Randomized phase-II trial evaluating induction therapy with idarubicin and etoposide plus sequential or concurrent azacitidine and maintenance therapy with azacitidine.

The aim of this randomized phase-II study was to evaluate the effect of substituting cytarabine by azacitidine in intensive induction therapy of patients with acute myeloid leukemia (AML). Patients were randomized to four induction schedules for two cycles: STANDARD (idarubicin, cytarabine, etoposide); and azacitidine given prior (PRIOR), concurrently (CONCURRENT), or after (AFTER) therapy with idarubicin and etoposide. Consolidation therapy consisted of allogeneic hematopoietic-cell transplantation or three courses of high-dose cytarabine followed by 2-year maintenance therapy with azacitidine in the azacitidine-arms. AML with CBFB-MYH11, RUNX1-RUNX1T1, mutated NPM1, and FLT3-ITD were excluded and accrued to genotype-specific trials. The primary end point was response to induction therapy. The statistical design was based on an optimal two-stage design applied for each arm separately. During the first stage, 104 patients (median age 62.6, range 18-82 years) were randomized; the study arms PRIOR and CONCURRENT were terminated early due to inefficacy. After randomization of 268 patients, all azacitidine-containing arms showed inferior response rates compared to STANDARD. Event-free and overall survival were significantly inferior in the azacitidine-containing arms compared to the standard arm (p < 0.001 and p = 0.03, respectively). The data from this trial do not support the substitution of cytarabine by azacitidine in intensive induction therapy.

Epidemiological, genetic, and clinical characterization by age of newly diagnosed acute myeloid leukemia based on an academic population-based registry study (AMLSG BiO).

We describe genetic and clinical characteristics of acute myeloid leukemia (AML) patients according to age from an academic population-based registry. Adult patients with newly diagnosed AML at 63 centers in Germany and Austria were followed within the AMLSG BiO registry (NCT01252485). Between January 1, 2012, and December 31, 2014, data of 3525 patients with AML (45% women) were collected. The median age was 65 years (range 18-94). The comparison of age-specific AML incidence rates with epidemiological cancer registries revealed excellent coverage in patients < 70 years old and good coverage up to the age of 80. The distribution according to the European LeukemiaNet (ELN) risk categorization from 2010 was 20% favorable, 31% intermediate-1, 28% intermediate-2, and 21% adverse. With increasing age, the relative but not the absolute prevalence of patients with ELN favorable and intermediate-1 risk (p < 0.001), with activating FLT3 mutations (p < 0.001), with ECOG performance status < 2 (p < 0.001), and with HCT-CI comorbidity index < 3 (p < 0.001) decreased. Regarding treatment, obesity and favorable risk were associated with an intensive treatment, whereas adverse risk, higher age, and comorbidity index > 0 were associated with non-intensive treatment or best supportive care. The AMLSG BiO registry provides reliable population-based distributions of genetic, clinical, and treatment characteristics according to age.

Characteristics and outcome of patients with therapy-related acute promyelocytic leukemia front-line treated with or without arsenic trioxide.

Therapy-related acute promyelocytic leukemia (t-APL) is relatively rare, with limited data on outcome after treatment with arsenic trioxide (ATO) compared to standard intensive chemotherapy (CTX). We evaluated 103 adult t-APL patients undergoing treatment with all-trans retinoic acid (ATRA) alone (n=7) or in combination with ATO (n=24), CTX (n=53), or both (n=19). Complete remissions were achieved after induction therapy in 57% with ATRA, 100% with ATO/ATRA, 78% with CTX/ATRA, and 95% with CTX/ATO/ATRA. Early death rates were 43% for ATRA, 0% for ATO/ATRA, 12% for CTX/ATRA and 5% for CTX/ATO/ATRA. Three patients relapsed, two developed therapy-related acute myeloid leukemia and 13 died in remission including seven patients with recurrence of the prior malignancy. Median follow-up for survival was 3.7 years. None of the patients treated with ATRA alone survived beyond one year. Event-free survival was significantly higher after ATO-based therapy (95%, 95% CI, 82-99%) as compared to CTX/ATRA (78%, 95% CI, 64-87%; P=0.042), if deaths due to recurrence of the prior malignancy were censored. The estimated 2-year overall survival in intensively treated patients was 88% (95% CI, 80-93%) without difference according to treatment (P=0.47). ATO when added to ATRA or CTX/ATRA is feasible and leads to better outcomes as compared to CTX/ATRA in t-APL.

