In vivo study of the effects of cryopreservation on heart valve xenotransplantation.

BACKGROUND: Recent reports have suggested that cryopreservation reduces the immunogenicity of donor tissue. The immunomodulation by cryopreservation might influence on the tissue durability after xenotransplantation. We investigated the in vivo morphologic changes in cryopreserved xenograft (CXG) heart valves. MATERIAL AND METHOD: We transplanted a fresh (fresh xenograft; FXG) and a cryopreserved (CXG) porcine aortic root and a cryopreserved canine (cryopreserved allograft; CAG) aortic root into the abdominal aorta of a dog without any immunosuppressive agents. Explanted grafts on the 21st to 49th days after implantation were analyzed morphologically with light microscopy using some special stains, immunohistochemical analysis, and scanning electron microscopy (SEM). RESULT: Light microscopy showed the absence of smooth muscle cells in the media of the aorta in any group after transplantation. FXG valves did not maintain any cellularity after transplantation. CXG valves contained cellular infiltration in themselves. CAG valves contained numerous fibroblasts, which showed the maintenance of tissue integrity without allowing cellular infiltration. The structure of elastic fibers was well maintained, even in the part of CXG valve with cellular infiltration. Immunohistochemical studies documented the infiltration of T lymphocytes in CXG valves that were labeled by anti-CD3 antibodies. SEM demonstrated that no endothelia were seen on the surface of the valves in any group after transplantation. CONCLUSION: We concluded that the cryopreservation method might provide an immunomodulation of xenogeneic heart valves for transplantation.     

Biotin deficiency in amino acid formula nutrition for an infant with milk protein allergy]

We reported a 4-month-old girl with biotin deficiency caused by amino acid formula. Two weeks after birth, she was diagnosed as having a milk protein allergy. After switching to amino acid formula from usual formula, her symptoms and laboratory findings became normal. About three weeks after the beginning of amino acid formula, she developed intractable skin erosions around the eyes, mouth, neck, and anogenital area. By measuring concentrations of some trace elements, she was diagnosed as having a biotin deficit, because of the organic aciduria and undetectable serum biotin concentration. Her serum biotinidase level was normal. Upon administration of oral biotin supplementation, all her symptoms and laboratory findings were dramatically improved. Since amino acid formula contains very few biotin, we should pay attention to biotin deficiency when infants receiving amino acid formula.     

Sustained interleukin-6 signalling leads to the development of lymphoid organ-like structures in the lung

A variety of pathological changes are seen in lymphoproliferative disorders of the lung but the histogenesis of these abnormalities is not yet fully understood. We previously showed that adenovirus vector-mediated transient expression of both the human interleukin-6 (IL-6) and IL-6 receptor (IL-6R) genes, but not the IL-6 gene alone, in the rat lung induced lymphocytic alveolitis. In the present study, we explored the lung pathology of human IL-6 and IL-6R double transgenic mice to elucidate the effects of prolonged IL-6 signalling on the lung. The transgenic animals developed mononuclear cell accumulation in peribronchovascular regions, but little infiltration into alveolar spaces. Immunohistochemical analysis revealed that the cellular accumulations contained not only mixtures of inflammatory cells but also lymphoid tissue-like structures. As the expression of CXCL13/BLC, the indispensable chemokine for lymphoid organogenesis, was recognized in the B cell follicles of the pulmonary lesions, we speculate that this chemokine plays an inductive role in the development of the lymphoid tissue-like structures. These structures were distinguished from bronchus-associated lymphoid tissues (BALTs) by their location and by the lack of lymphoepithelium, which is a characteristic of BALT. These findings imply that IL-6 signalling may play a role in the pathogenesis of lymphoproliferative disorders of the lung.     

Freeze-fracture studies of muscle plasma membranes in myopathic patients with hypo-and hyperthyroidism

To examine the influence of thyroid hormone on the skeletal muscle plasma membrane, we analyzed the changes in ultrastructural architecture and membrane area complexed with digitonin of muscle plasma membrane in myopathic patients with hypo-and hyperthyroidism by the conventional freeze-fracture (F-F) technique and F-F cytochemistry using the sterol-specific ligand digitonin. The densities of flask-shaped invaginations, which are mainly thought to correspond to caveolae, intramembranous particles, and orthogonal arrays, and the changes of digitoninreacted membrane areas in the muscle plasma membranes in three patients with hypothyroid myopathy and one patient with both myasthenia gravis and hyperthyroidism were compared with those in age-matched controls. In the conventional F-F study, the muscle plasma membrane of hypothyroid patients showed increased invagination density, whereas that of the hyperthyroid patient was normal ultrastructurally. In the F-F cytochemistry study, however, the ratio of digitonin-reacted membrane areas versus fractured membrane areas was not different between hypothyroid patients and controls, whereas that of the hyperthyroid patient was lowered in comparison with that of control. These results suggest that thyroid hormone may alter the biochemical properties and ultrastructural architecture of muscle plasma membrane.This study was presented at the 29th annual meeting of the Clinical Electron Microscopy Society of Japan, Sapporo, October 2–3, 1997     

Cell surface laminin-like substances and laminin-related carbohydrates of rat ascites hepatoma AH7974 and its variants with different lung-colonizing potential

