Sara Pagani

Analysis of immune reconstitution in children undergoing cord blood transplantation

OBJECTIVE The aim of this study was to investigate and compare immune reconstitution in allogeneic cord blood transplantation (CBT) and bone marrow transplantation (BMT) recipients. MATERIALS AND METHODS Twenty-three children underwent CBT from either human leukocyte antigen-identical siblings (11 cases) or unrelated donors (12 cases) were enrolled in the study, together with 23 matched children receiving BMT. Patients were analyzed 2-3 and 12-15 months after transplant. Recovery of T-, B-, and NK-lymphocyte subsets, proliferative in vitro response to mitogens, as well as cytotoxic activities, were investigated. RESULTS CBT recipients showed a marked increase in the number of B lymphocytes as compared with patients who underwent BMT (p < 0.001). The absolute number of CD3(+) and CD8(+) T cells, as well as the proliferative response to T-cell mitogens, recovered with time after transplantation, irrespective of the source of stem cells used. Recipients of unrelated CBT had a better recovery of CD4(+) T lymphocytes (p < 0.01). Among patients experiencing acute graft-versus-host disease (GVHD), children given CBT had a much greater production of CD4(+) CD45RA(+) T cells than BMT recipients (p < 0.005). Recovery of NK cell number and innate cytotoxic activities was fast, irrespective of the source of stem cells used. CONCLUSIONS Despite the much lower number of lymphocytes transferred with the graft, recovery of lymphocyte number and function toward normal in CBT recipients was rapid and comparable to that observed after transplantation of bone marrow progenitors. This prompt immune recovery possibly was favored by the reduced incidence and severity of GVHD observed in children who underwent CBT.

Variations in biological and immunological activity of growth hormone during the neonatal period.

Background/Aims: It was postulated that a high growth hormone (GH) bioactivity might explain the rapid growth rate of neonates. The aim of this study is to verify changes in serum GH biological potency (Bio-/Immuno-GH ratio) and their effects on serum growth factors during the first month of life in term and preterm babies. Methods: Blood samples were collected from 10 small-for-gestational-age preterm (SGAPT), 17 appropriate for gestational age preterm (AGAPT) and 26 AGA term (T) neonates on days 4, 15 and 30 of life to evaluate serum GH values measured by IFMA (IFMA-GH) and bioassay (Bio-GH), serum insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 (IGFBP-3). Results: High serum Bio-GH values on the first few days of life correspond to high IFMA-GH values, suggesting full biological potency of circulating GH. Furthermore, IGF-I/IGFBP-3 molar ratio values in preterm babies were higher than in full-term infants. Conclusions: These data confirmed the hypothesis that the higher growth velocity in the first month of life of preterm neonates is due to an increased bioavailability of IGF-I. A progressive maturation of the hypothalamic-pituitary-IGF-I axis without any alteration in the GH biological potency seems to underpin the increase of the growth factors early in life.

The shortness of Pygmies is associated with severe under-expression of the growth hormone receptor.

The stature is a highly heritable trait controlled by genetic and environmental factors. The African Pygmies represent a paradigmatic example of non-disease-related idiopathic short stature (ISS), showing a similar endocrine profile of Caucasian individuals with ISS. Pygmy children show normal anthropometric and endocrine parameters until puberty, while adult Pygmies show normal baseline and post-stimulation serum growth hormone (GH) levels but low values of baseline serum GH-binding protein (GHBP) and insulin-like growth factor-I (IGF-I). This discrepancy suggests a defective response to GH occurring in adulthood since Pygmies lack both the pubertal serum IGF-I surge and the growth spurt. However, sequencing of the key genes of the GH-IGF-I axis failed to identify Pygmy specific variants or haplotypes. We therefore aimed at assessing whether the quantitative gene expression profile of two key genes of the GH-IGF-I axis, GH and GHR, was also similar in low-stature and normal stature populations. We showed that the GH gene expression is 1.8-fold reduced and the GH receptor (GHR) gene expression is 8-fold reduced in adult Pygmies in comparison with sympatric adult Bantu, and that this reduction is not associated with sequence variants of the GHR gene. The marked decrease of the GHR expression in Pygmies is associated with reduced serum levels of the IGF-I and GHBP. Our results, documenting a markedly reduced GHR gene expression in adult Pygmies, could contribute to elucidate the mechanisms involved in ISS in Caucasoid subjects.