CCL4 as an adjuvant for DNA vaccination in a Her2/neu mouse tumor model.

Chemokines are key regulators of both innate and adaptive immune responses. CCL4 (macrophage inflammatory protein-1ß, MIP-1ß) is a CC chemokine that has a broad spectrum of target cells including immature dendritic cells, which express the cognate receptor CCR5. We asked whether a plasmid encoding CCL4 is able to improve tumor protection and immune responses in a Her2/neu+ mouse tumor model. Balb/c mice were immunized twice intramuscularly with plasmid DNA on days 1 and 15. On day 25, a tumor challenge was performed with 2 × 10(5) syngeneic Her2/neu+ D2F2/E2 tumor cells. Different groups of mice were vaccinated with pDNA(Her2/neu) plus pDNA(CCL4), pDNA(Her2/neu), pDNA(CCL4) or mock vector alone. Our results show that CCL4 is able to (i) improve tumor protection and (ii) augment a TH1-polarized immune response against Her2/neu. Although Her2/neu-specific humoral and T-cell immune responses were comparable with that induced in previous studies using CCL19 or CCL21 as adjuvants, tumor protection conferred by CCL4 was inferior. Whether this is due to a different spectrum of (innate) immune cells, remains to be clarified. However, combination of CCL19/21 with CCL4 might be a reasonable approach in the future, particularly for DNA vaccination in Her2/neu+ breast cancer in the situation of minimal residual disease.

CCL19 as an adjuvant for intradermal gene gun immunization in a Her2/neu mouse tumor model: improved vaccine efficacy and a role for B cells as APC.

The aim of this study was to evaluate the efficacy of the chemokine CCL19 (ELC) as an adjuvant for intradermal gene gun delivery of Her2/neu DNA and to investigate the role of B cells in CCL19-mediated enhancement of immune responses. Balb/c mice were immunized intramuscularly (i.m.) on days 1 and 15 with plasmid DNA (pDNA) (100 µg DNA) or intradermally (i.d.) by gene gun delivery (1-2 µg DNA). Administration of pDNA encoding Her2/neu (pDNA(Her2/neu) was compared with pDNA(Her2/neu) plus pDNA(CCL19), pDNA(CCL19), mock vector or uncoated gold particles/phosphate-buffered saline (PBS). Tumor challenge was performed subcutaneously on day 25 with syngeneic Her2/neu(+) tumor cells (D2F2/E2). Intradermal immunization by gene gun led to an enhancement of tumor protection by the DNA vaccine as compared with i.m. immunization. The protective effect of the vaccine was further enhanced by coadministration of pDNA(CCL19) both after i.m. and i.d. immunization. Tumor protection was associated with Her2/neu-specific T cell and humoral immune responses. Experiments in B-cell-deficient µMT mice showed that B cells are crucial for CCL19-mediated enhancement of tumor rejection, most likely as antigen-presenting B cells. DNA vaccines against Her2/neu may play a future role in the treatment of Her2/neu-positive breast cancer patients in a clinical situation of minimal residual disease.

CCL21 (SLC) improves tumor protection by a DNA vaccine in a Her2/neu mouse tumor model.

