Stem cell transplantation for primary immunodeficiencies.

BMT is curative in almost 75% of children affected by severe primary immunodeficiencies (PIDs). Recently, the chance of cure has increased thanks to the availability of matched unrelated donors (MUDs). Nevertheless, besides the conventional indications to BMT (profound or absent T-cell function, profound or absent natural killer function, known syndromes with T-cell deficiencies), indications to BMT for PIDs affecting the quality of life or having an expectation of life that does not exceed the third-fourth decade remain unclear. Infact, if it is evident that the survival rate in an infant grafted for a PID with a MUD is expected to be more than 80%, alternative treatments such as gene therapy are now available.

Growth hormone bioactivity and levels of growth hormone, growth hormone-binding protein, insulinlike growth factor I, and insulinlike growth factor-binding proteins in premature and full-term newborns during the first month of life.

OBJECTIVE: To assess the pattern of growth hormone bioactivity (GH-BIO) and the levels of GH-binding protein (GH-BP), insulin-like growth factor I (IGF-I), and insulin-like growth factor-binding proteins (IGFBPs) in the first month of life in premature and full-term (FT) newborns. PATIENTS AND METHODS: Serum samples were collected from 9 premature newborns who were small for gestational age, 18 premature newborns who were of appropriate size for gestational age, and 20 FT newborns on the 4th and 30th days of life to evaluate the GH-BIO using the Nb2 cell bioassay, the GH levels using a radio-immunoassay (GH-RIA), and the levels of GH-BP, IGF-I and IGFBPs. RESULTS: On day 4, the GH-RIA and GH-BIO values were increased in all newborns (P < .05) compared with values in the prepubertal control subjects. The GH-BP levels were low in all newborns, with the lowest values (P < .05) found in the premature newborns and positively correlated with gestational age (P < .001). The IGF-I levels were also low, with lower values (than those found in the FT newborns) (P < .005) found in the premature group and positively correlated with the GH-BP levels (P < .001) and gestational age (P < .001). The levels of IGFBP-1 and IGFBP-2 were high, with higher values found in the premature newborns than in the FT newborns (P < .05) and negatively correlated with gestational age (P < .005). The IGFBP-3 level was lower in the premature (P < .05) than in the FT newborns and positively correlated with gestational age (P < .005). During the first month of life, the GH-RIA and GH-BIO values were significantly decreased in all newborns (P < .001), while the IGF-I level was increased in the premature newborns (P < .005). The GH-BP levels were increased only in the FT newborns (P < .001). CONCLUSIONS: The elevated bioactive GH level seen in the first few days of life seemed to be attributable to a low IGF-I level secondary to a decreased number and/or function of the GH receptors. The decrease in the serum GH level observed thereafter seemed to be secondary to an increase in the IGF-I level in the premature newborns; however, other factors may have been involved in the FT newborns in whom no increase in the IGF-I level was observed.

Postnatal variations of growth hormone bioactivity and of growth hormone-dependent factors.

OBJECTIVE: To evaluate whether the low insulinlike growth factor I (IGF-I) levels that are observed in the neonate depend on the biological inactivity of the molecular forms of growth hormone (GH) or on the immaturity of the hepatic GH receptors during the early postnatal period. MATERIALS AND METHODS: Serum samples were collected from 60 normal full-term neonates on day 5 and at 1 and 4 months of age to evaluate the GH concentrations by using both an immunofluorometric assay and Nb2 cell bioassay, as well as the GH-binding protein, IGF-I, and IGF-binding protein 3 values by radioimmunoassay. RESULTS: Five-day-old neonates showed significantly higher (P < .001) mean +/- SEM GH levels that were measured by using the immunofluorometric assay (27.22 +/- 1.62 micrograms/L) and Nb2 cell bioassay (3.56 +/- 0.14 U/mL) compared with those levels in 11 prepubertal children who were studied as control subjects (1.26 +/- 0.28 micrograms/L and 0.74 +/- 0.08 U/mL, respectively). At 1 and 4 months of age, GH values that were measured by using both the immunofluorometric assay (9.15 +/- 0.89 and 2.58 +/- 0.32 micrograms/L, respectively) and Nb2 cell bioassay (2.52 +/- 0.11 and 1.71 +/- 0.15 U/mL, respectively) were decreased significantly (P < .001). In 5-day-old neonates, we observed significantly lower (P < .001) serum GH-binding protein (9.73% +/- 0.42%), IGF-I (67.63 +/- 5.20 ng/mL), and IGF-binding protein 3 (1.46 +/- 0.17 mg/L) concentrations compared with those in the prepubertal children (30.74% +/- 2.01%, 210 +/- 25 ng/mL, and 3.08 +/- 0.22 mg/L, respectively). At 1 month of age, serum GH-binding protein (16.00% +/- 0.70%) and IGF-binding protein 3 (2.96 +/- 0.30 mg/L) values were increased significantly (P < .001), while IGF-I levels (72.55 +/- 7.6 ng/mL, P = .09) were not increased. Serum IGF-I values were increased significantly (P < .005) at 4 months of age (97.94 +/- 9.68 ng/mL). CONCLUSION: The interaction of bioactive molecular forms of GH with the increased hepatic GH receptors induces the rise in postnatal IGF-I levels in early infancy.

