Matthew M. Hsieh

Allogeneic Hematopoietic Stem-Cell Transplantation for Sickle Cell Disease

BACKGROUND Myeloablative allogeneic hematopoietic stem-cell transplantation is curative in children with sickle cell disease, but in adults the procedure is unduly toxic. Graft rejection and graft-versus-host disease (GVHD) are additional barriers to its success. We performed nonmyeloablative stem-cell transplantation in adults with sickle cell disease. METHODS Ten adults (age range, 16 to 45 years) with severe sickle cell disease underwent nonmyeloablative transplantation with CD34+ peripheral-blood stem cells, mobilized by granulocyte colony-stimulating factor (G-CSF), which were obtained from HLA-matched siblings. The patients received 300 cGy of total-body irradiation plus alemtuzumab before transplantation, and sirolimus was administered afterward. RESULTS All 10 patients were alive at a median follow-up of 30 months after transplantation (range, 15 to 54). Nine patients had long-term, stable donor lymphohematopoietic engraftment at levels that sufficed to reverse the sickle cell disease phenotype. Mean (+/-SE) donor-recipient chimerism for T cells (CD3+) and myeloid cells (CD14+15+) was 53.3+/-8.6% and 83.3+/-10.3%, respectively, in the nine patients whose grafts were successful. Hemoglobin values before transplantation and at the last follow-up assessment were 9.0+/-0.3 and 12.6+/-0.5 g per deciliter, respectively. Serious adverse events included the narcotic-withdrawal syndrome and sirolimus-associated pneumonitis and arthralgia. Neither acute nor chronic GVHD developed in any patient. CONCLUSIONS A protocol for nonmyeloablative allogeneic hematopoietic stem-cell transplantation that includes total-body irradiation and treatment with alemtuzumab and sirolimus can achieve stable, mixed donor-recipient chimerism and reverse the sickle cell phenotype. (ClinicalTrials.gov number, NCT00061568.)

Prevalence of Neutropenia in the U.S. Population: Age, Sex, Smoking Status, and Ethnic Differences