Secondary lymphoid-tissue chemokine (SLC/CCL21) is a CC chemokine that is constitutively expressed in various lymphoid tissues and binds to chemokine receptor CCR7 on mature dendritic cells (DCs) and distinct T-and B-cell sub-populations. In vivo, CCL21 regulates the encounters between DC and T cells and thus is a key regulator of adaptive immune responses. We asked whether CCL21 is able to augment immunogenicity of a DNA-based vaccine against Her2/neu in a Balb/c mouse model with syngeneic Her2/neu+ tumor cells (D2F2/E2). Mice were vaccinated intramuscularly with plasmid DNA (pDNA) on day 1 and boosted on day 15; tumor challenge was performed subcutaneously on day 25. Coexpression of CCL21 and Her-2/neu resulted in induction of a TH1-polarized immune response and substantial improvement of the protective effect of the DNA vaccine. Coexpression of tumor antigen pDNA(Her2/neu) with both pDNA(GM-CSF) and pDNA(CCL21) as adjuvants led to further improvement of protection by the vaccine (70% tumor-free mice on day 35 vs 40% with either adjuvant alone vs 5-10% with tumor antigen alone). Our results show that CCL21 is a potent adjuvant for DNA vaccination, particularly in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF). Clinical use of a pDNA(Her2/neu/CCL21/GM-CSF) vaccine might be particularly promising in minimal residual Her2/neu+ breast cancer.

Allogeneic gene-modified tumor cells (RCC-26/IL-7/CD80) as a vaccine in patients with metastatic renal cell cancer: a clinical phase-I study.

Despite novel targeted agents, prognosis of metastatic renal cell cancer (RCC) remains poor, and experimental therapeutic strategies are warranted. Transfection of tumor cells with co-stimulatory molecules and/or cytokines is able to increase immunogenicity. Therefore, in our clinical study, 10 human leukocyte antigen (HLA)-A(*)0201(+) patients with histologically-confirmed progressive metastatic clear cell RCC were immunized repetitively over 22 weeks with 2.5-40 × 10(6) interleukin (IL)-7/CD80 cotransfected allogeneic HLA-A(*)0201(+) tumor cells (RCC26/IL-7/CD80). Endpoints of the study were feasibility, safety, immunological and clinical responses. Vaccination was feasible and safe. In all, 50% of the patients showed stable disease throughout the study; the median time to progression was 18 weeks. However, vaccination with allogeneic RCC26/IL-7/CD80 tumor cells was not able to induce TH1-polarized immune responses. A TH2 cytokine profile with increasing amounts of antigen-specific IL-10 secretion was observed in most of the responding patients. Interferon-γ secretion by patient lymphocytes upon antigen-specific and non-specific stimulation was substantially impaired, both before and during vaccination, as compared with healthy controls. This is possibly due to profound tumor-induced immunosuppression, which may prevent induction of antitumor immune responses by the gene-modified vaccine. Vaccination in minimal residual disease with concurrent depletion of regulatory cells might be one strategy to overcome this limitation.

In vivo proliferation of rat lamina propria T lymphocytes: general hyporesponsiveness but increased importance of the CD2 and CD28 pathways.

Lamina propria T lymphocytes (LPL-T) have a low proliferative potential in vitro. We asked whether LPL-T are also hyporesponsive in vivo and whether this is specific for the alphabeta T cell receptor (TCR). Mitogenic mAb directed at the alphabeta TCR, CD2, CD28, or control mAbs plus IL-2 were injected into rats. Proliferation and/or apoptosis were detected by double staining using 5-bromo-2'-deoxyuridine/TUNEL and the alphabeta TCR. LPL-T were hyporesponsive to various stimuli compared to other T cells. The strongest proliferation was found upon CD2/CD28 stimulation (LPL-T: 281 +/- 6%; spleen: 642 +/- 31%). LPL-T proliferation was only detectable at 24 h while proliferation in other compartments also occurred later. Hyporesponsiveness was not caused by enhanced T cell apoptosis upon alphabeta TCR stimulation. In conclusion, stimulation of LPL-T results in much shorter and weaker in vivo proliferation than in other lymphoid organs. Overall, CD2/CD28 costimulation is the strongest T cell stimulus in vivo.

Percutaneous embolization on hereditary hemorrhagic telangiectasia patients with severe epistaxis.

