In the Czech Republic is born to 5 % of SGA / IUGR children, about 5, 000 children a year, and their incidence is increasing. In 10 - 15 % does not occur postnatal compensation growth acceleration and growth retardation is then further deepens. Since 2003, these children can be treated by recombinant growth hormone in supraphysiological doses. Master thesis brings informations about SGA / IUGR children and their growth parameters during the first 2 years from the beginning of treatment when the monitoring was carried out at three - month intervals. The results are compared with the group of patients treated with growth hormone for growth hormone deficiency. Growth data was processed by a group of 57 SGA / IUGR children, which was compared with data of 34 children with growth hormone deficiency treated between 2003-2015 with growth hormone. Treatment of both groups of patients was carried out by a suitable therapeutic protocol, with the dose of GH by the respective diagnosis. These parameters were monitored: body height and weight, growth rate, BMI and weight - height ratio of patients. SGA / IUGR and GHD patients showed different growth profile in the initial phase of treatment when GHD patients had higher acceleration peak after 3 months of starting treatment and steeper increase in growth rate....

The present study deals with the comparison and establishment of methods for the detection of antibodies against recombinant human growth hormone (rhGH). Therefore, different methods for the detection of hGH-Abs were evaluated and compared in order to establish a test system that can be used for the detection of neutralizing antibodies against hGH, which could be developed under rhGH treatment. This manuscript describes in detail the validation of a newly developed biological assay, the neutralizing hGH-antibody assay (NAb assay). Therefore, a cell line transfected with the growth hormone receptor, that proliferates in the presence of hGH, was used. This proliferation was quantified by an increase of the optical density (OD/ absorbance) after addition of a colorimetric reagent, whereas the presence of hGH-antibodies leads to an inhibition of cell proliferation. To validate the test system for the detection of hGH-antibodies, we tested serum samples of 4 patients suffering from neurosecretory dysfunction (NSD) and samples taken from 6 patients with growth hormone deficiency (GHD) which were treated with rhGH and were highly suspected for a-hGH antibodies. These samples were tested in two different immunological assays, capable to screen sera for anti-hGH immunreactivity in the case of hGH-insensitivity during GH treatment. Using the NAb assay the neutralizing activity of specific hGH-antibodies was proved in serum samples of NSD and GHD type 1A patients. In case of neutralizing hGH-antibody activity, a clinically based decision can be made whether rhGH therapy should be stopped or the rhGH dosis should be increased. By the use of our test system, we offer the measurement of anti-hGH-antibody activity to other laboratories in cases when secondary hGH-insensitivity is assumed or observed.

Genetic and Hormonal Regulation of Children's Growth MUDr. Jan Vosáhlo Abstract Growth in childhood is a complex process of changing the body, which can be disrupted by various illnesses including endocrine disorders, particularly growth hormone deficiency. Tumors or other processes affecting hypothalamic-pituitary area can be a postnatal cause of GHD; prenatal causes include 1) developmental disorders of the pituitary as part of complex syndromes, 2) developmental disorders of the pituitary due to defects in regulatory genes and 3) defects in genes involved in the synthesis and secretion of GH. The first topic of the thesis was septo-optic dysplasia - a complex syndrome involving optic nerve hypoplasia, structural brain abnormalities and pituitary dysfunctions. We extensively described phenotype in 11 Czech patients; we observed both complete SOD and incomplete forms variously combining two of the three main components of the syndrome. The cohort then became a part of an international study of 68 patients, in which we studied the phenotype in dependence on the brain morphology. We found correlation between the severity of clinical symptoms and the degree of septum pellucidum abnormities and also a correlation between hippocampus and falx abnormities and neurological symptoms. As the second topic we studied...

Introdução: A incidência de baixa estatura devido a deficiência do hormônio do crescimento (DGH) ocorre em 1:4.000-10.000 nascidos vivos. Diversos fatores de transcrição são necessários para a diferenciação dos cinco tipos de células produtoras de 6 hormônios hipofisários. Mutações nos fatores de transcrição HESX1, GLI2, LHX3, LHX4, SOX2, SOX3, PROP1 e POU1F1 foram descritas em pacientes com deficiência hormonal hipofisária isolada ou múltipla associada ou não a outras malformações. Mutações no gene OTX2, um fator de transcrição responsável pela formação da vesicula ocular e pela hipófise, podem causar malformações oculares tais como anoftalmia e microftalmia, isoladamente ou em associação com DGH isolado (DGHI) ou deficiência hipofisária hormonal múltipla (DHHM). Recentemente, dois pacientes não relacionados com DHHM e neuroipófise ectópica, sem anormalidades oculares foram descritos com mutações em heterozigose no OTX2 sugerindo um papel deste gene na etiologia do hipopituitarismo sem outras características sindrômicas. Objetivo: O objetivo desse trabalho foi o de analisar o gene OTX2 em pacientes com DGHI ou DHHM e correlacionar os achados moleculares com o fenótipo. Pacientes: Foram estudados 125 pacientes com DHHM (6 filhos de pais consangüíneos e 33 com parentes