Generation and ex vivo expansion of cytotoxic T lymphocytes directed toward different types of leukemia or myelodysplastic cells using both HLA-matched and partially matched donors

OBJECTIVE Successful priming and in vitro expansion of anti-leukemia cytotoxic T lymphocytes (CTL) are preliminary conditions for designing approaches of adoptive immunotherapy in patients with hematological malignancies undergoing allogeneic hematopoietic stem cell transplantation (HSCT). In this study, we evaluated the possibility of generating and expanding in vitro CTL directed toward different types of either leukemia or myelodysplastic cells, using both HLA-matched and partially matched donors. PATIENTS AND METHODS Eleven donor/recipient pairs were enrolled; donor-derived dendritic cells, pulsed with patient blast cells, were used to generate CTL. RESULTS Anti-leukemia CTL lines were successfully obtained from 10 of 11 donors. After repeated rounds of stimulation, CTL lines showed, along with an increase in cytotoxic activity, a variable but continuous expansion of cultured cells. In order to increase the magnitude of CTL expansion, two anti-leukemia CTL lines were further stimulated using allogeneic feeder cells, anti-CD3, and low doses of interleukin-2 (IL-2). This stimulation gave rise to 150-fold to 270-fold expansion of the absolute number of cultured cells. Most cultures showed either absent or low reactivity of anti-leukemia CTL against patient non-leukemia cells. Three anti-leukemia CTL lines displayed a more pronounced cytotoxicity against nonmalignant recipient cells, which was always lower than that observed against leukemia blasts (LB). Spectratyping analysis of the TCR-Vbeta subfamilies revealed a preferential expansion of oligoclonal populations that persisted in CTL lines following repeated rounds of stimulation. CONCLUSIONS Results provide the biological background for designing protocols of adoptive immunotherapy for the control of minimal residual disease in patients with hematological malignancies given HSCT.

Role of adiponectin and leptin on body development in infants during the first year of life

BackgroundThe control of growth and nutritional status in the foetus and neonate is a complex mechanism, in which also hormones produced by adipose tissue, such as adiponectin and leptin are involved. The aim of this study was to evaluate levels of adiponectin, leptin and insulin in appropriate (AGA) and small for gestational age (SGA) children during the 1st year of life and to correlate these with auxological parameters.MethodsIn 33 AGA and 29 SGA infants, weight, length, head circumference, glucose, insulin, adiponectin and leptin levels were evaluated at the second day of life, and at one, six and twelve months, during which a portion of SGA could show catch-up growth (rapid growth in infants born small for their gestational age).ResultsBoth total and isoform adiponectin levels were comparable between AGA and SGA infants at birth and until age one year. These levels significantly increased from birth to the first month of life and then decreased to lower values at 1 year of age in all subjects. Circulating leptin concentrations were higher in AGA (2.1 ± 4.1 ng/ml) than in SGA neonates (0.88 ± 1.03 ng/ml, p < 0.05) at birth, then similar at the 1st and the 6th month of age, but they increased in SGA from six months to one year, when they showed catch-up growth. Circulating insulin levels were not statistically different in AGA and SGA neonates at any study time point. Insulin levels in both AGA and SGA infants increased over the study period, and were significantly lower at birth compared to one, six and 12 months of age.ConclusionsDuring the first year of life, in both AGA and SGA infants a progressive decrease in adiponectin levels was observed, while a difference in leptin values was correlated with the nutritional status.