OBJECTIVES: To evaluate the results of embolization in patients with hereditary hemorrhagic telangiectasia (HHT) because of severe epistaxis. METHODS: All HHT patients who underwent an embolization (between 1992 and 2006) were asked to participate in this retrospective study. Twelve patients who had in total 19 embolization procedures were interviewed. A questionnaire was used assessing the frequency, severity, duration of epistaxis and their Impact on Lifestyle (IoL). Haemoglobin values were collected from the patients' records. Embolization of the pathologically enhancing lesions was performed using PVA particles. RESULTS: The direct effect of the embolization is very good in 95% of patients. The Impact factor (daily frequency x severity) of epistaxis improved in the first month (p = 0.000) and one year after embolization (p = 0.009). Eleven embolizations (61%) were still associated with significant improvement. There was a reduction in the duration of epistaxis by 16 minutes per day one month after embolization (p = 0.005). However, this reduction was not found one year after embolization. Mean haemoglobin rose significantly after 1 year by an average of 0.8 mmol/l (p = 0.045). Impact on Lifestyle improved in 68% of the procedures and was unchanged in 32%. CONCLUSION: Embolizations remain a therapeutic option in experienced hands. The indication should be made carefully, because of possible (major) complications.

Activation of cortical and inhibited differentiation of medullary epithelial cells in the thymus of lymphotoxin-beta receptor-deficient mice: an ultrastructural study.

The reciprocal influences of thymic lymphocyte and nonlymphocyte populations, i.e. thymic cross-talk, are necessary for the proper maturation of thymocytes and the development/maintenance of thymic stromal microenvironments. Although the molecular influences exerted by thymic stromal cells on maturing thymocytes have been extensively studied, the identity of signalling molecules used by thymocytes to influence the thymic stromal cells is still largely unknown. Our study provides the first ultrastructural evidence that the functional lymphotoxin-beta receptor (LTbetaR) signalling pathway is engaged in the cross-talk between thymocytes and the thymic stromal cell population. We show that LTbetaR signalling is of the utmost significance for the preservation of the subcellular integrity of all thymic epithelial cells. In the absence of LTbetaR there is (1) hypertrophy and activation of cortical thymic epithelial cells, (2) the complete loss of fully differentiated medullary thymic epithelial cells, and (3) the inhibited differentiation of remaining medullary thymic epithelial cells with the appearance of prominent intercellular cysts in the thymic medulla.

[Study of medicine: "full" load as a parameter for the organization of a successful curriculum]. / Medizinstudium: "Gefühlte" Belastung als Parameter für die Organisation eines erfolgreichen Curriculums.

QUESTION: The licensing regulations for doctors (AAppO) allow medical faculties a wide range of possibilities in their implementation. Are there parameters which are easy to survey and which at the same time contribute to the speedy detection of possible undesirable developments? METHOD: The results of a retrospective student evaluation of the preclinical period clearly revealed that there are extreme fluctuations in the "perceived" stress of students in Semester one to four. In succession, classes were restructured so that stress was as equally balanced as possible throughout the four semesters. RESULTS: Over a period of half a decade, the "perceived" stress of students in the four semesters of the preclinical period was stabilised at an optimum level. At the same time, the students' satisfaction with organisation of curriculum increased and their exam results improved significantly. CONCLUSIONS: The parameter "perceived" stress has been conducive to developing the curriculum to such an extent that teaching in Lübeck has improved considerably. Simultaneously, new space for development has been created for both students and lecturers, which makes it possible to shape the academic aspect of medical studies in a more challenging manner.

CCL19 (ELC) as an adjuvant for DNA vaccination: induction of a TH1-type T-cell response and enhancement of antitumor immunity.

Coexpression of tumor antigens together with immunomodulatory molecules is a strategy in DNA vaccination aiming at an amplification of the antitumor immune response. Epstein-Barr virus-induced-molecule-1-ligand-chemokine (ELC/CCL19) is a CC chemokine that binds to the chemokine receptor CCR7. CCR7 is expressed on mature dendritic cells (DC) and distinct T- and B-cell subpopulations. CCL19 (ELC) is mainly expressed in secondary lymphoid organs and plays a central role in regulating the encounters between DC and T cells. We asked whether CCL19 is able to augment immunogenicity of a DNA vaccine in a C57BL/6 mouse model with syngeneic MCA205 (beta-gal) tumor cells. Mice were vaccinated twice intramuscularly on days 1 and 15 and tumor challenge was performed subcutaneously on day 25. Coadministration of plasmid DNA (pDNA) (beta-gal) plus pDNA (CCL19) was compared with pDNA (beta-gal), pDNA (CCL19), mock vector and phosphate-buffered saline (PBS) alone. Coexpression of CCL19 resulted in enhancement of a Th1-polarized immune response with substantial improvement of the protective effect of the DNA vaccine. Immunohistochemical staining revealed an increased CD8+ T-cell infiltration in the tumor tissue of mice that had been immunized with pDNA (beta-gal) plus pDNA (CCL19). We conclude that CCL19 is an attractive adjuvant for DNA vaccination able to augment antitumor immunity and that this effect is partially caused by enhanced CD8+ T-cell recruitment.