com baixa estatura) e 33 com DGHI (7 filhos de pais consangüíneos e 8 com parentes com baixa estatura). Materiais e métodos: Amostras de DNA dos pacientes foram submetidas à reação de polimerização em cadeia utilizando-se primers intrônicos desenhados para amplificar os 3 exons e as regiões flanqueadoras do gene OTX2. Os produtos de PCR foram purificados e sequenciados pelo método de Sanger. Resultados: Uma nova variante alélica c.689A>T, p.H230L em heterozigose no exon 5 foi encontrado em um único paciente com deficiência de GH, TSH, LH/FSH e ACTH associada a neuroipófise ectópica, sem malformação ocular. A histidina na posição 230 é altamente conservada em todas as espécies de vertebrados, e a análise in silico prediz um efeito prejudicial à estrutura da proteína. A análise da variante na família revelou 8 parentes não afetados como portadores heterozigotos, sugerindo uma doença autossômica dominante com padrão de penetrância incompleta. Esta variante não foi encontrada em 400 alelos de 200 controles brasileiros, porém foi descrito como polimorfismo no banco de dados de SNP em uma população européia americana, com incidência de 1 alelo T, entre 8600 alelos, sendo assim considerado raro. Encontramos também outras quatro variantes alélicas na casuística (c.98-70C> A; c.420G> C, p.P148P; c.435C> T, p.S145S; C * 10G> A), não conservadas entre as espécies. Duas delas levando a troca silenciosa de amino ácidos, sem efeito deletério no sítio exonic splice enhancer. Conclusão A nossa coorte de 158 pacientes é a maior população rastreada para mutações no OTX2 e a detecção de uma variante suspeita em heterozigose em um único paciente portador de hipopituitarismo e neuroipófise ectópica sugere que mutações no OTX2 são uma causa rara de DHHM ou DGHI sem malformação ocular na população estudada. O achado molecular da variante c.689A>T, p.H230L em heterozigose, com padrão de penetrância incompleta é consistente com a observação de que as características fenotípicas de camundongos heterozigotos com perda de função do Otx2 são fortemente influenciados pela background genético, não podendo dessa forma, descartar que outros moduladores genéticos possam ser responsáveis pela penetrância incompleta nessa família. Essa hipótese deverá ser investigada pela análise do exoma do paciente e seus familiares. O fato de a variante estar localizada numa região altamente conservada entre as espécies, sugere que a mesma seja causadora do fenótipo em questão, porém serão necessários os estudos funcionais de transfecção transitória para determinar se se trata de perda de função ou efeito negativo dominante de genes alvos expressos no ectoderme oral ou neural / Introduction: The incidence of short stature due to growth hormone deficiency (GHD) occurs in 1:4.000-10.000 live births. Several transcription factors are required for differentiation of five types of cells producing 6 pituitary hormones. Mutations in the transcription factors HESX1, GLI2, LHX3, LHX4, SOX2, SOX3, PROP1 and POU1F1 have been described in patients with isolated pituitary hormone deficiency or multiple associated or not with other malformations. Mutations in OTX2, a transcription factor responsible for the formation of the eye vesicle and the pituitary gland, can cause ocular malformations such as anophthalmia and microphthalmia, alone or in association with isolated GHD (IGHD) or combined pituitary hormone deficiency (CPHD). Recently, two unrelated patients with CPHD and ectopic neurohypophysis without ocular abnormalities were described with heterozygous mutations in OTX2 suggesting a role of this gene in the etiology of hypopituitarism without other syndromic features. Objective: The aim of this study was to analyze the OTX2 gene in patients with GHD or CPHD and correlate the molecular findings with the phenotype. Patients: We studied 125 patients with CPHD (6 children of consanguineous parents and 33 relatives with short stature) and 33 with IGHD (7 children of consanguineous parents and 8 relatives with short stature). Materials and methods: DNA samples from the patients were subjected to polymerase chain reaction using intronic primers designed to amplify the 3 exons and flanking regions of the gene OTX2. The PCR products were purified and sequenced by the Sanger method. Results: A new heterozygous allelic variant c.689A> T, p.H230L in exon 5 was found in one patient with deficiencies of GH, TSH, LH / FSH and ACTH associated with ectopic neurohypophysis without ocular malformation. The histidine at position 230 is strongly conserved in all vertebrate species, and in silico analysis predicts a detrimental effect on protein structure. The analysis of the variant in the family revealed eight unaffected relatives as heterozygous carriers, suggesting an autosomal dominant pattern with incomplete penetrance. This variant was not found in 400 alleles of 200 Brazilian controls, but it was described as a polymorphism in the SNP database in a European American population, with an incidence of 1 T allele among 8600 alleles, and therefore considered rare. We also found four other allelic variants in the samples (c.98-70C> A; c.420G> C, p.P148P; c.435C> T, p.S145S; C * 10G> A), not conserved between species. Two of them leading to a silent amino acid exchange, without deleterious effect in the exonic splice enhancer site. Conclusion Our cohort of 158 patients is the largest population screened for mutations in OTX2 and the detection of a suspected heterozygous variant in one patient with hypopituitarism and ectopic neurohypophysis suggests that OTX2 mutations are a rare cause of CPHD/IGHD without ocular malformation in the studied population. The molecular finding of a heterozygous variant c.689A> T, p.H230L, with incomplete penetrance pattern is consistent with the observation that the phenotypic characteristics of mice with heterozygous loss of function of Otx2 are strongly influenced by genetic background, suggesting that other genetic modulators may be responsible for the incomplete penetrance in this family. This hypothesis should be investigated by analysis of exoma in the patient and their family. The fact that the variant is located in a region highly conserved among species suggests that it is causing the phenotype in question, but it will require the functional studies with transient transfections to determine whether it is the loss of function or effect of dominant negative target genes expressed in neural or oral ectoderm