Ex vivo generation and expansion of anti-tumor cytotoxic T-cell lines derived from patients or their HLA-identical sibling

Successful ex‐vivo priming and long‐term maintenance of anti‐tumor cytotoxic T‐cell (CTL) lines are preliminary conditions for their use in approaches of adoptive immunotherapy for patients with cancer. We describe the results of a novel procedure for generating in vitro anti‐tumor CTL using CD8‐enriched peripheral blood mononuclear cells (PBMC) and dendritic cells (DC), pulsed with irradiated tumor cells (TC) as source of tumor antigen. Eight patients were enrolled in our study: 4 sarcoma, 2 renal cell carcinoma, 1 ovarian carcinoma and 1 breast carcinoma. Ten anti‐tumor CTL‐lines cytotoxic towards patient TC were generated. Five CTL‐lines were obtained using both DC and PBMC from the patients (autologous setting). For 5 CTL‐lines, DC derived from an HLA‐identical sibling were employed (allogeneic setting): patients or siblings PBMC were used to generate CTL‐lines in 2 and 3 cases, respectively,. After tumor‐specific rounds of stimulation, followed by antigen‐independent cycle of expansion, CTL‐lines obtained in both autologous and allogeneic setting showed an expansion of the absolute number of cultured cells. In 6 of 10 CTL‐lines, the majority of effector cells (>70%) were CD3+/CD8+, while in the remaining 4, 40–70% of effector cells were CD3+/CD4+. Both CD8+ and CD4+ T cells displayed anti‐tumor cytotoxic activity. Spectratyping analysis of the TCR‐Vβ subfamilies revealed a preferential expansion of oligoclonal populations in 18 of 24Vβ subfamily. Altogether these results demonstrate that our experimental approach is suitable for efficiently generating and expanding anti‐solid tumor CTL to be used for adoptive immunotherapy.

Human amniotic fluid cells are able to produce IL-6 and IL-8

Problem:  In order to investigate the role of amniotic fluid cells (AFC) in the establishment of feto–maternal immune relationship, we evaluated their phenotype and capacity to produce cytokines.

Differences in Serum GH Cut-Off Values for Pharmacological Tests of GH Secretion Depend on the Serum GH Method Clinical Validation from the Growth Velocity Score during the First Year of Treatment

Background: The serum GH cut-off value for pharmacological tests of GH secretion (PhT GH) depends on the type of test and also on the method used for determining serum GH. Cut-off serum GH values as different as 5–10 ng/ml, have been reported, and have been validated biochemically. We have used the growth velocity (GV)-standard deviation score (SDS) during the first year of treatment with rhGH to validate these cut-offs on a biological basis. Methods: Fifty pre-pubertal patients with short stature (height ≤–2 SDS and GV ≤–1.2 SDS) were studied. GH deficiency (GHD) was diagnosed in 39 patients, on the basis of clinical and auxological parameters and on the serum concentration of IGF-1, and non-GHD in the other 11 patients. Two PhT GH (arginine and clonidine) were carried out in the 50 patients. Serum GH was determined by two different methods: one detecting most of serum GH isoforms, named Total GH (HGH Bio-Tech, MAIA Clone), and another one, only detecting the 22 kDa GH, named 22K GH (GH-22K IFMA, Wallac). Results: Basal data: all patients with GHD and with non-GHD had maximal serum GH response (MaxR) values below and above the cut-off, respectively, for the serum Total GH and 22K GH. The mean 22K GH/Total GH ratio was similar to previous publications. Post-rhGH treatment data: the two groups improved their height SDS during the first year of treatment, particularly patients with GHD. A receiver-operator curve was used to define the best threshold for post-treatment GV-SDS that separates GHD from non-GHD patients. This value was 1.91 GV-SDS. A negative correlation between first year treatment GV-SDS and pre-treatment serum GH MaxR was found for the two assays (p < 0.001). Then, the best cut-off GV-SDS, previously calculated with the receiver-operator curve (1.91 SDS) was used to interpolate the corresponding serum GH values, as determined by the two methods. For Total GH, the value was 10.8 ng/ml, and for 22K GH, it was 5.4 ng/ml. Conclusion: The cut-off values calculated by biological means to separate GHD from non-GHD were remarkably similar to those calculated biochemically (10.0 and 4.8 ng/ml, respectively) for Total and 22K GH. This is a biological validation for using different cut-off values, appropriate for each assay, to diagnose GHD.

