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Background. Despite the promising therapeutic potential of regulatory T cells (Treg) in animal studies of graft-versus-host disease (GVHD), little is known about their effect on human GVHD. Whether Treg are capable of ameliorating GVHD tissue damage has never been demonstrated in humans. It is also unknown whether Treg modulation of GVH histopathologic damage relies on their presence during effector T-cell priming, or whether allogeneic Treg are safe to use clinically. Methods. To address these questions, we used an in vitro human skin explant GVHD model, which mimics the physiopathology of GVHD. First, "donor"-derived CD8 + T cells were stimulated with human leukocyte antigen-unmatched "recipient" dendritic cells (priming phase), then primed "donor" CD8 + T cells were co-cultured with "recipient" skin to induce GVH tissue damage (effector phase). "Donor"-derived Treg were added at the priming or effector phase of the GVH response. Histopathologic changes in the skin were evaluated using a clinically validated GVHD scoring system. Results. "Donor"-derived Treg significantly reduced the severity of GVH histopathologic damage when present during T-cell priming. In contrast, Treg failed to prevent GVH tissue damage when added to the skin co-culture (effector phase), concurrently with primed T cells. Importantly, "donor" Treg alone did not induce GVH tissue damage. Delayed Treg addition led to reduced and impaired Treg suppression of CD8 + T-cell activation and their cytolytic function. Conclusion. "Donor"-derived Treg effectively suppress CD8 + T-cell-mediated GVH tissue damage but are critically required during priming of effector T cells. "Donor"-derived Treg seem to be safe and do not induce GVH histopathologic damage.

The aim of this article is to highlight both experimental and clinical studies contributing to our present understanding of acute graft versus host disease.New cellular players and new molecules in induction, modulation and treatment of graft versus host disease are reported. The contribution of antigen presenting cells to induction of graft versus host disease and graft versus leukemia effects is well understood in animal models and now needs confirmation in human analyses. Both, regulatory T cells and mesenchymal stem cells are a new hope for prophylaxis and treatment. Receptors of innate immunity, homing molecules and chemokines regulate activation and trafficking of these cellular players and may be used for selective blockade, activation or cell selection.Better understanding of the early events involved in graft versus host disease should allow risk adapted and more precise early or even preemptive treatment of graft versus host disease.

BACKGROUND: Veno-occlusive disease (VOD) is one of the most serious complications following allogeneic hematopoietic stem cell transplantation (HSCT) and is associated with a high mortality. We conducted a large trial in order to investigate the value of plasminogen activator inhibitor-1 (PAI-1) plasma antigen levels in VOD patients as PAI-1 has been described as a possible diagnostic marker of VOD. METHODS: In all, 350 stem cell recipients were included in our study. PAI-1 levels were analyzed prior to conditioning therapy and then weekly until eight weeks after HSCT. Transplantation-related complications (TRC) including VOD, microangiopathic hemolytic anemia (MAHA), and graft-versus-host disease (GVHD) were recorded weekly throughout the study. RESULTS: Maximum PAI-1 antigen levels were increased in all patients with VOD (n=15; mean 248 ng/ml; 95% CI 183-314 ng/ml). Maximum PAI-1 levels above 120 ng/ml showed a sensitivity of 100% and a specificity of 30.6% for VOD after HSCT. CONCLUSION: Our study underlines that maximum PAI-1 plasma antigen levels not exceeding 120 ng/ml have a strong negative predictive value in the diagnosis of VOD and thus represent a helpful non-invasive tool for exclusion of VOD after HSCT.

BACKGROUND: Relapse and graft-versus-host disease (GVHD) represent major causes of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Although leukocyte and T-cell chimerism analyses are performed routinely suggesting a predictive value on the patients outcome, little is known about chimerism of dendritic cells (DC) representing strong initiators of immune responses. METHODS: In this prospective study, peripheral DC1 (CD11c+) and DC2 (CD123+) chimerism was determined in hematopoetic stem cell recipients. DCs were isolated from peripheral blood by fluorescence activated cell sorting. Chimerism analyses were performed by fluorescent in situ hybridization or by polymerase chain reaction-based typing of short tandem repeats. RESULTS: At time of engraftment, DC chimerism analyses showed complete chimerism in 76.3% (DC1)/79.5% (DC2), mixed chimerism (MC) in 21.0% (DC1)/17.9% (DC2) and no chimerism in 2.7% (DC1)/2.6% (DC2) of the patients. Peripheral DC chimerism had no significant effect on relapse-free or overall survival. Although acute GVHD was observed more often in patients with MC for DC1/DC2 and chronic GVHD occurred more often in patients with MC for DC2, there was no statistically significant correlation. CONCLUSIONS: Although DCs as antigen presenting cells are supposed to have an impact on the induction of GVHD, there was no significant correlation between incidence of GVHD and DC chimerism after HSCT.

