Interleukin-18 regulates acute graft-versus-host disease by enhancing Fas-mediated donor T cell apoptosis.

Interleukin (IL)-18 is a recently discovered cytokine that modulates both T helper type 1 (Th1) and Th2 responses. IL-18 is elevated during acute graft-versus-host disease (GVHD). We investigated the role of IL-18 in this disorder using a well characterized murine bone marrow transplantation (BMT) model (B6 --> B6D2F1). Surprisingly, blockade of IL-18 accelerated acute GVHD-related mortality. In contrast, administration of IL-18 reduced serum tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) levels, decreased intestinal histopathology, and resulted in significantly improved survival (75 vs. 15%, P < 0.001). Administration of IL-18 attenuated early donor T cell expansion and was associated with increased Fas expression and greater apoptosis of donor T cells. The administration of IL-18 no longer protected BMT recipients from GVHD when Fas deficient (lpr) mice were used as donors. IL-18 also lost its ability to protect against acute GVHD when interferon (IFN)-gamma knockout mice were used as donors. Together, these results demonstrate that IL-18 regulates acute GVHD by inducing enhanced Fas-mediated apoptosis of donor T cells early after BMT, and donor IFN-gamma is critical for this protective effect.

Ruxolitinib in corticosteroid-refractory graft-versus-host disease after allogeneic stem cell transplantation: a multicenter survey.

Despite major improvements in allogeneic hematopoietic cell transplantation over the past decades, corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) cause high mortality. Preclinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib. In this retrospective survey, 19 stem cell transplant centers in Europe and the United States reported outcome data from 95 patients who had received ruxolitinib as salvage therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grades III or IV) or SR-cGVHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1-7) and SR-cGVHD (1-10). The overall response rate was 81.5% (44/54) in SR-aGVHD including 25 complete responses (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3-90.7%, 95% confidence interval (CI)) and 97.4% (92.3-100%, 95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and cytomegalovirus-reactivation were observed during ruxolitinib treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Ruxolitinib may constitute a promising new treatment option for SR-aGVHD and SR-cGVHD that should be validated in a prospective trial.

Rapid engraftment after allogeneic ABO-incompatible peripheral blood progenitor cell transplantation complicated by severe hemolysis.

A patient with CML in accelerated phase received G-CSF-mobilized PBPC from an unrelated HLA genotypically matched donor. The blood groups of the patient and donor were bidirectionally incompatible. Hematologic recovery was rapid with > 500 PMN/microliter on day +9. Starting on day +5 bilirubin levels increased from 1.3 mg/dl up to a maximum of 18 mg/dl on day +14. Clinical signs and laboratory tests supported major hemolysis. Blood typing on day +16 revealed early blood-group change, consistent with donor-derived antibodies produced by passenger-lymphocytes which may have mediated severe hemolysis. The early onset and strong intensity of the hyperbilirubinemia could be a specific feature of ABO-incompatible allogeneic PBPC transplantation which would be difficult to differentiate from GVHD or VOD.

Encouraging results in the treatment of haemorrhagic cystitis with estrogen - report of 10 cases and review of the literature.

Haemorrhagic cystitis (HC) after allogeneic haematopoietic stem cell transplantation (HSCT) or high-dose cyclophosphamide (CP) chemotherapy is a severe side-effect and can cause significant morbidity and mortality. In this report, we describe the clinical courses of 10 patients with HC and review the literature. The patients were treated with oral conjugated estrogen in an attempt to improve severe haemorrhagic cystitis. In seven patients positive effects were seen, haematuria resolved in all, but residual symptoms of dysuria remained for longer periods. In one patient application of estrogen was interrupted because of hepatotoxicity. Two patients failed all treatment modalities including oral estrogen because of terminal illness. We conclude that in the management of HC the administration of oral conjugated estrogen should be considered.

Acceptance and feasibility of peripheral stem cell mobilisation compared to bone marrow collection from healthy unrelated donors.