Context Low neutrophil counts may be more common in certain ethnic groups and ages. Contribution When blood count data were analyzed from a national sample of presumably healthy persons, the authors found that neutrophil counts were lower and neutropenia was more prevalent in U.S. black persons compared with white persons. Smoking was associated with increased neutrophil counts, especially in white persons. Cautions Blood counts were measured only once and could have differed in a second measurement or changed over time. Implications Race and smoking status influence the number of blood neutrophils and should be taken into account when considering the need to evaluate abnormal counts. The Editors Neutrophils normally comprise most circulating leukocytes and are critical in providing antimicrobial activity against bacteria and fungi. An inverse quantitative relationship between neutrophil count and infection was established 4 decades ago in patients undergoing chemotherapy for acute leukemia (1). The risk for infection was highest when the neutrophil count was less than 0.5109 cells/L for a prolonged period. Risk is substantially reduced when the duration of neutropenia is shortened or when the neutrophil count is greater than 0.5109 cells/L. In healthy persons, reserves of neutrophils in bone marrow greatly outnumber circulating neutrophils, which implies that isolated minor reductions in peripheral neutrophils should not lead to increased risk for opportunistic infection (2). Asymptomatic or benign reductions in neutrophils are observed in individuals of all ethnic backgrounds but may be more common in those of African descent. Benign ethnic neutropenia has been described in Africans (3, 4), African-Caribbean persons (5), West Indians (6), Ethiopians, Yemenite Jews (7, 8), and certain Arabs (7). Prevalence estimates range from 10% to more than 30%. Asymptomatic leukopenia in African-American workmen was described in 1941 (9) and has subsequently been described in larger cohorts of persons in the United States (1013). Extensive evaluations of these neutropenic persons showed low neutrophil counts with normal subpopulations of lymphocytes and other leukocytes, normal bone marrow morphologic features and cellularity, and no increased risk for local or systemic infections (1417). Because previous reports describing neutropenia in African-American persons included only military personnel (12, 13) or patients having elective surgeries (10), or were limited to a single geographic location (10, 18), these findings have limited general applicability. In addition, there is no clear consensus regarding the definition of neutropenia; therefore, its prevalence in the United States is unknown. The National Cancer Institute has established a neutrophil count of 1.5109 cells/L as the threshold for neutrophil toxicity (19). As a result, this threshold has been adopted as the minimum count required to initiate or continue treatment in many therapeutic clinical trials, and a neutrophil count less than 1.5109 cells/L has become the commonly accepted definition of neutropenia. Although reports on neutrophil and granulocyte counts in the U.S. population exist (20, 21), we are unaware of any detailed analysis regarding the population-based prevalence of neutropenia. We used data from the 1999 to 2004 National Health and Nutritional Examination Survey (NHANES) to describe age-, sex-, ethnicity-, and smoking-related differences in blood counts in the United States, focusing on the neutrophil counts in black persons. Methods The NHANES consisted of an interview, an examination, and laboratory data that were collected from a complex, multistage, stratified, and clustered probability sample of civilian, noninstitutionalized persons. During the 1999 to 2004 examinations, black persons, Mexican-American persons, and all persons 15 to 19 years of age and older than 60 years of age were oversampled to ensure accurate estimates for these groups (22). Of the 29608 persons 1 year of age or older who were asked to come to the mobile examination centers, 1668 did not report to the centers and 2715 had missing hematologic laboratory values. After 3 additional persons were excluded because of unrealistic values, there were 25222 participants with complete blood counts and differentials. Smoking data were available for participants who were age 20 years or older for the first 4 of the 6 years of the study. Current smokers were defined as participants who reported smoking occasionally or every day during the past 7 days. We collected 3 mL or 5 mL K3 EDTA anticoagulated whole blood from all persons who were 1 year of age or older by using established venipuncture protocols and procedures. Complete blood counts were performed in duplicate on a Coulter MAXM counter (Beckman Coulter, Miami, Florida) (22). All analyses used weighted samples and considered the stratification and clustering of the design to derive estimates that were applicable to the