Adrenocortical tumors in children

Childhood adrenocortical tumors (ACT) are rare. In the USA, only about 25 new cases occur each year. In Southern Brazil, however, approximately 10 times that many cases are diagnosed each year. Most cases occur in the contiguous states of São Paulo and Paraná. The cause of this higher rate has not been identified. Familial genetic predisposition to cancer (p53 mutations) and selected genetic syndromes (Beckwith-Wiedemann syndrome) have been associated with childhood ACT in general but not with the Brazilian counterpart. Most of the affected children are young girls with classic endocrine syndromes (virilizing and/or Cushing). Levels of urinary 17-ketosteroids and plasma dehydroepiandrosterone sulfate (DHEA-S), which are abnormal in approximately 90% of the cases, provide the pivotal clue to a diagnosis of ACT. Typical imaging findings of pediatric ACT consist of a large, well-defined suprarenal tumor containing calcifications with a thin capsule and central necrosis or hemorrhage. The pathologic classification of pediatric ACT is troublesome. Even an experienced pathologist can find it difficult to differentiate carcinoma from adenoma. Surgery is the single most important procedure in the successful treatment of ACT. The role of chemotherapy in the management of childhood ACT has not been established although occasional tumors are responsive to mitotane or cisplatin-containing regimens. Because of the heterogeneity and rarity of the disease, prognostic factors have been difficult to establish in pediatric ACT. Patients with incomplete tumor resection or with metastatic disease at diagnosis have a dismal prognosis. In patients with localized and completely resected tumors, the size of the tumor has predictive value. Patients with large tumors have a much higher relapse rate than those with small tumors

ELISA for Determination of Human Growth Hormone: Recognition of Helix 4 Epitopes

Human growth hormone (hGH) signal transduction initiates with a receptor dimerization in which one molecule binds to the receptor through sites 1 and 2. A sandwich enzyme-linked immunosorbent assay was developed for quantifying hGH molecules that present helix 4 from binding site 1. For this, horse anti-rhGH antibodies were eluted by an immunoaffinity column constituted by sepharose-rhGH. These antibodies were purified through a second column with synthetic peptide correspondent to hGH helix 4, immobilized to sepharose, and used as capture antibodies. Those that did not recognize synthetic peptide were used as a marker antibody. The working range was of 1.95 to 31.25 ng/mL of hGH. The intra-assay coefficient of variation (CV) was between 4.53% and 6.33%, while the interassay CV was between 6.00% and 8.27%. The recovery range was between 96.0% to 103.8%. There was no cross-reactivity with human prolactin. These features show that our assay is an efficient method for the determination of hGH