Allogeneic peripheral blood stem cell transplantation leads to an earlier engraftment compared to BMT. The feasibility, acceptance and long-term side-effects of G-CSF mobilisation of PBSC in unrelated healthy donors needs to be evaluated. Forty unrelated healthy donors received G-CSF in a dose of 10 microg/kg bodyweight for 5 days and two aphereses were performed. The donors were monitored prospectively. The data were compared to bone marrow harvests from unrelated donors. Almost all stem cell donors reported some side-effects due to Filgrastim application. Bone pain (32), headache (20), chest pain (two) and night sweats (one) were complained of. By taking analgesics, the pain was relieved in most cases. No donor discontinued the filgrastim application. Bone pain and headache resolved within 2-4 days after termination of Filgrastim application. There was, as expected, a seven-fold increase in the number of total WBCs. There were no significant changes of platelet counts during G-CSF application. After 4 weeks haemoglobin concentration and platelet counts showed no significant differences compared to baseline values. The aphereses were mostly tolerated very well. Eighteen donors reported paraesthesia, one donor developed dizziness, two complained of nausea and vomiting. There was a significant decrease in platelet count (242 before, 98 x 10(9)/l after aphereses). Autologous platelets were transfused after the second aphereses in four donors. These data were compared to data from 245 unrelated bone marrow donors, who had on average, 14 days bone pain and tiredness after donation. The G-CSF mobilisation and apheresis of peripheral blood stem cells is an alternative to traditional bone marrow harvesting in unrelated healthy donors. It is well tolerated and the duration of side-effects on average is shorter than after the surgical procedure. So far no long-term effects have been observed in the follow-up.

Skin involvement as the first manifestation of systemic aspergillosis in patients after allogeneic hematopoietic cell transplantation.

Skin involvement due to a systemic infection with Aspergillus species in the course of allogeneic hematopoietic cell transplantation is extremely rare. We report the clinical course of two patients with hematologic malignancies who underwent allogeneic hematopoietic cell transplantation (HCT) and developed disseminated skin involvement as the first clinical symptom of a proven systemic Aspergillus infection.

CD49d blockade by natalizumab in patients with multiple sclerosis affects steady-state hematopoiesis and mobilizes progenitors with a distinct phenotype and function.

Therapeutic application of natalizumab, an anti-CD49d Ab, in patients with multiple sclerosis (MS) has been associated with increased levels of circulating CD34+ progenitors. We analyzed the frequency, phenotype and functional activity of CD34+ HSC in blood and BM of patients with MS who were treated with natalizumab. Compared with healthy controls and untreated MS patients, natalizumab treatment increased CD34+ cells in the peripheral blood 7-fold and in BM 10-fold. CD34+ cells derived from blood and marrow of natalizumab-treated patients expressed less of the stem cell marker CD133, were enriched for erythroid progenitors (CFU-E) and expressed lower levels of adhesion molecules than G-CSF-mobilized CD34+ cells. The level of surface CXCR-4 expression on CD34+ cells from patients treated with natalizumab was higher compared with that of CD34+ cells mobilized by G-CSF (median 43.9 vs 15.1%). This was associated with a more than doubled migration capacity toward a chemokine stimulus. Furthermore, CD34+ cells mobilized by natalizumab contained more mRNA for p21 and less for matrix metallopeptidase 9 compared with G-CSF-mobilized hematopoietic stem cell (HSC). Our data indicate that G-CSF and CD49d blockade mobilize different HSC subsets and suggest that both strategies may be differentially applied in specific cell therapy approaches.

Treatment of refractory acute GVHD with third-party MSC expanded in platelet lysate-containing medium.

Mesenchymal stem cells have been shown to mediate immunomodulatory effects. They have been used in patients with steroid-refractory acute GVHD (aGVHD), but their relevance as a therapeutic agent targeting aGVHD has still to be defined. In this case series, we report 13 patients with steroid-refractory aGVHD who received BM-derived MSC expanded in platelet lysate-containing medium from unrelated HLA disparate donors. MSC were characterized by their morphological, phenotypical and functional properties. All tested preparations suppressed the proliferation of in vitro activated CD4+ T cells. MSC were transfused at a median dosage of 0.9 x 10(6)/kg (range 0.6-1.1). The median number of MSC applications was 2 (range 1-5). Only two patients (15%) responded and did not require any further escalation of immunosuppressive therapy. Eleven patients received additional salvage immunosuppressive therapy concomitant to further MSC transfusions, and after 28 days, five of them (45%) showed a response. Four patients (31%) are alive after a median follow-up of 257 days, including one patient who initially responded to MSC treatment. In our patient cohort, response to MSC transfusion was lower than in the series reported earlier. However, our experience supports the potential efficacy of MSC in the treatment of steroid-refractory aGVHD.

Foscarnet--an alternative for cytomegalovirus prophylaxis after allogeneic stem cell transplantation?