Evaluation of growth hormone bioactivity using the Nb2 cell bioassay in children with growth disorders

The Nb2 cell bioassay could be a useful tool for evaluating human growth hormone (hGH) bioactivity, but is not specific for hGH since it relies on the ability of the hormone to produce effects by cross-reacting with the lactogenic receptor on Nb2 cells. We studied the biological activity of both endogenous and exogenous hGH in short patients using the Nb2 bioassay after inhibiting the mitogenic effect of the other lactogenic hormone, that is human prolactin (hPRL), by adding a specific antibody against hPRL to each assay. The in vitro study showed a significant (p<0.0001) increase in Nb2 cell proliferation when increasing concentrations of highly purified hGH were added to the cell culture. A complete inhibition of Nb2 cell replication was observed after adding a specific antibody against hGH. The in vivo study showed a significantly (p<0.0001) lower hGH bioactivity (4.90±0.28 ng/ml) evaluated during stimulation tests in 9 children with total idiopathic GH deficiency, mean age 9.25±1.99 years, in comparison with that found in 11 short children with normal growth velocity, mean age 8.22±1.41 years (12.25±1.19 ng/ml). Likewise, serum GH levels measured by immunofluorometric assay IFMA in the same serum samples were significantly (p<0.001) lower in the 9 GH-deficient (1.97±0.37 ng/ml) than in the 11 short children (21.85±2.71 ng/ml). Moreover, we evaluated GH concentrations using both IFMA and the Nb2 cell bioassay in serum samples collected from another 11 idiopathic GH-deficient children, mean age 10.71±1.18 years, before and then, 6 and 24 hours following the 1st injection of r-hGH (0.1 IU/kg sc). Serum GH values measured by both IFMA and Nb2 bioassay significantly (p<0.0001) increased 6 hours after r-hGH administration and decreased to reach basal levels after 24 hours. In conclusion, the Nb2 cell bioassay with minor modifications seems to provide a specific and sensitive assessment of hGH bioactivity.

Growth Hormone Immunoreactivity Does Not Reflect Bioactivity

In childhood, the largest secretory burst of GH occurs during nighttime, and consists of a complex mixture of molecular forms of GH that are thought to have different biologic activity. Standard GH assays cannot distinguish between bioactive and biologically inactive GH isoforms. To examine this relationship, overnight GH secretion was assessed by blood sampling every 30 min in 10 short prepubertal children (7 boys and 3 girls) to evaluate both the serum concentration and the biologic activity of GH. Serum GH concentrations were measured by an immunofluorometric assay and its biologic activity by the Nb2 cell bioassay. The 12-h (2000 h to 0800 h) and 6-h (2000 h to 0200 h and 0200 h to 0800 h) GH profiles were analyzed using the Pulsar program. When GH secretory pattern was measured by immunofluorometric assay, the area under the curve above the 0 line, the mean GH concentration, and the mean height of the secretory peaks were significantly higher during the first than during the second part of the night (29.17 ± 5.93 versus 16.29 ± 1.87 mIU/L, p < 0.05; 7.77 ± 1.28 versus 4.83 ± 0.33 mIU/L, p < 0.05; 4.61 ± 0.94 versus 2.68 ± 0.27 mIU/L, p < 0.05, respectively). In contrast, GH biologic activity was not significantly different during the two parts of the night. In conclusion, a dissociation between GH bioactivity and immunoreactivity is present in physiologic conditions, indicating that standard GH measurements do not provide any information on the biologic activity of the hormone. Although GH secretion is regulated by complex neuroendocrine mechanisms, the biologic activity of the hormone seems to be independent of them and is most probably regulated by peripheral mechanisms acting on its clearance or bioavailability to the target tissues.