Introdução: A incidência de baixa estatura devido a deficiência do hormônio do crescimento (DGH) ocorre em 1:4.000-10.000 nascidos vivos. Diversos fatores de transcrição são necessários para a diferenciação dos cinco tipos de células produtoras de 6 hormônios hipofisários. Mutações nos fatores de transcrição HESX1, GLI2, LHX3, LHX4, SOX2, SOX3, PROP1 e POU1F1 foram descritas em pacientes com deficiência hormonal hipofisária isolada ou múltipla associada ou não a outras malformações. Mutações no gene OTX2, um fator de transcrição responsável pela formação da vesicula ocular e pela hipófise, podem causar malformações oculares tais como anoftalmia e microftalmia, isoladamente ou em associação com DGH isolado (DGHI) ou deficiência hipofisária hormonal múltipla (DHHM). Recentemente, dois pacientes não relacionados com DHHM e neuroipófise ectópica, sem anormalidades oculares foram descritos com mutações em heterozigose no OTX2 sugerindo um papel deste gene na etiologia do hipopituitarismo sem outras características sindrômicas. Objetivo: O objetivo desse trabalho foi o de analisar o gene OTX2 em pacientes com DGHI ou DHHM e correlacionar os achados moleculares com o fenótipo. Pacientes: Foram estudados 125 pacientes com DHHM (6 filhos de pais consangüíneos e 33 com parentes com baixa estatura) e 33 com DGHI (7 filhos de pais consangüíneos e 8 com parentes com baixa estatura). Materiais e métodos: Amostras de DNA dos pacientes foram submetidas à reação de polimerização em cadeia utilizando-se primers intrônicos desenhados para amplificar os 3 exons e as regiões flanqueadoras do gene OTX2. Os produtos de PCR foram purificados e sequenciados pelo método de Sanger. Resultados: Uma nova variante alélica c.689A>T, p.H230L em heterozigose no exon 5 foi encontrado em um único paciente com deficiência de GH, TSH, LH/FSH e ACTH associada a neuroipófise ectópica, sem malformação ocular. A histidina na posição 230 é altamente conservada em todas as espécies de vertebrados, e a análise in silico prediz um efeito prejudicial à estrutura da proteína. A análise da variante na família revelou 8 parentes não afetados como portadores heterozigotos, sugerindo uma doença autossômica dominante com padrão de penetrância incompleta. Esta variante não foi encontrada em 400 alelos de 200 controles brasileiros, porém foi descrito como polimorfismo no banco de dados de SNP em uma população européia americana, com incidência de 1 alelo T, entre 8600 alelos, sendo assim considerado raro. Encontramos também outras quatro variantes alélicas na casuística (c.98-70C> A; c.420G> C, p.P148P; c.435C> T, p.S145S; C * 10G> A), não conservadas entre as espécies. Duas delas levando a troca silenciosa de amino ácidos, sem efeito deletério no sítio exonic splice enhancer. Conclusão A nossa coorte de 158 pacientes é a maior população rastreada para mutações no OTX2 e a detecção de uma variante suspeita em heterozigose em um único paciente portador de hipopituitarismo e neuroipófise ectópica sugere que mutações no OTX2 são uma causa rara de DHHM ou DGHI sem malformação ocular na população estudada. O achado molecular da variante c.689A>T, p.H230L em heterozigose, com padrão de penetrância incompleta é consistente com a observação de que as características fenotípicas de camundongos heterozigotos com perda de função do Otx2 são fortemente influenciados pela background genético, não podendo dessa forma, descartar que outros moduladores genéticos possam ser responsáveis pela penetrância incompleta nessa família. Essa hipótese deverá ser investigada pela análise do exoma do paciente e seus familiares. O fato de a variante estar localizada numa região altamente conservada entre as espécies, sugere que a mesma seja causadora do fenótipo em questão, porém serão necessários os estudos funcionais de transfecção transitória para determinar se se trata de perda de função ou efeito negativo dominante de genes alvos expressos no ectoderme oral ou neural / Introduction: The incidence of short stature due to growth hormone deficiency (GHD) occurs in 1:4.000-10.000 live births. Several transcription factors are required for differentiation of five types of cells producing 6 pituitary hormones. Mutations in the transcription factors HESX1, GLI2, LHX3, LHX4, SOX2, SOX3, PROP1 and POU1F1 have been described in patients with isolated pituitary hormone deficiency or multiple associated or not with other malformations. Mutations in OTX2, a transcription factor responsible for the formation of the eye vesicle and the pituitary gland, can cause ocular malformations such as anophthalmia and microphthalmia, alone or in association with isolated GHD (IGHD) or combined pituitary hormone deficiency (CPHD). Recently, two unrelated patients with CPHD and ectopic neurohypophysis without ocular abnormalities were described with heterozygous mutations in OTX2 suggesting a role of this gene in the etiology of hypopituitarism without other syndromic features. Objective: The aim of this study was to analyze the OTX2 gene in patients