enzymes (ADA, iNOS, lysozyme, acid phosphatase) became upregulated and anti-tumor effector molecules such as nitric oxide (NO) and TNF-alpha were found in the supernatant. NDV activated macrophages performed anti-tumor activity in vitro such as anti-tumor cytostasis and anti-tumor cytotoxicity. The cytotoxic anti-tumor activity was broad and active against all tumor lines tested including mammary carcinoma, lung carcinoma, mastocytoma and immune escape variants (lymphoma). Macrophage activation via BCG/LPS also caused a broad range anti-tumor cytotoxic activity while activation via mixed lymphocyte culture conditioned medium had restricted anti-tumor activity. Anti-tumor activity of NDV activated macrophages could be transfered in vivo. Transfer of macrophages which had not been appropriately activated exerted either no effect or a tumor growth augmenting effect. Repeated intravenous transfer of NDV activated macrophages exerted a significant suppressive effect on pulmonary metastases in a mammary carcinoma tumor model as well as in a lung carcinoma model. Taken together these results demonstrate that NDV can strongly activate macrophages to perform anti-tumor activities in vitro and in vivo.

T-cell-mediated antitumor effects play an important role clinically in allogeneic graft-versus-leukemia (GvL) reactivity, whereas T-cell-mediated antihost effects are associated with a risk of developing graft-versus-host (GvH) disease. GvL and GvH were compared in an animal tumor model system after the systemic transfer of allogeneic antitumor immune T lymphocytes from B10.D2 [H-2d; minor lymphocyte-stimulating antigen (Mls)b] mice into ESb-MP tumor-bearing or normal DBA/2 (H-2d; Mls(a)) mice. Here we demonstrate that this T-cell-mediated therapy involves the formation of clusters of donor CD4 and CD8 T cells with host macrophages, in particular, with a subpopulation expressing the lymphocyte adhesion molecule sialoadhesin. DBA/2 mice and the derived tumor ESb-MP express viral superantigen 7 (Mls(a)), an endogenous viral superantigen that is absent from B10.D2 mice. To test the contribution of viral superantigen 7-reactive Vbeta6 donor T cells in the GvL-mediated eradication of liver metastases, we performed immunohistological and transmission electron microscopy studies. Vbeta6+ CD4 and CD8 T cells from B10.D2 donors formed tight clusters with host sialoadhesin-positive macrophages, and transmission electron microscopy pictures revealed direct membrane-membrane interactions between T cells and macrophages. Clusters were more abundant and consisted of more cells in tumor-bearing hosts (GvL model) than in non-tumor-bearing hosts (GvH model). In addition, Vbeta6 T cells within the clusters showed a strong proliferation activity, indicating stimulation. Moreover, in an in vitro tumor cytostasis assay, primed as well as nonprimed purified Vbeta6 T cells from donor mice were able to inhibit the proliferation of superantigen-expressing ESb-MP lymphoma cells. This suggests that the transferred superantigen-reactive Vbeta6 T cells contribute to the eradication of metastases. The observed cell clusters might be sites for antigen presentation and the activation of tumor-reactive T cells.

This study demonstrates that systemic interleukin 2 (IL-2) can decrease the homing of syngeneic immune T cells to the target organ of metastases and accelerate unwanted side effects of allogeneic immune T cells. As a tumor system, we used the well-characterized highly aggressive DBA/2 mouse leukemia ESb and its less aggressive adhesion variant, ESb-MP. Systemic IL-2 treatment was performed with recombinant human interleukin-2 (Proleukin), which was slowly released via an implanted osmotic pump or was modified with polyethylene glycol (PEG-IL-2) to achieve constant plasma levels. Allogeneic B10.D2 antitumor immune spleen cells (ISPL cells) exerted strong graft-versus-leukemia (GvL) reactivity after adoptive transfer into late-stage ESb-MP tumor-bearing DBA/2 mice. Mls(a) superantigen-reactive vbeta6 donor T cells were not eliminated or tolerized by in vivo priming with the tumor cells and were present in active proliferation in liver infiltrates. When exogenous PEG-IL-2 or Proleukin was applied in addition to ISPL cells in such mice, the strong GvL-mediated protective immunity was converted into a fatal graft-versus-host disease. IL-2 treatment alone had no toxic effect and caused a moderate protection effect in the absence of an effect on local tumor growth. Potentiation of GvH reactivity of B10.D2 ISPL by PEG-IL-2 was proven in non-tumor-bearing DBA/2 mice, in which graft-versus-host disease was characterized by: (a) heavy hepatic lymphocytic infiltration, (b) irreversible increase of serum glutamate-oxalacetate-transaminase and glutamate-pyruvate-transaminase levels, (c) weight loss, and (d) death. Antagonistic effects of systemic IL-2 on GvL were observed with syngeneic DBA/2 anti-ESb immune peritoneal effector cells (PECs). There was a detrimental effect of systemic IL-2 on liver target organ infiltration by immune T cells causing, at day 6 after transfer, a drop from 20-30 CD4 or CD8 T cells per liver lobule in the PEC group to5 in the PEC plus IL-2 group. The results emphasize the importance of a better understanding of IL-2 function in vivo and of its interaction with immune cell function to improve protocols for optimal application in the clinic to achieve maximal GvL effects.

