John M. Cunningham

Outcomes of transplantation with matched-sibling and unrelated-donor bone marrow in children with leukaemia

BACKGROUNDnFor most conditions amenable to bone-marrow transplantation, grafts from HLA-matched but unrelated donors have yielded poorer results than those obtained from matched-sibling donors. We assessed this pattern in the light of improvements in donor selection and post-transplant supportive care.nnnMETHODSnWe reviewed transplant outcome in 103 consecutive patients with childhood leukaemia who underwent allogeneic bone-marrow transplantation with HLA-matched sibling marrow (n = 52) or matched unrelated donor marrow (n = 51) between May, 1990, and March, 1996, at St Jude Childrens Research Hospital.nnnFINDINGSnAnalysis of engraftment, frequency of procedure-related complications, and disease-free survival revealed no advantage from use of matched-sibling marrow. The 2-year disease-free survival estimate for standard-risk recipients of matched-sibling marrow was 81 [8.1]% compared with 73 [12.1]% in the unrelated donor marrow group (p = 0.77). In the high-risk category, patients with a matched-sibling donor had a 2-year disease-free survival of 31 [11.6]%, compared with 32 [15.1]% among recipients of matched unrelated donor marrow (p = 0.87).nnnINTERPRETATIONnWe believe this improved result with unrelated donor marrow is a consequence of recent innovations in histocompatibility matching, prevention of graft-versus-host disease (GvHD), and antiviral prophylaxis. We suggest that such grafts can now be used in patients at both standard and high risk without compromising treatment outcome.

Inositol- and folate-resistant neural tube defects in mice lacking the epithelial-specific factor Grhl-3

The neural tube defects (NTDs) spina bifida and anencephaly are widely prevalent severe birth defects. The mouse mutant curly tail (ct/ct) has served as a model of NTDs for 50 years, even though the responsible genetic defect remained unrecognized. Here we show by gene targeting, mapping and genetic complementation studies that a mouse homolog of the Drosophila grainyhead (grh) gene, grainyhead-like-3 (Grhl3), is a compelling candidate for the gene underlying the curly tail phenotype. The NTDs in Grhl3-null mice are more severe than those in the curly tail strain, as the Grhl3 alleles in ct/ct mice are hypomorphic. Spina bifida in ct/ct mice is folate resistant, but its incidence can be markedly reduced by maternal inositol supplementation periconceptually. The NTDs in Grhl3−/− embryos are also folate resistant, but unlike those in ct/ct mice, they are resistant to inositol. These findings suggest that residual Grhl3 expression in ct/ct mice may be required for inositol rescue of folate-resistant NTDs.

A highly conserved novel family of mammalian developmental transcription factors related to Drosophila grainyhead

The Drosophila transcription factor Grainyhead regulates several key developmental processes. Three mammalian genes, CP2, LBP-1a and LBP-9 have been previously identified as homologues of grainyhead. We now report the cloning of two new mammalian genes (Mammalian grainyhead (MGR) and Brother-of-MGR (BOM)) and one new Drosophila gene (dCP2) that rewrite the phylogeny of this family. We demonstrate that MGR and BOM are more closely related to grh, whereas CP2, LBP-1a and LBP-9 are descendants of the dCP2 gene. MGR shares the greatest sequence homology with grh, is expressed in tissue-restricted patterns more comparable to grh and binds to and transactivates the promoter of the human Engrailed-1 gene, the mammalian homologue of the key grainyhead target gene, engrailed. This sequence and functional conservation indicates that the new mammalian members of this family play important developmental roles.

The identification and characterization of human Sister-of-Mammalian Grainyhead (SOM) expands the grainyhead-like family of developmental transcription factors

The Drosophila gene grainyhead is the founding member of a large family of genes encoding developmental transcription factors that are highly conserved from fly to human. The family consists of two main branches, with grainyhead as the ancestral gene for one branch and the recently cloned Drosophila CP2 as the ancestral gene for the other. We now extend this family with the identification of another novel mammalian member, Sister-of-Mammalian Grainyhead (SOM), which is phylogenetically aligned with grainyhead. SOM is closely related to the other mammalian homologues of grainyhead, including Mammalian Grainyhead (MGR) and Brother-of-MGR, sharing a high degree of sequence identity with these factors in the functional DNA-binding, protein dimerization and activation domains. Protein interaction studies demonstrate that SOM can heterodimerize with MGR and Brother-of-MGR, but not with the more distant members of the family. Like grainyhead, the SOM gene too produces several distinct isoforms with differing functional properties through alternative splicing. The tissue distributions of these isoforms differ and all display highly restricted expression patterns. These findings indicate that SOM, like its family members, may play important roles in mammalian development.

Hemorrhagic Cystitis after Allogeneic Bone Marrow Transplantation in Children: Clinical Characteristics and Outcome

Hemorrhagic cystitis (HC) is a well-documented adverse event experienced by patients undergoing hematopoietic stem cell transplantation. When severe, HC causes significant morbidity, leads to renal complications, prolongs hospitalization, increases health-care costs, and occasionally contributes to death. We retrospectively studied the medical records of 245 children undergoing an initial allogeneic bone marrow transplantation for malignant disease at St. Jude Childrens Research Hospital between 1992 and 1999 to describe the clinical course of HC in all patients and to identify the risk factors for HC in this cohort. Conditioning regimens included cyclophosphamide, cytarabine, and total body irradiation. Grafts from unrelated or mismatched related donors were depleted of T lymphocytes, whereas matched sibling grafts were unmanipulated. All patients received cyclosporine as prophylaxis for graft-versus-host disease. Recipients of grafts from matched siblings also received pentoxifylline or short-course methotrexate. Severe HC developed in 27 patients (11.0%). The median duration of HC was 73 days (range, 5-619 days); 12 patients had ongoing HC at the time of death. In univariate analyses, patients were at increased risk of severe HC if they were male (P =.021) or had received T cell-depleted grafts (P =.017), grafts from unrelated donors (P =.021), a lower total nucleated cell dose (P =.032), or antithymocyte globulin (P =.0446). Multiple regression analysis revealed male sex (beta =.97; P =.027) and unrelated donor graft recipients (beta =.83; P =.039) to be significant factors.