U.S. population (22). To provide estimates for the entire 6 years, a 6-year, weight-variable sample was created by taking two thirds for the 4-year weight for each person who was sampled in 1999 to 2002 and one third for the 2-year weight for each person who was sampled in 2003 to 2004. All analyses were conducted in SUDAAN, version 9.0.1 (Research Triangle Institute, Research Triangle Park, North Carolina [23]) and SAS, version 9.1.3 (SAS Institute, Inc., Cary, North Carolina). Nonadjusted frequency distribution of leukocyte and neutrophil counts and nonadjusted prevalence of neutropenia regarding age groups, sex, and ethnicity were obtained. These age groups were commonly used in highly stratified NHANES analyses (21, 22). We obtained means and comparisons of age-adjusted and sex-adjusted blood counts by using linear regression, in which age is modeled as a continuous variable. Adjusted means in leukocyte and neutrophil counts in smokers and nonsmokers were compared. Logistic regression adjusting for age group and sex was then used to generate predictive marginals for having neutropenia. More complex models, which involved interaction between ethnicity and sex as well as between ethnicity and age (as a continuous variable), were also tested and different categorizations of age were considered. These models did not substantially add to the explanatory power of the model. Results The 25222 NHANES participants with valid hematologic indices represented 253.2 million noninstitutionalized residents of the United States. Among 2715 participants with missing data, 35% were white, 30% were black, and 26% were Mexican American; children 5 years of age or younger accounted for 35%, those 6 to 18 years of age accounted for 43%, and adults 18 years of age or older accounted for 32%. We compared age-adjusted and sex-adjusted mean blood counts across the major ethnic groups by using linear regression. Relative to white participants, black participants had lower mean leukocyte counts (mean difference, 0.89109/L; P< 0.001), lower neutrophil counts (0.83109 cells/L; P< 0.001), and similar lymphocyte counts (0.022109 cells/L; P= 0.36). Mexican-American participants had slightly higher mean leukocyte counts (0.16109 cells/L; P= 0.014), higher neutrophil counts (0.11109 cells/L; P= 0.026), and higher lymphocyte counts (0.095109 cells/L; P< 0.001). In addition, leukocyte, neutrophil, and lymphocyte counts were lower in males than in females (Table 1). Appendix Tables 1 and 2 summarize other basic hematologic variables in the 3 major ethnic groups for sex and for the 13 age groups. Table 1. Mean Hematologic Values, by Age, Sex, and Ethnic Group* Appendix Table 1. Mean Hematologic Values, by Age, Sex, and Ethnic Group* Appendix Table 2. Mean Hematologic Values in 3 Major Ethnic Groups, by Age* For participants who were age 18 years or older, the distribution of leukocyte and neutrophil counts suggested a downward shift of approximately 1.0109 cells/L among black persons compared with white persons and Mexican-American persons (Figure 1; Appendix Table 3). There was a slight upward shift for leukocyte counts among Mexican-American participants, but little difference for neutrophil counts. Similar shifts were seen in the distribution of leukocyte and neutrophil counts in participants who were younger than age 18 years (data not shown). Figure 1. Distribution of leukocyte ( top ) and neutrophil ( bottom ) counts in persons age 18 years or older from 3 ethnic groups. Appendix Table 3 shows the actual percentages and number of participants. Appendix Table 3. Number and Proportion of Participants, by Distribution of Leukocyte and Neutrophil Count Mean age-adjusted and sex-adjusted leukocyte and neutrophil counts were compared for smokers and nonsmokers (Table 2). The overall mean leukocyte and neutrophil counts were higher in smokers than in nonsmokers (1.38109 cells/L and 0.87109 cells/L, respectively). Smoking had the greatest effect on increments of neutrophil counts in white participants (0.87109 cells/L), less of an effect in black participants (0.50109 cells/L), and the least effect in Mexican-American participants (0.41109 cells/L). Table 2. Comparative Mean Leukocyte and Neutrophil Counts in Smokers and Nonsmokers Age 20 Years or Older The prevalence of neutropenia (neutrophil count <1.5109 cells/L) differed by age, sex, and ethnicity (Figure 2; Appendix Table 4). A total of 583 participants had neutrophil counts less than 1.5109 cells/L. The weighted prevalence was 1.2% (95% CI, 1.1% to 1.4%), which represented an estimated 3.1 million persons in the United States. Neutropenia was present in 4.5% (CI, 3.9% to 5.0%) of black participants, 0.79% (CI, 0.57% to 1.0%) of white participants, and 0.38% (CI, 0.24% to 0.52%) of Mexican-American participants. Across ethnic groups, males were more likely to have neutropenia: 6.65% for black males versus 3.57% for black females, 0.90% for white males versus 0.59% for white females, and 0