Flt-3 ligand as adjuvant for DNA vaccination augments immune responses but does not skew TH1/TH2 polarization.

Since transfection of dendritic cells (DC) plays a key role in DNA vaccination, in vivo expansion of DC might be a tool to increase vaccine efficacy. We asked whether Fms-like tyrosine kinase-3 ligand (Flt-3L), a growth factor for DC, can be used as an adjuvant for DNA vaccination. Beta-galactosidase (beta-gal) was used as a model antigen in C57BL/6 mice. Mice were immunized i.m. with DNA coding for beta-gal with or without additional injection of Flt-3L. In both cases, antigen-specific CD4+ and CD8+ T cells were detectable after vaccination. Compared with DNA alone, additional administration of Flt-3L led to a significant increase in the antigen-specific proliferative response. However, increased cytotoxicity by T cells was not observed. The cytokines secreted by splenocytes of immunized mice upon in vitro stimulation with antigen had a TH2 profile. Humoral responses against beta-gal preferentially consisted of IgG1 antibodies. Analysis of DC from Flt-3L-treated mice revealed an immature phenotype with low or absent expression levels of CD80, CD86 and CD40. We conclude that Flt-3L does not generally skew immune responses towards a TH1 type. More likely, factors determined by the antigen and/or the vaccination procedure itself are crucial for the resulting type of immune response. Flt-3L - under circumstances such as the one we have investigated - can also lead to suppression of TH1 T cell immunity, possibly by expansion of immature/unactivated DC.

Proliferating macrophages, dendritic cells, natural killer cells, T and B lymphocytes in the middle ear and Eustachian tube mucosa during experimental acute otitis media in the rat.

Although many studies focus on the increase of immunocompetent cells within the middle ear mucosa during acute otitis media it is poorly understood how this increase is mediated. The differentiation between two possible causes, i.e. immigration and local proliferation, would help to better understand the pathophysiology of this disease. Therefore, the number of proliferating macrophages, dendritic cells, natural killer cells and T and B lymphocytes was studied during acute otitis media in the rat middle ear mucosa (ME mucosa) and Eustachian tube mucosa (ET mucosa) by labelling proliferating leucocytes with the DNA precursor bromodeoxyuridine (BrdU). By removing the middle ear and Eustachian tube 24 h after BrdU injection, the contribution of immigrated newly formed cells was estimated. At this timepoint, many leucocytes in the ME and ET mucosa had incorporated BrdU (between 15 and 25% within the subsets). By analysing these tissues one hour after BrdU injection, the local proliferation rate was determined (between 2 and 9% within the subsets). Thus, the inflamed ME and ET mucosa are the destination of immunocompetent cells and, as our data show, the inflamed microenvironment supports local proliferation of immunocompetent cells.

Prolonged alpha-adrenergic stimulation causes changes in leukocyte distribution and lymphocyte apoptosis in the rat.

