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A decisive step for future space missions in the field of next-generation gravity missions was taken by launching the first and only inter-satellite interferometer aboard the GRACE-FO mission. Improving the LRI for NGGM is the next goal. Both microwave and LRI instruments provide ‘biased’ ranges with an unknown offset. Thus, absolute ranging that resolves the bias may open new avenues for orbit determination and data processing. Laser ranging involves precise pointing due to the narrow width of the laser beams. This complicates the initial link acquisition which requires the simultaneous alignment of two optical beams with respect to the line of sight, as well as matching the laser frequencies to enable interferometry. Even small changes in the pointing angle lead to enormous movement in the beam spot at the opposite spacecraft several 100 km away. In the LRI, the link acquisition procedure consists of synchronized spatial scans (two per spacecraft) performed by a steering mirror on each spacecraft together with a frequency scan of the laser on the transponder spacecraft. The whole procedure takes about nine hours. We investigate alternative strategies to improve link acquisition speed, robustness, autonomy, and compatibility with redundancy schemes. In particular, we evaluate the use of a dedicated non-coherent acquisition sensor, its capabilities and its interaction with the other optical elements. The sensor, likely an InGaAs CMOS camera, will measure the tilt of the incoming beam significantly reducing the initial acquisition time. Lastly, laser interferometry can be combined with techniques from telecommunications. With minimal extra hardware, auxiliary functions can be added to the existing laser link. These include the measurement of pseudo-ranges, i.e. combinations of absolute range and clock offsets that can be disentangled to measure the absolute range to centimeter accuracy and clock offsets to nanosecond accuracy. Both may be useful for gravity field recovery data processing. In addition, a data stream of up to dozens of kilobits per second can be transmitted in parallel with the same modulation. This would provide extra redundancy for the ground contacts and/or simplify ground operations, or maybe even enable additional operational features in real-time. Since noise sources and performance limitations directly depend on laser performance and stabilization technique, advanced frequency stabilization based on molecular iodine references may be integrated into the test bed in the future. In this poster, we show a LRI test bed comprising two hexapods capable to simulate satellite rotations, initially built to test different acquisition procedures. We address the methodology to improve the 5D link acquisition with realistic hardware and introduce a 3-stage control system for the FSM. Furthermore, we evaluated different optical layouts for NGGM and provide trade-off.

Aboard GRACE-FO, the first and up to now only intersatellite laser ranging interferometer (LRI) was launched in 2018. Developed as a technology demonstrator, it exceeds the performance of the actual main instrument, which is based on microwave ranging (MWI), by several orders of magnitude. Therefore, the LRI will most likely be the prime technology for next generations of gravity missions, opening up further optimization opportunities, but also new challenges. In a future mission, the central axis will not be occupied by a microwave system, allowing new interferometer layouts as e.g. an on-axis design. Beside the layout, the frequency reference of the laser plays a crucial role. Currently, optical cavities that provide high frequency stability are used, while at the same time only small portion of the available size- mass- and power-budget is required for the laser stabilization unit. However, relative frequency references also bring some disadvantages that make absolute references a possible alternative, especially when the MWI is no longer available. Relative references are dependent on external environmental influences, which can be suppressed on short timescales by a well-designed setup, while on long timescales they become apparent and influence the so-called scale-factor. The scale-factor corresponds to the absolute frequency of the laser and affects the conversion of the phase measurement into a length change and therefore the performance of the LRI. Absolute frequency references based on molecular iodine or hybrid references consisting of a cavity and a molecular iodine spectroscopy unit would suppress these dependencies and will simultaneously ease up the initial acquisition process of the LRI. We will discuss possible alternatives to optical cavities for next generation gravity missions and will outline the advantages and disadvantages of the different technologies. In addition, we analyze proposed technologies that would allow a readout of the absolute frequency of optical cavities in space and underline these with first measurement results from the laboratory.