with GHD or CPHD and correlate the molecular findings with the phenotype. Patients: We studied 125 patients with CPHD (6 children of consanguineous parents and 33 relatives with short stature) and 33 with IGHD (7 children of consanguineous parents and 8 relatives with short stature). Materials and methods: DNA samples from the patients were subjected to polymerase chain reaction using intronic primers designed to amplify the 3 exons and flanking regions of the gene OTX2. The PCR products were purified and sequenced by the Sanger method. Results: A new heterozygous allelic variant c.689A> T, p.H230L in exon 5 was found in one patient with deficiencies of GH, TSH, LH / FSH and ACTH associated with ectopic neurohypophysis without ocular malformation. The histidine at position 230 is strongly conserved in all vertebrate species, and in silico analysis predicts a detrimental effect on protein structure. The analysis of the variant in the family revealed eight unaffected relatives as heterozygous carriers, suggesting an autosomal dominant pattern with incomplete penetrance. This variant was not found in 400 alleles of 200 Brazilian controls, but it was described as a polymorphism in the SNP database in a European American population, with an incidence of 1 T allele among 8600 alleles, and therefore considered rare. We also found four other allelic variants in the samples (c.98-70C> A; c.420G> C, p.P148P; c.435C> T, p.S145S; C * 10G> A), not conserved between species. Two of them leading to a silent amino acid exchange, without deleterious effect in the exonic splice enhancer site. Conclusion Our cohort of 158 patients is the largest population screened for mutations in OTX2 and the detection of a suspected heterozygous variant in one patient with hypopituitarism and ectopic neurohypophysis suggests that OTX2 mutations are a rare cause of CPHD/IGHD without ocular malformation in the studied population. The molecular finding of a heterozygous variant c.689A> T, p.H230L, with incomplete penetrance pattern is consistent with the observation that the phenotypic characteristics of mice with heterozygous loss of function of Otx2 are strongly influenced by genetic background, suggesting that other genetic modulators may be responsible for the incomplete penetrance in this family. This hypothesis should be investigated by analysis of exoma in the patient and their family. The fact that the variant is located in a region highly conserved among species suggests that it is causing the phenotype in question, but it will require the functional studies with transient transfections to determine whether it is the loss of function or effect of dominant negative target genes expressed in neural or oral ectoderm

Atualmente, a maioria dos endocrinologistas pediátricos usa uma dose fixa de GH, calculada por quilo de peso ou área de superfície corporal, para todos os pacientes. Algumas crianças com DGH, tratadas com as doses atuais, não atingem uma estatura normal e outras não atingem a estatura-alvo geneticamente prevista pela altura dos pais. Além disso, algumas crianças com DGH desenvolvem características acromegalóides após o uso por longo prazo da medicação. A existência de um marcador preciso e eficiente seria útil para a individualização da dose de rGH. Esse marcador deveria ser mensurado em um período de tempo menor que a velocidade de crescimento (VC). Neste estudo usamos as concentrações de IGF-I como esse marcador. Durante 12 meses, acompanhamos trinta crianças portadoras de DGH grave, em tratamento prévio por cerca de quatro anos, divididas em dois grupos de 15, para comparar o tratamento com a dose de rGH baseada no peso versus o tratamento com a dose de rGH ajustada pelas concentrações de IGF-I para mantê-las em uma faixa alvo (entre 0 a +2 DP). Foi possível manter concentrações de IGF-I dentro de valores predeterminados pelo ajuste da dose de rGH em 13 dos 15 pacientes, mas a VC não foi diferente entre os grupos (6,8±2,6 vs. 6,9±2,7 cm/ano; p=NS); porém, quando considerados apenas os pacientes prépúberes que mantiveram concentrações de IGF-I entre 0 e +2DP em pelo menos 75% das dosagens, sua velocidade de crescimento foi maior em comparação com as crianças que mantiveram concentrações de IGF-I abaixo de 0DP em 50% ou mais das dosagens (8,8±1,8 vs. 6,3±2,9 cm/ano; p<0,05). Em paralelo, comparamos as concentrações de IGF-I de diferentes genótipos do exon 3 do receptor de GH (GHR), nessas trinta crianças, durante o tratamento. Duas das mais comuns isoformas em seres humanos são geradas pela retenção (full length GHR - GHRfl) e exclusão do exon 3 (exon 3 deleted GHR - GHRd3). A influência desse polimorfismo na resposta ao tratamento com rGH em pacientes com DGH tem sido alvo de controvérsia. No presente estudo, apesar de receberem a mesma dose de rGH (41±10 vs. 41±8 ug/kg d; p=NS), pacientes carreando ao menos um alelo-d3 GHR, como grupo, tiveram maiores níveis de IGF-I que aqueles homozigotos para o alelo GHR full-length (0,9±0,9 vs. -0,3±1,2 DP; p<0,05) , indicando uma melhor sensibilidade ao GH para o primeiro genótipo. A VC não foi diferente entre os grupos GHRd3 vs. GHRfl (7,3±1,9 vs. 6,4±3,1cm/ano; p=NS). / Currently, most pediatric endocrinologists use a fixed rGH dose calculated according to the weight or body surface area for all patients. Some children with GHD, treated with the present doses, do not achieve normal height, and some, even reaching normal height, do not achieve the genetic target height determined by their parents\' heights. At the same time, some children with GHD develop acromegalic characteristics after long-term treatment with rGH. The existence of a specific and effective marker to individualize rGH dose would be useful to control therapy of