Nonmyeloablative HLA-Matched Sibling Allogeneic Hematopoietic Stem Cell Transplantation for Severe Sickle Cell Phenotype

IMPORTANCE Myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) is curative for children with severe sickle cell disease, but toxicity may be prohibitive for adults. Nonmyeloablative transplantation has been attempted with degrees of preparative regimen intensity, but graft rejection and graft-vs-host disease remain significant. OBJECTIVE To determine the efficacy, safety, and outcome on end-organ function with this low-intensity regimen for sickle cell phenotype with or without thalassemia. DESIGN, SETTING, AND PARTICIPANTS From July 16, 2004, to October 25, 2013, 30 patients aged 16-65 years with severe disease enrolled in this nonmyeloablative transplant study, consisting of alemtuzumab (1 mg/kg in divided doses), total-body irradiation (300 cGy), sirolimus, and infusion of unmanipulated filgrastim mobilized peripheral blood stem cells (5.5-31.7 × 10(6) cells/kg) from human leukocyte antigen-matched siblings. MAIN OUTCOMES AND MEASURES The primary end point was treatment success at 1 year after the transplant, defined as a full donor-type hemoglobin for patients with sickle cell disease and transfusion independence for patients with thalassemia. The secondary end points were the level of donor leukocyte chimerism; incidence of acute and chronic graft-vs-host disease; and sickle cell-thalassemia disease-free survival, immunologic recovery, and changes in organ function, assessed by annual brain imaging, pulmonary function, echocardiographic image, and laboratory testing. RESULTS Twenty-nine patients survived a median 3.4 years (range, 1-8.6), with no nonrelapse mortality. One patient died from intracranial bleeding after relapse. As of October 25, 2013, 26 patients (87%) had long-term stable donor engraftment without acute or chronic graft-vs-host disease. The mean donor T-cell level was 48% (95% CI, 34%-62%); the myeloid chimerism levels, 86% (95% CI, 70%-100%). Fifteen engrafted patients discontinued immunosuppression medication with continued stable donor chimerism and no graft-vs-host disease. The normalized hemoglobin and resolution of hemolysis among engrafted patients were accompanied by stabilization in brain imaging, a reduction of echocardiographic estimates of pulmonary pressure, and allowed for phlebotomy to reduce hepatic iron. The mean annual hospitalization rate was 3.23 (95% CI, 1.83-4.63) the year before, 0.63 (95% CI, 0.26-1.01) the first year after, 0.19 (95% CI, 0-0.45) the second year after, and 0.11 (95% CI, 0.04-0.19) the third year after transplant. For patients taking long-term narcotics, the mean use per week was 639 mg (95% CI, 220-1058) of intravenous morphine-equivalent dose the week of their transplants and 140 mg (95% CI, 56-225) 6 months after transplant. There were 38 serious adverse events: pain and related management, infections, abdominal events, and sirolimus related toxic effects. CONCLUSIONS AND RELEVANCE Among 30 patients with sickle cell phenotype with or without thalassemia who underwent nonmyeloablative allogeneic HSCT, the rate of stable mixed-donor chimerism was high and allowed for complete replacement with circulating donor red blood cells among engrafted participants. Further accrual and follow-up are required to assess longer-term clinical outcomes, adverse events, and transplant tolerance. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00061568.

Allogeneic hematopoietic stem cell transplantation for sickle cell disease: the time is now

Although sickle cell disease (SCD) has a variable clinical course, many patients develop end-organ complications that are associated with significant morbidity and early mortality. Myeloablative allogeneic HSCT (allo-HSCT) is curative but has been historically performed only in children younger than 16 years of age. Modest modifications in the conditioning regimen and supportive care have improved outcome such that the majority of children with a suitable HLA-matched sibling donor can expect a cure from this approach. However, adult patients have been excluded from myeloablative allo-HSCT because of anticipated excess toxicity resulting from accumulated disease burden. Efforts to use nonmyeloablative transplantation strategies in adults logically followed but were initially met with largely disappointing results. Recent results, however, indicate that nonmyeloablative allo-HSCT in adult patients with SCD allows for stable mixed hematopoietic chimerism with associated full-donor erythroid engraftment and normalization of blood counts, and persistence in some without continued immunosuppression suggests immunologic tolerance. The attainment of tolerance should allow extension of these potentially curative approaches to alternative donor sources. Efforts to build on these experiences should increase the use of allo-HSCT in patients with SCD while minimizing morbidity and mortality.

Side population cells derived from adult human liver generate hepatocyte-like cells in vitro.

We sought to determine whether hepatic side population (SP) cells derived from adult human liver possess the potential of a novel candidate hepatic stem cell. Human cadaveric donor liver was subjected to collagenase perfusion and hepatocytes were separated from nonparenchymal cells by differential centrifugation. SP cells were isolated from the nonparenchymal portion after Hoechst 33342 staining. Since CD45 is a panleukocyte antigen, CD45-negative SP cells were separated from the vast majority of CD45-positive SP cells (90%), and hepatic growth medium was used to culture both groups. Both CD45-negative and CD45-positive hepatic SP cells generated colonies in the hepatic growth medium in 2–3 weeks. The colonies yielded large cells morphologically consistent with human hepatocytes, demonstrating granule-rich cytoplasm, dense, often double nuclei, and intracellular lipofuscin pigment. The cultured cells from both sources were positive for markers of human hepatocytes: HepPar, cytokeratin 8 (CK8), and human albumin. Reverse transcriptase–polymerase chain reaction (RT-PCR) performed on both groups demonstrated positivity for additional liver markers including human albumin, CK18, α-1 anti-trypsin, and the human cytochrome P450 enzyme CYP2B6. Double immunostaining (CD45 and HepPar) and RT-PCR confirmed that the hepatocyte-like cells derived from the CD45-negative SP cells acquired HepPar positivity but had no detectable CD45 antigen expression. In contrast, the cultured cells derived from the CD45-positive SP cells also acquired HepPar positivity, but only a minimal fraction expressed the CD45 antigen. We conclude that hepatic SP cells derived from the nonparenchymal portion of human liver are a potential source of human hepatocytes irrespective of their CD45 status, and further animal studies will be required to assess their regenerative potential.

