[Long-term experiences with corneal crosslinking in patients with progressive keratoconus at the University Eye Hospital in Tübingen, Germany]. / Langzeiterfahrungen des kornealen Crosslinkings bei Patienten mit progredientem Keratokonus an der Universitäts-Augenklinik Tübingen.

BACKGROUND AND PURPOSE: The corneal collagen crosslinking procedure has been clinically performed worldwide for approximately 20 years. The aim of the study was to analyze the long-term outcomes of corneal crosslinking at the University Eye Hospital in Tübingen. METHODS: In this retrospective study 136 consecutive eyes with progressive keratoconus from 107 patients were included. The observational period was from December 2008 to March 2018. The parameters analyzed were best corrected visual acuity (BCVA) and the keratometry values from elevation maps measured using a Scheimpflug camera. RESULTS: The study population of 107 patients included 96 (90%) male and 11 (10%) female subjects and the mean age was 23 ± 8 years. The mean observation time between corneal collagen crosslinking and the last follow-up examination was 42 ± 29 months. The mean postoperative BCVA showed a significant improvement from baseline at each point of postoperative measurement. The mean astigmatism and the mean Kmax showed a statistically significant reduction in the first 6 postoperative months. The mean thinnest point of the cornea showed a significant reduction in the first 6 months, after which the cornea thickness stabilized. No serious adverse events relating to the treatment were registered. CONCLUSION: Long-term outcomes after corneal collagen crosslinking showed a significant reduction of the keratometry values and the BCVA was significantly higher compared to the preoperative value at all times of observation. In conclusion, corneal collagen crosslinking was shown to be an effective and safe treatment for progressive keratoconus.

Role of the RET proto-oncogene in sporadic hyperparathyroidism and in hyperparathyroidism of multiple endocrine neoplasia type 2.

Parathyroid tumors occur either sporadically or as part of inherited syndromes such as multiple endocrine neoplasia (MEN) types 2A and 2B. The development of both of these familial syndromes has been related to specific germline gain-of-function mutations predominantly in exons 10 and 11 (MEN 2A) and 16 (MEN 2B) of the RET proto-oncogene. The same mutations have also been implicated in the pathogenesis of sporadic medullary thyroid carcinoma and sporadic pheochromocytoma. The RET mutations are thought to have a transforming effect only in cells of neural crest origin such as thyroid parafollicular (C-cells) and adrenal chromaffin cells, which normally express the RET proto-oncogene. Expression of RET messenger RNA has not yet been studied in the parathyroid, however, we demonstrate in this study by a sensitive, semiquantitative RT-PCR technique and in situ hybridization, that RET is expressed in MEN 2A parathyroid tumors and in sporadic adenomas. Although DNA from a parathyroid tumor of a MEN 2A patient displayed an expected mutation, none of the previously described MEN 2A or 2B mutations were found in DNA of 34 sporadic adenomas. Our data suggest that parathyroid disease is an integral part of the MEN 2A syndrome, but that MEN 2 mutations in RET rarely play a part in the pathogenesis of sporadic parathyroid tumors.

Atrial natriuretic factor binding sites in rat area postrema: autoradiographic study.

The area postrema (AP) is a brain stem circumventricular organ implicated, among other functions, in central cardiovascular (CV) regulation. Competition binding analysis performed by quantitative in vitro autoradiography demonstrated specific, high-affinity (Kd, 0.32 +/- 0.11 nM), low-capacity (Bmax, 57.5 +/- 10.9 fmol/mg protein) atrial natriuretic factor (ANF) binding sites in the AP. C-ANF [des-(Gln116-Gly120)ANF-(Arg102-Cys121)-NH2] and ANF-(Phe106-Ile113)-NH2 (two ligands endowed with selectivity for the ANF-C receptor), as well as C-type natriuretic peptide (CNP), did not compete noticeably at pathophysiological concentrations for 125I-ANF binding. 125I-[Tyr0]CNP bound to the AP to a much lower extent than 125I-ANF. Electron microscopic autoradiography in vivo disclosed that 125I-ANF was preferentially bound to axon, dendrite, and astrocyte plasmalemma. These studies demonstrate that the AP contains natriuretic peptide binding sites with pharmacological characteristics of the ANF-A and ANF-B but not of the ANF-C receptor subtype. In the AP, ANF interacts with those sites resembling ANF-A receptors. Cellular localization of these binding sites may relate to their possible involvement in the centrally mediated salt and water regulation and/or CV effects of circulating ANF.

Autoradiographic visualization of the natriuretic peptide receptor-B in rat tissues.

Natriuretic peptide receptor-B (NPRB) was visualized in rat tissues by in vitro autoradiography, using its putative physiological agonist C-type natriuretic peptide (CNP). In initial studies, we determined that atrial natriuretic peptide (ANP) is not a suitable ligand for labeling the NPRB: in tissues reported to contain NPRB transcripts, CNP did not inhibit [125I]ANP binding except to NPRC sites. Therefore, to visualize the NPRB we used 125I[Tyr(o)]-CNP as a radioligand with an excess of NPRC-blocking peptide: C-ANP. With this approach we detected the highest number of NPRB-like sites in the pars intermedia of the pituitary gland. A large number of these sites were present in pituitary neural and anterior lobes, area postrema, adrenal medulla and cortex. A moderate NPRB population was observed in the subfornical organ, plexiform layer of the olfactory bulb and kidney. Low concentrations of NPRB were noted in the cerebellum and cerebrum but not in the choroid plexus and pia-arachnoid. Saturation experiments performed on cerebellum sections revealed a very low concentration (Bmax 4.8 fmol/mg protein) of high affinity (Kd 1.2 nM) NPRB-like sites. This study is the first demonstration of 125I[Tyr(o)]-CNP binding sites with characteristics of the NPRB in intact tissues.

