Eva C. Guinan

Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.

Background. Marrow stromal cells (MSC) can differentiate into multiple mesenchymal tissues. To assess the feasibility of human MSC transplantation, we evaluated the in vitro immunogenicity of MSC and their ability to function as alloantigen presenting cells (APC). Methods. Human MSC were derived and used in mixed cell cultures with allogeneic peripheral blood mononuclear cells (PBMC). Expression of immunoregulatory molecules on MSC was analyzed by flow cytometry. An MSC-associated suppressive activity was analyzed using cell-proliferation assays and enzyme-linked immunoassays. Results. MSC failed to elicit a proliferative response when cocultured with allogeneic PBMC, despite provision of a costimulatory signal delivered by an anti-CD28 antibody and pretreatment of MSC with &ggr;-interferon. MSC express major histocompatibility complex (MHC) class I and lymphocyte function-associated antigen (LFA)-3 antigens constitutively and MHC class II and intercellular adhesion molecule (ICAM)-1 antigens upon &ggr;-interferon treatment but do not express CD80, CD86, or CD40 costimulatory molecules. MSC actively suppressed proliferation of responder PBMC stimulated by third-party allogeneic PBMC as well as T cells stimulated by anti-CD3 and anti-CD28 antibodies. Separation of MSC and PBMC by a semipermeable membrane did not abrogate the suppression. The suppressive activity could not be accounted for by MSC production of interleukin-10, transforming growth factor-&bgr;1, or prostaglandin E2, nor by tryptophan depletion of the culture medium. Conclusions. Human MSC fail to stimulate allogeneic PBMC or T-cell proliferation in mixed cell cultures. Unlike other nonprofessional APC, this failure of function is not reversed by provision of CD28-mediated costimulation nor &ggr;-interferon pretreatment. Rather, MSC actively inhibit T-cell proliferation, suggesting that allogeneic MSC transplantation might be accomplished without the need for significant host immunosuppression.

Results of the Cord Blood Transplantation Study (COBLT): clinical outcomes of unrelated donor umbilical cord blood transplantation in pediatric patients with hematologic malignancies

Outcomes of unrelated donor cord blood transplantation in 191 hematologic malignancy children (median age, 7.7 years; median weight, 25.9 kg) enrolled between 1999 and 2003 were studied (median follow-up, 27.4 months) in a prospective phase 2 multicenter trial. Human leukocyte antigen (HLA) matching at enrollment was 6/6 (n = 17), 5/6 (n = 58), 4/6 (n = 111), or 3/6 (n = 5) by low-resolution HLA-A, -B, and high-resolution (HR) DRB1. Retrospectively, 179 pairs were HLA typed by HR. The median precryopreservation total nucleated cell (TNC) dose was 5.1 x 10(7) TNC/kg (range, 1.5-23.7) with 3.9 x 10(7) TNC/kg (range, 0.8-22.8) infused. The median time to engraftment (absolute neutrophil count > 500/mm(3) and platelets 50 000/muL) was 27 and 174 days. The cumulative incidence of neutrophil engraftment by day 42 was 79.9% (95% confidence interval [CI], 75.1%-85.2%); acute grades III/IV GVHD by day 100 was 19.5% (95% CI, 13.9%-25.5%); and chronic GVHD at 2 years was 20.8% (95% CI, 14.8%-27.7%). HR matching decreased the probability of severe acute GVHD. The cumulative incidence of relapse at 2 years was 19.9% (95% CI, 14.8%-25.7%). The probabilities of 6-month and 2-year survivals were 67.4% and 49.5%. Unrelated donor cord blood transplantation from partially HLA-mismatched units can cure many children with leukemias. The study was registered at www.clinicaltrials.gov as #NCT00000603.