BACKGROUND: Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication of allogeneic bone marrow transplantation (BMT). IPS is associated with elevated bronchoalveolar lavage (BAL) fluid levels of tumor necrosis factor-alpha and lipopolysaccharide, both of which are potent activators of endothelial cells (ECs). EC expression of the adhesion molecule CD54 (intercellular adhesion molecule [ICAM]-1) has been shown to be a major regulator of pulmonary inflammation in various experimental models. METHODS: Using a well-established murine BMT system in which lung injury and graft-versus-host disease (GvHD) are induced by minor histocompatibility antigenic differences between donor and host, the RNase Protection Assay, mice deficient in ICAM-1 expression, and a monoclonal blocking antibody to ICAM, we evaluated the role of the pulmonary vascular expression of CD54 in the development of IPS. RESULTS: Enhanced pulmonary vascular expression of ICAM-1 coincided with the development of IPS. When ICAM-1 -/- mice were used as allogeneic BMT recipients, IPS severity (measured by lung histopathology, BAL cellularity, and cytokine expression) was significantly reduced compared with wild-type controls. Similar results were also observed when wild-type recipients were treated with a monoclonal blocking antibody to ICAM-1. Surprisingly, ICAM-1 had differential effects on leukocyte infiltration into GvHD target organs; ICAM-1 deficiency had no impact on intestinal histopathology, whereas ICAM-1-/- BMT recipients had significantly enhanced hepatic injury. CONCLUSIONS: These data demonstrate that although the expression of ICAM-1 is critical for the development of IPS, different mechanisms of leukocyte recruitment are operative in other GvHD target organs.

Background Idiopathic pneumonia syndrome (IPS) is a frequent and often fatal complication of allogeneic bone marrow transplantation (BMT). We have previously shown that experimental IPS is associated with alloreactive donor T cells and the inflammatory mediators TNF-alpha and lipopolysaccharide. Both TNF-alpha and lipopolysaccharide are known contributors to endothelial injury. Although damage to vascular endothelia has been associated with other complications after BMT, its relationship to lung injury has not been explored. Methods We used a well-established murine BMT system, in which lung injury and graft-versus-host disease are induced by minor histocompatibility antigenic differences between donor and host, and the DNA terminal transferase nick-end labeling (TUNEL) procedure to evaluate whether significant pulmonary vascular endothelial cell (EC) apoptosis is present during the development of IPS. Results Our data demonstrate that pulmonary histopathology after allogeneic BMT is accompanied by significant EC apoptosis and the appearance of activated caspase 3. Further evaluation reveals that EC injury coincides with the onset of pulmonary pathology, is associated with elevations in bronchoalveolar lavage fluid tumor necrosis factor (TNF)-alpha levels, and is accompanied by evidence for EC activation. Administration of a soluble TNF-alpha binding protein (recombinant human TNF-alpha receptor:Fc) from week 4 to week 6 after allogeneic BMT significantly reduces EC apoptosis and lung histopathology observed in this setting. Conclusions EC damage mediated by TNF-alpha is directly linked to the development of experimental IPS. Methods that protect or maintain the integrity of the pulmonary vascular endothelium may therefore prove effective in reducing the severity of lung injury after BMT.

Background Hemostatic complications are not uncommon after bone marrow transplantation (BMT). However, little is known about the frequency, localization, determinants, and outcome of hemostatic events in autologous and allogeneic BMT. Methods Four hundred forty-seven patients (364 allogeneic, 83 autologous transplants) were evaluated retrospectively for the presence of hemostatic complications (bleeding, thrombosis, hepatic veno-occlusive disease [VOD], microangiopathic hemolytic anemia) from the start of conditioning therapy until June 2000. Results A total of 83.2% of the patients presented with at least one hemostatic complication during the investigational period. Most bleeding episodes occurred within the first 4 weeks after transplantation and were relatively mild. However, 27.1% of the patients hemorrhaged severely, generally doubling the overall mortality of the BMT recipients. Fatal gastrointestinal or intracerebral hemorrhages contributed to 1.1% of the events. Bleeding was strongly associated with prolonged thrombocytopenia and graft-versus-host disease (GVHD). Hemorrhagic cystitis may additionally have been triggered by the preceding conditioning regimens containing cyclophosphamide. Thromboembolic events occurred most frequently in allogeneic transplant recipients, for whom the incidence was 14.6%. Chronic GVHD and treatment with steroids were the major determining factors. The incidence of hepatic VOD in 4.7% of the allogeneic transplant recipients was associated with a high fatality rate. Busulfan conditioning increased the VOD risk 2.6-fold. Moderate or severe microangiopathic hemolytic anemia was associated with GVHD and occurred in 14.6% of the allogeneic transplant recipients, leading to an increased overall mortality. Conclusion Hemostatic disturbances, commonly found in the course of transplantation, are associated with a high transplantation risk and closely related to thrombocytopenia and immunologic complications.

