[Descemet membrane endothelial keratoplasty (DMEK): two-year results]. / Queratoplastia endotelial de membrana de Descemet (DMEK): resultados a dos años.

PURPOSE: To report the two-year results of Descemet membrane endothelial keratoplasty (DMEK) for managing corneal endothelial disorders. METHODS: Non-randomized prospective clinical trial. A DMEK was performed in ten patients with Fuchs' endothelial dystrophy or bullous keratopathy. A 3.5 mm clear corneal incision was made and "under air" DM was stripped off from the posterior stroma. A 9.0 mm diameter, organ cultured donor DM roll was inserted into a recipient anterior chamber, positioned into the posterior stroma and secured by completely filling the anterior chamber with air for 30 minutes. RESULTS: Three eyes showed complete detachment of the tissue; this was managed by a secondary Descemet stripping endothelial keratoplasty procedure. The remaining seven eyes had a best corrected visual acuity of >or=0.7 in three eyes (43%) at one month, in five eyes (71%) at six months, and in six eyes (86%) at one and two years. At six months, the endothelial cell density averaged 2039 (+/-373) cells/mm2 (n=7), at one year 1925 (+/-267) cells/mm2 (n=7) and at two years 1730 (+/-400) cells/mm2 (n=6). CONCLUSIONS: DMEK may provide quick and nearly complete visual rehabilitation. Since the donor tissue can be stripped from donor corneo-scleral rims, the procedure may be readily accessible to most corneal surgeons.

GATA-3 is involved in the development of serotonergic neurons in the caudal raphe nuclei.

The GATA-3 transcription factor shows a specific and restricted expression pattern in the developing and adult mouse brain. In the present study we investigated the role of GATA-3 in the caudal raphe system, which is known to operate as a modulator of motor activity. We demonstrate that virtually all neurons in the caudal raphe nuclei that express GATA-3 also produce serotonin. Absence of GATA-3, as analyzed in chimeric -/- mice, affects the cytoarchitecture of serotonergic neurons in the caudal raphe nuclei. As a result the chimeras show a serious defect in their locomotor performance on a rotating rod. In sum, we conclude that GATA-3 plays a major role in the development of the serotonergic neurons of the caudal raphe nuclei, and that it is crucial for their role in locomotion.

Developmental expression and differential regulation by retinoic acid of Xenopus COUP-TF-A and COUP-TF-B.

COUP-TFs (Chicken Ovalbumin Upstream Promoter Transcription Factors) have been proposed to be negative regulators of retinoid receptor-mediated transcriptional activation. In a previous paper we reported the cloning of a Xenopus (x) COUP-TF (Matharu, P.J. and Sweeney, G.E. (1992) Biochim. Biophys. Acta 1129, 331-334). Here we describe the cloning of a second xCOUP-TF. Amino acid sequence comparison between these two Xenopus COUP-TFs revealed a high level of similarity. Extensive amino acid sequence conservation was found among all Drosophila, Xenopus, zebrafish and mammalian COUP-TF genes examined. Phylogenetic tree analyses indicate that the vertebrate COUP-TFs fall into three classes. The two Xenopus COUP-TF genes show similar temporal expression patterns: both are expressed from the end of gastrulation. In situ hybridization studies reveal complex expression patterns in the developing central nervous system (CNS), besides expression in the eye and in some mesodermal tissues. Retinoic acid (RA) treatment enhances xCOUP-TF-A expression in neurula stage embryos, whereas the expression of xCOUP-TF-B is inhibited during the same developmental period. The strictly conserved amino acid sequences and the strong similarities between the expression patterns of the two different xCOUP-TFs on the one hand, and other vertebrate COUP-TF homologues on the other, make it likely that COUP-TFs have a conserved role in patterning the nervous system.

Descemet membrane endothelial keratoplasty (DMEK) for Fuchs endothelial dystrophy: review of the first 50 consecutive cases.