A spontaneously metastasizing, well-defined mouse lymphoma was chosen as an in vivo model to study the effect of tumor-host interaction on gene expression in liver sinusoidal endothelial cells. Forty-nine bovine aortic endothelial cell (BAEC) genes, recently isolated by a differential screening approach of a cDNA library enriched for tumor necrosis factor-alpha (TNF-alpha) suppressed genes, were investigated. Four of these genes were finally selected because they were affected differentially by host immuno-competence, TNF-alpha, and tumor cells. Sequence analysis showed them to encode the bovine polyubiquitin (A4), elongation factor 1alpha (B2), the acidic ribosomal phosphoprotein PO (C3), and the ribosomal protein S2 (E10). Gene expression was analyzed by dot-blot or Northern blot analysis. TNF-alpha and tumor cell conditioned supernatant suppressed the genes additive in BAEC but not in other endothelial cells except for bovine capillary endothelial cells. Ex vivo-isolated liver endothelial cells of tumor-bearing syngeneic DBA/2 mice showed strong downregulation of these four genes in comparison to normal control values. In contrast, endothelial cells of tumor-bearing immuno-incompetent Balb/c (nu/nu) mice showed no downregulation but upregulation of these genes. Consistently, all four genes were also downregulated when BAEC were incubated with supernatants derived from ex vivo-isolated liver metastases from immuno-competent but not from -incompetent mice. Thus, the expression of a group of genes involved in protein translation and processing was more profoundly altered in endothelial cells in vivo than in vitro, suggesting that microenviromental factors and cell-cell and cell-matrix interactions play an important role.

Here we investigated the effects of the second messenger molecule NO at various concentrations on the activation of transcription factor NF-kappaB, IkappaB-alpha kinase (IKK-alpha), Jun N-terminal kinase (JNK) and apoptosis in murine endothelial cells. Low concentrations of NO alone failed to activate NF-kappaB, IKK-alpha and JNK. When NF-kappaB was prestimulated by TNF-alpha or phorbol 12-myristate 13-acetate, the addition of NO at low concentrations enhanced the activation of NF-kappaB. This provides a mechanism for a self-amplifying signal in the inflammatory response, since the inducible NO synthase in endothelial cells is regulated by NF-kappaB. The co-stimulatory effect of NO on NF-kappaB activation was also evident from IKK-alpha kinase assays and reporter gene experiments in endothelial cells. High doses of NO impaired the TNF-alpha-induced DNA-binding activity of NF-kappaB. Accordingly, these high amounts of NO also repressed the TNF-alpha-induced transactivation by NF-kappaB as efficient as dexamethasone. The doses of NO required for the inhibition of NF-kappaB are not cytotoxic for the endothelial cells, enabling the establishment of an autoregulatory loop for NF-kappaB signaling.

