Kazushige Takehana

Chlamydial infection in canine atherosclerotic lesions.

We attempted to detect chlamydial antigens in canine atherosclerotic lesions from seven dogs by immunohistochemical technique using anti-Chlamydia psittaci (C. psittaci) polyclonal and anti-C. pneumoniae monoclonal antibodies. Immunopositive signals to both antibodies were recognized in the atherosclerotic lesions of the aortas, coronary and splenic arteries of all dogs. Positive signals were found in the foamy cytoplasm of infiltrated macrophages and extracellular matrices in the lesions. In some lesions, cytoplasm of the endothelial cells and smooth muscle cells was also immunopositive against both antibodies. By electron microscopy, chlamydial microorganisms were found in the cytoplasm of endothelial cells and smooth muscle cells. Using polymerase chain reaction (PCR), detection of C. pneumoniae DNAs were performed in the spleen, heart (coronary arteries) and kidney in one of the seven dogs. Positive 314 bp PCR products were obtained in all samples of the dog. These results confirmed the presence of viable Chlamydiae in atheromas and supported the conclusion that the organism may be an active factor in the pathogenesis of canine, as well as human atherosclerosis.

Graded Arrangement of Collagen Fibrils in the Equine Superficial Digital Flexor Tendon

By using ultramorphological and biochemical methods, we analyzed the regional differences between the three parts of the equine superficial digital flexor tendon (SDFT), namely, the myotendinous junction (MTJ), middle metacarpal (mM), and osteotendinous junction (OTJ). Cross-sectional images showed unique distributions of collagen fibrils of varying diameters in each region. Small collagen fibrils (diameter <100 nm) were distributed predominantly in the MTJ region, and the OTJ region was relatively rich in large collagen fibrils (diameter >200 nm). In the mM region, the collagen fibrils were intermediately distributed between the MTJ and OTJ. The results indicate a graded arrangement of collagen fibrils in the tendon. Type V collagen was detected preferentially in the MTJ region. Since type V collagen is believed to be one of the collagens regulating collagen fibril formation, its possible functionality in the MTJ region in terms of fibril formation and fibril arrangement in the tendon has been discussed here.

Comparative study of the characteristics and properties of tendinocytes derived from three tendons in the equine forelimb.

The aim of this study was to determine the characteristic differences in tendinocytes derived from tendons in the equine forelimb, superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT) and common digital extensor tendon (CDET), in morphology, proliferation, collagen production ability and ability for synthesis of matrix metalloproteinases (MMPs). Significant differences were observed in cell number in vivo. The cellular number was largest in the SDFT and smallest in the CDET. The values of in vitro proliferation ratios and ability for synthesis of collagen and MMPs were largest in the SDFT and smallest in the CDET. Addition of TNFalpha to culture of all three types of tendinocytes increased the synthesis of both proMMP-9 (except CDET) and collagen and decreased proMMP-13 synthesis and had no effect on proMMP-2 synthesis. Flexor tendons in forelimbs (SDFT and DDFT) restore energy during locomotion and are more easily injured than are extensor tendons. This structural property would cause active ECM and MMPs synthesis. And CDET have very low turnover potential; in the small number of cells, low cellular proliferation, lower ability for synthesis of collagen and MMPs. The isolated tendinocytes provided much information on the characteristics and properties of tendons for the ECM turnover system and responsiveness of tendinocytes to complex inflammatory responses in tendinopathy.

Downregulation of Decorin and Transforming Growth Factor-β1 by Decorin Gene Suppression in Tendinocytes

Scars formed after tendonitis result in altered tissue mechanical properties after injury. The interaction of collagen molecules with decorin affects collagen fibrogenesis, and scar tissue is fragile as a consequence of a large amount of decorin in the scar. We hypothesized that scar formation could be prevented by controlling decorin expression in tendinocytes. As a preliminary experiment, we treated tendinocytes with decorin antisense oligodeoxynucleotides (ODNs). Tendinocytes were isolated from Achilles tendons of New Zealand white rabbits and treated with ODN. When tendinocytes were transfected with decorin sense ODN, there was no alteration, whereas decorin antisense ODN-treated tendinocytes showed suppression of transforming growth factor (TGF)-β1 production. Decorin and TGF-β1-production of tendinocytes is regulated by decorin gene suppression. The results showed that the antisense approach is an attractive therapeutic strategy not only for preventing decorin deposition in scar tissue, which decreases collagen fibril diameter, but also for controlling TGF-β1 production, which leads to organ fibrosis.