children with GHD. This marker ideally should be obtained in a shorter time interval than the growth velocity (GV). In the present study, we measured insulin-like growth factor (IGF-I) as this marker. During one year, we followed 30 children with severe GHD, treated previously with rGH for an average of 4yrs, divided in two groups of 15, to compare weight-based versus IGF-I-based rGH dosing to reach IGF-I levels between a target range (0 and +2 SDS). It was feasible to maintain IGF-I levels in this predetermined range by adjusting the rGH dose in 13 of 15 patients, but growth velocity was not different in groups with weight-based or IGF-I based rGH dose (6.8±2.6 vs. 6.9±2.7cm/y; p=NS); however, in prepubertal children who reached predetermined target IGF-I levels in 75% of the measurements GV was higher than in those who did not (8.8±1.8 vs. 6.3±2.9 cm/y; p<0.05). In parallel, we compared IGF-I levels of different GH receptor (GHR) exon 3 genotypes in these 30 children during treatment. Two of the more common GHR isoforms in humans are generated by retention (full length GHR - GHRfl) and by exclusion of exon 3 (exon 3 deleted GHR - GHRd3). The influence of this polymorphism on the response to rGH treatment in patients with GH deficiency has been controversial. In the present study, despite receiving similar rGH doses (41±10 vs. 41±8 ug/kg d; p=NS), patients carrying at least one GHR d3-allele, as a group, had higher IGF-I levels than those homozygous for the GHR full-length allele (0.9±0.9 vs. -0.3±1.2 SDS; p<0.05), indicating a greater GH sensitivity with the former genotype. GV was not different between groups GHRd3 vs. GHRfl (7.3±1.9 vs. 6.4±3.1cm/y; p=NS).

The objective of this thesis was to investigate the effects of the somatotrope GH/IGF-1 axis upon the thymus. This work included two parts: 1. Translational research study: Thymus function in adult GH deficiency (AGHD) with and without GH treatment Background: Despite age-related adipose involution, T cell generation in the thymus (thymopoiesis) is maintained beyond puberty in adults. In rodents, growth hormone (GH), insulin-like growth factor-1 (IGF-1), and GH secretagogues reverse agerelated changes in thymus cytoarchitecture and increase thymopoiesis. GH administration also enhances thymic mass and function in HIV-infected patients. Until now, thymic function has not been investigated in adult GH deficiency (AGHD). The objective of this clinical study was to evaluate thymic function in AGHD, as well as the repercussion upon thymopoiesis of GH treatment for restoration of GH/IGF-1 physiological levels. Methodology/Principal Findings: Twenty-two patients with documented AGHD were enrolled in this study. The following parameters were measured: plasma IGF-1 concentrations, signal-joint T-cell receptor excision circle (sjTREC) frequency, and sj/b TREC ratio. Analyses were performed at three time points: firstly on GH treatment at maintenance dose, secondly one month after GH withdrawal, and thirdly one month after GH resumption. After 1-month interruption of GH treatment, both plasma IGF-1 concentrations and sjTREC frequency were decreased (p,0.001). Decreases in IGF-1 and sjTREC levels were correlated (r = 0.61, p,0.01). There was also a decrease in intrathymic T cell proliferation as indicated by the reduced sj/b TREC ratio (p,0.01). One month after reintroduction of GH treatment, IGF-1 concentration and sjTREC frequency regained a level equivalent to the one before GH withdrawal. The sj/b TREC ratio also increased with GH resumption, but did not return to the level measured before GH withdrawal. Conclusions: In patients with AGHD under GH treatment, GH withdrawal decreases thymic T cell output, as well as intrathymic T cell proliferation. These parameters of thymus function are completely or partially restored one month after GH resumption. These data indicate that the functional integrity of the somatotrope GH/IGF-1 axis is important for the maintenance of a normal thymus function in human adults. 2. Fundamental study: intrathymic expression of members of the GH/IGF-1 axis and effects of GH on T-cell differentiation in murine fetalthymic organ cultures (FTOC). We here address the question of expression and role of GH/IGF axis in the thymus. Methods: Using RT-qPCR, the expression profile of various components of the somatotrope GH/IGF axis was measured in different thymic cell types and during thymus embryogenesis in Balb/c mice. Effect of GH on T-cell differentiation was explored through thymic organotypic culture. Results: Transcription of Gh, Igf1, Igf2 and their related receptors predominantly occurred in thymic epithelial cells (TEC), while a low level of Gh and Igf1r transcription was also evidenced in thymic T cells (thymocytes). Gh, Ghr, Ins2, Igf1, Igf2, and Igfr1, displayed distinct expression profiles depending on the developmental stage. The protein concentration of IGF-1 and IGF-2 were in accordance with the profile of their gene expression. In fetal thymus organ cultures (FTOC) derived from Balb/c mice, treatment with exogenous GH resulted in a significant increase of double negative CD4-CD8- T cells and CD4+ T cells, with a concomitant decrease in double positive CD4+CD8+ T cells. These changes were inhibited by concomitant treatment with GH and GHR antagonist pegvisomant. However, GH treatment also induced a significant decrease in FTOC Gh, Ghr and Igf1 expression. Conclusion: These data show that the thymotropic properties of the somatotrope GH/IGF-1 axis involve an interaction between exogenous GH and GHR expressed by TEC. Since thymic IGF-1 is not increased by GH treatment, the effects of GH upon T-cell differentiation could implicate a different local growth factor or cytokine.