Hepatic Veno-Occlusive Disease following Stem Cell Transplantation: Incidence, Clinical Course, and Outcome

The occurrence of hepatic veno-occlusive disease (VOD) has been reported in up to 60% of patients following stem cell transplantation (SCT), with incidence varying widely between studies depending on the type of transplant, conditioning regimen, and criteria used to make the diagnosis. Severe VOD is characterized by high mortality and progression to multiorgan failure (MOF); however, there is no consensus on how to evaluate severity. This review and analysis of published reports attempts to clarify these issues by calculating the overall mean incidence of VOD and mortality from severe VOD, examining the effect of changes in SCT practice on the incidence of VOD over time, and discussing the methods used to evaluate severity. Across 135 studies performed between 1979 and October 2007, the overall mean incidence of VOD was 13.7% (95% confidence interval [CI]=13.3%-14.1%). The mean incidence of VOD was significantly lower between 1979-1994 than between 1994-2007 (11.5% [95% CI, 10.9%-12.1%] vs 14.6% [95% CI, 14.0%-15.2%]; P <.05). The mortality rate from severe VOD was 84.3% (95% CI, 79.6%-88.9%); most of these patients had MOF, which also was the most frequent cause of death. Thus, VOD is less common than early reports suggested, but the current incidence appears to be relatively stable despite recent advances in SCT, including the advent of reduced-intensity conditioning. The evolution of MOF in the setting of VOD after SCT can be considered a reliable indication of severity and a predictor of poor outcome.

Defibrotide for the treatment of severe hepatic veno-occlusive disease and multiorgan failure after stem cell transplantation: a multicenter, randomized, dose-finding trial.

Therapeutic options for severe hepatic veno-occlusive disease (VOD) are limited and outcomes are dismal, but early phase I/II studies have suggested promising activity and acceptable toxicity using the novel polydisperse oligonucleotide defibrotide. This randomized phase II dose-finding trial determined the efficacy of defibrotide in patients with severe VOD following hematopoietic stem cell transplantation (HSCT) and identified an appropriate dose for future trials. Adult and pediatric patients received either lower-dose (arm A: 25 mg/kg/day; n = 75) or higher-dose (arm B: 40 mg/kg/day; n = 74) i.v. defibrotide administered in divided doses every 6 hours for > or =14 days or until complete response, VOD progression, or any unacceptable toxicity occurred. Overall complete response and day +100 post-HSCT survival rates were 46% and 42%, respectively, with no significant difference between treatment arms. The incidence of treatment-related adverse events was low (8% overall; 7% in arm A, 10% in arm B); there was no significant difference in the overall rate of adverse events between treatment arms. Early stabilization or decreased bilirubin was associated with better response and day +100 survival, and decreased plasminogen activator inhibitor type 1 (PAI-1) during treatment was associated with better outcome; changes were similar in both treatment arms. Defibrotide 25 or 40 mg/kg/day also appears effective in treating severe VOD following HSCT. In the absence of any differences in activity, toxicity or changes in PAI-1 level, defibrotide 25 mg/kg/day was selected for ongoing phase III trials in VOD.

Polysaccharide conjugate vaccine responses in bone marrow transplant patients

Bone marrow transplant patients have impaired responses to pure polysaccharide (PS) vaccines and are at an increased risk for disease caused by PS encapsulated pathogens such as Haemophilus influenzae type B (HIB) and Streptococcus pneumoniae. We immunized 35 BMT patients (21 allogeneic and 14 autologous) ages 2-45 years with pure PS pneumococcal (Pnu-imune 23) HIB-conjugate (HibTITER), and tetanus toxoid vaccines. Patients were assigned to receive vaccines at either 12 and 24 months after transplantation or at 24 months only. Only 19% of all enrolled patients developed protective antibody concentrations (> or = 0.300 microgram antibody nitrogen/ml) to the 6 pneumococcal serotypes measured after the 24-month immunization. Poor response to pneumococcal vaccine was not different for the 2 study groups and was similar to previous studies. In contrast, HIB-conjugate vaccine elicited protective concentrations of antibody (> or = 1.0 microgram/ml) in 56% of patients after 1 dose and in 80% after 2 doses. The group that received 2 doses of HIB-conjugate vaccine had a significantly higher geometric mean antibody concentration of 14.5 micrograms/ml as compared with 1.43 micrograms/ml for those receiving only 1 dose (P = 0.012). Responses to tetanus toxoid vaccine were similar to HIB-conjugate vaccine, with a booster response documented after the second dose. In summary, 2 doses of HIB-conjugate vaccine given at 12 and 24 months after transplantation produced protective antibody concentrations in 80% of patients. While the response to pure PS pneumococcal vaccine was poor, the results with HIB-conjugate vaccine suggest that future pneumococcal conjugate vaccines may also benefit BMT patients.

