Inhibition of the proliferation of human periodontal ligament fibroblasts by hyaluronidase.

Hyaluronan (HA) exists in various living tissues as one of the major matrix macromolecules, and is well known to play an integral role in cell differentiation and proliferation. The present study was conducted to elucidate whether or not the proliferation of periodontal ligament (PDL) cells are affected specifically by the degradation of HA by hyaluronidasze (HAase). Human PDL fibroblasts were isolated and cultured with and without 15-150U/ml bovine testicular HAase from 1 to 11 days after seeding. The cells were also cultured with anti-CD44 antibody of 2 microg/ml. For the control against the anti-CD44 antibody treatment, 2 microg/ml IgG was used. The HA-dependent pericellular matrix was visualized by particle-exclusion assay. The number of cells was counted by MTT assay during the proliferation. The mRNA levels of HA synthases (HASs), HAases (HYALs) and CD44s were examined by a quantitative real-time PCR analysis. The cell proliferation was inhibited by the treatment with HAase and anti-CD44 antibody in cultured PDL fibroblasts. HASs mRNAs were down-regulated, whereas HYALs mRNAs were up-regulated significantly by the treatment with HAase and anti-CD44 antibody. The CD44s mRNA level exhibited no significant changes. These results suggest that HA may contribute to modulate the proliferation of cultured human PDL cells through a CD44-mediated mechanism.

Polyarginine-mediated protein delivery to dendritic cells presents antigen more efficiently onto MHC class I and class II and elicits superior antitumor immunity.

Protein transduction domains (PTDs) have been used increasingly to deliver reagents to a variety of cell types in vitro and in vivo. We have previously shown that HIV TAT-PTD-containing whole protein antigens (Ags)-transduced dendritic cells (DCs) stimulated Ag-specific CD8+ and CD4+ T cells. Although the cytotoxic T lymphocytes (CTL) activity generated was sufficient to prevent engraftment of mice with Ag-expressing tumors, treatment of tumor-bearing mice with TAT-PTD Ag-transduced DCs resulted in tumor regression in some animals. Recently, several other PTDs were reported to promote higher transduction efficiencies than TAT-PTD. To evaluate the role of individual PTDs in induction of immune responses in tumor vaccination studies, we engineered recombinant fusion Ovalbumin (OVA) that contained three differrent PTDs, including the most efficacious known PTD (polyarginine (R9)-PTD). Our results demonstrated that R9-PTD-containing OVA transduced DCs most efficiently, and that transduction efficacy was closely correlated with the extent of Ag-specific CD4+ and CD8+ T-cell activation in vitro and in vivo. Repeated vaccination with R9-PTD-OVA-transduced DC in (OVA-expressing) tumor-bearing mice induced enhanced antitumor immunity, and elicited complete rejection of tumors when DC was co-injected with adjuvants. This vaccination strategy may be clinically applicable, and offers theoretical and practical advantages to those that are in current use.

[Measurement of half-value layer in mammography].

The half-value layer (HVL) of an X-ray beam for film-screen mammography is considered an important parameter for image quality and patient dose. Thus, HVL must be measured in accordance with The Manual of Accuracy for Mammography printed by the Japanese Society of Radiological Technology. The manual prescribes exactly the geometry of measurement, chamber position of measurement in the field, selection of chamber, and so on. However, the measurement of HVL is difficult in the actual clinical setting. This study examined the results of failure to perform the measurement of HVL in accordance with the manual for measuring HVL in the clinical setting. The investigation indicated that serious problems do not arise when measuring HVL for routine quality control even if the chamber in the field is not always set according to the manual and if a chamber for radiotherapy or diagnosis is used that is not recommended for soft X-ray by the manual.

Modulation of hyaluronan catabolism in chondrocytes by mechanical stimuli.

Hyaluronan (HA) is a component of the extracellular matrices of cartilage contributing to the structural and functional integrity. HA metabolism is regulated by both anabolic and catabolic processes; however, a great deal more of the detail has been unknown yet. The purpose of this study was to clarify the effect of excessive mechanical load on the expression and activity of hyaluronidase (HYAL) in chondrocytes with a special reference to the expressions of IL-1beta and tumor necrosis factor (TNF)-alpha. A cyclic tensile load of 22.8% cell elongation, regarded as an excessive mechanical stimulus, was applied to cultured rabbit knee articular chondrocytes. HYAL1, HYAL2, IL-1beta, and TNF-alpha mRNA levels were examined by quantitative real-time PCR analysis. The HYAL activity in culture medium was examined by HA zymography. Both HYAL1 and HYAL2 mRNA levels were upregulated significantly by the loading in cultured chondrocytes. HYAL activity was also enhanced as compared with unloaded controls. The IL-1beta mRNA level was upregulated significantly by the loading, and TNF-alpha mRNA level was slightly upregulated. HYAL1 and HYAL2 mRNA levels were upregulated significantly by IL-1beta treatment, resulting in a slight increase in HYAL activity. These results show that the expression of HYAL1 and HYAL2 in articular chondrocytes is enhanced by excessive mechanical stimuli and affected in part by induction of IL-1beta, leading to HA catabolism in articular cartilage.

Effects of mechanical load on the expression and activity of hyaluronidase in cultured synovial membrane cells.