We have previously shown in the rat model that acutely or chronically increased peripheral catecholamines lead to suppression of lymphocyte responsiveness via alpha(2)-adrenoceptor activation. Here we investigated the effects of alpha-adrenergic treatment on total leukocyte numbers and proportions of leukocyte subsets in peripheral blood and lymphoid tissues. It was found that a 12-h treatment with subcutaneously implanted tablets, one containing norepinephrine (NE) and one propranolol, leads to an increase in total blood leukocyte counts, due to a pronounced increase in granulocytes. In contrast, the numbers of all classes of lymphocytes other than NK cells were decreased. This decrease in blood lymphocytes is apparently not due to redistribution, since in the thymus, spleen, mesenteric and peripheral lymph nodes, the total numbers of lymphocytes were decreased as well, without any changes in subpopulations. Analogous results were obtained with rats adrenalectomized before the catecholamine treatment. Animals that received the alpha-adrenergic treatment displayed significantly more apoptotic cells in the lymphoid organs, as determined by the TUNEL technique. In the spleen, the enhanced rate of apoptosis was confined to the white pulp; red pulp areas exhibited significantly fewer apoptotic cells. Thus, an increased alpha-adrenergic tone in rats led to a general loss of lymphocytes due to lymphocyte directed apoptosis that was independent of glucocorticoids.

Splenectomy of rats selectively reduces lymphocyte function-associated antigen 1 and intercellular adhesion molecule 1 expression on B-cell subsets in blood and lymph nodes.

Splenectomy increases the number of B cells in the blood of humans and animals. It is unknown whether this is due to changes in migration, proliferation, or both. The numbers of naïve (IgD(+)IgM(+)), memory (IgD(-)IgM(high)), newly formed (IgM(high)CD90(high)), early recirculating follicular (IgM(low)CD90(high)), recirculating follicular (IgM(low)CD90(-)), and marginal zone (IgM(high)CD90(-)) phenotype B cells were determined in control and splenectomized rats by flow cytometry. All subsets increased significantly in the blood after splenectomy. Because surface molecules are involved in the regulation of migration and proliferation, their expression (lymphocyte function-associated antigen 1 [LFA-1], intercellular adhesion molecule 1 (ICAM-1), L-selectin, alpha4-integrins, CD44, major histocompatability complex class II, interleukin 2 receptor-alpha chain) was determined on B- and T-cell subsets of both groups. B cells, but not T cells, showed a significantly reduced LFA-1 and ICAM-1 expression in blood and lymph nodes, whereas the expression of the other surface molecules analyzed remained unchanged. The down-regulation of these molecules did not influence the adherence of B cells to high endothelial venules in vitro. In vivo, however, ICAM-1(low)-expressing B cells migrated significantly faster through lymph nodes (ICAM-1(low) 41 +/- 5 hours versus ICAM-1(high) 58 +/- 3 hours), whereas proliferation of B cells in bone marrow, lymph node, and blood remained unchanged. Thus, the presence of one organ is necessary for appropriate expression of LFA-1 and ICAM-1 on B cells in other, distant organs. The more rapid transit of ICAM-1(low) B cells through lymph nodes may be responsible for the increased B-cell number in the blood after splenectomy.

Acute laryngotracheitis in the rat induced by Sendai virus: the influx of six different types of immunocompetent cells into the laryngeal mucosa differs strongly between the subglottic and the glottic compartment.

OBJECTIVES: Acute laryngotracheitis is a disease in which mainly the subglottic area is infected, whereas adjacent parts of the larynx, especially the narrow glottic fold, remain unaffected. The reason for the difference between these two directly adjacent regions is unknown. Therefore, in the present study the influx of dendritic cells, neutrophils, T and B lymphocytes, natural killer cells, and macrophages into the mucosa of different laryngeal compartments was investigated after Sendai virus infection in the rat. The aims were to study both the influx of immunocompetent cells and the adhesion of the pathogen and to correlate them to the different reactions of the laryngeal areas during pseudocroup. METHODS: Acute laryngotracheitis was induced by intranasal application of Sendai virus in brown Norway rats. This virus is exclusively pneumotropic in rodents and belongs to the parainfluenza virus type 1, the main pathogen of acute laryngotracheitis in children. The numbers of dendritic cells, neutrophils, T and B lymphocytes, natural killer cells, and macrophages were determined in the supraglottic, glottic, subglottic, and tracheal mucosa on days 2, 5, 7, and 14 after virus application. Furthermore, the nucleoprotein of the virus and major histocompatibility complex (MHC) Class II expression were detected immunohistologically on the laryngeal epithelium. RESULTS: All cell subsets entered the laryngeal mucosa during inflammation. The highest influx was detected among dendritic cells subglottically. This was accompanied by a strong virus adhesion and MHC Class II expression on the subglottic epithelium. In contrast, only a few immunocompetent cells entered the adjacent glottic mucosa, and on the glottic epithelium staining for virus nucleoprotein and MHC Class II expression was weak. CONCLUSIONS: The inflammatory response of the laryngeal mucosa shows great regional differences in this animal model during experimental viral infection. The response was characterized by a strong subglottic and a weak glottic reaction. A possible reason for this difference might be region-specific viral adhesion on the epithelium of the laryngeal areas, as well as differences in MHC Class II expression. Thus, these data agree with the clinical observation during acute laryngotracheitis and may explain why the subglottic part of the larynx is affected preferentially during pseudocroup. The molecular mechanisms mediating the different reactions await clarification.