Space offers virtually unlimited free-fall in gravity. Bose-Einstein condensation (BEC) enables ineffable low kinetic energies corresponding to pico- or even femtokelvins. The combination of both features makes atom interferometers with unprecedented sensitivity for inertial forces possible and opens a new era for quantum gas experiments. On January 23, 2017, we created Bose-Einstein condensates in space on the sounding rocket mission MAIUS-1 and conducted 110 experiments central to matter-wave interferometry. In particular, we have explored laser cooling and trapping in the presence of large accelerations as experienced during launch, and have studied the evolution, manipulation and interferometry employing Bragg scattering of BECs during the six-minute space flight. In this letter, we focus on the phase transition and the collective dynamics of BECs, whose impact is magnified by the extended free-fall time. Our experiments demonstrate a high reproducibility of the manipulation of BECs on the atom chip reflecting the exquisite control features and the robustness of our experiment. These properties are crucial to novel protocols for creating quantum matter with designed collective excitations at the lowest kinetic energy scales close to femtokelvins., 6 pages, 4 figures

Several space missions, such as quantum optical experiments or quantum sensors, require ultrahigh vacuum (UHV) systems to perform their experiments and fulfill the scientific mission goals. Most of these experiments use standard ConFlat (CF) flanges for the design of the UHV system. One of the biggest challenges for these vacuum systems during a space mission is the mechanical loads. These loads are applied during launch, stage separation, etc., as a combination of static and dynamic loads. When loads are applied, the UHV system can experience an increased leakage rate, which decreases the vacuum quality. Investigation of the rise of the leakage rate due to static loads is an important step to allow a proper design process of space suitable UHV systems. In this paper, CF DN40 flange connections will be experimentally tested for standard aluminum (AluVaC) and stainless steel (316LN-ESR, 14429-ESU) tubes. Additionally, three different bolt pretension torques of 10, 12.5, and 15 N m are tested and compared. The presented results can be used to improve the design and assembly procedure for future UHV systems, such as the MAIUS (Matter-Wave Interferometry under Microgravity) sounding rocket missions.

Positioning currently relies heavily on Global Navigation Satellite Systems (GNSS). Combined with classical accelerometers and gyroscopes, precise determination of orientation and position at any given time become available. However, but the availability of GNSS (e.g. GPS) is limited and not guaranteed at all times.In this paper we present an alternative based on atom interferometry using cold atom ensembles. An inertial sensor based on cold atoms allows, in theory, for nearly drift-free measurements of inertial forces with accuracies unreached by classical sensors, but the technology is still locked away in large physics laboratories [1].This paper introduces a compact device called SECAMP, which is capable of cooling atoms down to μ-Kelvin. SECAMP has the potential to measure inertial acceleration in three degrees of freedom. In the following, we present the current experimental setup of the apparatus and outline the next steps for the inertial sensor.

We present a diode laser system optimized for laser cooling and atom interferometry with ultra-cold rubidium atoms aboard sounding rockets as an important milestone towards space-borne quantum sensors. Design, assembly and qualification of the system, combing micro-integrated distributed feedback (DFB) diode laser modules and free space optical bench technology is presented in the context of the MAIUS (Matter-wave Interferometry in Microgravity) mission. This laser system, with a volume of 21 liters and total mass of 27 kg, passed all qualification tests for operation on sounding rockets and is currently used in the integrated MAIUS flight system producing Bose-Einstein condensates and performing atom interferometry based on Bragg diffraction. The MAIUS payload is being prepared for launch in fall 2016. We further report on a reference laser system, comprising a rubidium stabilized DFB laser, which was operated successfully on the TEXUS 51 mission in April 2015. The system demonstrated a high level of technological maturity by remaining frequency stabilized throughout the mission including the rocket's boost phase.