Late onset of renal dysfunction in survivors of bone marrow transplantation

Between 1980 and 1986, 44 children with acute lymphoblastic leukemia (ALL) or Stage IV neuroblastoma (NB) underwent allogeneic or autologous bone marrow transplantation (BMT). Twenty-nine of these patients were alive and in remission 3 months post BMT and were evaluable for this analysis of whom eleven have developed renal dysfunction. Six of 17 (35%) evaluable ALL patients developed renal dysfunction (3.5 to 6 months post BMT). This group was transplanted for CALLA positive ALL and received an autologous transplant. Preparation included tenopiside (VM 26) cytosine arabinoside, and cyclophosphamide followed by total body irradiation (TBI). One patient received 850 cGy in a single fraction, while all other patients received fractionated TBI (1200-1400 cGy in 6-8 fractions over 3-4 days). Five of 7 (71%) evaluable patients who received a BMT for NB have developed late renal problems (4-7 months after BMT). The preparation for NB patients included VM 26, cis-platinum, melphalan, cyclophosphamide, and fractionated TBI (1200-1296 cGy). All seven NB patients had received cis-platinum as induction treatment prior to transplantation. All patients presented with anemia, hematuria, and elevations of BUN and creatinine. Two patients underwent renal biopsies which were consistent with radiation nephropathy or hemolytic uremic syndrome. In conclusion, a high incidence of renal dysfunction has occurred 3 to 7 months after BMT for children with NB and ALL. The clinical and laboratory features are consistent with either acute radiation nephropathy or hemolytic-uremic syndrome. These patients were prepared for BMT with multiple chemotherapeutic agents as well as TBI. The relatively young age of these patients and conditioning with intensive multi-agent chemotherapy may decrease the tolerance of the kidney to radiation injury.

Diamond-Blackfan anemia. Natural history and sequelae of treatment.

To define further the natural history of treated Diamond-Blackfan anemia (DBA), a congenital anemia characterized by a paucity of erythroid precursors, we analyzed 76 patients diagnosed or followed at Childrens Hospital, Boston, between 1931 and 1992. Although DBA is generally defined as macrocytic, we found that mean corpuscular volume in infants aged <5 months rarely exceeded the normal range and is of little diagnostic value. Macrocytosis in patients aged > or = 5 months was more striking. Nine of 16 patients who never received steroids experienced remissions, sometimes after years of transfusions. Thirty-one of 56 patients receiving corticosteroids responded. Of these 31, 13 experienced remissions, 11 remained steroid dependent and 7 later required transfusions. Most nonresponders maintained transfusion dependence. Patients frequently (64%) experienced treatment-related morbidity, most commonly hemosiderosis. The relative risk of leukemia was profoundly elevated (RR, 200; 95% CI, 54.5-512.1) and hematologic parameters at long-term follow-up were often abnormal. While potential responsiveness to steroids and relative ease of red cell transfusion make DBA one of the most treatable congenital marrow failure syndromes, both disease-related and treatment-related factors contributed to a limited prognosis (median survival, 38 years). Patients should be carefully monitored longitudinally for evidence of leukemia and adverse effects of therapy, and alternative treatment strategies should be considered on an individual basis.