Hyaluronan (HA) has lubricating and buffering functions in joints. Mechanical load is a regulatory factor of HA metabolism in joints, and HA synthesis by synovial membrane cells is modulated by mechanical load. However, the effects of mechanical load on HA catabolism by hyaluronidase (HYAL) in synovial membrane cells remain unclear. The purpose of this study was to evaluate the effects of cyclic tensile load on the expression and activity of HYAL in synovial membrane cells. A cyclic tensile load of 22.8% cell elongation was applied to cultured rabbit synovial membrane cells for 3 to 48 h with or without cycloheximide. HYAL1 and HYAL2 mRNA levels were evaluated by means of real-time polymerase chain reaction (PCR) analysis. HYAL activity in the cell culture was analyzed by means of HA zymography with or without HYAL2-small interfering (si) RNA. Levels of both HYAL1 and HYAL2 mRNA were up-regulated significantly (p < 0.01) by the cyclic tensile load with or without cycloheximide. HYAL activity was detected in the loaded cell cultures and was suppressed substantially by HYAL2-siRNA. HYAL activity was undetectable in unloaded cell cultures. These results show that a cyclic tensile load induces the expression and activity of HYAL in synovial membrane cells, suggesting that increased HYAL by mechanical load affects HA catabolism in synovial fluid.

Mechanisms underlying the dysfunction of melanocytes in vitiligo epidermis: role of SCF/KIT protein interactions and the downstream effector, MITF-M.

Little is known about the mechanisms involved in the dysfunction of melanocytes in vitiligo epidermis. It is hypothesized that some cytokine/receptor interactions may be affected, resulting in dysfunction and/or loss of melanocytes. This study has compared the expression of endothelin (ET)-1, the ET-1 receptor (ET(B)R), granulocyte macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), the SCF receptor (KIT protein), tyrosinase, and S100 alpha between lesional and non-lesional vitiligo epidermis. Analysis by reverse transcription-polymerase chain reaction (RT-PCR) and by western blotting for ET-1 and SCF unexpectedly demonstrated up-regulated expression of these cytokines in lesional vitiligo epidermis. Immunohistochemistry with antibodies to melanocyte markers revealed that at the edge of the lesional epidermis, melanocytes remain and express tyrosinase, S100 alpha and ET(B)R, but not KIT protein or melanocyte-specific microphthalmia-associated transcription factor (MITF-M). Quantitation of the staining revealed a slight or moderate decrease in the number of S100 alpha, tyrosinase, and ET(B)R-positive cells at the edge of the lesional epidermis. In contrast, the number of cells expressing KIT protein was markedly decreased at the edge of the lesional epidermis compared with the non-lesional epidermis. At the centre of the lesional epidermis, there was complete loss of melanocytes expressing KIT protein, S100 alpha, ET(B)R, and/or tyrosinase. Western blotting revealed down-regulated expression of c-kit and MITF-M proteins at the edge of the lesional epidermis in vitiligo. These findings suggest that reduction in the expression of KIT protein by melanocytes and its downstream effectors, including MITF-M, may be associated with the dysfunction and/or loss of melanocytes in vitiligo epidermis.

Approaches to repigmentation of vitiligo skin: new treatment with ultrasonic abrasion, seed-grafting and psoralen plus ultraviolet A therapy.

Vitiligo vulgaris is a common disease throughout the world although its pathogenesis is not yet known. The most frequent treatment used for vitiligo is PUVA (psoralen plus ultraviolet A) and topical steroids but against stable refractory vitiligo, various other surgical techniques have been developed such as autografting, epidermal grafting with suction blisters, epithelial sheet grafting, and transplantation of cultured melanocytes. We have discovered a new method using ultrasonic abrasion, seed-grafting and PUVA therapy. The ultrasonic surgical aspirator abrades only the epidermis of recipient sites. This easily and safely removes only the epidermis, even on spotty lesions or intricate regions which are difficult to remove using a conventional motor-driven grinder or liquid nitrogen. Epidermal seed-grafting can cover more area than sheet-grafting, and subsequent PUVA treatment can enlarge the area of pigmentation with coalescence of adjacent grafts. In this article, we provide a general overview of the current surgical therapies including our method for treating stable refractory vitiligo.

Hyaluronidase expression in cultured growth plate chondrocytes during differentiation.

Hyaluronan (HA) is a major component of the extracellular matrix of cartilage, contributes to its structural and functional integrity, and has various important roles in the differentiation of chondrocytes. HA metabolism is regulated by both anabolic and catabolic processes; however, the details have not yet been clarified. The purpose of this study was to clarify the expression patterns of hyaluronidase (HAase) mRNAs (from the relevant HAase genes: the HYALs) and HAase activity during chondrocyte differentiation. Cartilage tissue and growth plate chondrocytes were isolated from the ribs of 4-week-old male Japanese rabbits. The expression of HYAL mRNAs in cartilage was analyzed by in situ hybridization. The expression levels of HYAL mRNAs in the culture were analyzed for each of the chondrocyte differentiation stages by means of quantitative real-time polymerase chain reaction analysis. Enzymatic activity in the conditioned medium from the cultures was examined by using HA zymography and an enzyme-linked immunosorbent-like assay. The expression levels of HYAL1 and HYAL2 mRNAs were enhanced about 2.8-fold and 3.2-fold at the maximum during the early matrix forming stage, respectively, and by about 3.2-fold and 2.0-fold at the maximum in the hypertrophic stage, respectively. HYAL3 mRNA was not detected throughout the experimental period. HAase activity was enhanced at the early matrix forming and hypertrophic stages. These results suggest that selective expression of HYALs is essential for extracellular HA metabolism during chondrocyte differentiation.