Ultrasound prediction of Zika virus-associated congenital injury using the profile of fetal growth.

Zika virus (ZIKV) is a mosquito-transmitted flavivirus, recently linked to microcephaly and central nervous system anomalies following infection in pregnancy. Striking findings of disproportionate growth with a smaller than expected head relative to body length have been observed more commonly among fetuses with exposure to ZIKV in utero compared to pregnancies without ZIKV infection regardless of other signs of congenital infection including microcephaly. This study's objective was to determine the diagnostic accuracy of femur-sparing profile of intrauterine growth restriction for the identification of ZIKV-associated congenital injuries on postnatal testing. A retrospective cohort study of pregnant women with possible or confirmed ZIKV infection between January 1, 2016 and December 31, 2017 were included. Subjects were excluded if no prenatal ultrasound was available. A femur-sparing profile of growth restriction determined using INTERGROWTH-21st sonographic standard for head circumference to femur length (HC: FL). Congenital injuries were determined postnatally by imaging, comprehensive eye exam and standard newborn hearing screen. A total of 111 pregnant women diagnosed with ZIKV infection underwent fetal ultrasound and 95 neonates had complete postnatal evaluation. Prenatal microcephaly was detected in 5% of fetuses (6/111). Postnatal testing detected ZIKV-associated congenital injuries in 25% of neonates (24/95). A HC: FL Z-score ≤ -1.3 had a 52% specificity (95% CI 41-63%), 82% negative predictive value (NPV, 95% CI 73-88%) for the detection of ZIKV-associated congenital injuries in the neonatal period. A more stringent threshold with a Z-score ≤ -2 was associated with a 90% specificity (95% CI 81-95%), 81% NPV (95% CI 77-85%). Excluding cases of fetal microcephaly, HC: FL (Z-score ≤ -2) demonstrated a similar specificity (89%, 95% CI 81-95%) with superior NPV (87%, 95% CI 84-90%). The sonographic recognition of a normally proportioned fetus may be useful prenatally to exclude a wider spectrum of ZIKV-associated congenital injuries detected postnatally.

Zika virus and the nonmicrocephalic fetus: why we should still worry.

Zika virus is a mosquito-transmitted flavivirus and was first linked to congenital microcephaly caused by a large outbreak in northeastern Brazil. Although the Zika virus epidemic is now in decline, pregnancies in large parts of the Americas remain at risk because of ongoing transmission and the potential for new outbreaks. This review presents why Zika virus is still a complex and worrisome public health problem with an expanding spectrum of birth defects and how Zika virus and related viruses evade the immune response to injure the fetus. Recent reports indicate that the spectrum of fetal brain and other anomalies associated with Zika virus exposure is broader and more complex than microcephaly alone and includes subtle fetal brain and ocular injuries; thus, the ability to prenatally diagnose fetal injury associated with Zika virus infection remains limited. New studies indicate that Zika virus imparts disproportionate effects on fetal growth with an unusual femur-sparing profile, potentially providing a new approach to identify viral injury to the fetus. Studies to determine the limitations of prenatal and postnatal testing for detection of Zika virus-associated birth defects and long-term neurocognitive deficits are needed to better guide women with a possible infectious exposure. It is also imperative that we investigate why the Zika virus is so adept at infecting the placenta and the fetal brain to better predict other viruses with similar capabilities that may give rise to new epidemics. The efficiency with which the Zika virus evades the early immune response to enable infection of the mother, placenta, and fetus is likely critical for understanding why the infection may either be fulminant or limited. Furthermore, studies suggest that several emerging and related viruses may also cause birth defects, including West Nile virus, which is endemic in many parts of the United States. With mosquito-borne diseases increasing worldwide, there remains an urgent need to better understand the pathogenesis of the Zika virus and related viruses to protect pregnancies and child health.

The Aftermath of Zika: Need for Long-Term Monitoring of Exposed Children.

Pregnancy infections with Zika virus are associated with a spectrum of fetal brain injuries beyond microcephaly. Nonmicrocephalic children exposed to Zika virus in utero or early life should undergo neurodevelopmental testing to identify deficits and allow for early intervention. Additionally, long-term monitoring for higher order neurocognitive deficits should be implemented.