Tumor necrosis factor-alpha regulates the expression of inducible costimulator receptor ligand on CD34(+) progenitor cells during differentiation into antigen presenting cells.

The inducible costimulator receptor (ICOS) is a third member of the CD28 receptor family that regulates T cell activation and function. ICOS binds to a newly identified ligand on antigen presenting cells different from the CD152 ligands CD80 and CD86. We used soluble ICOSIg and a newly developed murine anti-human ICOS ligand (ICOSL) monoclonal antibody to further characterize the ICOSL during ontogeny of antigen presenting cells. In a previous study, we found that ICOSL is expressed on monocytes, dendritic cells, and B cells. To define when ICOSL is first expressed on myeloid antigen presenting cells, we examined ICOSL expression on CD34(+) cells in bone marrow. We found that CD34(bright) cells regardless of their myeloid commitment were ICOSL(-), whereas ICOSL was first expressed when CD34 expression diminished and the myeloid marker CD33 appeared. However, acute myeloid leukemia cells were ICOSL-negative, whereas among B-cell malignancies only some cases of the most mature tumors such as prolymphocytic leukemia and hairy cell leukemia were positive. Next, we investigated purified CD34(+) hematopoietic progenitor cells that did not constitutively express ICOSL but were induced to express ICOSL within 12 h after granulocyte/macrophage colony-stimulating factor/tumor necrosis factor alpha (TNF-alpha) stimulation. Interestingly, ICOSL was induced prior to CD80/CD86 induction on CD34(+) cells so that ICOSL was expressed in the absence of CD80/CD86. This suggests that ICOSL is an early differentiation marker along the monocytic/dendritic maturation pathway. Induction of ICOSL was dependent on TNF-alpha and was regulated via NF-kappa B as revealed by use of inhibitors specific for I kappa B alpha phosphorylation such as BAY 11-7082 and BAY 11-7085. The antigen presenting capacity of TNF-alpha stimulated CD34(+) cells was strongly inhibited by ICOSIg fusion proteins or by NF-kappa B inhibition. Thus, TNF-alpha-induced ICOSL expression seemed to be functionally important for the costimulatory capacity of CD34(+) hematopoietic progenitor cells.

Rapid exchange of large numbers of donor- and host leukocytes after human liver transplantation.

After liver transplantation, the release of donor leukocytes into the host and the uptake of host leukocytes by the graft is one of the earliest immunologic interactions between donor and host. Using three-color flow cytometry, these interactions were investigated in eight patients from 5 min-24 h after receiving HLA unmatched liver grafts. Five minutes after reperfusion, 5.0 % +/- 1.4 % of all blood leukocytes in the host were of donor origin, decreasing to 1.1 % +/- 0.8 % after 24 h. Donor granulocytes preferentially disappeared from the host circulation, whereas no differences were found between NK-cells and various B- and T cell subpopulations. Furthermore, host granulocytes were preferentially retained in the donor liver. Thus, despite extensive pre-operative perfusion, more than 10(9) donor leukocytes quickly leave the liver graft while host granulocytes preferentially accumulate there. A better understanding of the molecular mechanisms mediating these early interactions might help to develop new strategies for diagnosis and therapy of liver graft rejection.