Atualmente, a maioria dos endocrinologistas pediátricos usa uma dose fixa de GH, calculada por quilo de peso ou área de superfície corporal, para todos os pacientes. Algumas crianças com DGH, tratadas com as doses atuais, não atingem uma estatura normal e outras não atingem a estatura-alvo geneticamente prevista pela altura dos pais. Além disso, algumas crianças com DGH desenvolvem características acromegalóides após o uso por longo prazo da medicação. A existência de um marcador preciso e eficiente seria útil para a individualização da dose de rGH. Esse marcador deveria ser mensurado em um período de tempo menor que a velocidade de crescimento (VC). Neste estudo usamos as concentrações de IGF-I como esse marcador. Durante 12 meses, acompanhamos trinta crianças portadoras de DGH grave, em tratamento prévio por cerca de quatro anos, divididas em dois grupos de 15, para comparar o tratamento com a dose de rGH baseada no peso versus o tratamento com a dose de rGH ajustada pelas concentrações de IGF-I para mantê-las em uma faixa alvo (entre 0 a +2 DP). Foi possível manter concentrações de IGF-I dentro de valores predeterminados pelo ajuste da dose de rGH em 13 dos 15 pacientes, mas a VC não foi diferente entre os grupos (6,8±2,6 vs. 6,9±2,7 cm/ano; p=NS); porém, quando considerados apenas os pacientes prépúberes que mantiveram concentrações de IGF-I entre 0 e +2DP em pelo menos 75% das dosagens, sua velocidade de crescimento foi maior em comparação com as crianças que mantiveram concentrações de IGF-I abaixo de 0DP em 50% ou mais das dosagens (8,8±1,8 vs. 6,3±2,9 cm/ano; p<0,05). Em paralelo, comparamos as concentrações de IGF-I de diferentes genótipos do exon 3 do receptor de GH (GHR), nessas trinta crianças, durante o tratamento. Duas das mais comuns isoformas em seres humanos são geradas pela retenção (full length GHR - GHRfl) e exclusão do exon 3 (exon 3 deleted GHR - GHRd3). A influência desse polimorfismo na resposta ao tratamento com rGH em pacientes com DGH tem sido alvo de controvérsia. No presente estudo, apesar de receberem a mesma dose de rGH (41±10 vs. 41±8 ug/kg d; p=NS), pacientes carreando ao menos um alelo-d3 GHR, como grupo, tiveram maiores níveis de IGF-I que aqueles homozigotos para o alelo GHR full-length (0,9±0,9 vs. -0,3±1,2 DP; p<0,05) , indicando uma melhor sensibilidade ao GH para o primeiro genótipo. A VC não foi diferente entre os grupos GHRd3 vs. GHRfl (7,3±1,9 vs. 6,4±3,1cm/ano; p=NS). / Currently, most pediatric endocrinologists use a fixed rGH dose calculated according to the weight or body surface area for all patients. Some children with GHD, treated with the present doses, do not achieve normal height, and some, even reaching normal height, do not achieve the genetic target height determined by their parents\' heights. At the same time, some children with GHD develop acromegalic characteristics after long-term treatment with rGH. The existence of a specific and effective marker to individualize rGH dose would be useful to control therapy of children with GHD. This marker ideally should be obtained in a shorter time interval than the growth velocity (GV). In the present study, we measured insulin-like growth factor (IGF-I) as this marker. During one year, we followed 30 children with severe GHD, treated previously with rGH for an average of 4yrs, divided in two groups of 15, to compare weight-based versus IGF-I-based rGH dosing to reach IGF-I levels between a target range (0 and +2 SDS). It was feasible to maintain IGF-I levels in this predetermined range by adjusting the rGH dose in 13 of 15 patients, but growth velocity was not different in groups with weight-based or IGF-I based rGH dose (6.8±2.6 vs. 6.9±2.7cm/y; p=NS); however, in prepubertal children who reached predetermined target IGF-I levels in 75% of the measurements GV was higher than in those who did not (8.8±1.8 vs. 6.3±2.9 cm/y; p<0.05). In parallel, we compared IGF-I levels of different GH receptor (GHR) exon 3 genotypes in these 30 children during treatment. Two of the more common GHR isoforms in humans are generated by retention (full length GHR - GHRfl) and by exclusion of exon 3 (exon 3 deleted GHR - GHRd3). The influence of this polymorphism on the response to rGH treatment in patients with GH deficiency has been controversial. In the present study, despite receiving similar rGH doses (41±10 vs. 41±8 ug/kg d; p=NS), patients carrying at least one GHR d3-allele, as a group, had higher IGF-I levels than those homozygous for the GHR full-length allele (0.9±0.9 vs. -0.3±1.2 SDS; p<0.05), indicating a greater GH sensitivity with the former genotype. GV was not different between groups GHRd3 vs. GHRfl (7.3±1.9 vs. 6.4±3.1cm/y; p=NS).