RDP58 does not prevent graft-versus-host disease after dog leukocyte antigen-nonidentical canine hematopoietic cell transplantation.

Graft-versus-host disease (GVHD) remains a cause of substantial morbidity for patients undergoing allogeneic hematopoietic cell transplantation (HCT). The present study was undertaken to investigate the effectiveness of RDP58, a peptide derived from the human leukocyte antigen class I heavy chain, in preventing GVHD in the established dog leukocyte antigen (DLA)-nonidentical canine model. Dogs underwent HCT from unrelated DLA-nonidentical donors after conditioning with 920 cGy total body irradiation. Engraftment and achievement of full donor chimerism was seen in five of six dogs, whereas one dog showed rejection and died of marrow aplasia. All five dogs with engraftment developed acute GVHD and were euthanized at an average of 20.6 days after HCT. Compared with historical controls, the Suse of RDP58 neither prevented acute GVHD nor significantly prolonged survival of DLA-nonidentical HCT recipients.

Use of multigeneration-family molecular dog leukocyte antigen typing to select a hematopoietic cell transplant donor for a dog with T-cell lymphoma.

CASE DESCRIPTION: A 7-year-old Golden Retriever was examined because of anorexia, lethargy, vomiting, and gradual weight loss. CLINICAL FINDINGS: Splenomegaly, pancytopenia, high serum calcium concentration, and high alkaline phosphatase activity were detected. Magnetic resonance imaging revealed an enlarged mesenteric lymph node and increased signals from the bone marrow of the ilium and vertebral bodies. Histologic examination and immunophenotyping of biopsy specimens confirmed a stage V (b) T-cell malignant lymphoma. TREATMENT AND OUTCOME: Clinical remission was attained by use of 2 chemotherapy cycles, followed by an allogeneic hematopoietic cell transplant performed at 18 weeks after diagnosis. A donor was identified by molecular dog leukocyte antigen typing methods. The patient was conditioned with 2 fractions of 4 Gy total body irradiation delivered 3 hours apart at 7 cGy/min, followed by an IV infusion of recombinant canine granulocyte colony-stimulating factor mobilized leukapheresis product and postgrafting immunosuppression with cyclosporine. Chimerism analyses revealed full donor engraftment that has been maintained for at least 58 weeks after transplant. Remission has been confirmed by normal results of serum thymidine kinase assays and the absence of peripheral blood clonal T-cell receptor gene rearrangements. CLINICAL RELEVANCE: Systemic chemotherapy induces remissions; however, most dogs succumb to disease recurrence because of multidrug resistance. Outcome of allogeneic hematopoietic cell transplantation in dogs can be excellent because of improved donor-recipient selection by use of molecular dog leukocyte antigen typing, compared with early attempts, and better prevention of graft versus host disease, better supportive care, and substitution of peripheral blood mononuclear cells for bone marrow.

Durable engraftment of AMD3100-mobilized autologous and allogeneic peripheral-blood mononuclear cells in a canine transplantation model.

Peripheral-blood mononuclear cells (PBMCs) mobilized with AMD3100, a CXCR4 antagonist, combined with granulocyte colony-stimulating factor (G-CSF) have reconstituted autologous hematopoiesis in cancer patients following myeloablative conditioning. The engraftment potential of PBMCs mobilized with AMD3100 alone, however, has remained unproven. We therefore studied AMD3100-mobilized PBMCs in a canine model. Four dogs received 920 cGy total body irradiation (TBI) before infusion of autologous AMD3100-mobilized PBMCs (median CD34 cell count, 3.9 x 10(6)/kg). Neutrophil (> 0.5 x 10(9)/L [500/microL]) and platelet (> 20 x 10(9)/L [> 20 000/microL]) recoveries occurred at medians of 9 (range, 7-10) days and 25 (range, 23-38) days, respectively, after TBI, and all dogs had normal marrow function at 1 year after transplantation. To evaluate the long-term engraftment potential of AMD3100-mobilized PBMCs, 5 dogs were given 920 cGy TBI followed by infusion of AMD3100-mobilized PBMCs (median CD34 cell dose, 4.7 x 10(6)/kg) from their dog leukocyte antigen (DLA)-identical littermates. Neutrophil and platelet recoveries occurred at medians of 8 (range, 8-10) days and 26 (range, 26-37) days, respectively, after TBI. With a median follow-up of 53 (range, 33-61) weeks, recipients' marrow function was normal, and blood-donor chimerism levels were 97% to 100%. In summary, both autologous and allogeneic AMD3100-mobilized PBMCs led to prompt and durable engraftment in dogs after 920 cGy TBI.