ELISA for Determination of Human Growth Hormone: Recognition of Helix 4 Epitopes

Human growth hormone (hGH) signal transduction initiates with a receptor dimerization in which one molecule binds to the receptor through sites 1 and 2. A sandwich enzyme-linked immunosorbent assay was developed for quantifying hGH molecules that present helix 4 from binding site 1. For this, horse anti-rhGH antibodies were eluted by an immunoaffinity column constituted by sepharoserhGH. These antibodies were purified through a second column with synthetic peptide correspondent to hGH helix 4, immobilized to sepharose, and used as capture antibodies. Those that did not recognize synthetic peptide were used as a marker antibody. The working range was of 1.95 to 31.25 ng/mL of hGH. The intra-assay coefficient of variation (CV) was between 4.53% and 6.33%, while the interassay CV was between 6.00% and 8.27%. The recovery range was between 96.0% to 103.8%. There was no cross-reactivity with human prolactin. These features show that our assay is an efficient method for the determination of hGH. INTRODUCTION Diagnosis of growth hormone deficiency (GHD) is usually based upon assessment of anthropometric parameters in patients who present reduced levels of growth hormone in response to a pharmacological test Most of the commercially available immunoassays do not describe the precise epitope of human growth hormone (hGH) recognized by its primary antibodies, neither do they take into consideration the possibility of an assessment of the biological activity of the hGH isoforms. One single hGH molecule binds to two receptors through hGH binding sites 1 and 2 An IFA usually presents low immunoreactivity to mutant hGH isoforms, which, by contrast, may be in normal or high levels revealed by an assay that employs a primary polyclonal antibody. These assays have employed different markers. The immunoradiometric assay (IRMA) This work was designed to develop a sensitive and specific horseradish peroxidase (HRPO) enzyme-linked immunosorbent assay (ELISA) for hGH determination. Several small sequences of amino acids form the binding site 1 domain MATERIALS AND METHODS Animal immunization One adult female horse was immunized with rhGH (Genotropin, Pharmacia Diagnostics AB, Upsala, Sweden) through subcutaneous applications at 15-day interval. The emulsion for the first injection was prepared using 750 µg of rhGH dissolved in 2.5 mL of 0.05 M phosphate buffer saline pH 7.4 (PBS) and 2.5 mL of Freund's complete adjuvant. The remaining 7 applications were prepared using Freund's incomplete adjuvant. Optimal animal immunization was indicated by high serum concentration of anti-rhGH employing two different protocols, immunodiffusion test and ELISA. Animal sera were obtained (1.5 L) and immunoglobulins were precipitated in a saturated ammonium sulfate solution. Purification of anti-rhGH antibodies Specific anti-rhGH antibodies were purified through immunoaffinity columns. One gram of cyanogen bromide-activated sepharose (CNBr-sepharose) was coupled to 21.5 mg of rhGH according to the manufacturer's instructions (Pharmacia). Conjugated sepharose-rhGH was packed into a 6.0 mL polystyrene column, washed and filled with PBS and 0.05% sodium azide, and maintained at 4 • C. Aliquots of horse immunoglobulins dissolved in PBS circulated through the column at a flow rate of 20 mL/h overnight at 4 • C. Afterwards, the column was washed with PBS until the absorbance (280 nm) of the eluted solution had returned to baseline. Recovery of the immunoglobulins bound to the sepharose-rhGH column was performed washing the column with 0.1 M glycineHCl 0.15 M NaCl, pH 5.0, until an immunoglobulin peak had been obtained. Finally, the column was washed with PBS until the absorbance returned to baseline. Solution containing anti-rhGH antibodies was dialyzed overnight at 4 • C in PBS. Antibodies anti-helix 4 of hGH A number of peptides were generously provided by the Peptide Laboratory from UNIFESP (São Paulo, SP). The technique used by this laboratory was reported by Kates and Albericio Each synthetic peptide (20 mg) was immobilized to 1.0 g of CNBr-sepharose according to the manufacturer's instructions (Pharmacia). Sepharose-helix 4 peptide was packed into a 6.0 mL polystyrene column, washed and filled with PBS and 0.05% sodium azide. This column was maintained at 4 • C. Aliquots with purified anti-rhGH antibodies circulated through the sepharose-helix 4 peptide column (or through other columns prepared with other peptides) at a flow rate of 20 mL/h. Unbound anti-rhGH antibodies that did not recognize the helix 4 peptide were eluted and separated to be conjugated with HRPO (later used as second anti-hGH antibody). The column was washed with PBS until the absorbance (280 nm) of the eluate returned to baseline. Samples of eluted anti-rhGH were dialysed overnight at 4 • C in PBS. To collect anti-helix 4 peptide antibodies, the column pH was reduced with 0.1 M glycineHCl 0.15 M NaCl, pH 5.0, and the eluted solution was after a