Femtosecond and excimer laser-assisted endothelial keratoplasty (FELEK): a new technique of endothelial transplantation.

PURPOSE: To describe a new technique of endothelial keratoplasty (EK) that improves the quality of lamellar dissection of donor cornea. METHODS: We compared four techniques of donor cornea preparation for lamellar dissection on 8 donor corneas: mechanical dissection with a microkeratome, a single femtosecond laser lamellar cut, a double femtosecond laser lamellar cut and combined femtosecond laser lamellar dissection with excimer laser surface photoablation. The quality of the donor cornea interface was assessed and compared using scanning electron microscopy (SEM), and the most satisfactory technique was employed for EK on three patients. The postoperative anatomic results were analyzed with anterior segment spectral-domain optical coherence tomography (SD-OCT) and in vivo confocal microscopy (IVCM). RESULTS: The smoothest stromal interface was observed on SEM with the combined use of femtosecond laser dissection and excimer photoablation. The surgical procedures performed with donor cornea prepared by a combination of femtosecond and excimer lasers resulted in clear corneas after 1 month. SD-OCT showed good attachment of the endothelial graft and a hyperreflective interface. On IVCM, subepithelial haze, honeycomb-like activated keratocytes and needle-shaped particles were visible in the recipient corneal stroma as well as numerous hyperreflective particles on the donor-recipient interface. CONCLUSION: A new technique, femtosecond and excimer laser-assisted endothelial keratoplasty (FELEK), which refines the current limitations observed in Descemet-stripping automated endothelial keratoplasty (DSAEK), is described. Femtosecond laser dissection provides a thin and reproducible endothelial graft cut with a high level of safety and accuracy, while excimer photoablation yields a smooth, high-quality interface.

Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness.

BACKGROUND: New models continue to be required to improve our understanding of colorectal cancer progression. To this aim, we characterised in this study a three-dimensional multicellular tumour model that we named colospheres, directly obtained from mechanically dissociated colonic primary tumours and correlated with metastatic potential. METHODS: Colorectal primary tumours (n=203) and 120 paired non-tumoral colon mucosa were mechanically disaggregated into small fragments for short-term cultures. Features of tumours producing colospheres were analysed. Further characterisation was performed using colospheres, generated from a human colon cancer xenograft, and spheroids, formed on agarose by the paired cancer cell lines. RESULTS: Colospheres, exclusively formed by viable cancer cells, were obtained in only 1 day from 98 tumours (47%). Inversely, non-tumoral colonic mucosa never generated colospheres. Colosphere-forming capacity was statistically significantly associated with tumour aggressiveness, according to AJCC stage analysis. Despite a close morphology, colospheres displayed higher invasivity than did spheroids. Spheroids and colospheres migrated into Matrigel but matrix metalloproteinase (MMP)-2 and MMP-9 activity was detected only in colospheres. Mouse subrenal capsule assay revealed the unique tumorigenic and metastatic phenotype of colospheres. Moreover, colospheres and parental xenograft reproduced similar CD44 and CD133 expressions in which CD44+ cells represented a minority subset of the CD133+ population. CONCLUSION: The present colospheres provide an ex vivo three-dimensional model, potentially useful for studying metastatic process.

First discrete autoradiographic distribution of aminopeptidase N in various structures of rat brain and spinal cord using the selective iodinated inhibitor [125I]RB 129.