Bose-Einstein-Condensates (BECs) can be used as a very sensitive tool for experiments on fundamental questions in physics like testing the equivalence principle using matter wave interferometry. Since the sensitivity of these experiments in ground-based environments is limited by the available free fall time, the QUANTUS project started to perform BEC interferometry experiments in micro-gravity. After successful campaigns in the drop tower, the next step is a space-borne experiment. The MAIUS-mission will be an atom-optical experiment that will show the feasibility of experiments with ultra-cold quantum gases in microgravity in a sounding rocket. The experiment will create a BEC of 10(5) (87)Rb-atoms in less than 5 s and will demonstrate application of basic atom interferometer techniques over a flight time of 6 min. The hardware is specifically designed to match the requirements of a sounding rocket mission. Special attention is thereby spent on the appropriate magnetic shielding from varying magnetic fields during the rocket flight, since the experiment procedures are very sensitive to external magnetic fields. A three-layer magnetic shielding provides a high shielding effectiveness factor of at least 1000 for an undisturbed operation of the experiment. The design of this magnetic shielding, the magnetic properties, simulations, and tests of its suitability for a sounding rocket flight are presented in this article.

The sounding rocket mission MAIUS-1 has the objective to create the first Bose–Einstein condensate in space; therefore, its scientific payload is a complete cold atom experiment built to be launched on a VSB-30 sounding rocket. An essential part of the setup is an ultrahigh vacuum system needed in order to sufficiently suppress interactions of the cooled atoms with the residual background gas. Contrary to vacuum systems on missions aboard satellites or the international space station, the required vacuum environment has to be reached within 47 s after motor burn-out. This paper contains a detailed description of the MAIUS-1 vacuum system, as well as a description of its qualification process for the operation under vibrational loads of up to 8.1 gRMS (where RMS is root mean square). Even though a pressure rise dependent on the level of vibration was observed, the design presented herein is capable of regaining a pressure of below 5 × 10−10 mbar in less than 40 s when tested at 5.4 gRMS. To the authors' best knowledge, it is the first UHV system qualified for operation on a sounding rocket.

A new processing route for porous b-Si3N4 –ceramics suitable as substrates for Si3N4 functionally graded materials, FGM`s with an environmental barrier coating, EBC, is described. Such materials could be useful for operation at temperatures up to 1650°C in combustion engines, where oxidation and corrosion under hydrothermal conditions cause severe damage of known dense Si3N4-ceramics. The new process is based on microwave sintering of a-Si3N4–Yb-silicate green parts to obtain up to 60% open porous b-Si3N4 pre-forms equilibrated at high temperature with respect to the RE-silicate content, followed by Si-melt infiltration. A sufficient wetting by molten silicon is achieved by coating the inner surface of the porous pre-form with amorphous carbon prior to infiltration. The influence of different green processing parameters and different heating methods - microwave heating and conventional heating – on the porosity of the pre-form is reported. Infiltration is performed by microwave heating of the carbon coated pre-forms, whereas for nitridation both heating processes are combined.

The purpose of the present study was to investigate the effect of interleukin-12 on apoptosis of chronic lymphatic leukemia (CLL) B cells. Apoptotic indices were determined in highly purified CD5(+) B lymphocytes isolated from peripheral blood of seven patients with histologically confirmed CLL. Interleukin-4 as a known inhibitor of apoptosis was used as control. Quantitative analysis of apoptosis was determined by cell death detection ELISA. Our findings indicate that interleukin-12 inhibits ex vivo apoptosis in a large proportion of B-CLL patients and may be closely involved in the pathogenesis of disease. Therefore, our results may help identify potential new therapeutic targets in this malignancy.

The MAIUS experiment is a high precision quantum optics experiment about to fly on a VSB-30 sounding rocket in spring 2015. The scientific objective of the mission is to demonstrate the feasibility of creating a Bose-Einstein Condensate and performing atom interferometry aboard a sounding rocket with Rubidium 87 atoms. This paper will summarize the thermal and mechanical design of the payload and its subsystems. Moreover the qualification procedures and the results of the vacuum qualification test will be presented in this paper.