What role is there for antithymocyte globulin in allogeneic nonmyeloablative canine hematopoietic cell transplantation?

We investigated whether pretransplantation immunosuppression with canine-specific rabbit antithymocyte globulin (ATG), combined with a suboptimal dose of 1 Gy of total body irradiation (TBI), would permit engraftment of canine dog leukocyte antigen-identical marrow. Cumulative ATG doses of 2 to 5 mg/kg produced a T-cell depletion of 1 log in the peripheral blood and 50% in the lymph nodes. Serum levels of ATG peaked on days 4 to 6 after initiation of therapy and became undetectable by day 13 as a result of canine antibody responses to ATG. ATG prolonged allogeneic skin graft survival to 14 days (n = 5), compared with 8 days in control dogs (P = .0003). Five dogs were given marrow transplants after ATG (3.5-5 mg/kg) and 1 Gy of TBI. Posttransplantation immunosuppression consisted of mycophenolate mofetil and cyclosporine. All dogs showed initial engraftment, with maximum donor chimerism levels of 25%. However, only 1 dog achieved sustained engraftment, and 4 rejected their grafts. The duration of engraftment ranged from 8 to > or = 36 weeks (median, 11 weeks), and this is comparable to that in 6 historical controls not given ATG (range, 3-12 weeks; median, 10 weeks; P = .20). The total nucleated cell doses in the marrow grafts had the highest correlation coefficient with the duration of engraftment: 0.82 (P = .09). We concluded that administering ATG before an otherwise suboptimal conditioning dose of 1 Gy of TBI failed to secure uniform stable hematopoietic engraftment.

Kinetics of engraftment following allogeneic hematopoietic cell transplantation with reduced-intensity or nonmyeloablative conditioning.

Nonmyeloablative or reduced-intensity conditioning regimens have been used to condition elderly or ill patients with hematological malignancies for allogeneic hematopoietic cell transplantation (HCT). Initial mixed donor/host chimerism (i.e. the coexistence of hematopoietic cells of host and donor origin) has been observed in most patients after such transplants. Here, we describe both factors affecting engraftment kinetics in patients given a nonmyeloablative or a reduced-intensity conditioning, and associations between peripheral blood cell subset chimerism levels and HCT outcomes.

Molecular cloning and characterization of canine ICOS.

Inducible costimulatory receptor (ICOS) is one recently identified member of the CD28 family of costimulatory molecules. Evidence suggests ICOS functions as a critical immune regulator and, to evaluate these effects, we employed the canine model system that has been used to develop strategies currently in clinical use for hematopoietic stem cell transplantation. To investigate the effects of blocking the ICOS pathway in the canine hematopoietic cell transplantation model, we tested existing murine and human reagents and cloned the full length of the open reading frame of canine ICOS cDNA to allow the development of reagents specific for the canine ICOS. Canine ICOS contains a major open reading frame of 624 nucleotides, encoding a protein of 208 amino acids, and localizes to chromosome 37. Canine ICOS shares 79% sequence identity with human ICOS, 70% with mouse, and 69% with rat. Canine ICOS expression is limited to stimulated PBMC.

Hematopoietic stem cell transplantation does not restore dystrophin expression in Duchenne muscular dystrophy dogs.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene on the X-chromosome that result in skeletal and cardiac muscle damage and premature death. Studies in mice, including the mdx mouse model of DMD, have demonstrated that circulating bone marrow-derived cells can participate in skeletal muscle regeneration, but the potential clinical utility of treating human DMD by allogeneic marrow transplantation from a healthy donor remains unknown. To assess whether allogeneic hematopoietic cell transplantation (HCT) provides clinically relevant levels of donor muscle cell contribution in dogs with canine X-linked muscular dystrophy (c-xmd), 7 xmd dogs were given hematopoietic cell (HC) transplants from nonaffected littermates. Compared with the pretransplantation baseline, the number of dystrophin-positive fibers and the amount of wild-type dystrophin RNA did not increase after HCT, with observation periods ranging from 28 to 417 days. Similar results were obtained when the recipient dogs were given granulocyte colony-stimulating factor (G-CSF) after their initial transplantation to mobilize the cells. Despite successful allogeneic HCT and a permissive environment for donor muscle engraftment, there was no detectable contribution of bone marrow-derived cells to either skeletal muscle or muscle precursor cells assayed by clonal analyses at a level of sensitivity that should detect as little as 0.1% donor contribution.

Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning.

We analyzed the kinetics of donor engraftment among various peripheral blood cell subpopulations and their relationship to outcomes among 120 patients with hematologic malignancies given hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning consisting of 2 Gy total body irradiation (TBI) with or without added fludarabine. While patients rapidly developed high degrees of donor engraftment, most remained mixed donor/host chimeras for up to 180 days after HCT. Patients given preceding chemotherapies and those given granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell (G-PBMC) grafts had the highest degrees of donor chimerism. Low donor T-cell (P = .003) and natural killer (NK) cell (P = .004) chimerism levels on day 14 were associated with increased probabilities of graft rejection. High T-cell chimerism on day 28 was associated with an increased probability of acute graft-versus-host disease (GVHD) (P = .02). Of 93 patients with measurable malignant disease at transplantation, 41 achieved complete remissions a median of 199 days after HCT; 19 of the 41 were mixed T-cell chimeras when complete remissions were achieved. Earlier establishment of donor NK-cell chimerism was associated with improved progression-free survival (P = .02). Measuring the levels of peripheral blood cell subset donor chimerisms provided useful information on HCT outcomes and might allow early therapeutic interventions to prevent graft rejection or disease progression.

Adoptive immunotherapy to increase the level of donor hematopoietic chimerism after nonmyeloablative marrow transplantation for severe canine hereditary hemolytic anemia.

Severe hemolytic anemia in Basenji dogs secondary to pyruvate kinase deficiency can be corrected by allogeneic hematopoietic cell transplantation (HCT) from littermates with normal hematopoiesis after conventional myeloablative or nonmyeloablative conditioning regimens. If the levels of donor chimerism were low (<20%) after nonmyeloablative HCT, there was only partial correction of the hemolytic anemia. We next addressed whether allogeneic cell therapy after nonmyeloablative HCT would convert mixed to full hematopoietic chimerism, achieve sustained remission from hemolysis, and prevent progression of marrow fibrosis and liver cirrhosis. Three pyruvate kinase-deficient dogs were given HCT from their respective dog leukocyte antigen-identical littermates after nonmyeloablative conditioning with 200 cGy of total body irradiation. Postgrafting immunosuppression consisted of mycophenolate mofetil and cyclosporine. All 3 dogs engrafted and had mixed hematopoietic chimerism with donor levels ranging from 12% to 55% in bone marrow. In 2 of the 3 dogs, there were decreases in the levels of donor chimerism so that at 25 weeks after nonmyeloablative HCT, hemolysis recurred that was associated with increased reticulocyte counts. All 3 dogs then had 2 serial infusions of donor lymphocytes (DLI) from their respective donors at least 20 weeks apart to convert from mixed to full donor chimerism. Both dogs with recurrence of hemolytic anemia after nonmyeloablative HCT achieved higher levels of donor chimerism, with donor contributions ranging from 47% to 62% in the bone marrow and 50% to 69% and 16% to 25% in the granulocyte and mononuclear cell fractions of the peripheral blood, respectively, and with remission of the hemolytic anemia. One dog responded after the first DLI, and 5 weeks after the second DLI, the other dog converted to full donor chimerism. At last follow-up, all these dogs showed clinical improvement, as determined by increasing hematocrits and normal reticulocyte counts. Analysis of the marrow 3 years after HCT showed normal cellularity, a normal myeloid-erythroid ratio, and no or minimal marrow fibrosis. Liver biopsies demonstrated normal histologies with no or minimal fibrosis. We conclude that DLI after nonmyeloablative HCT can increase the levels of donor cells contributing to hematopoiesis in recipients, inducing remissions of the hemolytic process and preventing complications associated with iron overload.