Granulocyte Colony-Stimulating Factor (G-CSF) Administration in Individuals with Sickle Cell Disease: Time for a Moratorium?

Granulocyte colony-stimulating factor (G-CSF) is used commonly in an attempt to reduce the duration of neutropenia and hospitalization in patients undergoing chemotherapy and to obtain hematopoietic stem cells (HSC) for transplantation applications. Despite the relative safety of administration of G-CSF in most individuals, including subjects with sickle cell trait, severe and life-threatening complications have been reported when used in individuals with sickle cell disease (SCD), including those who were asymptomatic and undiagnosed prior to administration. The administration of G-CSF has now been reported in a total of 11 individuals with SCD. Seven developed severe adverse events, including vaso-occlusive episodes, acute chest syndrome, multi-organ system failure and death. Precautions, including minimizing the peak white blood cell count, dividing or reducing the G-CSF dose and red blood cell transfusions to reduce sickle hemoglobin (HbS) levels, have been employed with no consistent benefit. These reported data indicate that administration of G-CSF in individuals with SCD should be undertaken only in the absence of alternatives and after full disclosure of the risks involved. Unless further data demonstrate safety, routine usage of G-CSF in individuals with SCD should be avoided.

Modeling tumor–host interactions of chronic lymphocytic leukemia in xenografted mice to study tumor biology and evaluate targeted therapy

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental factors for proliferation and survival. In particular, the B-cell receptor (BCR) and nuclear factor- κB (NF-κB) pathways are activated in the lymph node (LN) microenvironment. Thus, model systems mimicking tumor–host interactions are important tools to study CLL biology and pathogenesis. We investigated whether the recently established NOD/scid/γcnull (NSG) mouse xenograft model can recapitulate the effects of the human microenvironment. We assessed, therefore, tumor characteristics previously defined in LN-resident CLL cells, including proliferation, and activation of the BCR and NF-κB pathways. We found that the murine spleen (SP) microenvironment supported CLL cell proliferation and activation to a similar degree than the human LN, including induction of BCR and NF-κB signaling in the xenografted cells. Next, we used this model to study ibrutinib, a Bruton’s tyrosine kinase inhibitor in clinical development. Ibrutinib inhibited BCR and NF-κB signaling induced by the microenvironment, decreased proliferation, induced apoptosis and reduced the tumor burden in vivo. Thus, our data demonstrate that the SP of xenografted NSG mice can, in part, recapitulate the role of the human LN for CLL cells. In addition, we show that ibrutinib effectively disrupts tumor–host interactions essential for CLL cell proliferation and survival in vivo.

Development of a Human Immunodeficiency Virus Type 1-Based Lentiviral Vector That Allows Efficient Transduction of both Human and Rhesus Blood Cells

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5α and APOBEC3G, which target HIV-1 capsid and viral infectivity factor (Vif), respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV), including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (χHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34+ hematopoietic stem cells (HSCs), the χHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34+ HSCs, the χHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques, the χHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus, 15 to 30% versus 1 to 5%; second rhesus, 7 to 15% versus 0.5 to 2%, respectively) 3 to 7 months postinfusion. In summary, we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application.

Mixed haematopoietic chimerism for sickle cell disease prevents intravascular haemolysis.