The selective and potent aminopeptidase N inhibitor [125I]RB 129 has been used for the radioautographic localization of this enzyme in rat brain, spinal cord and intestine. Brain microvessels and intestine brush-border cells were shown to present a high concentration of aminopeptidase N. Moreover, a labeling of various brain structures was observed. A very high level of binding occurred in the meninges, choroid plexus, pineal gland, paraventricular nucleus and pituitary gland. Moderate to high labeling was also observed in the cortex, caudate-putamen, subthalamic nucleus, central periaqueductal gray, thalamus, as well as in the dorsal and ventral horn of the spinal cord, which are known to contain a high concentration of enkephalins, opioid receptors and neutral endopeptidase. This co-localization confirms the physiological implication of aminopeptidase N in the inactivation of enkephalins accounting for the requirement of dual inhibition of neutral endopeptidase and aminopeptidase N to observe highly significant morphine-like effects induced by the protected endogenous opioid peptides. Aminopeptidase N was also visualized in moderate to high levels in other brain structures such as the hippocampus, nucleus accumbens, substantia nigra, hypothalamus (dorsomedial and ventromedial nuclei), raphe nucleus, pontine nucleus, inferior olive, and in high concentration in the granular layer of cerebellum. In summary, aminopeptidase N has been visualized for the first time in numerous brain areas using the selective inhibitor [125I]RB 129. This iodinated probe could allow the ex vivo and in vivo localization of aminopeptidase N in various tissues to be investigated and may also be used to evaluate quantitative changes in aminopeptidase N expression in pathological situations. Aminopeptidase N, which preferably removes NH2-terminal neutral amino acids from peptides, has probably a host of substrates. Nevertheless, a certain in vivo selectivity could be achieved by the presence of the enzyme in structures where the peptide effector and its receptors are also co-localized.

Characterization of human villous and extravillous trophoblasts isolated from first trimester placenta.

Trophoblasts of the human placenta differentiate along two pathways to give either extravillous cytotrophoblasts (EVCT) with invasive properties and that are implicated in the implantation process, or villous cytotrophoblasts (VCT) that by cell fusion form multinucleated syncytiotrophoblasts. We report the first isolation and purification of these two cell types from the same chorionic villi of first trimester human placenta. We also studied their differentiation in vitro. Electron microscopy showed that in contrast to VCT, EVCT had no microvilli but contained large fibrinoid inclusions. EVCT cultures required a matrix to invade, and as previously established, VCT cultured on plastic dishes aggregated and fused to form syncytiotrophoblasts. These differentiation processes were characterized by a particular pattern of gene expression as assessed by real-time PCR and confirmed by immunocytochemical analysis of the corresponding proteins. EVCT cultured in vitro expressed high levels of HLA-G, c-erbB2, human placental lactogen, and very little human chorionic gonadotropin. Interestingly, TGFbeta2 was a marker of EVCT in vitro and in situ. These data offer a new tool for cell biologists to study the molecular mechanisms involved in human placental development and its pathology.

Binding properties of a highly potent and selective iodinated aminopeptidase N inhibitor appropriate for radioautography.

Aminopeptidase N (APN) is a zinc metallopeptidase involved in the inactivation of biologically active peptides. The knowledge of its precise distribution is crucial to investigate its physiological role. This requires the use of appropriate probes such as the recently developed highly potent and selective radiolabeled APN inhibitor 2(S)-benzyl-3-[hydroxy(1'(R)-aminoethyl)phosphinyl]propanoyl-L-3-[ (12 5)I]iodotyrosine ([(125)I]RB 129). Its binding properties were investigated using rat brain homogenates (K(d)=3.4 nM) or APN expressed in COS-7 cells (K(d)=0.9 nM). The specific binding was 95% at [K(d)], and preliminary autoradiography in intestine is promising. The decreased affinity of [(125)I]RB 129 (=10(-6) M) for the E(350)D APN mutant, supports the critical role of E(350) in the amino-exopeptidase action of APN.

Phagocytic and tumor necrosis factor alpha response of human mast cells following exposure to gram-negative and gram-positive bacteria.

Recent studies have implicated rodent mast cells in the innate immune response to infectious bacteria. We report that cord blood-derived human mast cells (CBHMC) obtained from culture of cord blood progenitors phagocytozed and killed various gram-negative and gram-positive bacteria and simultaneously released considerable amounts of tumor necrosis factor alpha. Overall, the extent of the endocytic and exocytic response of CBHMC correlated with the number of adherent bacteria. Thus, human mast cells are intrinsically capable of mediating microbial recognition and of actively contributing to the host defense against bacteria.

Antinociception produced by the peptidase inhibitor, RB 101, in rats with adrenal medullary transplant into the spinal cord.