PURPOSE: To evaluate the clinical outcome and complications of Descemet membrane endothelial keratoplasty (DMEK), using Descemet-stripping endothelial keratoplasty (DSEK) as a back-up procedure, in the management of Fuchs endothelial dystrophy. DESIGN: Non-randomised prospective clinical study. METHODS: The first fifty consecutive eyes that underwent DMEK, that is, transplantation of an isolated donor Descemet membrane carrying its endothelium, for Fuchs endothelial dystrophy were evaluated. In all eyes, the best-corrected visual acuity (BCVA) as well as the endothelial cell density (ECD) was measured before and at 6 months after surgery, as clinical outcome parameters. RESULTS: Ten patients required a secondary DSEK for failed DMEK. In the remaining 40 DMEK eyes, 95% had a BCVA of > or = 20/40 (> or = 0.5) and 75% > or = 20/25 (> or = 0.8) at 6 months after surgery. ECD averaged 2618 (+ or - 201) cells/mm(2) before, and 1876 (+ or - 522) cells/mm(2) at 6 months after surgery (n = 35). When the outcomes of DMEK and secondary DSEK procedures were combined, 94% reached a BCVA of > or = 20/40 (> or = 0.5) and 66% > or = 20/25 (> or = 0.8) (n = 47), and ECD averaged 2623 (+ or - 193) cells/mm(2) before, and 1815 (+ or - 578) cells/mm(2) at 6 months after surgery (n = 43). CONCLUSION: With DSEK as a back-up procedure, DMEK may provide relatively quick and complete visual rehabilitation in a majority of patients operated on for Fuchs endothelial dystrophy. Endothelial cell survival may be similar to earlier types of (lamellar) keratoplasty. Early graft detachment was the main complication in this first series of DMEK surgeries for Fuchs endothelial dystrophy.

Floating device for donor corneas in organ culture.

AIM: To introduce a new floating device for donor corneas to avoid accumulation of debris onto the endothelial surface during organ culture and to facilitate handling of the tissue during preservation and surgery. METHODS: From 11 donors, one randomly chosen cornea was stored in organ culture attached to a floating device, while the contralateral cornea was attached to the lid of the phial by a suture ("hanging by suture"). Endothelial cell density (ECD) was evaluated prior to tissue storage and after 2-3 weeks of culture. Furthermore, we compared ECD in a larger group of corneas sent off for transplantation with the device (n = 281) to a historical group of control corneas "hanging by suture" (n = 444). RESULTS: There was no significant difference in ECD between corneas attached to the floating device or "hanging by suture" (n = 11; p > or = 0.1). Similarly, no different ECDs were observed between corneas sent off for transplantation with the device (n = 281) and the historical group of control corneas "hanging by suture" (n = 444) (p > or = 0.1). CONCLUSION: The use of the floating device may not affect tissue quality. Since its introduction, the use of the device has been uneventful and greatly facilitated tissue handling.

GATA3 haploinsufficiency causes a rapid deterioration of distortion product otoacoustic emissions (DPOAEs) in mice.

Human HDR (hypoparathyroidism, deafness and renal dysplasia)-syndrome is caused by haploinsufficiency of zinc-finger transcription factor GATA3. The hearing loss due to GATA3 haploinsufficiency has been shown to be peripheral in origin, but it is unclear to what extent potential aberrations in the outer hair cells (OHCs) contribute to this disorder. To further elucidate the pathophysiological mechanism underlying the hearing defect in HDR-syndrome, we investigated the OHCs in heterozygous Gata3-knockout mice at both the functional and morphological level. While the signal-to-noise ratios of distortion product otoacoustic emissions (DPOAE) in wild type mice did not change significantly during the first half-year of live, those in the heterozygous Gata3 mice decreased dramatically. In addition, both light microscopic and transmission electron microscopic analyses showed that the number of OHCs containing vacuoles was increased in the mutants. Together, these findings indicate that outer hair cell malfunctioning plays a major role in the hearing loss in HDR-syndrome.

Clearence of antibiotics from the intestines after termination of antibiotic decontamination.

The clearance of neomycin and kanamycin from the intestines after stopping oral supply has been determined in mice. Both antibiotics, although given in different doses, were excreted in essentially the same way; the clearance being a little faster than logarithmically in both cases. The importance of this observation with regard to isolation and the moment of reconventionalization is discussed.