To investigate the prognostic value of interleukin 6 (IL-6), procalcitonin (PCT), and C-reactive protein (CRP) in critically ill patients during the first increase of fever, serum levels were measured in 38 patients admitted to intensive care unit of the Department of Medicine, Klinikum Grosshadern, University of Munich, immediately after increase of body temperature more than 38.3 degrees C. Ten healthy controls were also included for comparison. The onset of fever was accompanied by elevated circulating levels of all the 3 markers in comparison with healthy controls. However, only IL-6 levels were significantly higher (P < 0.05) in nonsurvivors (n = 21) compared with survivors. Sensitivity, specificity, positive, and negative predictive values calculated from median levels was higher for IL-6 compared with PCT and CRP. Areas under receiver characteristic operating curves revealed the highest area under the curve for IL-6 in contrast to PCT and CRP. These data suggest that IL-6 rather than PCT or CRP may be an early predictor of mortality in patients with onset of fever and identify patients, who need intensive monitoring to initiate appropriate therapy at an early stage.

University of Munich, and To extend previous studies on the anti-inflammatory role of interleukin (IL)-10 in vivo, mice pretreated with IL-10 were subjected to ionizing radiation (IR), lipopolysaccharide (LPS), or both and assessed for the expression of the intercellular adhesion molecule 1 (ICAM-1) in immunohistochemical analyses. IL-10 was able to almost fully protect LPS+IR-treated animals against ICAM-1 up-regulation. Because LPS and IR also increased adhesion of peripheral blood mononuclear cells, transendothelial migration assays were performed to investigate the functional significance of these findings. IR was found to induce transendothelial migration, and this effect could be enhanced by cotreatment with LPS, in the same fashion as peripheral blood mononuclear cell adhesion. Also in this system, IL-10 proved to act as a potent LPS antagonist. Finally, in vivo immunohistochemical analyses revealed an infiltration of CD3 + T lymphocytes into organs that were the target of transplant-related complications after LPS+IR treatment. This infiltration could also be completely reversed by IL-10 pretreatment.

Chimerism and tolerance after bone marrow transplantation provide excellent conditions for adoptive immunotherapy with T cells of the marrow donor. We studied adoptive immunotherapy in dog leukocyte antigen-identical canine littermate chimeras. Mixed chimeras were produced by conditioning treatment with total body irradiation of a dose of 10 Gy, a uniformly lethal dose in dogs, and infusion of between 1 x 10(8) and 2 x 10(8)/kg mononuclear marrow cells treated with absorbed antithymocyte globulin for inactivation of T cells. Donors were of opposite sex. Persistent mixed chimerism was induced in six of nine dogs, chimerism was complete in one dog, and only transient in two dogs. Tolerance to donor skin grafts was demonstrated in eight dogs, including a dog without cytogenetic evidence of chimerism. Lymphocytes of the marrow donor (between 3.2 x 10(8)/kg and 4.1 x 10(8)/kg) were transfused at various times after transplantation. Nontransfused dogs survived without graft-versus-host disease (GVHD), whereas dogs transfused on days 1 and 2 and dogs transfused on days 21 and 22 developed GVHD and died. In contrast, dogs transfused on days 61 and 62 or later survived without GVHD. Chimerism converted from mixed to complete in six of six transfused dogs and in one of eight nontransfused dogs (P