FTY720 does not abrogate acute graft-versus-host disease in the dog leukocyte antigen-nonidentical unrelated canine model.

BACKGROUND: Acute graft-versus-host disease (GVHD) remains a significant impediment to successful hematopoietic stem-cell transplantation (HSCT). Here, we examined the effectiveness of 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride (FTY720), an immunosuppressant that retraffics activated lymphocytes to secondary lymphoid organs, for the treatment of acute GVHD in an established dog leukocyte antigen-nonidentical unrelated canine HSCT model. METHODS: Dogs were given HSCT after conditioning with 920 cGy total body irradiation. The dogs received methotrexate 0.4 mg/kg/day on days 1, 3, 6, and 11 and FTY720 (5 mg/kg/day orally) after developing GVHD. RESULTS: Five of six dogs achieved engraftment, developed acute GVHD, and were treated with FTY720. FTY720 resulted in a profound decrease in lymphocytes and a temporary mitigation of clinical GVHD; however, GVHD recurred in all dogs. Four of five dogs were euthanized because of severe GVHD and the fifth because of severe inanition associated with moderate GVHD. CONCLUSIONS: Compared with controls, treatment of GVHD with FTY720 did not control this complication or significantly increase survival.

Postgrafting immunosuppression with sirolimus and cyclosporine facilitates stable mixed hematopoietic chimerism in dogs given sublethal total body irradiation before marrow transplantation from DLA-identical littermates.

We studied the value of postgrafting immunosuppression with sirolimus (SRL) and cyclosporine (CSP) in enhancing engraftment of dog leukocyte antigen-identical littermate marrow after nonmyeloablative conditioning in a canine model. Dogs received either 2 Gy (n=7) or 1 Gy (n=5) total body irradiation (TBI), followed by postgrafting immunosuppression with SRL and CSP. In the first cohort, all 7 dogs showed rapid initial engraftment. One engrafted dog died on day 21 due to hemorrhagic pneumonitis. Durable engraftment was seen in 5 of 6 remaining dogs, with a median follow-up of >48 (range, >32 to >56) weeks. The sixth dog rejected the marrow graft (as assessed by variable number of tandem repeats) at 11 weeks; however, a subsequent skin graft from the same marrow donor did not undergo acute cellular rejection, suggesting donor-specific tolerance. In the second cohort, all 5 dogs rejected the marrow graft at a median of 9 weeks (range, 3-11 weeks). We conclude that SRL/CSP is as effective as a previously studied combination of mycophenolate mofetil and CSP at establishing durable marrow engraftment after sublethal conditioning.

FLT3 ligand promotes engraftment of allogeneic hematopoietic stem cells without significant graft-versus-host disease.

BACKGROUND: Graft-versus-host (GVH) reactions contribute to stable engraftment of allogeneic hematopoietic stem cell transplants. It was hypothesized that the in vivo expansion of recipient dendritic cells (DC) with the administration of ligand for Flt3 (FL) could promote allogeneic engraftment after reduced-intensity conditioning by enhancing the GVH effect. METHODS: FL was first administered to three nonirradiated healthy dogs for 13 days at a dosage of 100 microg/kg/day. Next, nine dogs received 4.5 Gy total-body irradiation (TBI) and unmodified marrow grafts from dog leukocyte antigen (DLA)-identical littermates without posttransplant immunosuppression. FL was administered to the recipients at a dosage of 100 microg/kg/day from day -7 until day +5. RESULTS: In normal dogs, FL produced significant increases in monocytes (CD14+) and neutrophils in the peripheral blood, a marked increase in CD1c+ cells with DC-type morphology in lymph nodes, and increased alloreactivity of third-party responders to peripheral blood mononuclear cells in mixed lymphocyte reactions (P<0.001). Sustained engraftment was observed in eight of nine (89%) FL-treated dogs compared with 14 of 37 (38%) controls (P=0.02, logistic regression). All engrafted FL-treated dogs became stable complete (n=2) or mixed (n=6) hematopoietic chimeras without significant graft-versus-host disease (GVHD). Recipient chimeric dogs (n=4) were tolerant to skin transplants from their marrow donors but rejected skin grafts from unrelated dogs within 7 to 9 days (median, 8 days). CONCLUSIONS: In this study, the authors showed that FL administered to recipients promotes stable engraftment of allogeneic marrow from DLA-identical littermates after 4.5 Gy TBI without significant GVHD.

