David D. Martin

Transplantation of a combination of CD133+ and CD34+ selected progenitor cells from alternative donors.

Positive selected haematopoietic stem cells are increasingly used for allogeneic transplantation with the CD34 antigen employed in most separation techniques. However, the recently described pentaspan molecule CD133 appears to be a marker of more primitive haematopoietic progenitors. Here we report our experience with a new CD133‐based selection method in 10 paediatric patients with matched unrelated (n = 2) or mismatched‐related donors (n = 8). These patients received a combination of stem cells (median = 29·3 × 106/kg), selected with either anti‐CD34 or anti‐CD133 coated microbeads. The proportion of CD133+ selected cells was gradually increased from patient to patient from 10% to 100%. Comparison of CD133+ and CD34+ separation procedures revealed similar purity and recovery of target populations but a lower depletion of T cells by CD133+ selection (3·7 log vs. 4·1 log, P < 0·001). Both separation procedures produced >90% CD34+/CD133+ double positive target cells. Engraftment occurred in all patients (sustained primary, n = 8; after reconditioning, n = 2). No primary acute graft versus host disease (GvHD) ≥ grade II or chronic GvHD was observed. The patients showed a rapid platelet recovery (median time to independence from substitution = 13·5 d), whereas T cell regeneration was variable. Five patients are alive with a median follow‐up of 10 months. Our data demonstrates the feasibility of CD133+ selection for transplantation from alternative donors and encourages further trials with total CD133+ separated grafts.

Clinical grade generation of hexon-specific T cells for adoptive T-cell transfer as a treatment of adenovirus infection after allogeneic stem cell transplantation.

Adenovirus infection after allogeneic hematopoietic stem cell transplantation is still causing significant morbidity and mortality, especially in children. It has been demonstrated that a sufficient host T-cell response is essential to clear the virus. Adoptive transfer of specific T-cell immunity from the donor to the recipient has become a new treatment option for patients with systemic adenoviral infection who lack specific T-cell responses. The adenoviral hexon protein was shown to be an immunodominant T-cell target. We describe here a Good Manufacturing Practice-compatible generation of hexon-specific T cells developed by isolating interferon-γ–secreting T cells after stimulation of mononuclear cells ex vivo with hexon protein. Phenotypical and functional characterization of the generated, specific T-cell product resulted in a mixed population of CD4 and CD8-positive T cells with an intermediate effector memory phenotype. Isolated hexon-specific T cells showed high expansion potential in vitro and specific cytotoxicity. T-cell lines, directed against type 5 hexon protein showed good crossreactivity against viral strains from other adenovirus species. The availability for isolation of hexon-specific T cells among 76 hematopoietic stem cell transplantation donors showed in >72% a sufficient T-cell response (0.05% of T cells). In conclusion, Good Manufacturing Practice-grade selection of adenovirus-specific T cells for adoptive immunotherapy by hexon-induced secretion of interferon-γ has been established. Adoptive T-cell transfer could potentially restore T-cell immunity against adenovirus after allogeneic stem cell transplantation.

The Endocrine Phenotype in Silver-Russell Syndrome Is Defined by the Underlying Epigenetic Alteration

CONTEXT Around 50% of children with Silver-Russell syndrome (SRS) carry a hypomethylation of the imprinting control region 1 at the IGF2/H19 locus on 11p15, the functional significance of which is unknown. OBJECTIVE We aimed to compare the genotype in SRS with the endocrine phenotype. DESIGN The retrospective study included all SRS children who were treated during the last 18 yr at our hospital and for comparison a cohort of GH treated nonsyndromic short children born small for gestational age (SGA). PATIENTS The 61 patients with SRS included were defined by the presence of intrauterine growth retardation, lack of catch-up growth, and at least two of the criteria: typical face, relative macrocephaly, and skeletal asymmetry. Routine karyotype and GH secretion was normal in all children studied. A subgroup of 53 patients was treated with GH. MATERIALS AND METHODS Genomic DNA was available from 44 children. Multiplex ligation probe-dependent amplification analysis was performed to detect hypomethylation at the imprinting control region 1 on 11p15. Uniparental disomy of chromosome 7 (UPD7) was analyzed by short tandem repeats typing. Serum levels of GH, IGF-I, and IGF-binding protein (IGFBP)-3 were measured by RIA. RESULTS Epimutations at 11p15 were found in 19 of 44, UPD7 in five of 44, and small structural aberrations of the short arm of chromosome 11 in two of 44 children. Of 44 cases, 18 were negative for any genetic defect known (41%). The most severe phenotype was found in children with 11p15-SRS. Children with UPD7-SRS had a significantly higher birth length (P < 0.004) but lost height sd score (SDS) postpartum, whereas children with 11p15-SRS showed no change in height SDS. IGF-I and IGFBP-3 serum levels were inadequately high in 11p15-SRS at -0.02 SDS (1.07, sd) and +1.38 SDS (1.01), compared with the low levels in UPD7-SRS and in the cohort of 58 nonsyndromic SGA children (P < 0.0009). During GH therapy, IGFBP-3 serum levels increased above normal values in 11p15-SRS (P < 10(-4)), whereas IGF-I increase was moderate. There was a trend toward more height gain in children with UPD7 than in those with 11p15 epimutation under GH therapy (+2.5 vs. +1.9 height SDS after 3 yr) (P = 0.08). CONCLUSIONS Children with SRS and an 11p15 epimutation have IGFBP-3 excess and show endocrine characteristics suggesting IGF-I insensitivity, whereas children with SRS and UPD7 were not different from nonsyndromic short children born SGA. This phenotype-genotype correlation implicates divergent endocrine mechanisms of growth failure in SRS.