Concerted and adaptive alignment of decorin dermatan sulfate filaments in the graded organization of collagen fibrils in the equine superficial digital flexor tendon

The equine superficial digital flexor tendon (SDFT) has a graded distribution of collagen fibril diameters, with predominantly small‐diameter fibrils in the region of the myotendinous junction (MTJ), a gradual increase in large‐diameter fibrils toward the osteotendinous junction (OTJ), and a mixture of small‐ and large‐diameter fibrils in the middle metacarpal (MM) region. In this study, we investigated the ultrastructure of the SDFT, to correlate the spatial relationship of the collagen fibrils with the graded distribution. The surface‐to‐surface distances of pairs of fibrils were found to be almost constant over the entire tendon. However, the center‐to‐center distances varied according to fibril diameter. Decorin is the predominant proteoglycan in normal mature tendons, and has one dermatan sulfate (DS) or chondroitin sulfate (CS) filament as a side chain which is associated with the surfaces of the collagen fibrils via its core protein. We identified a coordinated arrangement of decorin DS filaments in the equine SDFT. The sizes of the decorin DS filaments detected by Cupromeronic blue staining showed a unique regional variation; they were shortest in the MM region and longer in the MTJ and OTJ regions, and a considerable number of filaments were arranged obliquely to adjacent collagen fibrils in the MTJ region. This regional variation of the filaments may be an adaptation to lubricate the interfibrillar space in response to local mechanical requirements. The results of this study suggest that the MTJ region, which receives the muscular contractile force first, acts as a buffer for mechanical forces in the equine SDFT.

Fine Structural and Histochemical Study of Equine Paneth Cells

Ultrastructure. lysozyme and glycoconjugate activity in duodenal Paneth cells were observed concurrently in the horse. Paneth cells were seen to uniformly line the base of the equine intestinal glands. The round secretory granules have centrally located electron densities with peripherally located electron lucent halos. Histochemically, the peripheral halo layer was positively stained for carbohydrates by the periodic acid‐thiocarbohydrazide‐silver protein‐physical development (PA‐TCH‐SP‐PD) method and the entire granules reacted positively to the WGA. The central core area reacted with anti‐lysozyme. We identified a young (Type I) and an old (Type II) cell population in the same crypt, but we suggest that the observed populations are variations of the same cell type with the varied appearance due to aging of the secretory granules.

Morphological characterization of gland cells of the glandular sac area in the complex stomach of the Bactrian camel (Camelus bactrianus).

The morphology of the gland cells in the glandular sac area of the Bactrian camel and the composition of secretory substances were examined by histochemical methods. It was found that the gland cells of the glandular sac area were of the same type and size as those of the cardiac glands. The composition of secretory substances from the glandular sac area was the same as that of secretory substances from the cardiac glands. Moreover, secretory substances from the gland cells of the glandular sac area contained a great deal of acid glycoconjugates, such as sialic acid, in addition to neutral saccharides (fucose, mannose, glucose, N‐acetyl‐D‐glucosamin, galactose and N‐acetylgalactosamin). Furthermore, immunohistochemical examination showed that progastricsin was present in the gland cells of the glandular sac area and the cardiac gland. In this study, histological analysis suggested that the stomach of the Bactrian camel is a single cavity stomach, formed as a result of multiple differentiation and growth of cardiac glands through the process of evolution.

Efficacy of self-assembled hydrogels composed of positively or negatively charged peptides as scaffolds for cell culture.

KASEA16(+) and KASEA16(−) peptides, the net charges of which are positive and negative, respectively, under a neutral condition could undergo self-assembly into nanofibers and form transparent hydrogels without peptide aggregation upon rapid pH neutralization. The numbers of NIH3T3 cells attached to the KASEA16(+) hydrogel and KASEA16(−) hydrogel were similar, and cells proliferated with time on both hydrogels. Cells on the KASEA16(+) hydrogel had spindle-like morphology, while cells on the KASEA16(−) hydrogel formed clusters without extending cytoplasmic processes. Comparison of differently charged peptides under a neutral condition suggested that the charges of the scaffolds should be taken into consideration for the best design and selection of scaffolds for cell culture. Since the KASEA16(+) peptide could form a stable hydrogel under a neutral condition and the hydrogel served as a scaffold for cell proliferation, the KASEA16(+) hydrogel will be a useful scaffold for cell culture.

A preliminary study of direct application of atelocollagen into a wound lesion in the dog cornea.

Purpose: To evaluate the effectiveness of atelocollagen for canine corneal wound healing. Materials and Methods: Atelocollagen was used to fill a transplant bed in the central cornea, which was then covered with a contact lens. The wound healing process was analyzed clinically, morphologically, and biochemically. Results: At the early healing stage, both the pupillary zone and details of the iris were observed. The stromal collagen fibrils normalized in a time-dependent manner. Type III collagen in the wound area was detected faintly throughout the experimental period. Conclusions: This novel method is advantageous for accelerating wound healing without causing inflammation.

Morphometric Analysis of Collagen: a Comparative Study in Cow and Pig Skins

The detailed ultrastructure of skin has not yet been determined, partly because of the problems involved in quantifying age variation in the collagen fibril diameter distribution with increasing depth below the epidermis and in quantifying the variations that occur with anatomical site. In the present study, we investigated the correlation between subfibrillar architecture and collagen fibril diameter in dermis samples taken from three different regions of the skin of clinically normal adult animals. However, further studies are needed to determine the correlation between the age variation in collagen fibril diameters and the subfibrillar architecture.