Background: The most widely accepted criteria for the evaluation of prognosis of malignant melanoma are histopathologic and clinical presentation. No currently available laboratory tests provide additional prognostic information. It has recently been suggested that reverse transcription and polymerase chain reaction (RT-PCR)-based detection of tyrosinase messenger RNA (mRNA) in peripheral blood might be useful in the early detection of circulating tumor cells, since tyrosinase is thought to be a melanocyte-specific marker. Purpose: To further evaluate the clinical relevance of this potential marker, we examined peripheral blood samples from patients with malignant melanoma in different stages of disease for the presence of tyrosinase mRNA. Methods: Total cellular RNA was extracted from heparinized peripheral blood cells from 64 patients with malignant melanoma, from five healthy control subjects, and from four patients with other cancers using the RNAzol A method. For analysis of tyrosinase mRNA, RT-PCR was performed as previously described by Smith et al.; the sensitivity of this assay was tested using RNA extracted from human melanoma cells (SK-mel 1 and SK-mel 3 cell lines) serially diluted with peripheral blood obtained from healthy control subjects. Two additional human melanoma cell lines (SK-mel 30 and RPMI-7951) served as positive controls for RT-PCR detection of tyrosinase mRNA. Overall patient survival curves were constructed using Kaplan-Meier estimates. Results: Tyrosinase mRNA was detected by RTPCR assay of all four of the established melanoma cell lines tested. Nine of the 64 patients with malignant melanoma were found to have detectable tyrosinase mRNA in their peripheral blood cells (tyrosinase-positive patients). The 16 patients with localized primary melanoma did not have detectable tyrosinase mRNA in their peripheral blood cells. Among the 48 patients with metastatic disease, all 27 patients who exhibited no evidence of disease progression were tyrosinase negative. Notably, all nine tyrosinase-positive patients had visceral metastases and were found to exhibit disease progression at the time of the sampling. Four of the nine tyrosinase-positive patients were also found to test negative at times without evidence of progressive disease; one patient became negative after achieving stable disease and three became positive for tyrosinase transcripts on disease progression. The probability of survival from time of sampling was significantly lower in the nine tyrosinase-positive patients when tested versus the 23 patients with comparable disease but without detectable tyrosinase mRNA (two-sided; P

We investigated immunohistochemically the leukocyte infiltrate [CD3, CD4, CD8, CD11a, CD11b, CD14, CD56, VLA-4 and platelet endothelial cell adhesion molecule-1 (PECAM-1)] and the endothelial expression of cell adhesion molecules (PECAM-1, VCAM-1, ICAM-1 and ICAM-2) in 23 renal cell carcinoma tumor tissues. Tumors with a moderate or high density of PECAM-1 positive endothelia showed a stronger infiltration with PECAM-1-positive leukocytes as compared to tumors with a low density of positive endothelia (p0.0085). Additionally, overall survival of patients who presented with tumors exhibiting a moderate or high density of PECAM-1 endothelia alone or in combination with a PECAM-1-positive infiltrate was extended (median survival: 23.5 months) as compared to patients without these tumor characteristics (median survival: 6.5 months). These results suggest an involvement of PECAM-1 in the process of leukocyte migration and a potential role as a prognostic marker in renal cell carcinoma.

We evaluated 28 patients with advanced renal cell carcinoma for the initial expression of P-glycoprotein (MDR1 gene product) employing immunocytochemistry. Tumor specimens were obtained upon primary tumor nephrectomy. In all patients, progression-free survival time following nephrectomy was evaluated and correlated statistically with the staining results. Progression-free survival of patients with no or very few (1%) P-glycoprotein-positive tumor cells (n = 8, median survival 27.0 months) was significantly extended (p0.04) as compared to patients with 1% or more P-glycoprotein-positive tumor cells (n = 20, median survival 4.0 months). Correlations with histopathological tumor characteristics were insignificant. These results suggest a potential role for P-glycoprotein as a biologic parameter predictive of tumor progression in renal cell carcinoma patients.