The course of neonatal cholestasis in congenital combined pituitary hormone deficiency.

BACKGROUND Neonatal cholestatic hepatitis is frequently associated with congenital combined pituitary hormone deficiency (CCPHD). Data on the course of this hepatopathy are scarce. AIM We retrospectively analyzed the data of all CCPHD infants with cholestasis who presented at the University Childrens Hospital, Tuebingen. RESULTS All infants (n = 9; 2 females) presented with early and prolonged jaundice, failure to thrive and recurrent hypoglycemia. All males had micropenis and 3/7 cryptorchidism. Median age at diagnosis was 1.4 months. Cholestasis began at a median age of 13 days (range 5-31) and resolved at 88 days (54-174). Maximum direct bilirubin level was 6.9 mg/dl (2.4-11.6). Peaks of ALP (median 721 U/l), ALT (148 U/l) and AST (195 U/l) occurred 2-4 weeks later, while GGT levels were elevated in only two infants (167 U/l). Functional liver parameters were always normal. Liver biopsies (n = 4) showed canalicular cholestasis and mild portal eosinophilic infiltration. TEBIDA radioisotope excretion into the intestinal tract was blocked. Substitution with Lthyroxine, hydrocortisone and growth hormone seemed to accelerate the cure from cholestasis. Liver function at follow-up (median 4 yr) stayed normal. CONCLUSION Cholestasis in CCPHD follows the course described here, frequently with normal GGT levels.

Clinical and Biological Heterogeneity in Pseudohypoparathyroidism Syndrome

Pseudohypoparathyroidism (PHP) is a rare inherited syndrome frequently associated with Albright’s hereditary osteodystrophy (AHO). We conducted a multicenter study including 71 PHP children and 77 rel

Muscle Function Improves during Growth Hormone Therapy in Short Children Born Small for Gestational Age: Results of a Peripheral Quantitative Computed Tomography Study on Body Composition

BACKGROUND Short small for gestational age (SGA) children can be affected by a lack of muscle mass rather than fat mass. They also face a high risk of the metabolic syndrome developing after childhood. It is not known whether low muscle mass influences muscle function. AIM Our aim was to investigate muscle-fat distribution and muscle function before and during GH treatment in short SGA children. PATIENTS A total of 34 prepubertal short SGA children (11 females, seven with Silver-Russell syndrome) were included in the study. Mean values were: age at GH start 7.3 yr; height sd score (SDS) -3.3; and birth weight SDS -2.7. METHODS Investigations over 24 months on GH treatment (57 microg/kg.d) were performed. Body composition, including fat area and muscle area (MA), was assessed through peripheral quantitative computed tomography (XCT 2000; Stratec, Inc., Pforzheim, Germany). Maximal isometric grip force was performed with a Jamar dynamometer (Preston, Jackson, MI). Comparison with height-dependent reference values (SDS(Height)) was calculated. RESULTS MA SDS(Height) at GH start was -1.8 and increased to -0.8 (P < 0.001) and -0.8, and fat area SDS(Height) decreased from -0.6 to -2.0 (P < 0.001) and -1.5 after 12 and 24 months on GH. Maximal isometric grip force SDS(Height) increased from -0.9 to 0.3 (P < 0.001) and 0.5. MA at start correlated negatively with height velocity (R = -0.54; P < 0.001) and MA SDS at start and Delta-height SDS during the first year of GH treatment (R = -0.40; P < 0.001). CONCLUSIONS Short stature in SGA children is associated with low muscle mass and function. Supraphysiological GH doses led to a concomitant increment in height, muscle mass, and function, whereas fat mass decreased. Furthermore, body composition at GH start gives insight into GH responsiveness and the individual risk of metabolic syndrome.

The Use of Bone Age in Clinical Practice – Part 2

If height-limiting treatment is being considered for a child with tall stature, skeletal maturity is invaluable in the selection of appropriate patients for treatment, determining appropriate age of treatment commencement, monitoring progress of treatment, and determining the expected treatment effect on adult height. In precocious puberty, bone maturation can be usefully assessed at initial diagnosis and start of treatment and at regular intervals thereafter during treatment monitoring. Together with height, bone maturation is an essential parameter for long-term treatment monitoring in congenital adrenal hyperplasia. Bone age (BA) determination in children with skeletal dysplasia is only feasible in a few disorders and estimations should be treated with caution. Radiographs of the left hand and wrist are, however, essential in the diagnosis of many skeletal disorders. Bone mineralization and measures of bone lengths, width, thickness and cortical thickness should always be evaluated in relation to a child’s height and BA, especially around puberty. The use of skeletal maturity, assessed on a radiograph alone to estimate chronological age for immigration authorities or criminal courts is not recommended.

A paediatric bone index derived by automated radiogrammetry

SummaryHand radiographs are obtained routinely to determine bone age of children. This paper presents a method that determines a Paediatric Bone Index automatically from such radiographs. The Paediatric Bone Index is designed to have minimal relative standard deviation (7.5%), and the precision is determined to be 1.42%.IntroductionWe present a computerised method to determine bone mass of children based on hand radiographs, including a reference database for normal Caucasian children.MethodsNormal Danish subjects (1,867), of ages 7–17, and 531 normal Dutch subjects of ages 5–19 were included. Historically, three different indices of bone mass have been used in radiogrammetry all based on

IGF-I and IGF Binding Protein-3 Levels during Initial GH Dosage Step-Up Are Indicators of GH Sensitivity in GH-Deficient Children and Short Children Born Small for Gestational Age

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