Tumor necrosis factor-alpha (TNF alpha) is a central mediator in the pathogenesis of sepsis. It also interferes with the hemostatic system and exerts and a net procoagulant effect. Since TNF alpha may contribute to thrombotic complications in sepsis patients, we determined markers of thrombin activation, parameters of the fibrinolytic system (D-dimer, tissue plasminogen activator antigen (tPA) urinary type plasminogen activator antigen (uPA), plasminogen activator inhibitor antigen (PAI-1) and von Willebrand factor antigen (vWF) in 30 patients with sepsis or septic shock. All patients were treated with standard therapy, but 14 patients were treated additionally with an anti-TNF alpha monoclonal antibody (MAK 195F); 16 patients served as historical controls. No significant effect of the antibody on the parameters of the hemostatic system could be determined. Our data speak against a modulation of coagulation or the fibrinolytic system by the monoclonal anti-TNF alpha antibody MAK 195F in this cohort of sepsis patients.

Previous work from our group has contributed to demonstrate the role of conditioning related release of proinflammatory cytokines in induction of acute graft-versus-host disease (GVHD) following allogeneic bone marrow transplantation (BMT). In the present report we show that ionizing radiation (IR) in a clinical relevant dose upregulates intercellular adhesion molecule 1 (ICAM-1) on cultured human microvascular endothelial cells (HMEC). Bacterial endotoxin (lipopolysaccharide, LPS) in a concentration corresponding to serum levels seen during clinical endotoxemia, is capable of further enhancing ICAM-1 expression on irradiated cells. Adhesion assays with freshly isolated peripheral blood mononuclear cells (PBMC) revealed that increased ICAM-1 expression on IR-treated endothelial cells led to an increased adhesion of PBMC. Again, this effect could be superinduced by LPS. Recombinant human interleukin 10 (IL-10), an antagonistic cytokine known to function as an LPS antagonist, was able to counteract the LPS-mediated enhancement of IR-triggered ICAM-1 induction and PBMC adhesion. In contrast, IL-10 could not inhibit irradiation caused effects. IL-10 seemed to interfere with the translocation of preformed intracellular ICAM-1 to the cell membrane. To investigate whether this superinductive function of IR and LPS on endothelial cells is of clinical relevance, mice were treated with total body irradiation (TBI) and inoculated with a single dose of LPS. Immunohistochemical analyses of murine tissues demonstrated that LPS superinduces IR-triggered ICAM-1 also in vivo. These findings may be of clinical importance as they suggest that the endothelium is activated after radiotherapy or TBI used for conditioning in bone marrow transplantation. The activated endothelium in turn may facilitate the accumulation of effector cells at sites of inflammation.

In an uncontrolled clinical trial the effects of repeated administration of the F(ab')2 fragment of a murine monoclonal anti-tumor necrosis factor α (TNFα)-antibody (MAK 195F) on cytokine levels and the cardiovascular system were studied in 20 patients with severe sepsis. Patients were treat

In 20 patients with severe sepsis, skeletal muscle pO2 was continuously measured in order to assess whether a decrease of skeletal muscle pO2 was accompanied by an improvement of sepsis after repeated administration of F(ab')2 fragments of a murine anti-TNF alpha-antibody. Abnormally high skeletal muscle pO2 decreased from 43.5 +/- 10.9 mmHg (day 0) to 36.4 +/- 10.1 mmHg within 24 h after the first administration of anti-TNF alpha-antibody (day 1, p = .006, n = 20) and remained at 34.6 +/- 7.7 mmHg thereafter (mean day 2-7, p = .004). The decrease of skeletal muscle pO2 within 24 h exceeded 5 mmHg (-7 to -19 mmHg) in 11 patients in contrast to nine patients (-4 to +4 mmHg). Only in the patients showing a decrease of skeletal muscle pO2 did sepsis improve as determined by Elebute score, APACHE II score, and interleukin-6 serum levels. The change of skeletal muscle pO2 within 24 h was associated with a change of interleukin-6 serum levels within 24 h (r = .5, n = 20), with a change of Elebute score (r = .7, n = 20) and of APACHE II score (r = .62). These data suggest that a decrease of skeletal muscle pO2 might be an early indicator of improvement of sepsis after administration of anti-TNF alpha-antibodies.

Article abstract— Neurologic manifestation of graft-versus-host disease (GvHD) after allogeneic bone marrow transplantation (BMT) has until now been limited to rare neuromuscular syndromes. Investigating cerebral findings using a murine BMT model, the authors found parenchymal lymphocytic inflammation, microglia activation, and mild cerebral angiitis-like changes in allogeneic transplanted animals but not in syngeneic controls. These findings suggest that cerebral involvement during GvHD may be a new neurologic complication after BMT.