Minor histocompatibility antigen-specific cytotoxic T lymphocytes generated with dendritic cells from DLA-identical littermates.

Donor cytotoxic T lymphocytes (CTL) specific for minor histocompatibility antigens (mHA) mediate the graft-versus-host effect whereas host mHA-specific CTL mediate graft rejection in the setting of major histocompatibility complex identical allogeneic hematopoietic stem cell transplantation. Development of a large animal model from which mHA-specific CTL can be isolated would accelerate translation in clinical studies to improve control of the graft-versus-host effect as well as prevention of graft rejection in sensitized hosts. The aims of the current study were to isolate mHA-specific CTL from dog leukocyte antigen-identical littermate nonsensitized recipients before transplantation, from stable mixed hematopoietic chimeras, and from dogs sensitized to mHA after graft rejection. Donor dendritic cells (DCs) were cultured from bone marrow-derived CD34(+) cells and were used to stimulate recipient T lymphocytes on days 1, 10, and 20 of CTL culture. We reliably generated and expanded mHA-specific CTL ex vivo from sensitized dogs that were given a donor-specific blood transfusion to boost immune recall after graft rejection after a nonmyeloablative transplantation. The mHA-specific cytotoxicity measured by (51)Cr release assay was enriched from less than 5% in the starting population of sensitized peripheral blood mononuclear cells to a median of 63% after 4 weeks in CTL culture. The expanded mHA-specific CTLs were not tissue-specific: hematopoietic cells, fibroblast, and stromal cell lines were lysed in an mHA-specific manner. Allogeneic DCs, but not peripheral blood mononuclear cells, were necessary for stimulating ex vivo expansion of mHA-specific CTL. We were unable to generate mHA-specific CTL from nonsensitized dogs before transplantation, from previously sensitized dogs but without recent recall immunization, or from stable mixed hematopoietic chimeras. We conclude that after recent in vivo sensitization, large-scale ex vivo expansion of mHA-specific CTL was feasible using allogeneic DCs.

Adoptive immunotherapy against kidney targets in dog-leukocyte antigen-identical mixed hematopoietic canine chimeras.

BACKGROUND: Stable mixed-donor-host-hematopoietic chimerism can serve as a platform for adoptive immunotherapy. Infusions of donor lymphocytes (DLI) sensitized against hematopoietic cells converted mixed hematopoietic into full-donor chimerism in dog-leukocyte antigen (DLA)-identical littermates. Whether sensitization against tissue of solid organs leads to organ-specific immunity that can be transferred by DLI was unknown and was investigated in these experiments using the kidney as target. METHODS: DLA-identical recipients with established stable mixed-donor-host-hematopoietic chimerism were used. In five pairs, hematopoietic stem-cell transplant (HSCT) donors were sensitized by kidney transplantation from the respective chimeras. In a second group, five HSCT donors received vaccinations that were generated from kidney cells of the respective mixed chimeras. Twenty-eight days after sensitization, DLI were administered to the mixed-hematopoietic chimeras. RESULTS: All HSCT donors rejected their kidney grafts from their mixed-chimeric recipients within 22 to 45 days. DLI caused no sustained graft-versus-kidney effects in the mixed-chimeric recipients. However, DLI donors sensitized by kidney transplantation converted 4 of 5 mixed chimeras into virtually complete (>95%) donor-type chimeras, compared with 1 of 5 mixed chimeras given DLI by vaccination from sensitized donors. CONCLUSION: Although DLA-identical kidney grafts from mixed-hematopoietic chimeras were readily rejected by their HSCT donors, subsequent transfusions of sensitized-donor lymphocytes into mixed chimeras converted mixed to all-donor chimerism but failed to induce graft-versus-kidney effects. Vaccination strategies in lieu of kidney grafts failed to convert mixed chimerism.

Hematopoietic cell transplantation: five decades of progress.