Atrial natriuretic factor receptor subtypes in the rat central nervous system.

In this study we investigated the presence and anatomical location of atrial natriuretic factor (ANF) receptor subtypes in the rat central nervous system using in vitro autoradiographic and cross-linking techniques. 125I-ANF-(Ser99-Tyr126) served as a labeled ligand, whereas ANF-(Ser99-Tyr126) and two peptides endowed with selectivity for ANF-C receptor--namely, C-ANF (des-[Gln116-Gly120] ANF-[Asp102-Cys121]-NH2) and ANF-(Phe106-Ile113)-NH2--were used as displacing agents. Distribution studies revealed the presence of specific ANF binding sites in a number of central nervous system areas examined. C-ANF at 10(-6) M competed for 125I-ANF binding to a much lower extent than ANF in many of those structures, whereas ANF-(106-113)-NH2 at 10(-6) M did not have a significant effect on the radioligand binding except in the choroid plexus, pia-arachnoid, and olfactory bulb. Analysis of the competition curves revealed that in the choroid plexus, pia-arachnoid, olfactory bulb, subfornical organ, area postrema, and habenular nucleus, ANF interacts with its binding sites with high affinity (IC50, 0.46-0.77 nM). In contrast, C-ANF and ANF-(106-113)-NH2 competed for 125I-ANF binding with high potency (IC50, 2-16 nM) in the choroid plexus and pia-arachnoid only, where they were able to displace 60-70% of the radioligand binding. 125I-ANF cross-linking to olfactory bulb membranes resolved a specific 120-kDa band corresponding to the high molecular weight receptor but did not disclose a specifically labeled band corresponding to the low molecular mass receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain natriuretic peptide binding sites in rats: in vitro autoradiographic study.

Brain natriuretic peptide (BNP) is a recently discovered family of natriuretic peptides highly homologous to atrial natriuretic factor (ANF). Quantitative in vitro autoradiography with a computerized microdensitometer demonstrated that the distribution of BNP binding sites is similar to the known distribution pattern of ANF binding sites in rat tissues. Analysis of saturation and competition curves disclosed that the maximal binding capacity for BNP-(Asp-81--Tyr-106) and ANF-(Ser-99--Tyr-126) is similar within the plexiform layer of the olfactory bulb, the choroid plexus, and the adrenal zona glomerulosa. Examination of the competition curves of BNP-(Asp-81--Tyr-106), ANF-(Ser-99--Tyr-126), and des-(Gln-116--Gly-120)ANF-(Asp-102--Cys-121)NH2 (C-ANF, a ligand highly specific for ANF-R2 receptors) for 125I-labeled BNP-(Asp-81--Tyr-106) and 125I-labeled ANF-(Ser-99--Tyr-126) binding revealed that ANF fully displaced 125I-BNP binding and, conversely, BNP completely displaced 125I-ANF binding in these tissues, whereas C-ANF partially displaced 125-BNP and 125-ANF binding. Angiotensin II, insulin, glucagon, and substance P had no influence on 125I-BNP binding in the above tissues. These results support the view that BNP and ANF share the same binding sites in rats.

Localization and characterization of binding sites for circulating and cerebroventricular atrial natriuretic factor in rat choroid plexus.

In vitro autoradiographic studies showed that high-affinity atrial natriuretic factor (ANF) binding sites are present on rat choroid plexus (Kd = 83.8 pM, Bmax = 22.9 fmol/mg protein). Guanylate cyclase-coupled receptors (ANF-R1) represent 30% and non-guanylate cyclase-coupled ANF receptors (ANF-R2) represent 70% of the total ANF receptors present in this tissue. To provide detailed cellular localization of the binding sites, the technique of electron-microscopic autoradiography was applied using 125I-ANF (Ser 99-Tyr 126) as an in vivo ligand. In order to identify possible binding sites at the basolateral and the apical aspects of the choroid plexus, the ligand was injected into the carotid artery or into the lateral cerebral ventricles, respectively. Light-microscopic autoradiography demonstrated that ANF binds specifically to choroid plexus regardless of its route of administration. Electron-microscopic autoradiography showed that silver grains were localized primarily on epithelial cells of the choroid plexus (96-99%) and marginally on endothelial and pial cells. In choroidal epithelial cells, ultrastructural analysis of silver grain distribution revealed that, at 2 min after intracarotid or intracerebroventricular 125I-ANF injection, lysosomes were the most distinctly labeled organelle (highest relative specific radioactivity). HPLC Chromatographic analysis disclosed that 96-99% of choroid plexus-bound ANF was already degraded 2 min after injection and that at least 63-66% of the degradation took place at the plasma membrane. These results indicate that ANF binding sites are present on both aspects of choroidal epithelium, and suggest that ANF is very quickly degraded in choroid plexus by membrane-associated as well as lysosome-associated processes.