Hormonal control of T-cell development in health and disease.

The physiology of the thymus, the primary lymphoid organ in which T cells are generated, is controlled by hormones. Data from animal models indicate that several peptide and nonpeptide hormones act pleiotropically within the thymus to modulate the proliferation, differentiation, migration and death by apoptosis of developing thymocytes. For example, growth hormone and prolactin can enhance thymocyte proliferation and migration, whereas glucocorticoids lead to the apoptosis of these developing cells. The thymus undergoes progressive age-dependent atrophy with a loss of cells being generated and exported, therefore, hormone-based therapies are being developed as an alternative strategy to rejuvenate the organ, as well as to augment thymocyte proliferation and the export of mature T cells to peripheral lymphoid organs. Some hormones (such as growth hormone and progonadoliberin-1) are also being used as therapeutic agents to treat immunodeficiency disorders associated with thymic atrophy, such as HIV infection. In this Review, we discuss the accumulating data that shows the thymus gland is under complex and multifaceted hormonal control that affects the process of T-cell development in health and disease.

Properties of cryptic epitopes and their corresponding antibodies as indicated by the study of human and ovine growth hormones.

Antibodies (Ab) directed to hidden antigenic determinants (cryptotopes) are undesirable because they are not neutralizing. Additionally, we have previously demonstrated a close association between the extent of Ab to cryptic determinants and the expression of autoantibodies (autoAb) under some experimental conditions. Thus, the first objective of this work was to establish the physicochemical characteristics of Ab to cryptotopes and the second one was to examine the structural features of cryptic epitopes themselves. Using human and ovine growth hormones (hGH and oGH) as antigenic models and competition ELISA under different conditions of temperature, pH or ionic strength, we did not find any difference between the binding properties of anti-cryptic epitope antibodies (Ab) and anti-native epitope Ab. Then, using synthetic peptides and tryptic digests and direct and competition ELISAs we studied the structures of cryptic hGH and oGH epitopes. Isolated peptides either in solution or adsorbed on microplates failed to react. Partially digested hGH was recognized only when insolubilized on microplates, and anti-oGH Ab only reacted with a large fragment of the hormone either in solution or insolubilized. These results indicate that, at least in the case of hGH and oGH, cryptic epitopes are not simple linear sequences, as commonly referred without any evidence, but new exposed conformational structures different from those found in the native antigen.

Dose dependency of the serum bio/immuno GH ratio in children during pharmacological secretion tests.

Dissociation between GH bioactivity (bio-GH) and GH immunoactivity (immuno-GH) is due to the heterogeneity of the molecule: the measurements do not always provide reliable information on the bio-GH. We studied the ratio of bio-GH and immuno-GH during pharmacological secretion tests in 211 sera to study the concentration-response curve of the assay (C1), 16 samples of normally growing subjects with idiopathic short stature (C2), 13 samples from patients with GH deficiency (GHD1) and 6 samples of 3 patients with GHD and normal provocative tests (GHD2). GH bioactivity was determined by the Nb2 cell proliferation assay (bio-GH) and immuno-GH by a time-resolved immunofluorometric assay (IFMA) (immuno-GH). A non-linear negative relationship between the serum bio-GH/immuno-GH ratio and serum immuno-GH was observed in C1. In log-log plotting representation, two cut-off lines were drawn: a vertical cut-off line separating above-below cut-off serum peak immuno-GH values in provocative tests, and a diagonal cut-off line separating normal-abnormal serum bio-GH/immunoGH ratio; four areas were defined. GHD1 had normal ratios, but below cut-off peak immuno-GH responses. P2 and P3 of Group GHD2 had abnormal ratios in samples with low serum immuno-GH but only P2 had autosomal dominant mutation. P1 had the same autosomal dominant isolated GHD as P2 but a low normal ratio. Our data underline the importance of relatively low serum GH concentrations in mediating GH biological actions. An abnormal serum bio-GH/immuno-GH ratio might explain certain cases of GHD and might be useful in detecting abnormal circulating isoforms of GH in patients with growth failure.

Effects of various adjuvants and a viral infection on the antibody specificity toward native or cryptic epitopes of a protein antigen.

An immunization protocol that induces antibodies (Abs) directed to cryptic epitopes of a protein antigen (Ag) reduces the efficacy of vaccines that ideally should induce Abs against native epitopes. We have shown earlier that viral infections concomitant with immunization against a protein tend to shift the Ab specificity toward cryptic epitopes and tend to induce the production of autoantibodies (autoAbs). Here, we show the effects of three adjuvants on the Ab specificity in the absence or presence of a viral infection (lactate dehydrogenase-elevating virus or LDV), with human growth hormone (hGH) being, as before, the protein Ag. Pathogen-free CBA/Ht and BALB/c mice were immunized with hGH in the presence of complete Freund's adjuvant (CFA), monophosphoryl lipid A (MPL) or alum, with the animals being either infected with LDV or not infected with LDV. Conventional and competition enzyme-linked immunosorbent assays (ELISAs) indicated that in noninfected mice, CFA induced higher titres of anti-hGH Ab than did MPL or alum, with the Ab being almost totally directed to cryptic hGH epitopes. Strikingly, CFA plus LDV infection in CBA/Ht mice shifted the specificity of the anti-hGH Ab toward native epitopes, whereas the virus decreased the Ab titre when MPL or alum was used. Our Western blot results showed that 70% of mice immunized with hGH in the presence of any adjuvant produced autoAbs against a variety of tissue Ags. The amount of autoAb and the concentration of Ab to hGH cryptic epitopes did correlate, suggesting a relationship between both kinds of Ab. Significant differences were observed in the various effects of adjuvants and the viral infection between the two mouse strains used in this work.