During the past 50 years, the role of allogeneic hematopoietic cell transplantation (HCT) has changed from a desperate therapeutic maneuver plagued by apparently insurmountable complications to a curative treatment modality for thousands of patients with hematologic diseases. Now, cure rates following human leukocyte antigen (HLA) allogeneic HCT with matched siblings exceed 85% for some otherwise lethal diseases, such as chronic myeloid leukemia, aplastic anemia, or thalassemia. In addition, the recent development of non-myeloablative conditioning and stem cell transplantation has opened the way to include elderly patients with a wide variety of hematologic malignancies. Further progress in adoptive transfer of T cell populations with relative tumor specificity would make the transplant procedure more effective and would extend the use of allogeneic HCT for treatment of non-hematopoietic malignancies.

Low-dose total body irradiation (TBI) and fludarabine followed by hematopoietic cell transplantation (HCT) from HLA-matched or mismatched unrelated donors and postgrafting immunosuppression with cyclosporine and mycophenolate mofetil (MMF) can induce durable complete chimerism and sustained remissions in patients with hematological diseases.

Toxicities of high-dose conditioning regimens have limited the use of conventional unrelated donor hematopoietic cell transplantation (HCT) to younger, medically fit patients. Based on preclinical studies, an HCT approach has been developed for elderly or medically infirm patients with HLA-matched or mismatched unrelated donors. In this study, 52 patients with hematological diseases were included. Most (88%) had preceding unsuccessful conventional HCT or refractory/advanced disease. Patients were treated with fludarabine 30 mg/m(2)/d from days -4 to -2, 2 Gy total body irradiation on day 0, cyclosporine at 6.25 mg/kg twice daily from day -3, and mycophenolate mofetil at 15 mg/kg twice daily from day 0. Durable donor chimerism was attained in 88% of the patients. By day 28, a median of 100% of CD56(+) cells were of donor origin. Granulocyte and T-cell donor chimerism increased to medians of 100% on day 56 and day 180 (range, 55%-100%), respectively. Acute GVHD, grade II, was seen in 42% (CI, 29%-56%); grade III in 8% (CI, 0%-15%); and grade IV in 13% (CI, 4%-23%) of patients; it was fatal in 9%. The 100-day transplantation-related mortality was 11%. Complete remissions, including molecular remissions, were seen in 45% of patients with measurable disease before transplantation. Mortality from disease progression was 27% at one year. With a median follow-up of 19 months, 18 of the 52 patients (35%) were alive and 25% were in remission. HCT from HLA-matched or mismatched unrelated donors can be performed with a reduced intensity conditioning regimen in patients ineligible for conventional HCT.

Engraftment of early erythroid progenitors is not delayed after non-myeloablative major ABO-incompatible haematopoietic stem cell transplantation.

We hypothesized that patients undergoing major ABO-incompatible non-myeloablative haematopoietic stem cell transplantation (nm-HSCT) might experience prolonged haemolysis after transplant due to the delayed disappearance of host plasma cells producing anti-donor isohaemagglutinins (HAs). To address this question, we analysed data from 107 consecutive patients transplanted with allogeneic peripheral blood stem cells from human leucocyte antigen-matched (related, n = 84; unrelated, n = 23) donors after non-myeloablative conditioning (200 cGy total body irradiation +/- fludarabine). In total, 23 out of the 107 patients received major or major/minor ABO-incompatible transplants. Red blood cell (RBC) transfusion requirements during the first 120 d post transplant were higher in major ABO-mismatched than in ABO-matched recipients (0.12 vs 0.03 median units RBC concentrate/d, P = 0.04). Two patients developed transient pure red cell aplasia, which had resolved spontaneously by 9 months after transplant. Major ABO incompatibility did not influence rates of engraftment. Patients with sustained engraftment experienced gradual declines of anti-donor HAs, and the estimated median time to reaching IgM and IgG titres of < 1:1 was at least 133 d in evaluable patients, approximately twice longer than reported after myeloablative conditioning. There was a strong correlation between degrees of donor chimaerism in erythroid burst-forming units, granulocyte macrophage colony-forming units and granulocytes, indicating that donor erythroid engraftment, defined by early erythroid progenitors, was as prompt as myeloid engraftment. In conclusion, our data suggest that major ABO-incompatibility is not a barrier to successful non-myeloablative HSCT.