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Idiopathic Short Stature is a heterogeneous set of conditions without obvious hormonal changes or defined etiologies. It may include the partial insensitivity to GH (PGHI) and the moderate GH deficiency (MGHD), both of them with low concentrations of growth factor like insulin type I (IGF-I) and hyper or sub-answer of GH on tests of stimulation, respectively. The objective of this study is to assess the response to treatment with GH in PGHI and MGHD, comparing them twith the results obtained in a group with severe GH deficiency (SGHD). It was studied in PGHI (GH peak ≥18 ng/ml) 20 individuals (14 boys), 12.07 (2.57-year-old); in MGHD (GH peak between 5 and 10 ng/ml) 12 (7 boys), 10.73 (1.79-year-old); and in SGHD (GH peak lower than 5 ng/ml) 19 (10 boys), 10.90-(3.51) yearold, treated with GH for one to ten years. The initial and present GH doses were, respectively, 43.33(10.00) and 49.61 (12.90) μg/kg/day in SGHD, 50.27 (11.76) and 57.27 (15.83) μg/kg/day in MGHD; and50.18 (6.99) and 55.66 (9.61) μg/kg/day in PGHI. The standard deviation score (SDS) of initial height of the SGHD group was lower than MGHD group (p< 0.01) and PGHI group (p <0.001).The SDS of initial IGF-I of SGHD was similar to MGHD and lower than PGHI group (p<0.0001).The initial bone age (BA) in SGHD group was similar to MGHD and lower than PGHI group GH (p < 0.01). GH treatment has brought about a lower increase in the height SSD and in the IGF-I SSD (both, p < 0.05) and in the bone age (p< 0.01) in the PGHI group than in the SGHD group, probably due to the more accentuated height deficit in the SGHD group and lower IGF-I increase in the PGHI group. Treatment response was similar in MGHD and SGHD groups. The lowest height gain in the PGHI suggests that the partial GH insensitivity needs higher GH doses to be effective. / A baixa estatura idiopática é um conjunto heterogêneo de condições sem evidentes alterações hormonais ou etiologias definidas. Pode incluir a insensibilidade parcial ao GH (IPGH) e a deficiência moderada de GH (DMGH), ambas com concentrações baixas de fator de crescimento semelhante à insulina tipo I (IGF-I) e hiper ou sub-resposta do GH aos testes de estímulo, respectivamente. O objetivo do presente trabalho é avaliar a resposta ao tratamento com GH na IPGH e DMGH, comparando-as com os resultados obtidos na deficiência grave de GH (DGGH). Foram estudados no grupo IPGH (pico de GH ≥18 ng/ml), 20 indivíduos (14 meninos), com 12,0 (2,57) anos de idade; no grupo DMGH (pico de GH entre 5 e 10 ng/ml), 12 (sete meninos), com 10,73 (1,79) anos; e no DGGH (pico de GH < 5 ng/ml), 19 (10 meninos), com 10,90 (3,51) anos, tratados com GH por um a dez anos. As doses de GH iniciais e atuais foram, respectivamente, 43,33 (10,00) e 49,61 (12,90) μg/kg/dia no DGGH; 50,27 (11,76) e 57,27 (15,83) μg/kg/dia no DMGH; e 50,18 (6,99) e 55,66 (9,61) μg/kg/dia no IPGH. O escore de desvio-padrão (EDP) da altura inicial do grupo DGGH foi menor do que no DMGH (p< 0,01) e no IPGH (p <0, 001). O EDP do IGF-I inicial do grupo DGGH foi similar ao DMGH e menor do que o do IPGH (p< 0, 0001). A idade óssea inicial no grupo DGGH foi similar ao DMGH e menor do que a do grupo IPGH (p < 0,01). O tratamento com GH propiciou um menor incremento no EDP da altura e no EDP do IGF-I (ambos, p < 0,05) e na IO (p< 0,01) no grupo IPGH do que no DGGH, provavelmente refletindo o maior déficit estatural no grupo com DGGH e menor aumento do IGF-I no grupo IPGH. A resposta ao tratamento nos grupos DMGH e DGGH foi semelhante. O ganho estatural menor no grupo IPGH sugere que a insensibilidade parcial ao GH necessitaria de doses mais altas de GH para ser vencida.