Recent studies suggest that persistent intravascular haemolysis, a defining feature of sickle cell disease (SCD), is associated with severe long-term consequences, including pulmonary hypertension, which carries a 2-year mortality rate of up to 50% (Castro et al, 2003). Thus, the development of therapies that correct intravascular haemolysis and its complications are increasingly recognized as important for the long-term prognosis of the SCD patient. Haematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for SCD, and recent improvements in supportive care and the development of reduced-intensity conditioning regimens have substantially lessened the severity of the immediate toxicities of the transplant procedure (Locatelli, 2006). When lower intensity conditioning regimens are utilized, host haematopoiesis is incompletely eliminated, and mixed haematopoietic chimerism frequently results. The effects of persistent recipient erythro-poiesis on intravascular haemolysis are unknown, but would be anticipated to perpetuate intravascular haemolysis. Several serum biomarkers have been recently identified to strongly correlate with endothelial damage, pulmonary hypertension and prospective early mortality in SCD patients. These include increased soluble vascular cellular adhesion molecule 1 (sVCAM-1) levels, increased nitric oxide (NO) consumption, increased plasma free haemoglobin (Hb), and inverted argi-nine/ornithine ratio (Solovey et al, 1997; Reiter et al, 2002; Morris et al, 2005). Using these parameters, we examined the potential of partial donor engraftment to correct intravascular haemolysis in nine SCD patients, and hence to potentially avert the development of long-term SCD-associated complications. Of the 19 patients who underwent matched related donor HSCT for SCD across four transpalnt centres, each with a different conditioning regimen, nine developed mixed haematopoietic chimerism and were studied in detail. Heparinized blood was obtained from these nine patients upon enrolment onto Institutional Review Board-approved protocols. Overall mononuclear cell engraftment for all nine patients was measured by standard analysis of short tandem repeats from genomic DNA extracted from peripheral blood mononuclear cells. Indications for HSCT ranged from acute chest syndrome and strokes to intractable recurrent pain crises. As shown in Table I, patients 1–7 and 9 underwent nonmyeloablative transplant, while patient 8 underwent a fully myeloablative transplant. For patients 1–3, this period of partial donor engraftment, with levels ranging from 20% to 50%, lasted 5–11 months following HSCT. For patients 4–9, stable mixed chimerism was achieved with levels ranging from 42% to 90%. For patients 4–8, mixed chimerism was maintained even off immunosuppression. Patients 7 and 8 continue to stably demonstrate 55% and 42% peripheral blood donor engraftment, respectively, >4 years following transplant. Patient 9 has been continuously maintained on immunosuppression, and remains a mixed chimera. None of the subjects required red blood cell (RBC) transfusion beyond 1 month post-transplant unless they relapsed and none, while engrafted, experienced any SCD-related events. Table I Patient clinical characteristics. Despite differences in the conditioning regimens, all patients similarly demonstrated dramatic improvements in parameters of intravascular haemolysis while partially donor-engrafted. The median pretransplant values for indices of intravascular haemolysis of our cohort were elevated, similar to the general SCD population (Reiter et al, 2002). Following transplantation, significant normalization of these parameters was observed for each patient, to levels similar to normal donors. As shown in Fig 1A, lactate dehydrogenase (LDH) and free Hb concentration decreased from a median of 375 U/l (range: 255–1699) to 184 U/l (range: 122–260; P = 0.02), and from 19.7 μmol/l (range 5.5–75.9) to 1.6 μmol/l (range: 0.2–7.3; P = 0.02) respectively. Haptoglobin increased from a median value of 50 mg/l (range: 50–200) to 330 mg/l (range: 70–2420; P = 0.02). Fig 1 (A) Pre- and post-transplant plasma values for haptoglobin; LDH; free Hb concentration; nitric oxide (NO) consumption; sVCAM-1; and arginine/ornithine ratio. Median levels for each parameter are represented by the black filled diamonds. The dotted line ... Parameters of vascular function post-transplant similarly improved. Plasma NO consumption and sVCAM-1 levels significantly decreased from a median of 7.6 μmol/l (range: 1.9–49.7) to 1.3 μmol/l (range: 0.6–8.6; P = 0.02), and 952.1 ng/ml (range: 498.2–1067) to 543.1 ng/ml (range: 193.9–724; P = 0.02) respectively. The median arginine/ornithine ratio increased from 0.6 (range: 0.1–1.5) to 1.8 (range: 0.6–2.3; P = 0.06) following HSCT. The observed improvements in parameters of intravascular haemolysis and endothelial function were not related to presence of immunosuppression, which is routinely administered in the early post-transplant period as graft-vs.-host disease prophylaxis. Patients 4 and 6 maintained unchanged or improved levels of donor chimerism and extent of intravascular haemolysis and vascular function, both on and off immunosuppression (Fig 1B). We had sufficient samples from two of the three patients who experienced graft rejection (patients 1 and 3) to analyse in detail the impact of absence or presence of donor erythropoiesis on intravascular haemolysis. Temporary normalization of these parameters occurred with the presence of donor haematopoiesis, with reversion to abnormal baseline values following loss of the donor graft (Fig 1B). Direct indices of intravascular haemolysis (haptoglobin and total bilirubin) were the first to revert to abnormal, coincident with loss of donor engraftment. Free Hb concentration and NO consumption subsequently rose, consistent with the loss of the haemoglobin binding and clearance capacity of haptoglobin. Finally, a few months after graft rejection, a slight rise in LDH was observed. These findings directly demonstrate the dependence upon donor cells to achieve improvement in intravascular haemolysis and potentially on vascular function. To reconcile these results in the face of persistent recipient haematopoiesis, erythroid lineage-specific chimerism was analysed by RNA β-globin pyrosequencing. This method evaluates chimerism by quantitative sequencing of donor vs. host-derived erythroid lineage transcripts, based on detection of the sickle mutation (Wu et al, 2005). Peripheral blood RBCs were fully replaced by donor-derived erythrocytes for patients with at least 50% donor nucleated cell engraftment (Table I). For patients with <50% engraftment, 70–85% donor RBCs expression was observed. Post-transplant donor RBC enrichment relative to the degree of nucleated cell engraftment is probably related to factors such as decreased survival of SS erythrocytes and intrinsic SCD-associated ineffective erythropoiesis (Kean et al, 2003; Wu et al, 2005). Fig 1B shows that normalization of the parameters of haemolysis and endothelial dysfunction was coincidental with expression of donor RBCs, thus directly demonstrating the beneficial effect of donor erythrocytes. Although patients 1, 3 and 8 continued to produce recipient RBCs, which would be expected to perpetuate intravascular haemolysis, this degree of haemolysis is apparently within the buffering capacity of the system. Supporting these findings, RBC exchange and hyper-transfusion that reduce peripheral blood %HbS to 20–30% are clinically protective (Swerdlow, 2006). Taken together, our data mechanistically support mixed chimerism as a suitable endpoint of stem cell-based therapies for SCD.