The pathology, diagnosis, and treatment of hepatic veno-occlusive disease: current status and novel approaches.

The clinical syndrome of hepatic veno-occlusive disease (VOD) after haemopoietic stem cell transplantation (SCT) is characterized by liver enlargement and pain, ̄uid retention, weight gain, and jaundice (McDonald et al, 1984; 1987). Its onset is typically by day 30 after SCT, although later onset has been described (Lee et al, 1997). Other causes of hepatomegaly, weight gain, and jaundice must be excluded in as far as possible in order to make the clinical diagnosis with any degree of certainty. Because the diagnosis is based on clinical criteria, and due to differences in conditioning regimens and patient characteristics, the incidence reported and severity by different programmes is variable and ranges from 10% to 60% (Carreras et al, 1998; McDonald et al, 1993). Mild disease is de®ned by no apparent adverse effect from liver dysfunction with complete resolution of symptoms and signs. Moderate disease is characterized by adverse effects of liver dysfunction requiring therapy such as diuresis for ̄uid retention and analgesia for painful hepatomegaly but with eventual complete resolution. In contrast, severe disease results in adverse effects from liver dysfunction which fail to resolve despite treatment and the patient dies (McDonald et al, 1993). Assuming that up to one-quarter of patients readily ®t the diagnostic criteria as applied by most practitioners, it is estimated that approximately 5000 individuals of the 20 000 who were transplanted in the United States during 1998 would have been predicted to have symptoms and signs which met the criteria for VOD, and of these between 800 and 1600 persons were likely to have developed fatal illness.

Prize-based contests can provide solutions to computational biology problems

Advances in biotechnology have fuelled the generation of unprecedented quantities of data across the life sciences. However, finding individuals who can address such “big data” problems effectively has become a significant research bottleneck. Historically, prize-based contests have had striking success in attracting unconventional individuals who can solve difficult challenges. To determine whether this approach could solve a real “big data” biologic algorithm problem, we used a complex immunogenomics problem as the basis for a two-week online contest broadcast to participants outside academia and biomedical disciplines. Participants in our contest generated over 600 submissions containing 89 novel computational approaches to the problem. Thirty submissions exceeded the benchmark performance of NIH’s MegaBLAST. The best achieved both greater accuracy and speed (x1000). Here we show the potential of using online prize-based contests to access individuals without domain-specific backgrounds to address big data challenges in life sciences.

Bone marrow transplantation for myelodysplasia and secondary acute nonlymphoblastic leukemia.

Twenty-three patients with primary myelodysplasia (MDS) or secondary myelodysplasia/acute nonlymphocytic leukemia (MDS/ANLL) were treated with allogeneic or syngeneic bone marrow transplantation (BMT). Only one patient was in a chemotherapy-induced hematologic remission. Graft-versus-host disease prophylaxis included methotrexate, methotrexate plus cyclosporine, cyclosporine, or T-cell depletion using one of two anti-CD5 monoclonal antibodies. For patients with primary MDS, the median age was 19 years (range, 11 to 41 years) and the actuarial disease-free survival was 56% +/- 21% (median follow-up, 2 years; range, 0.8 to 5 years). There were three graft failures (two with autologous recovery) and two early deaths. Outcome appeared to be related to French-American-British (FAB) classification. For patients with secondary MDS/ANLL, the median age was 28 years (range, 3 to 16 years) and the actuarial disease-free survival was 27% +/- 13% (median follow-up, 5 years; range, 2.5 to 8.5 years). There were no graft failures, two relapses, and four early deaths. The presence of marrow fibrosis per se did not predict for graft failure (P = .21); however, the use of T-cell depleted marrow in patients with marrow fibrosis resulted in graft failure in three of five individuals. Our results suggest that in patients with primary MDS or secondary MDS/ANLL, BMT should be considered early in the course of the disease, and that attempts at inducing a remission prior to BMT appeared to be unnecessary. In MDS patients with marrow fibrosis, T-cell depletion should be avoided.