The present research has the objective to determinate the seric PCR concentrations in the monogenic isolated deficiency of the growth hormone (DIGH), test the hypothesis that DIGH is associated to the exacerbation of the inflammatory profile, identify the PCR elevation predictors, and evaluate the existence of an association between PCR and premature atherosclerosis. The growth hormone (GH) has as its main function the post-natal longitudinal growth; it interferes in the bone apposition, muscle mass growth, opposes to the action of insulin in the carbohydrates and lipids metabolism, and, in the cardiovascular system, helps the vascular and myocardial remodeling. IGF-I, GH primary mediator, plays a fundamental role in the growth regulation, cellular apoptosis and differentiation. The GH/IGF-I axis acts in the resistance to insulin and phenotypical expression of cardiovascular risk factors, associated to metabolic syndrome. IGF-I avoids the endothelial dysfunction, causes the increase of sensitivity to insulin, and avoids post-prandial dyslipidemia, besides presenting anti-inflammatory and anti-apoptotic activities. The decrease of IGF-I is associated to premature atherosclerosis and high cardiovascular risk. IGF-I role is controversial and its increase is related to the premature atherosclerosis in carotids. The deficiency of GH is associated to the increase of cardiovascular and brain vascular mortality. The inflammation plays an essential role in the atherosclerosis physiopathology from its initial phase up to atherotrombotic events in acute coronary syndromes. C - reactive protein, acute phase reagent of the inflammation, is produced by the liver, due mainly to the interleukin-6 stimulus. As a predictor and a mediator of atherosclerosis, among all circulating inflammatory markers, it is the most stable, the most studied and the one which presented the most constant relationship to future cardiovascular risk in various clinical situations, including asymptomatic individuals. In Itabaininha, Sergipe state we described a population with DIGH, with extremely low levels of IGF-I, high LDL-c and systolic arterial pressure and central obesity, a cluster of risk factors, highly susceptible to atherosclerosis ideal to this research. From this population, eighteen individuals were studied, eight male and ten female, with an average age of 45, compared to a control group composed of twenty individuals of the same region. After the clinical and metabolic characteristics were analyzed, the most relevant results were: PCR showed a meaningful difference between the groups (4,9 mg/l (4,7) vs 1,4 mg/l (2,2)); and IGF-I extremely low (1,0 ng/ml (1,0) vs 164,0 ng/ml (135,0)). The group predicts that PCR is independent from the other metabolic variables (R² = 0, 42), and that IGF-I is the main responsible for the increase of PCR in the DIGH. No association between PCR and the intimatemedia thickness of the carotids could be observed. Conclusion: it was demonstrated that the DIGH present high levels of PCR when compared to the control group; the variable group predicts this variation and IGF-I is the main responsible for the PCR variability. High PCR is not associated to premature atherosclerosis in this high risk differentiated group. / Esta pesquisa demonstra de forma original o grau de inflamação relacionado à deficiência isolada monogênica do hormônio de crescimento (DIGH) através da determinação das concentrações plasmáticas da proteína C reativa (PCR), testa a hipótese de a DIGH estar associada à exacerbação do perfil inflamatório, identifica os preditores da elevação da PCR e avalia a associação de inflamação com aterosclerose precoce. O hormônio de crescimento (GH) tem como função principal, o crescimento longitudinal pós-natal; interfere na aposição óssea, crescimento da massa muscular, opõe-se à ação da insulina no metabolismo dos carboidratos e lipídios e, no aparelho cardiovascular, atua no remodelamento miocárdico e vascular. A sua deficiência está associada ao aumento de mortalidade por doenças cardio e cérebro vasculares. O IGF-I, mediador primário do GH, desempenha papel fundamental na regulação do crescimento, diferenciação e apoptose celular. O eixo GH/IGF-I interfere quanto à resistência à insulina e na expressão fenotípica dos fatores de risco cardiovasculares, associados à síndrome metabólica. O IGF-I evita a disfunção endotelial, promove o aumento da sensibilidade à insulina previne a dislipidemia pós-prandial, além de possuir atividade anti-inflamatória e antiapoptótica. A diminuição do IGF-I está associada à aterosclerose prematura e elevado risco cardiovascular. O papel do IGF-I é controverso e o seu aumento está associado ao aparecimento precoce de aterosclerose em carótidas. A inflamação exerce papel fundamental na fisiopatologia da aterosclerose. A PCR, reagente de fase aguda da inflamação é produzida pelo fígado, em decorrência principalmente do estímulo da interleucina-6. Entre os marcadores inflamatórios circulantes, é a mais estável, a mais estudada e a que apresentou relação mais constante com o risco cardiovascular futuro em diversas situações clínicas, incluindo indivíduos assintomáticos; A PCR é considerada preditora e mediadora da aterosclerose. Em Itabaianinha, Sergipe, foi descrita uma população com DIGH, níveis extremamente baixos de IGF-I, LDL e pressão arterial sistólica elevados, obesidade central, portanto, uma população com múltiplos fatores de risco, altamente susceptível à aterosclerose. Este estudo inseriu 18 indivíduos DIGH, oito do sexo masculino e dez do feminino, idade média de 45 anos, e 20 controles (CO) da mesma região. Analisadas as características clínicas e metabólicas, o grupo DIGH apresentou PCR de (4,9 mg/l (4,7) vs controles (CO) 1,4 mg/l (2,2) com importante diferença significativa (p<0,0001); o IGF-I dos DIGH foi extremamente baixo, 1,0 ng/ml (1,0) vs 164,0 ng/ml (135,0) dos CO, p< 0,0001. O grupo é que prediz ser a PCR independente das outras variáveis metabólicas (R2 = 0,42) e o IGF- I é o principal responsável pelo aumento da PCR nos DIGH. Não houve nenhuma relação de associação entre a PCR e a espessura média-íntima das carótidas do DIGH. Conclusão: ficou demonstrado que os DIGH apresentam níveis muito elevados de PCR que denota um perfil inflamatório exacerbado; o grupo é que prediz esta variação e, o IGF-I é o principal responsável pelo variabilidade da PCR. A PCR elevada na DIGH não está associada à aterosclerose precoce.