Busulfan Produces Efficient Human Cell Engraftment in NOD/LtSz-Scid IL2RγNull Mice†‡§

Xenografting immunodeficient mice after low‐dose irradiation has been used as a surrogate human hematopoietic stem cell (HSC) assay; however, irradiation requires strict and meticulous animal support and can produce significant mortality rates, limiting the usefulness of this model. In this work, we examined the use of parenteral busulfan as an alternative conditioning agent. Busulfan led to dose‐dependent human HSC engraftment in NOD/LtSz‐scid/IL2Rγnull mice, with marked improvement in survival rates. Terminally differentiated B and T lymphocytes made up most of the human CD45+ cells observed during the initial 5 weeks post‐transplant when unselected cord blood (CB) products were infused, suggesting derivation from existing mature elements rather than HSCs. Beyond 5 weeks, CD34+‐enriched products produced and sustained superior engraftment rates compared with unselected grafts (CB CD34+, 65.8% ± 5.35%, vs. whole CB, 4.27% ± 0.67%, at 24 weeks). CB CD34+ group achieved significantly higher levels of engraftment than mobilized CD34+‐enriched peripheral blood (PB CD34+). At 8 weeks, all leukocyte subsets were detected, yet human red blood cells (RBCs) were not observed. Transfused human red cells persisted in the chimeric mice for up to 3 days; an accompanying rise in total bilirubin suggested hemolysis as a contributing factor to their clearance. Recipient mouse‐derived human HSCs had the capacity to form erythroid colonies in vitro at various time points post‐transplant in the presence of human transferrin (Tf). When human Tf was administered singly or in combination with anti‐CD122 antibody and human cytokines, up to 0.1% human RBCs were detectable in the peripheral blood. This long evasive model should prove valuable for the study of human erythroid cells. STEM CELLS 2009;27:175–182