Insulin-like growth factor-I treatment in two children with growth hormone gene deletions.

Two unrelated Brazilian patients had homozygous 6.7 kb deletions in the GH-1 gene (girl and boy, 1.8 and 3.3 yr, heights -7.9 and -6.0 SDS, respectively). Desensitization using small amounts of exogenous GH (0.033 IU/kg body weight/week, divided into daily s.c. injections) was attempted, but anti-GH antibodies appeared. Replacement with usual doses of hGH induced only transient increase in growth. IGF-I therapy with increasing doses resulted in catch-up growth without side-effects. Growth velocity was 7.5 cm/yr in the first year and 8.4 cm/yr in the next 6 months in patient 1, and 6.7 cm/yr in the first year, 5.9 cm/yr in the second year and 7.9 cm/yr in the third year of IGF-I treatment in patient 2, when the daily dose of 240 micrograms/kg was divided into three injections. IGFBP-3 levels were low (0.55 and 0.40 mg/I) and did not increase after IGF-I treatment, suggesting that this GH effect is not mediated by IGF-I, and injected IGF-I had a rapid disappearance rate. We conclude that IGF-I promotes growth by endocrine mechanisms and constitutes an effective treatment for patients with GH insensitivity secondary to GH antibodies.

Changes in the specificity of antibodies in mice infected with lactate dehydrogenase-elevating virus.

Infection with lactate dehydrogenase-elevating virus (LDV) modifies the isotypic distribution of antibodies (Ab) directed to several antigenic proteins with a preferential production of IgG2a. Because it was not known whether the virus could also affect the Ab specificity, the authors addressed this point using human growth hormone (hGH) as a model antigen. Anti-hGH monoclonal antibodies (MoAb) were used as probes to study the occurrence of Ab to three native hGH epitopes (3C11, F11 and 10D6) in sera from LDV-infected CBA/Ht and BALB/c mice immunized with hGH. Competition ELISA was used to determine the extent of Ab directed to cryptic hGH epitopes, i.e. antigenic determinants hidden in the native hormone. Results indicated that in LDV-infected CBA/Ht mice the titres of anti-hGH Ab were lower than in controls, although a consistent isotypic shift to IgG2a subclass was observed. Concurrently, the presence of Ab to epitopes 3C11, F11 and/or 10D6 were markedly reduced in infected animals and most anti-hGH Ab were directed to hGH cryptic epitopes. By contrast, LDV infection increased the amount of anti-KLH Ab elicited by CBA/Ht mice and did not affect Ab specificity, whilst control and LDV-infected BALB/c mice showed similar concentrations of anti-hGH Ab. Furthermore, the proportion of Ab to cryptic hGH epitopes did not change in infected animals even though an important shift to IgG2a was detected. Thus, data presented herein suggest that LDV infection modifies Ab specificity depending on the mice genetic background and on the antigenic characteristics of the immunogen.

Macroadenomas näo-funcionantes da hipófise: estudo imuno-histoquímico / Functionless pituitary macroadenomas: immunohistochemical study

Cinqüenta e nove adenomas hipofisários, do tipo nao-funcionante, foram analisados pelo método imuno-histoquímico. Os tumores foram estudados através da técnica da peroxidase - avidina - biotina (ABC) e pesquisados os seguintes hormônios, com anticorpos primários específicos: prolactina (PRL), hormônio do crescimento (HS), corticotrofina (ACTH), gonadotrofinas (HCG) e tireotrofina (TSH). Trinta e oito tumores nao reagiram a nenhum anticorpo, 8 tumores apresentaram imunorreaçao positiva à prolactina, 5 tumores eram imunorreativos ao HS, 6 eram imunorreativos ao ACTH, e 10 tumores eram imunorreativos às gonadrotofinas, sendo que 5 ao LH e 5 ao FSH. Dos tumores que apresentaram imunorreatividade ao hormônio do crescimento e à prolactina houve manifestaçao clínica em apenas um, cujo paciente evoluiu com altas taxas de prolactina sangüínea.

Obtención de un antisuero específico contra la forma 22K de la hormona de crecimiento humana (hGH) / Obtention of a antiserum specific against the form 22K of the human growth hormone (hGH)

En el presente trabajo se describe la obtención del anticuerpo policlonal específico contra la fracción monomérica 22K de la hormona de crecimiento humana (hGH). La hormona fue obtenida a partir de hipófisis humanas congeladas, mediante un esquema de extracción basado en diferencias de solubilidad y la fracción 22K separada por cromatografía de filtración por gel. El antisuero específico fue obtenido por inoculación del antígeno en conejos raza Nueva Zelanda, cuya producción fue controlada valorando el título por RIA. La caracterización parcial del antisuero se realizó mediante la determinación del título óptimo, especificidad. afinidad, y su aplicabilidad para cuantificación en términos de curva estándar y control de calidad, en comparación con el antisuero de referencia. Los resultados indicaron que el antisuero puede emplearse en la cuantificación de hGH por RIA