GVHD is still one of the major complications after allogeneic stem cell transplantation. Whereas murine data have clearly shown the beneficial effects of regulatory T cells (Tregs) on the prevention of GVHD, data from the human system are rare. Here, we present a comparative dynamic analysis of CD4+CD25hiCD127lo/− Tregs from patients with and without GVHD analyzing the whole genome profile over the first 6 months after stem cell transplantation, representing the most sensitive time window for tolerance induction. The Treg transcriptome showed a high stability. However, the comparison of Treg transcriptomes from patients with and without GVHD uncovered regulated gene transcripts highly relevant for Treg cell function. The confirmative protein analyses demonstrated a significantly higher expression of granzyme A, CXCR3, and CCR5 in Tregs of immune tolerant patients. These results point to a reduced suppressive function of Tregs from GVHD patients with diminished migration capacity to the target organs.

Defects in central and peripheral tolerance are thought to contribute to life-threatening graft-versus-host disease (GvHD), a severe complication following allogeneic stem cell transplantation (SCT). Recent investigations have demonstrated regulatory T cells (Tregs) to suppress allogeneic immune reactions. Therefore, SCT patients with no or critically low numbers of Tregs may have an increased risk of GvHD. To address this hypothesis, we analyzed the recovery of CD4(+)CD25(high)CD127(low/-) Tregs in the peripheral blood of patients who have never developed GvHD (n = 6), patients who developed acute/chronic GvHD (n = 18), and patients who developed chronic GvHD without an earlier acute manifestation (n = 5) every 30 days for the first 6 months after peripheral blood SCT (PBSCT). The number of Tregs continuously improved in acute/chronic GvHD patients, but always remained lower than Tregs quantified in patients who never developed a GvHD. In contrast, chronic GvHD patients who did not develop acute GvHD earlier displayed significantly increased Treg cell numbers at the timepoint of chronic inflammation. These results indicate that numerically deficient Tregs following PBSCT are associated with the development of acute but not chronic GvHD.

Background Aplastic anemia (AA) is a bone marrow failure syndrome mostly characterized by an immune-mediated destruction of marrow hematopoietic progenitor/stem cells. The resulting hypocellularity limits a detailed analysis of the cellular immune response. To overcome this technical problem we performed a microarray analysis of CD3+ T-cells derived from bone marrow aspirates and peripheral blood samples of newly diagnosed AA patients and healthy volunteers. Two AA patients were additionally analyzed after achieving a partial remission following immunosuppression. The regulation of selected candidate genes was confirmed by real-time RT-PCR. Results Among more than 22.200 transcripts, 583 genes were differentially expressed in the bone marrow of AA patients compared to healthy controls. Dysregulated genes are involved in T-cell mediated cytotoxicity, immune response of Th1 differentiated T-cells, and major regulators of immune function. In hematological remission the expression levels of several candidate genes tend to normalize, such as immune regulators and genes involved in proinflammatory immune response. Conclusion Our study suggests a pivotal role of Th1/Tc1 differentiated T-cells in immune-mediated marrow destruction of AA patients. Most importantly, immune regulatory genes could be identified, which are likely involved in the recovery of hematopoiesis and may help to design new therapeutic strategies in bone marrow failure syndromes.