Cytomegalovirus (CMV) disease is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT) and is associated with high morbidity and mortality. Early detection of the disease by antigenemia testing and polymerase chain reaction (PCR) along with pre-emptive antiviral therapy has been shown to be very effective in decreasing the incidence of CMV. We performed an uncontrolled observational study in 21 patients after HSCT (14 related, 7 unrelated donors) to evaluate the efficacy and toxicity of foscarnet administered as prophylaxis for CMV reactivation. Ten patients received bone marrow, and eleven patients received peripheral blood stem cells. All patients received foscarnet prophylaxis to study side effects, incidence of CMV reactivation, CMV disease, and transplant-related mortality. Foscarnet (90 mg/kg) was given every 12 h, day +11 to day +16. Thereafter, foscarnet (90 mg/kg) was given once per day, three times per week until day +60. The incidence of CMV reactivation detected by antigenemia (pp65 antigen) or PCR was 23.8% (5 of 21 patients). Two patients developed CMV disease and one patient died of CMV-pneumonia. Seventeen patients (81%) reported severe side effects, such as gastrointestinal disturbance, headache, and urethritis. In eight patients (38%), the dose of foscarnet had to be reduced and, in six patients (28.5%), foscarnet application was discontinued because of side effects. Compared with other groups, we believe that the potential benefit of foscarnet administration in this early setting is outweighed by the risks of severe toxicity.

[The Dresden Cord Blood Bank. Experiences of the Cord Blood Bank in Dresden, promoted by the German bone Marrow Donor Registry]. / Dresdner Nabelschnurblutbank. Erfahrungen der Nabelschnurblutbank in Dresden, unterstützt durch die Deutsche Knochenmarkspenderdatei.

BACKGROUND: Allogeneic bone marrow and peripheral blood stem cell transplantation is the treatment of choice for a number of malignant hematological diseases, marrow failure syndromes and severe congenital immunodeficiency states. As a new, valuable source of hematopoietic stem cells, cord blood has become increasingly attractive to the medical community. More than 1500 related and unrelated cord blood transplantations have already been performed worldwide. Cord blood can be a particularly good alternative source of stem cells for pediatric patients, if no HLA-identical donor can be found. MATERIAL AND METHODS: In August 1997 the Cord Blood Bank at the University Hospital of Dresden initiated the collection, processing and cryopreservation of placental blood. This Cord Blood bank is promoted by the German bone marrow donor registry DKMS in Tübingen/Germany collaborating with 8 gynecological clinics in Dresden, Bautzen and Erlabrunn. Before cryopreservation, volume reduction of cord blood units is routinely performed by centrifugation and by separation of the buffy coat. RESULTS: As of March 2000, more than 2200 cord blood units have been collected. 60% of the samples had to be discarded because of insufficient quality (low volume and/or cell count, bacterial contamination, positive infectious disease markers). However, more than 800 cord blood units met all quality control criteria and were cryopreserved. CONCLUSION: These data from the Cord Blood Bank at the University Hospital of Dresden are comparable with results from other cord blood banks. Efforts directed toward the cryopreservation and banking of increased numbers of cord blood units are being continued worldwide and should be supported by the general public.

Experiences of the Dresdner Cord Blood Bank, supported by the Deutsche Knochenmarkspenderdatei.

Allogeneic bone marrow and peripheral blood stem cell transplantation is the treatment of choice for some malignant hematologic diseases, marrow failure syndromes, and severe congenital immunodeficiency states. Since Gluckman et al reported in 1988 the first successful human leukocyte antigen (HLA)-matched sibling umbilical cord blood stem cell transplantation, it has been known that cord blood is a valuable source of hematopoietic stem cells. The Cord Blood Bank at the University Hospital of Dresden was founded in 1997 and started collecting, processing, and cryoconserving umbilical cord blood in August 1997. The cord blood bank is supported by the largest German donor registry: Deutsche Knochenmarkspenderdatei (DKMS) in Tubingen, Germany. With the informed consent of the mothers, the collection is performed in collaboration with six hospitals in Dresden, Berlin, and Bautzen. We routinely perform a volume reduction by centrifuging the blood bag and expressing the leukocyte-rich supernatant. Routinely, sterility, total nucleated cells (TNC), CD34+ cell count, HLA class I and II, ABO/Rh blood group, and colony-forming units are evaluated. The maternal blood is screened for anti-immunodeficiency virus (anti-HIV), anti-hepatitis C virus (anti-HCV), anti-hepatitis B surface antigen (HBsAg), anti-hepatitis B surface (anti-HBs), anti-hepatitis B core (anti-HBc), anticytomegalovirus (anti-CMV), and toxoplasmosis and with Treponema pallidum hemagglutination assay (TPHA). More than 1,000 cord blood units could be collected. Because of the required volume and cell count and because of sterility, 50% of the collected units had to be discharged. Our results are comparable with data of other cord blood banks: mean volume 79 mL; cell count after volume reduction-TNC, 7.16 x 10(8); mononucleated cells (MNC), 3.75 x 10(8); CD34+ cells, 1.95 x 10(6); colony-forming units (CFU), 67.1 x 10(4). To increase the pool of potential umbilical cord blood units and in order to evaluate the possibility for unrelated transplants, cryopreservation and banking of large numbers of cord bloods are necessary.