while dialysed at 4 • C in PBS, overnight. Finally, the immunoaffinity column was washed with PBS until the absorbance of the eluted solution returned to baseline. Preparation of anti-rhGH antibodies for ELISA Anti-rhGH antibodies that did not recognize the helix 4 peptide, eluted from the affinity column, were conjugated to HRPO according to the procedure reported by Nakane and Kawaoi A 96-well Nunc MaxiSorp plate (Nalge Nunc International, Roskilde, Denmark) was coated overnight at 4 • C with 100 µL of a 10 µg/mL solution of anti-helix 4 antibodies in 0.05 M carbonate buffer, pH 9.6. Afterwards, the wells were washed with wash buffer (0.05% Tween 20 in saline). Each well was filled with 120 µL of blocking buffer (2% casein in PBS) and the plate was incubated for 1 hour at 37 • C. After washing, serial dilution of rhGH (125 ng/mL to 0.98 ng/mL) in dilution buffer (0.25% casein, 0.05% Tween 20, PBS) was added to the wells starting from the first row. After incubating the plate for 1 hour at 37 • C, it was washed and anti-rhGH HRPO conjugated in dilution buffer was added to the wells with final dilutions of 1 : 250, 1 : 500, 1 : 1000, 1 : 2000, 1 : 4000, and 1 : 8000, starting from the first column (left to right). After incubation for 1 hour at 37 • C, the solution was removed and the wells were washed at least six times and 100 µL of an orthophenilenediamine solution (0.33 mg/mL in 0.5 M citrate buffer, pH 5.2, and 0.4% hydrogen peroxide) were added to each well. After 15 minutes at room temperature, protected from light, the enzymatic reaction was stopped through the addition of 20 µL of 2 M sulfuric acid. The absorbance (492 nm) was measured using a Bio-Tek EL X 800 reader. Samples This study was approved by the Ethics Committee from the Hospital of Clinics from the Federal University of Parana, and serum samples from 73 boys (10.9 ± 3.0 years) and 36 girls (10.1 ± 3.2 years) were collected after obtaining written consent from their parents. The patients were submitted to the GH test (GH released 2004(GH released :3 (2004 ELISA for hGH 145 after clonidine application) according to the established protocol for growth retardation at the Division of Pediatric Endocrinology of Hospital of Clinics. Time course of sample collection for each patient: baseline (before clonidine), 60, 90, and 120 minutes after clonidine administration. Sandwich ELISA was used to quantify hGH from these patients. The absorbance values from each serum were plotted against the standard curve obtained with rhGH and the results were compared with the previously known hGH measurements from IRMA (MaiaClone, Biodata Diagnostics, Rome, Italy). RESULTS Production, purification, and titration of antibodies Serum from rhGH-immunized horse was tested by immunodiffusion in the presence of rhGH 1 mg/mL and the results were positive up to 1 : 4 dilutions. After treating the animal with one extra injection of rhGH, serum titres were reanalyzed by ELISA two weeks later, when adequate immunization was revealed by titres of 1 : 256000. After ammonium sulfate precipitation and dialysis of whole immunoglobulins, these polyclonal antibodies were purified by sepharose-rhGH column and the final concentration was 1.6 mg/mL. Anti-rhGH antibodies were eluted through a second column with helix 4 peptide immobilized to sepharose. After dehydration the antibodies final concentration was 0.948 mg/mL. This antibody was used to capture hGH and rhGH. The antibodies that did not recognize helix 4 peptide were conjugated to HRPO and the best dilution used in all ELISAs was 1 : 1000. Sandwich ELISA The rhGH saturation curve was constructed with the absorbance data obtained using fresh dilutions of rhGH (Genotropin, Pharmacia) preparations Intra-assay precision control was assessed by measuring 4 groups of sera pools corresponding to time points basal (B), 60, 90, and 120 minutes. Each pool was measured 16 times in the same plate

An inherited p53 mutation that contributes in a tissue-specific manner to pediatric adrenal cortical carcinoma

The incidence of pediatric adrenal cortical carcinoma (ACC) in southern Brazil is 10–15 times higher than that of pediatric ACC worldwide. Because childhood ACC is associated with Li-Fraumeni syndrome, we examined the cancer history and p53 status of 36 Brazilian patients and their families. Remarkably, 35 of 36 patients had an identical germ-line point mutation of p53 encoding an R337H amino acid substitution. Differences within intragenic polymorphic markers demonstrated that at least some mutant alleles arose independently, thus eliminating a founder effect. In tumor cells, the wild-type allele was deleted, and mutant p53 protein accumulated within the nuclei. Although these features are consistent with Li-Fraumeni syndrome-associated adrenal tumors, there was no history of increased cancer incidence among family members. Therefore, this inherited R337H p53 mutation represents a low-penetrance p53 allele that contributes in a tissue-specific manner to the development of pediatric ACC