Objective Acquired aplastic anemia (AA) is a rare disorder characterized by pancytopenia and hypocellular bone marrow. Though experimental and clinical data suggest that AA represents a T cell–mediated disease, neither the immune response nor the nature of inciting antigen(s) have been characterized so far. The identification of a restricted T cell repertoire by PCR techniques in total lymphocyte populations supports an antigen-driven T cell response. In order to investigate the clonal composition, we analyzed the gene rearrangements of the T cell receptor (TCR) variable β chain (Vβ) at the single-cell level. Patients and Methods CD3 + T lymphocytes were micromanipulated from peripheral blood and bone marrow samples of 8 AA patients and healthy controls. Subsequently amplified VDJ gene segments of the TCRVβ chain were analyzed for functional rearrangements. More than 500 functionally rearranged TCR loci were studied for Vβ/Jβ gene segment usage and molecular composition of the complementary-determining region 3 (CDR3). Results In comparison to healthy controls, the Vβ sequences confirmed a highly restricted T cell repertoire in AA patients at the single-cell level. Both in bone marrow and peripheral blood a predominance of Vβ13 and Jβ2S7 was observed. Furthermore, individual clonal T-cell expansion was identified in the majority of patients. However, deduced CDR3 amino acid sequences revealed a high variability without common motifs among the 8 patients. Conclusion Individual clonal T-cell expansion with high diversity of the antigen-binding sites among the analyzed patients argues for the predominance of private inciting epitopes in AA.

The presence or absence of melanoma cells in human peripheral blood has recently been shown to be associated with disease prognosis, including overall survival. The detection of tyrosinase mRNA-positive circulating melanoma cells by reverse transcription-polymerase chain reaction (RT-PCR) has been limited to disseminated tumours expressing measurable amounts of this melanocyte-specific enzyme. To biologically classify both melanotic and amelanotic melanomas and to evaluate the clinical and prognostic relevance of tumour cell microcontamination, we examined autologous peripheral blood stem cell (PBSC) harvests from patients with advanced malignant melanoma prior to dose-escalated chemotherapy. To assay heterogeneous melanoma cell antigen expression, we developed a highly sensitive RT-PCR using four melanoma- and one tumour-associated antigen as molecular markers. Expression of the melanocyte-associated transcripts of tyrosinase, MART1/Melan-A, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) as well as the tumour-specific transcript of MAGE-3 was analysed by RT-PCR in PBSC harvests from 31 patients. Seven of the 31 PBSC harvests tested positive for one or more molecular markers: two patients for tyrosinase only, and one patient for MAGE-3 only, one patient for tyrosinase and MAGE-3, one for tyrosinase and MART1/Melan-A, and two patients for tyrosinase, MART1/Melan-A, TRP-2 and MAGE-3. mRNA-positive patients exhibited a significantly impaired overall survival (P = 0.0032), with a median survival of 3 months as opposed to 10 months in PBSC mRNA-negative patients. In conclusion, the use of this multiple-marker microcontamination assay allowed for molecular and prognostic classification of advanced malignant melanoma.

Expression of P-glycoprotein (P-gp) mediated multidrug resistance (MDR) has been suggested to be associated with an impaired clinical outcome in several malignancies. In contrast to P-gp itself, further phenotypical and functional alterations related to MDR are poorly characterized. In this in vitro study, we analyzed two Burkitt's lymphoma cell lines (Raji and Daudi) for the beta 1 integrin phenotype prior to and after induction of MDR via co-cultivation with vincristine. A significant loss of the VLA-3 (CD49c/CD29) adhesion receptor was observed whereas all other intergins analyzed lacked considerable changes. We conclude that induction of P-gp mediated MDR does not only affect resistance to cytotoxic drugs but also induces cellular changes with potential relevance for migratory and/or adhesive properties of malignant cells.

The detection of melanocyte-specific messenger RNA in patients with malignant melanoma suggests the potential contamination of peripheral blood stem cell (PBSC) harvests by neoplastic cells. In this study, the melanocyte-specific transcripts of tyrosinase and Melan-A/MART-1 were used to detect neoplastic cells in PBSC harvests of nine metastatic malignant melanoma patients. Only one patient’s PBSC harvest tested positive for tyrosinase. All harvests were negative for Melan-A/MART-1. Our results suggest that contamination of PBSC harvests with neoplastic cells may not contribute to disease progression following high-dose chemotherapy in advanced malignant melanoma.