This study was undertaken to investigate the effects induced by the systemic administration of RB 101 [N-[(R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyl dithio]-1-oxoprpyl]-L-phenylalanine benzyl ester], a mixed inhibitor of the enkephalin catabolism able to cross the blood-brain barrier, in antinociception produced by adrenal medullary tissue transplanted in the rat spinal subarachnoid space. For this purpose, the antinociceptive responses induced by intravenous (i.v.) administration of RB 101 were evaluated in the tail-flick in rats transplanted 28 and 56 days before the test. Systemic administration of RB 101 induced antinociceptive effects in sham-operated rats, as previously reported. RB 101 also enhanced significantly the antinociception produced by the autotransplant 28 and 56 days after surgery. The antinociceptive responses of RB 101 in both sham-operated and autotransplanted rats were blocked by naloxone, but were not modified by the noradrenergic antagonist, phentolamine, suggesting a selective involvement of opioid mechanisms. The present results indicate that the inhibitors of enkephalin catabolism enhance the antinociception induced by adrenal medullary transplants.

[Antiviral activity of hydroalcoholic extract from Haemanthus albiflos on the Moloney murine leukemia virus and the human immunodeficiency virus]. / Activité antivirale d'un extrait hydro-alcoolique d'Haemanthus albiflos sur le virus de la leucémie murine de Moloney et le virus du syndrome de l'immunodéficience acquise.

We have used a biological test on the microplates of cellular cultures in order to investigate the toxicity and the antiviral properties against different viruses: defective Moloney Murine Leukemia virus (MoMLV) derived from the SVX shuttle and expressing resistance to the G418 antimitotic, and Human Immunodeficiency Virus (HIV) of a hydroalcoholic extract from Haemanthus albiflos (Amaryllidacae). The toxicity was assessed through coloric test evaluation of fixed cells stained with crystal violet. In a population of NIH 3T3 cells (Fibroblasts mouse), the toxicity found with 2, 7, 14 and 28 microliters/ml of lyophilisat extract corresponding at: 0.23, 0.81, 1.62 and 3.24 mg of plant dry, was 32, 50, 63 and 70% respectively. With regards to the antiviral properties, the plant extracts showed an inhibition of 88% on the formation of G418 resistant 3T3 clones. The assay on HIV infected lymphotic cells (P4) showed an IC50 of 4 microliters/ml for this extract plant. Therefore, the toxic effect was similar to the antiviral response.

Development of [125I]RB104, a potent inhibitor of neutral endopeptidase 24.11, and its use in detecting nanogram quantities of the enzyme by "inhibitor gel electrophoresis".

Neutral endopeptidase 24.11, also known as the common acute lymphoblastic leukemia antigen, is a zinc metallopeptidase involved in the inactivation of biologically active peptides, such as the enkephalins and atrial natriuretic peptide. The highly potent radiolabeled inhibitor 2-((3-[125I]iodo-4-hydroxy)phenylmethyl)-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)propyl]amino-4-oxobutanoic acid ([125I]RB104; Ki = 30 pM) has been developed for the enzyme. [125I]RB104 is highly specific, its Ki for another widely distributed zinc peptidase, angiotensin-converting enzyme, being 15 microM. In binding studies using rat brain slices, [125I]RB104 was shown to have a high affinity (Kd = 300 +/- 20 pM) and high specific binding at the Kd concentration (90%). With rat brain homogenates the Kd of [125I]RB104 was 26.8 +/- 0.9 pM, close to the kinetically derived Kd, 7.0 +/- 0.8 pM. Using the inhibitor, we have developed a simple, rapid, and quantitative technique to detect low nanogram quantities of the endopeptidase directly from tissue extracts after SDS/PAGE. The method has been used to show the presence of low quantities of the enzyme in rabbit bone marrow. Apart from its sensitivity, "inhibitor gel electrophoresis" using [125I]RB104 has the advantage over immunohistochemical methods of being able to label the enzyme in all tissues and species. It will therefore be of great value in determining the exact role of this important regulatory peptidase in a number of biological systems. Moreover, this one-step characterization of neutral endopeptidase 24.11 could be extended to other zinc metallopeptidases such as angiotensin-converting enzyme or collagenases, and inhibitors with affinities as high as RB104 could open the way to visualization of zinc metallopeptidases in different tissues by electron microscopy.