Development of a Rotation Device for Microvascular Endothelial Cell Seeding

A rotation device (RD) specifically designed to achieve sterile endothelial cell (EC) seeding of vascular grafts has been developed. The basic characteristics of the RD include: small dimensions, fully autoclavable components, and perfectly sealed graft holders. These features make it possible to maintain sterility during all the steps of EC seeding. This was documented by negativity of all bacteriological assays performed . Moreover, the RD can simultaneously support three vascular grafts with different lengths (20, 40, and 60 cm) and diameters (4-8 mm). EC seeding is performed in the climatized chamber (37 °C; 5 % C02) with constant rotation (0.1 -3 rpm). The rotation cycle can be completed automatically. The practical efficacy of the RD was investigated by seeding 2 x 105/cm2 of human microvascular EC on 20 cm length, 4 mm internal diameter (ID) fibronectin- coated polytetrafluoroethylene (ePTFE) grafts for 24 and 48 hours respectively . Further, the effect of a highly viscous plasma expander, i.e., haemagel, on cell retention was also evaluated. Results were not as favorable as expected. However, it should be emphasized that after 48 hours of eel! incubation by using the RD, 42 % of the initially seeded EC were still present and approximately 15 % were fully spread over the graft surface. Moreover, the 10 minute perfusion with haemagel did not decrease the number of adherent microvascular EC

Electron Microscopy of Lipid Deposits in Human Atherosclerosis

The filipin probe associated with tannic acid stain was used to study intra-and extracellular lipids in surgically removed human atherosclerotic lesions (n = 20). In particular, intimal thickenings, fatty streaks and fibrolipidic plaques have been investigated by using mainly transmission and scanning electron microscopy. In the intimal thickenings, the lipid deposits were mainly localized in the subendothelial space as homogeneously sized particles (40-140 nm) and more heterogeneous uni-multilamellar vesicles (35-700 nm). Intermediate lipid forms were also observed. In the fatty streaks, the lipid deposits were intracellular and mainly observed in cells with a monocyte/macrophagic phenotype. Lipid inclusions, lipid lysosomal bodies and intracellular cholesterol crystals very similar to those observed in experimentally induced atherosclerosis were documented. In the fibrolipidic plaque the lipid deposits were found both in the intracellular and in the extracellular compartments. Lipids accumulated within arterial macrophages and smooth muscle cells, usually as lipid droplets. Clusters of lipoprotein-like particles (50 nm in diameter) as well as larger uni-multilamellar lipids (700 nm) with an occasional compound appearance were particularly observed bound to elastic tissue and collagen fibers. These morphological observations outline the complexity of lipid metabolism in the various histological aspects of human atherosclerosis

Visual evoked potentials in succinate semialdehyde dehydrogenase (SSADH) deficiency

In mammals, increased GABA in the central nervous system has been associated with abnormalities of visual evoked potentials (VEPs), predominantly manifested as increased latency of the major positive component P100. Accordingly, we hypothesized that patients with a defect in GABA metabolism, succinate semialdehyde dehydrogenase (SSADH) deficiency (in whom supraphysiological levels of GABA accumulate), would manifest VEP anomalies. We evaluated VEPs on two patients with confirmed SSADH deficiency. Whereas the P100 latencies and amplitudes for binocular VEP analyses were within normal ranges for both patients, the P100 latencies were markedly delayed for left eye (OS) (and right eye (OD), patient 1) and monocular OS (patient 2): 134-147 ms; normal <118 ms. We hypothesize that elevated GABA in ocular tissue of SSADH patients leads to a use-dependent downregulation of the major GABAergic receptor in eye, GABA(C), and that the VEP recordings' abnormalities, as evidenced by P100 latency and/or amplitude measurements, may be reflective of abnormalities within visual systems. This is a preliminary finding that may suggest the utility of performing VEP analysis in a larger sample of SSADH-deficient patients, and encourage a neurophysiological assessment of GABA(C) receptor function in Aldh5a1(-/-) mice to reveal new pathophysiological mechanisms of this rare disorder of GABA degradation

Are Long Term Cryopreservation and Patency of Vein Allograft Truly Achievable?

Despite extensive experimental work, neither the effect of long term cryopreservation on vein graft architecture nor the failure of alloveins due to graft rejection have yet been investigated. Herein, we investigated ultrastructurally: a) the integrity of rabbit jugular veins following 1, 2 and 3 months of cryopreservation; b) the outcome of the three-month cryopreserved vein auto- and allografts after 1 month of implantation in the rabbit carotid artery; and c) the immunologic response to cryopreserved vein allografts with and without seeded autologous endothelium. Prior to implantation, the cryopreserved rabbit veins were well-maintained except for endothelial cell damage. Following implantation, the cryopreserved vein autografts were comparable to fresh veins with a complete endothelial lining. Conversely, only one of the allograft was still patent with features of acute rejection. After seeding with autologous endothelium , these explants failed shortly after surgery. We found absence of endothelium and necrosis of the media components with neutrophil infiltration. Although three months of cryopreservation does not affect vein graft architecture significantly, endothelial cells are damaged irrespective of the time of cryopreservation. Vein autografts promptly healed after one month of implantation at which time a viable endothelial cell lining was restored from the host artery. Conversely, vein allografts, with and without seeded autologous endothelium, failed due to graft rejection. This study highlights that current methods of cryopreservation do not reduce antigenicity of venous allografts significantly

The hyperornithinemia-hyperammonemia-homocitrullinuria syndrome

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is a rare autosomal recessive disorder of the urea cycle. HHH has a panethnic distribution, with a major prevalence in Canada, Italy and Japan. Acute clinical signs include intermittent episodes of vomiting, confusion or coma and hepatitis-like attacks. Alternatively, patients show a chronic course with aversion for protein rich foods, developmental delay/intellectual disability, myoclonic seizures, ataxia and pyramidal dysfunction. HHH syndrome is caused by impaired ornithine transport across the inner mitochondrial membrane due to mutations in SLC25A15 gene, which encodes for the mitochondrial ornithine carrier ORC1. The diagnosis relies on clinical signs and the peculiar metabolic triad of hyperammonemia, hyperornithinemia, and urinary excretion of homocitrulline. HHH syndrome enters in the differential diagnosis with other inherited or acquired conditions presenting with hyperammonemia

CUGC for hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome

From 1999 to date, 50 affecting function variants have been identified and associated to HHH syndrome [1–5]. As it is not available in the literature a complete up-to-date list of disease-causing variants for SLC25A15 gene, we included this information as a Supplementary Excel sheet (See Supplementary Material File #1): this list was created by using LOVD and ClinVar databases and liked to the relevant literature reference. Reported variants consist of: 29 missense variants, 4 frameshift, 11 nonsense, 2 splicing, 2 small deletion, 1 in frame insertion, 1 gross deletion