Graft-versus-leukemia (GVL) and Graft-versus-host (GVH) reactions were compared after systemic transfer of allogeneic antitumor immune T lymphocytes from B10.D2 (H-2d; Mls(b)) into DBA/2 (H-2d; Mis(a)) mice. Before immune cell transfer, recipient DBA/2 mice were sublethally irradiated with 5 Gy to prevent host-versus-graft reactivity. Recipients were either bearing syngeneic metastatic ESb lymphomas (GVL system) or were normal, non-tumor-bearing mice (GVH system). We previously reported that this adoptive immunotherapy protocol (ADI) had pronounced GVL activity and led to immune rejection of even advanced metastasized cancer. In this study, monoclonal antibodies were used for immunohistochemical analysis of native frozen tissue sections from either spleen or liver to distinguish donor from host cells, to differentiate between CD4 and CD8 T lymphocytes, and to stain sialoadhesin-positive macrophages at different time points after cell transfer. The kinetics of donor cell infiltration in spleen and liver differed in that the lymphoid organ was infiltrated earlier (days 1 to 5 after transfer) than the nonlymphoid organ (days 5 to 20). After reaching a peak, donor cell infiltration decreased gradually and was not detectable in the spleen after day 20 and in the liver after day 30. The organ-infiltrating donor immune cells were mostly T lymphocytes and stained positive for CD4 or CD8 T-cell markers. A remarkable GVL-associated observation was made with regard to a subset of macrophages bearing the adhesion molecule sialoadhesin (SER+ macrophages). In the livers of tumor-bearing mice, their numbers increased between days 1 and 12 after ADI by a factor greater than 30. Double-staining for donor cell marker and SER showed that the sialoadhesin-expressing macrophages were of host origin. The SER+ host macrophages from GVL livers were isolated by enzyme perfusion and rosetting 12 days after ADI, when they reached peak values of about 60 cells per liver lobule, and were tested, without further antigen addition, for their capacity to stimulate an antitumor CD8 T-cell response. The results of this immunologic analysis suggest that these cells in the liver function as scavengers of the destroyed metastases and as antigen-processing and -presenting cells for antitumor immune T cells.

A concept and preliminary feasibility analysis of a divertor with the third-order poloidal field null is presented. The third-order null is the point where not only the field itself but also its first and second spatial derivatives are zero. In this case, the separatrix near the null-point has eight branches, and the number of strike-points increases from 2 (as in the standard divertor) to six. It is shown that this magnetic configuration can be created by a proper adjustment of the currents in a set of three divertor coils. If the currents are somewhat different from the required values, the configuration becomes that of three closely spaced first-order nulls. Analytic approach, suitable for a quick orientation in the problem, is used. Potential advantages and disadvantages of this configuration are briefly discussed.

Although intradermal primary tumor growth and spontaneous liver metastasis of ESbL-lacZ lymphoma in syngeneic DBA/2 mice are progressive and malignant, they are characterized by a transient plateau period with a constant tumor diameter and a low number of metastasized cells in the liver. This period, which was shown to be immune dependent, was followed by a second expansion phase characterized by a preferential localization of tumor cells in the periportal areas of liver lobules (mosaic phenotype). To elucidate possible mechanisms leading to the plateau period as well as for the mosaic-like metastasis pattern, we investigated, using flow cytometry analysis, alterations in costimulatory and adhesion molecule expression in liver sinusoidal cells as well as in tumor cells isolated directly ex vivo throughout the kinetics of metastasis. In tumor and sinusoidal cells, we found up-regulation in the expression of MHC class II and B7 molecules during the plateau period. These molecules, which facilitate cell-mediated immune responses, were again down-regulated during the final exponential tumor growth and metastasis. In the final expansion phase, in which the mosaic phenotype of liver metastasis is seen, we detected a significant increase of leukocyte function-associated antigen-1/intercellular adhesion molecule-1 expression in both tumor and sinusoidal cells, suggesting tumor cell-sinusoidal cell interactions. vascular cell adhesion molecule-1/very late activated antigen-4 did not show any modification during the whole metastatic process. In vivo application of monoclonal antibodies directed to leukocyte function-associated antigen-1 and intercellular adhesion molecule-1 appeared to block the spread of metastasis, while no effect was seen with monoclonal antibodies directed to vascular cell adhesion molecule-1 and very late activated antigen-4. This study reveals in situ expression changes of cell surface molecules in tumor and host cells during metastasis. The changes seen during the plateau phase and during the second expansion phase differ, suggesting associations with mechanisms of immune control and tumor immune evasion, respectively.

Sialoadhesin (SER) is a newly described macrophage-restricted adhesion molecule with a sequence similarity to CD22 on B cells and to myelin-associated glycoprotein on Schwann cells. We describe here a functional role of SER+ spleen macrophages in antigen processing and presentation to T lymphocytes. In two syngeneic murine tumour systems (ESb-MP and lacZ transduced ESbL T-lymphoma cells), the activation state of SER+ macrophages (tested by activity of marker enzymes adenosine deaminase and 5'-nucleotidase) correlated with the arrest of lymphoma metastasis. Furthermore, this macrophage subpopulation became activated upon anti-tumour immunization as well as upon adoptive transfer of immune T lymphocytes into tumour-bearing hosts. We suggest that in situ-activated SER+ macrophages contribute to host resistance against metastasis.