Brain parenchymal damage after haematopoietic stem cell transplantation for severe sickle cell disease

Prospective magnetic resonance imaging (MRI), magnetic resonance angiography (MRA), neuropsychological testing and neurological examinations were performed to determine the long‐term effect of successful haematopoietic stem cell transplantation on the neurological status of nine children with sickle cell disease. A scoring system for severity of brain parenchymal and vascular lesions was developed and applied. Neurological examinations and neuropsychometric tests were stable, but MRI and MRA studies were not. Transient changes occurred early in two patients. Persistent changes occurred in five. Parenchymal lesions occurred in zero of two patients without prior lacunae or infarcts and in all seven with prior lacunae or infarcts (Pu2003=u20030·0278).

BMP4-dependent expression of Xenopus Grainyhead-like 1 is essential for epidermal differentiation.

Morphogen-dependent epidermal-specific transacting factors have not been defined in vertebrates. We demonstrate that a member of the grainyhead transcription factor family, Grainyhead-like 1 (XGrhl1) is essential for ectodermal ontogeny in Xenopus laevis. Expression of this factor is restricted to epidermal cells. Moreover, XGrhl1 is regulated by the BMP4 signaling cascade. Disruption of XGrhl1 activity in vivo results in a severe defect in terminal epidermal differentiation, with inhibition of XK81A1 epidermal keratin gene expression, a key target of BMP4 signaling. Furthermore, transcription of the XK81A1 gene is modulated directly by binding of XGRHL1 to a promoter-localized binding motif that is essential for high-level expression. These results establish a novel developmental role for XGrhl1 as a crucial tissue-specific regulator of vertebrate epidermal differentiation.

Outcome of hematopoietic stem cell transplantation for pediatric patients with therapy‐related acute myeloid leukemia or myelodysplastic syndrome

Therapy‐related myelodysplastic syndrome (t‐MDS) and acute myeloid leukemia (t‐AML) carry a poor prognosis. We analyzed the results of allogeneic HSCT in 38 children to determine which factors, if any, affected outcome.

The role of p22 NF-E4 in human globin gene switching.

The human stage selector protein, a complex containing the ubiquitous transcription factor CP2 and the erythroid-specific factor p22 NF-E4, facilitates the interaction of the γ-globin genes with the locus control region in fetal erythroid cells. Enforced expression of p22 NF-E4 in K562 cells and human cord blood progenitors increases fetal globin gene expression, and in progenitors, reduces β-globin expression. To examine the role of NF-E4 in an in vivo model of hemoglobin switching, we enforced the expression of p22 NF-E4 in transgenic mice carrying the human β-globin locus yeast artificial chromosome. Although murine erythropoiesis and globin gene expression is unaffected in these mice, the expression profile of the human globin genes is altered. All three transgenic lines displayed an increased γ:β-globin ratio in E12.5–14.5 fetal liver, resulting in a delay in the fetal/adult switch. At E12.5, this is primarily due to a reduction of β-gene expression, whereas at E14.5, the increased γ:β ratio is due to enhanced γ-gene expression. Despite this, the switch in globin subtype is fully completed in the adult bone marrow. These findings indicate that p22 NF-E4 is capable of influencing human globin gene expression in vivo but is incapable of overriding the intrinsic mechanisms governing γ-gene silencing in this context.

Improved cerebrovascular patency following therapy in patients with sickle cell disease: Initial results in 4 patients who received HLA-identical hematopoietic stem cell allografts

To test whether magnetic resonance angiography can document the evolution of vasculopathy in patients with sickle cell disease, we reviewed records to identify all patients who underwent magnetic resonance angiography from 1993 to 1999. Of 512 angiographies performed, 105 were of sickle cell disease patients, and 24 sickle cell disease patients 7 years of age or older underwent baseline and follow‐up examinations. Films were paired by patient, blinded as to examination date and treatment, and quantitatively compared. Four patients who received allogeneic bone marrow transplantation were compared to 7 patients who received other therapy and to 13 untreated patients. Quantitative analysis revealed a 10% increase in the measured diameter of 64 vessels (p = 0.001) following any treatment. Patients who had undergone allogeneic bone marrow transplantation exhibited a 12% increase in the lumen of 22 vessels (p = 0.041), whereas patients treated with chronic transfusion or hydroxyurea exhibited an 8% increase in 42 vessels (p = 0.016). In 2 patients with severe stenosis, the artery normalized after transplantation, and the blood flow rate was reduced in all patients who underwent transplantation. In untreated patients, there was a trend for the size of the arterial lumen to decrease, which is consistent with disease progression. Results suggest that treatment can reverse progression of vasculopathy. Bone marrow transplantation may enable stenoses to heal and can substantially reduce cranial blood velocity, suggesting that allogeneic bone marrow transplantation may prevent infarction or brain damage. Ann Neurol 2001;49:222–229