Serum concentrations of Interleukin-6 (IL-6) were determined in renal cell carcinoma patients treated with low-dose subcutaneous human recombinant interleukin-2 (rIL-2). In all patients, administration of rIL-2 resulted in a significant increase in IL-6 serum levels to peak values within 4 to 6 hours as measured by enzyme-linked immunosorbent assays (ELISA). Repetitive administration of rIL-2 induced significantly lower IL-6 serum peaks when compared to the initial administration of rIL-2. Cumulative IL-6 release, as expressed by the area under the concentration curve (AUC), appeared to be independent of rIL-2 dose distribution (10 million IU rIL-2/m2 versus 20 million IU rIL-2/m2), and IL-6 serum peaks showed no direct dose dependency. Prior rIL-2 immunotherapy had no measurable effect on rIL-2 induced IL-6 release, while steroids resulted in a significant suppression of secondary IL-6. Release of IL-6 did not correlate with response to rIL-2 therapy or survival of rIL-2 treated renal cell carcinoma pati...

The use of NDV as biological adjuvant in vaccines against human cancer is still actual in several clinical treatment protocols. In this study, we have investigated in vitro-effects of Newcastle disease virus (NDV) strain 73-T on isolated mononuclear blood cells and cultured tumor cells. Cellular cytotoxicity of PBMC freshly isolated from healthy donors against tumor cells was enhanced significantly (p0.01) after coincubation of NDV with effector cells. NDV failed to enhance cytotoxicity of effector cells when PBMC were stimulated three days with 500 IU recombinant interleukin-2 (rIL-2) per ml prior to coincubation with the virus. No significant enhancement of cellular lysis was seen when only target cells were coincubated with NDV. As shown by depletion of various lymphocyte subsets, NK cells were the predominant mediator of lysis. Enhancement of cytotoxicity correlated with the induction of interferon-alpha (IFN-alpha) in PBMC by NDV. NDV also induced high amounts of tumor necrosis factor-alpha (TNF-alpha) in PBMC. Induction of interferon-gamma (IFN-gamma) was weak. A direct cytopathic effect (CPE) of NDV on different target cells was detected by colorimetric measurement of metabolic cell activity. The human tumor cell lines A-498, A-704, Caki-1, Caki-2, and K-562 and the fibroblast line MRC-5 showed progressive cellular destruction 48 h after infection with NDV, whereas PBMC and Daudi cells remained unaffected during the observation period. The nontransformed monkey kidney cell line CV-1 and the transformed monkey kidney cell line COS-1 were both lysed by NDV with marginal difference in time course of CPE. Our results indicate a reasonable potential of pleiotropic modifications of the immune response against tumors by NDV.

Natural killer (NK) cells are believed to contribute to the clinical efficacy of cancer immunotherapy using recombinant interleukin-2 (rIL-2) in humans. In previous trials of high-dose i.v. rIL-2, however, no correlation has been established between circulating NK cells and treatment response. Between January 1989 and October 1990, we treated a total of 47 outpatients with advanced tumors using low-dose s.c. rIL-2 and interferon-alpha-2 (rIFN-alpha). Therapy consisted of a 2-day rIL-2 pulse at 18 million IU/m2/day, followed by 6 weeks of rIL-2 (3.6 x 10(6)-4.8 x 10(6) IU/m2/day x 5 days/week) and rIFN-alpha (5 x 10(6)-6 x 10(6) U/m2 x 3/week). Before and after therapy, we phenotypically evaluated circulating lymphocytes and correlated them with clinical response. During 6-week therapy, peripheral blood lymphocytes bearing the CD56 (NK-cell-associated) surface antigen were increased significantly (por = 0.005) in treatment responders [complete response (CR) and partial response (PR), n = 10; 3.8-fold] and stable disease (SD) patients (n = 20; 2.1-fold), while patients with progressive disease (PD, n = 17) exhibited no significant expansion of circulating NK cells (p0.1). After one 6-week treatment cycle, CR/PR patients had significantly more peripheral NK cells, when compared with patients in SD (1.6-fold) and PD (1.9-fold) (p0.04). The overall number of circulating lymphocytes was also increased upon therapy (1.6-fold; por = 0.001), but remained independent of response (p0.4). These data demonstrate that s.c. rIL-2 and s.c. rIFN-alpha produce a significant increase in peripheral blood NK cells; this expansion correlates significantly with treatment response in advanced tumor patients receiving long-term combination immunotherapy at outpatient doses.