Retinoid signalling and axial patterning during early vertebrate embryogenesis.

There are many indications that active retinoids are regulatory signals during vertebrate embryogenesis. Treating vertebrate embryos with retinoids can cause teratogenic defects, including specific derangements of the main body axis. Other data show that early vertebrate embryos contain physiologically relevant concentrations of active retinoids and express retinoid binding proteins and receptors; that knockouts of retinoid receptors can induce homeotic defects; and that relevant developmental control genes are regulated by retinoid response elements. Here, we discuss the possibility that retinoids are developmental signals which regulate axial patterning in the early vertebrate embryo.

Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis.

A series of adenovirus type 5 precursor terminal protein (pTP) and DNA polymerase (Ad pol) genes with linker insertion mutations were separately introduced into the vaccinia virus genome under the control of a late vaccinia virus promoter. The recombinant viruses were used for overexpression of the mutant genes in HeLa cells. In total, 22 different mutant pTP and 10 different Ad pol vaccinia virus recombinants were constructed, including some that expressed carboxyl-terminus-truncated forms of both proteins and one that produced the mutant H5ts149 Ad pol. To investigate the structure-function relationships of both proteins, extracts from cells infected with the recombinant viruses were tested for in vitro complementation of the initiation and elongation steps in adenovirus DNA replication. The results were in accordance with those of earlier in vivo experiments with these insertion mutants and indicate that multiple regions of both proteins are essential for adenovirus DNA replication. The carboxyl termini of both pTP and Ad pol were shown to be essential for proper functioning of these proteins during initiation of adenovirus DNA replication. Three different DNA replication-negative pTP mutants were shown to have residual activity in the initiation assay, suggesting not only that pTP is required for initiation but also that it may play a role in DNA replication after the deoxycytidylation step.

Graded retinoid responses in the developing hindbrain.

The purpose of this study was to make an explicit test of the idea that a retinoid could act as a morphogen, differentially activating genes and specifying anteroposterior (a-p) level in the developing vertebrate central nervous system (CNS). Our approach was to characterize the concentration-dependent effects of retinoic acid (RA) on the neural expression of a set of a-p patterning genes, both in vivo and in an in vitro system for neural patterning. Our results indicate that a retinoid is unlikely to specify a-p level along the entire CNS. Instead, our data support the idea that the developing hindbrain may be patterned by a retinoid gradient. Sequentially more posterior hindbrain patterning genes were induced effectively by sequentially higher RA concentration windows. The most posterior CNS level induced under our RA treatment conditions corresponded to the most posterior part of the hindbrain.

Inhibition of retinoic acid receptor-mediated signalling alters positional identity in the developing hindbrain.

Retinoids regulate gene expression via nuclear retinoic acid receptors, the RARs and RXRs. To investigate the functions of retinoid receptors during early neural development, we expressed a dominant negative RARbeta in early Xenopus embryos. We obtained evidence that dominant negative RARbeta specifically inhibits RAR/RXR heterodimer-mediated, but not RXR homodimer-mediated, transactivation. Both all-trans- and 9-cis-RA-induced teratogenesis were, however, efficiently opposed by ectopic expression of dominant negative RARbeta, indicating that only RAR/RXR transactivation is required for retinoid teratogenesis by each of these ligands. Experiments with two RXR-selective ligands confirmed that activation of RXR homodimers does not cause retinoid teratogenesis. Dominant negative RARbeta thus specifically interferes with the retinoid signalling pathway that is responsible for retinoid teratogenesis. Dominant negative RARbeta-expressing embryos had a specific developmental phenotype leading to disorganization of the hindbrain. Mauthner cell multiplications in the posterior hindbrain, and (both anteriorly and posteriorly) expanded Krox-20 expression domains indicated (partial) transformation of a large part of the hindbrain into (at least partial) rhombomere 3, 4 and/or 5 identity. In contrast, the fore- and midbrain and spinal cord appeared to be less affected. These data indicate that RARs play a role in patterning the hindbrain.