The Effect of Antibody and Gene Therapy for Transforming Growth Factor- 1 on Scar Formation

BACKGROUND: Transforming growth factor (TGF)- has a large variety of biological functions, including the modulation of inflammation and the immune system, and is presumed to play important roles in repairing wounds and reducing scarring. The objective of this study is to examine the effects of TGF-1 on healing wounds and reducing scarring. We have also analysed the ability of the hemagglutinating virus of Japan (HVJ) liposome mediated antisense oligodeoxynucleotides (ODNs) to specifically inhibit wound-induced expressions of TGF-1 proteins and mRNA in the rat skin. METHODS: Skin wounds were created on the backs of 80 anesthetized rats. The first group of wounds, as the controls, was unmanipulated. The second group of wounds, as positive controls or an excessive scarring model, was injected with TGF-1 subcutaneously. The third group of wounds was injected with anti-TGF-1 antibody subcutaneously. The fourth group of wounds was injected with HVJ liposome mediated antisense ODNs for TGF-1 subcutaneously. The wounds of all groups were bisected and analysed histologically 5, 10, 15, 30, and 50 days after the wounds were made. RESULTS: All control wounds (TGF-1 or no injection) healed with scarring, whereas the wounds treated with the antibody or antisense ODNs healed with less scar formation compared to the control group. The wounds treated with the antibody or antisense ODNs had fewer macrophages, less collagen and fibronectin contents than the other wounds. Northern blotting and in situ hybridization analysis showed that wound sites treated with HVJ liposome mediated antisense ODNs for TGF-1 exhibited decreased levels of TGF-1 mRNA after injury. CONCLUSIONS: These findings suggest an important new approach to controlling scarring in normal wound healing, complementing the practice of adding exogenous growth factors to chronic wounds in the attempt to inhibit collagen deposition.

Expression of Transforming Growth Factor-beta1 in Cyclosporine-Induced Nephropathy in Rats

Cyclosporine nephropathy was induced by intraperitoneal injection of cyclosporine 25 mg/kg in Sprague-Dawley rats daily for 1, 4, 8, and 12 weeks to clarify the relationship between cyclosporine nephropathy and the expression of TGF-beta1 with extracellular matrix deposition. On light microscopic examination, the kidneys in the 12 week cyclosporine-treated rats showed focal or striped fibrosis, vacuolization of tubular cells, and injury of endothelial cells. Immunohistochemically, TGF-beta1 protein was strongly expressed in the cyclosporine-treated rat kidneys, especially in the glomerular endothelial cells, interstitial endothelial cells, tubular epithelial cells, and parietal cells in the Bowman's capsule of the glomerulus as well as the periglomerular arterioles. The amount of TGF-beta1 expression was correlated with the morphological change in the cyclosporine-treated rats. Extracellular matrix, such as fibronectin and collagen IV, was also expressed in the endothelial cells of the glomerulus and the interstitium. It can be concluded, therefore that TGF-beta1 protein is probably involved in the early stage of fibrogenesis in cyclosporine nephropathy. It can be postulated that cyclosporine nephropathy results from the accumulation of extracellular matrix associated with the increase of TGF-beta1 transcription. Therefore, these results could be used in reducing fibrosis in cyclosporine nephropathy.

Subgenus classification of Acanthamoeba by riboprinting

Subgenus classification of Acanthamoeba remains uncertain. Twenty-three reference strains of Acanthamoeba including 18 (neo)type-strains were subjected for classification at the subgenus level by riboprinting. PCR/RFLP analysis of 18S rRNA gene (rDNA). On the dendrogram reconstructed on the basis of riboprint analyses, two type-strains (A. astronyxis and A. tubiashi) of morphological group 1 diverged early from the other strains and were quite distinct from each other. Four type-strains of morphological group 3, A. culbertsoni, A. palestinensis, A. healyi were considered taxonomically valid, but A. pustulosa was regarded as an invalid synonym of A. palestinensis. Strains of morphological group 2 were classified into 6 subgroups. Among them, A. griffini which has an intron in its 18S rDNA was the most divergent from the remaining strains. Acanthamoeba castellanii Castellani, A. quina Vil3, A. lugdunensis L3a, A. polyphaga Jones, A. triangularis SH621, and A. castellanii Ma strains belonged to a subgroup, A. castellanii complex. However, A. quina and A. lugdunensis were regarded as synonyms of A. castellanii. The Chang strain could be regarded as A. hatchetti. Acanthamoeba mauritaniensis, A. divionensis, A. paradivionensis could be considered as synonyms of A. rhysodes. Neff strain was regarded as A. polyphaga rather than as A. castellanii. It is likely that riboprinting can be applied for rapid identification of Acanthamoeba isolated from the clinical specimens and environments.

Bacterial endosymbiosis within the cytoplasm of Acanthamoeba lugdunensis isolated from a contact lens storage case

Transmission electron microscopy of an Acanthamoeba isolate (KA/L5) from a contact lens case revealed bacterial endosymbionts within cytoplasm of the amoebae. The Acanthamoeba isolate belonged to the morphological group II. Based on the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of 18S ribosomal RNA coding DNA (rDNA), the isolate was identified as A. lugdunensis. Strain typing by isoenzyme analysis using isoelectric focusing (IEF) and mitochondrial (Mt) DNA RFLP revealed that the isolate was closely related with KA/L1, the most predominant type of isolates from contact lens storage cases, KA/E2, a clinical isolate, KA/W4, previously reported to host endosymbionts, and L3a strains of A. lugdunensis. The endosymbionts were similar to those of KA/W4 in aspects that they were randomly distributed in both trophozoites and cysts, and were rod-shaped bacteria measuring approximately 1.38 x 0.50 microns. But the number of endosymbionts per amoeba was significantly lower than that of KA/W4. They were neither limited by phagosomal membranes nor included in lacunaelike structure.

Close relatedness of Acanthamoeba pustulosa with Acanthamoeba palestinensis based on isoenzyme profiles and rDNA PCR-RFLP patterns

The taxonomic validity of morphological group III Acanthamoeba spp. is uncertain. In the present study, six type strains of group III Acanthamoeba spp., Acanthamoeba culbertsoni, A. healyi, A. pustulosa, A. palestinensis, A. royreba and A. lenticulata were subjected for the evaluation of their taxonomic validity by comparison of the isoenzyme patterns by isoelectic focusing on polyacrylamide gels, mitochondrial DNA (Mt DNA) restriction fragment length polymorphism (RFLP), and small subunit ribosomal DNA (ssu rDNA) PCR-RFLP patterns. The Mt DNA RFLP patterns were heterogeneous between species. The type strains of A. palestinensis and A. pustulosa showed almost identical patterns of isoenzymes and rDNA PCR-RFLP with an estimated sequence divergence of 2.6%. The other species showed heterogeneous patterns of isoenzymes and rDNA PCR-RFLP. It is likely that A. pustulosa is closely related with A. palestinensis and that the former may be regarded as a junior synonym of the latter.