Abstract 3748 Graft-versus-host disease (GvHD) remains the major clinical complication in hematopoietic stem cell transplantation (SCT) resulting in severe morbidity and significant mortality. This alloreactive immune response is mainly induced by donor T cells transplanted with the graft. Regulatory T cells (Tregs) play an essential role in the induction and maintenance of peripheral tolerance. In addition, data from murine models have shown that Tregs can prevent GvHD while preserving the graft-versus-leukemia effect. In order to functionally and dynamically characterize human Tregs after allogeneic SCT, we analyzed CD4+CD25highCD127dim T cells isolated from the peripheral blood of more than 80 patients with hematological malignancies every 30 days over half a year following SCT. Patients were divided into the following clinical groups: (A) no signs of acute or chronic GvHD, (B) acute GvHD, (C) chronic GvHD and (D) acute GvHD passed into chronic GvHD. Human peripheral blood lymphocytes were separated by Ficoll gradient and CD4+CD14−CD25highCD127dim T cells were isolated by MoFlow cell sorting. Isolated RNA was pooled and microarray analysis was performed by using Affymetrix HG_U133_Plus2.0 Arrays. Results were verified by using quantitative realtime RT-PCR. Additionally, Tregs were phenotypically analyzed by FACS. We monitored a continous but slower recovery of Tregs in GvHD within the first 6 months following PBSCT. Manifestation of acute and chronic GvHD correlated with significantly reduced frequencies of peripheral Tregs in the first month after PBSCT compared to patients without GvHD. Microarray data revealed a high stability of the Treg transcriptome in the first half year representing the most sensitive time window for tolerance induction. Moreover, comparison of the Treg gene expression profiles from patients with and without GvHD point to a reduced suppressive function of Tregs with diminished migration capacity to the target organs likely contributing to the development of GvHD. Our findings corroborate the impact of human Tregs in the pathophysiology of GvHD and identify novel targets for the manipulation of Tregs to optimize strategies for prophylaxis and treatment of life-threatening GvHD. Disclosures: No relevant conflicts of interest to declare.

The use of recombinant human cytokines has yielded promising results in the treatment of advanced-stage tumor patients for whom no standard therapy is available. In metastatic renal cell carcinoma, objective remissions were reported in patients who had received high-dose intravenous bolus recombinant interleukin-2 (rIL-2) in conjunction with autologous lymphokine-activated killer cells [1]. In this and subsequent clinical trials, however, the severity of adverse reactions (life-threatening fluid retention, hypotension, and pulmonary edema) has limited the intravenous use of rIL-2 to the inpatient and/or intensive care setting, depending on the schedule used [1–6].

Plasma IL-1Ra levels and cellular BCL-2 oncoprotein expression were measured in a total of forty blood samples obtained from twenty-eight B-CLL patients and four healthy subjects. High IL-1Ra plasma levels (as defined by mean + three times standard deviation of normal controls) were observed in eleven samples (ten patients) which showed a significantly decreased cellular expression of BCL-2 protein (14.7 +/- 16.3% of cells as determined by immunofluorescence) when compared to B-CLL samples with no elevated IL-1Ra (BCL-2, 31.0 +/- 18.6%; p < or = 0.0115). Albeit correlational only, our results may encourage further experiments to elucidate potential regulatory effects of IL-1Ra for cellular BCL-2 oncoprotein expression.