Rat ascites hepatoma AH7974 cells strongly expressed antilaminin antibody-reactive substances (laminin-like substances) andGriffonia simplicifolia isolectin B4 (GS)-reactive carbohydrate (alpha)-d-galactose; alpha-Gal) on their cell surface. The alpha-Gal expression was not apparently influenced by the pretreatment of cells with methanol. The cell membrane laminin-like substances had approximate molecular weights of 150, 62 and 56 kDa in denaturating reducing conditions, of which the 62 and 56 kDa bands were stained with GS. The cell membrane molecules bearing alpha-Gal were 62 and 56 kDa and were stained with antilaminin antibody. Therefore, the major molecules bearing alpha-Gal residues of AH7974 cell membrane are considered to be laminin-like substances. To determine the role of the substances in metastasis, we selected four cell lines (74AD, 74AD-f, 74FL, 74FL-a) from AH7974 in culture. 74AD and 74FL-a are adherent lines and 74AD-f and 74FL are floating lines. All of these cell lines strongly expressed laminin-like substances, but a marked difference was found in expression of alpha-Gal, which was most strongly expressed by 74FL, followed by 74AD, and rarely by 74AD-f and 74FL-a; the staining intensity was positively correlated with their experimental lung-colonizing potential. Cell membrane laminin-like substances were 200, 97, 62, 56 and 46 kDa and among them 62 and 56 kDa molecules were glycosylated with alpha-Gal. The pretreatment of 74FL cells with antilaminin antibody or with human type A serum (containing natural antibody to alpha-Gal epitope) depressed remarkably the lung-colonizing potential of the cells. These results suggest that the expression of 62 and 56 kDa laminin-like substances with alpha-Gal residues on tumor cell surfaces is one of the determinants associated with lung-colonizing potential of these cells.     

Immune Abnormalities Induced by Human Endogenous Retroviral Peptides: With Reference to the Pathogenesis of Systemic Lupus Erythematosus

P15E is a specific sequence among the envelope gene (env)-encoded transmembrane proteins of exogenous and endogenous retroviruses. A synthetic peptide (CKS-17) that shows homology to this p15E region in several species of retrovirus is known to induce immune abnormalities. In this study, we examined the effect of a synthetic peptide derived from a region of human endogenous retrovirus (HERV) clone 4-1 ( 4-1) similar to sequences of CKS-17 on the induction of systemic lupus erythematosus (SLE)-related immune abnormalities. Our results indicated that this peptide could induce T-cell activation and anergy in normal peripheral blood mononuclear cells, and the peptide could also promote the production of interleukins IL-6 and IL-16. These phenomena are representative immune abnormalities observed in SLE patients. Thus, our findings support the possibility that HERV acts as a pathogen in human SLE.     

The induction of intestinal metaplasia in rats by pyloroplasty or pyloroplasty plus vagotomy

The influence of increased gastric pH on induction of intestinal metaplasia in the stomach of male JCL/SD rats was examined following surgical procedures of pyloroplasty or pyloroplasty and vagotomy. Twelve months after pyloroplasty plus vagotomy serum gastric concentrations were significantly higher (group II) than in rats receiving only pyloroplasty (group I) or in sham operated animals (group III). Additionally, trehalase activity was also higher in group II than in groups I and III. The incidence of intestinal metaplasia was significantly higher in groups I and II compared with the sham operated control animals (group III). This study indicates that intestinal metaplasia could be induced by surgical procedures such as pyloroplasty with or without vagotomy. Furthermore, elevation of the pH on gastric mucosa by such procedures may play a significant role in the subsequent development of metaplasia in the stomach.     

Evolution of interleukin-18 binding proteins and interleukin-1 receptor, type II proteins

Interleukin-18 (IL-18) is one of the pivotal cytokines controlling the defense mechanism called inflammation. As a first step to develop proteins for controlling the IL-18 level, we initiated a study of IL-18-binding proteins (IL-18BPs). Twenty-four IL-18BP family members, 11 from vertebrates and 13 from chordopoxviruses, were picked from the NCBI database. Eight of these vertebrate IL-18BPs and two of the chordopoxvirus IL18-BPs were identified here and characterized as new members of the IL-18BP family. Their IL-18 binding domains were aligned and the distribution of highly conserved critical amino acid residues was analyzed and used to construct a phylogenetic tree. From this tree it was inferred that at least two independent events created two different ancestral viral IL-18BP genes by retroposition of IL-18BP genes from the vertebrate lineage. These two events are estimated to have occurred after an ancient mammalian IL-18BP gene diverged from birds, and before the mammalian IL-18BP gene diverged into human, ungulate and rodent IL-18BP genes. Moreover, our results suggest that IL-18BP and interleukin-1 receptor, type II (IL-1R2) had a common ancestral gene and diverged from the ancestral gene into IL-18BP and IL-1R2 genes in the fish period.     

Candidate interneurons mediating peripherally evoked disynaptic inhibition of masseter motoneurons of both sides

Location and axonal projection of interneurons presumed to mediate disynaptic inhibition evoked from the trigeminal sensory nerve in the ipsi- and contralateral masseter motoneurons were studied in pentobarbital anesthetized cats. Neurons monosynaptically excited from the periphery and antidromically activated from the contralateral trigeminal motor nucleus at low current intensity, hence probably terminating there, were found in the supratrigeminal region. Intracellular staining of such cells revealed collaterals terminating in the ipsilateral masseter motor nucleus. It is suggested that both the crossed and uncrossed disynaptic inhibition of masseter motoneurons are at least in part relayed by the same neurons in the supratrigeminal region.