[Prenatal and postnatal transplantation of hematopoietic stem cells in children with primary immunodeficiency]. / Il trapianto prenatale e postnatale di cellule staminali emopoietiche in bambini affetti da immunodeficienza primitiva.

Primary immunodeficiencies are inherited diseases characterized by impaired immune responses. In case of severe impairment of immunity bone marrow transplantation is the only therapeutic option. The molecular defect is known for several primary immunodeficiencies allowing prenatal diagnosis. This paper summarizes the clinical experience treating these pathologies by bone marrow transplantation.

Effects of growth hormone administration on neurotensin release in children with growth delay.

Neurotensin (NT), a tridecapeptide originally isolated from bovine hypothalamus, has numerous actions on endocrine functions. Since intravenous injection of NT in the rat stimulated the release of growth hormone (GH) among several pituitary hormones, the aim of our study was to investigate in humans the effects of GH injection on NT plasma levels. Plasma samples were obtained from 13 children with growth delay (7 boys and 6 girls; age range 5 years 1 month-14 years 1 month; mean +/- SE 10 years 9 months +/- 7 months) to evaluate NT and GH values before treatment and 4, 12 and 24 h after a subcutaneous rhGH injection (0.15 IU/kg). Plasma was extracted on a SEP-PAC C18 column and NT was eluted with propanol. NT concentrations were measured by a specific RIA and expressed as fmol/ml plasma. GH (ng/ml) and somatomedin C (SMC; U/ml) were evaluated by RIA using commercial kits. Free fatty acids (FFA; mEq/l) were measured using a colorimetric peroxidase technique. Before GH administration, NT levels were 7.19 +/- 1.01 fmol/ml. A significant increase in NT values was found 4 h (36.5 +/- 9.62, p < 0.001), 12 h (40.85 +/- 6.64, p < 0.001) and 24 h (19.5 +/- 3.48, p < 0.05) after GH injection. This increase was significantly correlated with the circulating GH levels 4 h after GH administration and with the circulating SMC levels 24 h after GH administration. No correlation was found between NT and FFA values.(ABSTRACT TRUNCATED AT 250 WORDS)

Does growth hormone treatment increase chromosomal abnormalities?

OBJECTIVE: The relationship between growth hormone (GH) therapy and malignancy, including leukaemias, remains controversial. In order to study this possible relationship further, we have investigated whether GH treatment induces chromosomal abnormalities in peripheral blood lymphocytes. DESIGN: Open, prospective study in a University Hospital to examine peripheral blood mononuclear cells in subjects with GH-deficiency (GHD) before and during GH treatment. SUBJECTS: Twelve idiopathic GHD patients, aged 1.8-12.5 years, were evaluated before and after 3, 6 and 12 months of GH therapy (0.6 IU/kg per week subcutaneously). Two additional GHD patients, aged 16.6 and 18 years, were studied 1 year after long-term GH therapy had been discontinued, and 12 age-matched healthy subjects were evaluated as controls. METHODS: We examined the incidence of chromosome and chromatid breaks, fragments, structural rearrangements and aneuploidies in 100 metaphases for each blood sample. A total of 5300 cells was analysed in the 14 patients. RESULTS: The proportion of cells with chromatid and chromosome breaks ranged from 0% to 6% in patients before treatment and from 1% to 5% in controls. During GH therapy the incidence of aneuploid metaphases ranged from 0% to 7% and was comparable with values observed in controls. Chromosomal loss and gain was random. CONCLUSIONS: We observed no increase in chromosomal abnormalities in GH-treated patients when compared with age-matched healthy controls.