Efficacy and tolerability of prophylactic treatment with intravenous piperacillin/tazobactam in patients undergoing hematopoietic stem cell transplantation.

BACKGROUND: Infections remain a major cause of morbidity and mortality in patients undergoing autologous or allogeneic hematopoietic stem cell transplantation (HSCT). About 80% of patients experience fever during aplasia and early engraftment despite oral antibacterial chemoprophylaxis. METHODS: In a pilot study, 50 patients undergoing autologous or allogeneic HSCT received a prophylactic antibacterial treatment with intravenous piperacillin/tazobactam beginning on day of stem cell or bone marrow transfusion. They were analyzed retrospectively for frequencies of fever of unknown origin (FUO), documented infection, bacteremia and death because of infection. Furthermore, data from microbiological monitoring and tolerability were evaluated. RESULTS: Among 28 autologous transplanted patients, 10 (36%) developed fever more than 38.5 degrees C; 9/10 FUO, 1/28 pulmonary infiltrates. Eighteen patients (64%) remained without any symptom of infection. In the allogeneic group (n = 22), there were eight patients (36%) with FUO, and five patients (23%) with documented infections (pneumonia 2, enteritis 1, pyelonephritis 1, Escherichia coli bacteremia 1). In nine patients (41%), escalation of antimicrobial treatment was not necessary. The majority of detected microbes in cultures of throat and nose secretions, blood, urine and stool were gram-positive bacteria (77.8%), among them Staphylococcus epidermidis (23.5%), streptococci (group A, B, C; 21.0%) and enterococci (10.6%). Incidence of gram-negative bacteria and fungi was similar with 11.8% and 10.4%, respectively. The most frequent gram-negative strains were Escherichia coli (6.5%) and Pseudomonas aeruginosa (1.7%). There was no severe toxicity or hypersensitivity. CONCLUSION: Compared to oral decontamination and chemoprophylaxis, an intravenous prophylactic regimen as described above could be an effective and well-tolerated approach in prevention of bacterial infections and related complications, with a higher acceptance in recipients of bone marrow or stems cell grafts. Further evaluation in comparison with fluoroquinolone prophylaxis regarding efficacy, development of resistances as well as cost-benefit analyses is warranted.

Defining optimum conditions for the ex vivo expansion of human umbilical cord blood cells. Influences of progenitor enrichment, interference with feeder layers, early-acting cytokines and agitation of culture vessels.

Ex vivo expansion of human umbilical cord blood cells (HUCBC) is explored by several investigators to enhance the repopulating potential of HUCBC. We performed experiments using either Ficoll-separated or CD34+-selected HUCBC from the same donation in serum-free medium. CD34-purified HUCBC were cultured on either human umbilical vein endothelial cells (HUVEC) or irradiated bone marrow-derived stroma cells (BMSC) with addition of different cytokines. In addition, we tested the expansion of HUCBC in culture vessels with continuous rotation. CD34 enrichment led to a significant increase in the expansion factor of CD34+ cells compared with unmanipulated HUCBC. BMSC were more efficient in amplifying early progenitors than HUVEC. Optimum results were reached by a combination of SCF, FLT-3L at 300 ng/ml and IL-3 at 50 ng/ml. No significant improvement in the expansion of CD34+/38- primitive progenitors could be obtained with other combinations. Addition of megakaryocyte-derived growth and development factor to each growth factor cocktail improved the expansion results. Continuous rotation of culture vessels did not ameliorate the expansion rate of the analyzed subsets. Culture conditions separating stroma and HUCBC by a semipermeable membrane improved the expansion factors of CD34+, CD34+/38-, and CD34+/41+ cells and CFU-GM compared with contact cultures. These data might be useful when designing culture systems for clinical scale ex vivo expansion of HUCBC.