Functional depletion of T cells by vincristine and methylprednisolone as an in vitro model for the prevention of graft versus host disease.

BACKGROUND AND METHODS. The outcome of mismatched bone marrow transplantation is still severely hampered by graft versus host disease (GVHD) and graft rejection. Procedures for the recognition and selective elimination of T cells are still unsatisfactory due to the increased incidence of graft failure and late rejection. Lymphocyte proliferation and generation of cytotoxic T cells (CTL) in response to allogeneic cells are considered good in vitro correlates of GVHD and have been used in the present study to asses the capacity of two drugs (vincristine, VCR, and methylprednisolone, MP) to affect the T cells involved in these reactions. RESULTS AND CONCLUSION. Treatment in vitro with VCR and MP has been shown to inhibit the functional capacity of peripheral blood lymphocytes to proliferate (mean reduction 95.8%) and to generate CTL in response to haploidentical stimulator cells. Responsiveness to antigens and mitogens was affected to a minor extent (mean reduction 40% and 65.5%, respectively), and the method allowed recovery of hemopoietic precursors. The results suggest that treatment of donor bone marrow with VCR and MP is worth studying as a new approach to the prevention of GVHD in haploidentical bone marrow transplantation.

Haploidentical peripheral blood and marrow stem cell transplantation in nine cases of primary immunodeficiency.

Bone marrow transplantation (BMT) is the treatment of choice in children affected by primary immunodeficiency (PID). Because only 10-15% of affected children have a familial HLA-identical donor alternative therapeutic options are BMT from a matched unrelated donor or an haploidentical BMT. In our experience only 40% of these children find a donor within the International Registry. Therefore, the remaining 50% children affected by PID are candidates for haploidentical BMT. Unfortunately, in PID other than sever-combined immunodeficiency (SCID), low engraftment rates have been reported because of minimal residual immunity. In order to enhance engraftment rate in haploidentical BMT in PID we suggest a protocol with addition of donor peripheral stem cells after mobilization with granulocyte colony-stimulating factor (G-CSF) (16 micrograms/kg for 5 days) and bone marrow cells. This procedure increases the cell load, which allows intensification of the conditioning regimen for induction of faster engraftment. The separation of CD34+ cells from leukapheresis products was achieved in the first 6 patients by the Isolex 300 system (Baxter) with a CD34+ cell purity range of 80-95% and in another three patients by the Clinimacs System (Miltenyi). The peripheral blood stem cells were cryopreserved until BMT, 15 days after G-CSF stimulation when the bone marrow was harvested, processed and T-cell depleted with Campath 1-M in the first 6 cases while the Clinimacs System was used in the remaining cases and no T-cell depletion was required. We included 9 patients in the study protocol: SCID (4), Omenn's syndrome (3), LAD (1) and CID (1). The mean value of peripheral CD34+ cells infused was 13.42 x 10(6)/kg and the mean CD3+ cells number was 0.385 x 10(5)/kg; the mean value of BM CD34+ cells infused was 10.62 x 10(6)/kg and the mean CD3+ cell number was 2.39 x 10(5)/kg. The mean number of infused CFU was 8.1 x 10(5)/kg for PBSC and 3.59 x 10(5)/kg for BM. The 9 patients achieved more than 0.5 x 10(9) peripheral blood neutrophils/L at a mean of 14.6 days (range: 6-22 days). One patient affected by SCID showed complete chimerism, but he died after BMT of systemic CMV infection; the other 8 patients are alive and well and 4 of them show complete chimerism in all cell lines. Split chimerism was documented in 2 SCID cases (CD3+ lymphocytes were of donor origin, monocytes were autologous and granulocytes were mainly autologous); 1 patient affected by Omenn's syndrome received 3 transplants (1 from the mother and 2 from the father, T-cells alone and bone marrow) and achieved engraftment with complete chimerism after the third transplant; the patient affected by LAD also received 3 transplants (2 bone marrow infusions and 1 PBSC infusion) achieving complete chimerism after the third one. In conclusion, the engraftment achieved in all treated patients, and the acceptable conditioning-related toxicity suggest that this approach could be successfully applied to children affected by PID and candidates for haploidentical BMT.