Establishment and characterization of two human mixed mesodermal tumor cell lines from the same patient.

A cell line designated HIRS -BM was established from fluid aspirated from the sternal bone marrow of a 16-year-old female. Another cell line ( HIRS -PB) was derived from the peripheral blood of the same patient. Both lines grew well, multilayering rapidly without contact inhibition, and 62 serial passages were successively done within 28 months. Both cultures contained spindle- or fibrous-shaped cells that revealed neoplastic and pleomorphic features, and these cells were characterized as possessing cross-striations in the cytoplasm. The cross-striations were detected by phosphotungstic acid hematoxylin stain. Some elongated cells were stained positively with anti-myoglobin by use of periodic acid-Schiff methods. The primary tumor in the uterus was diagnosed as a mixed mesodermal tumor composed of adenocarcinoma and rhabdomyosarcoma cells. The karyotype exhibited hyperploidy and large submetacentric marker chromosomes, and the modal chromosome number was 84. No difference was found between the 2 cell lines except for growth behavior and heterotransplantability . HIRS -BM cells grew more rapidly and were highly transplantable. The HIRS -BM cells were transplanted into the subcutis of BALB/c nude mice and produced mixed mesodermal tumors resembling the uterine tumor, while the HIRS -PB cells could not be transplanted. Due to the histogenesis of the mixed mesodermal tumor being's obscure with histologic observations only, this study was performed to obtain data by tissue culture of the tumor and resulted in support of the combination theory reported in the literature in regard to tumor.

Hydrocortisone increases the numbers of KL1+ cells, a discrete thymic epithelial cell subset characterized by high molecular weight cytokeratin expression.

The thymic epithelium, a major component of the thymic microenvironment, is a heterogeneous tissue bearing distinct monoclonal antibody-defined subsets. Among these, KL1+ cells represent a mouse medullary subpopulation characterized by high mol wt cytokeratin expression. Given the fact that thymic epithelial cells (TEC) express glucocorticoid receptors and that glucocorticoid hormones are known to modulate the expression of keratins, we decided to study the in vivo effects of hydrocortisone on KL1+ cells in normal and autoimmune mice. Within 24 h after a single injection of this steroid we observed a significant increase in the number of KL1+ cells. Interestingly, this effect was reversible and was no longer detected 7 days after treatment. Parallel studies analyzing the effects of hydrocortisone on the secretion of thymulin, a chemically defined thymic hormone revealed a transient decrease in serum levels of this hormone, but with different kinetics than the effects on KL1+ cells. Ontogenetic studies showed that the responsiveness of TEC to hydrocortisone, in terms of high mol wt cytokeratin expression, appeared late in fetal life and disappeared in aging animals. Importantly, aging, but also young adult, autoimmune mice were not responsive. In vitro experiments using a mouse TEC line confirmed the data observed in vivo demonstrating that the increase in KL1+ cells is a direct effect of hydrocortisone on TEC. The bulk of the data presently reported demonstrates that glucocorticoid hormone can act on TEC modulating the expression of both secretory and cytoskeletal protein families.

Promotion of the osteogenetic activity of recombinant human bone morphogenetic protein by prostaglandin E1.

We investigated the promotive effect of prostaglandin (PG) E1 on the osteogenetic activity of bone morphogenetic protein (BMP) using porous chemically synthesized hydroxyapatite ceramic (HAP) pellets as the carrier. After treating the pellets with recombinant human (rh) BMP-2 with or without PGE1, both of which were used at two different concentrations, they were inserted beneath the cranial periosteum of a rabbit. The degree of osteogenesis and osteoconductivity was then examined histopathologically as well as by means of an image-analyzing procedure. Results showed that there was extensive bone formation around the pellets as early as 3 weeks after insertion in the group, which received pellets treated with a relatively large amount of rhBMP alone as well as in the group receiving pellets treated with a small amount of rhBMP combined with PGE1. In the later group, the extent of bone formation was dose-dependent. Subsequent osteogenesis within the pores of the pellets in these groups slowly progressed over time, and by 9 weeks after the insertion, most of the pellet pores had filled with newly generated bone. In addition, the groups which received pellets treated with PGE1 alone also showed osteogenesis as demonstrated by osteoconduction, which was not observed in the group that received the phosphate buffered saline (PBS) treated pellets. The group that received pellets treated with a small amount of rhBMP plus a high concentration of PGE1 exhibited significantly greater bone induction than the group which received the pellets treated with a small amount rhBMP alone, and there was no statistically significant difference between the former group and the group that received a high rhBMP dose. These results clearly indicated that PGE1 has a strong and dose-dependent promotive effect on the osteogenetic activity of rhBMP and that it also promotes osteoconduction, even when used alone. This suggests that enormous reductions can be made in the amount of rhBMP administered when PGE1 is used in conjunction with porous HAP. This should prove to be very advantageous and practical in the clinical application of these materials.

The second carcinoma of the anterior two-thirds of the tongue after successful radiotherapy to the tongue.

Of 221 patients with carcinoma of the mobile tongue treated by radiotherapy, 129 survived without local recurrence for 5 years or longer after initial treatment. These 129 patients were studied to determine the incidence of second carcinoma of the tongue which first appeared more than 5 years after the initial treatment. Modalities of irradiation were radium needle implantation and intraoral electron irradiation for 105 and 24 patients, respectively. Twenty-two of the patients were found to have second carcinoma of the tongue. The incidence appeared to increase as the amount of radiation given increased. No obvious relationships were observed between the second carcinoma and modality of irradiation, T classification, presence of leukoplakia before the treatment, degree of histological differentiation, or degree of radiation injury. There were moderate to severe radiation injuries in 50% of the patients treated by radium needle implantation. It is likely that most of the second carcinomas of the tongue represent the late appearance of one of the multicentric foci of the tumor, not a regrowth of the residual primary tumor. In spite of the rather high rate of second carcinoma of the tongue, radiotherapy remains an excellent modality of treatment when patients are properly selected.

Sertoli-stromal cell tumor of the ovary: immunohistochemical, ultrastructural, and genetic studies.

The Sertoli-stromal cell tumor (SSCT) of the ovary shows a histologic resemblance to developing or adult testes and is often associated with virilization caused by tumor-produced androgenic hormone. In spite of the unique manifestation of SSCT, detailed characteristics of this tumor are still obscure. The mechanism by which SSCT occurs has not yet been determined. Six SSCTs were studied immunohistochemically, ultrastructurally, and by polymerase chain reaction (PCR) for the presence of sex-determining region Y (SRY) gene and the X chromosome activation state. Immunohistochemically, Sertoli-like cells of SSCT were positive not only for alpha-inhibin but also low-molecular-weight cytokeratin. In control testes, the expression of alpha-inhibin and cytokeratin was limited to a Sertoli cell component and rete testis, respectively. Ultrastructurally, tumor cells composing hollow tubules had an elongated nucleus with deep indentation and annulate lamellae, which are characteristic structures of mature Sertoli cells. In addition, they had studded microvilli on the apical surface and frequent desmosomes, which are structures noted in the cells of rete testis. Histologically, tumor cells of hollow tubules sometimes pouted into the lumen, as did the cells of tubulae rete, entrance into rete testis from seminiferous tubules. All of these findings indicate that some tumor cells of a SSCT show simultaneous differentiation into both Sertoli cells and cells of rete testis. SRY gene was not detected in any cases, and the X chromosome activation pattern was the same as that of the female control.

Evaluation of a high density polyethylene fixing system for hydroxyapatite ceramic implants.

A fixing system made of high density polyethylene (mini ROC fastener system, Innovasive Devices Inc., Marlborough, MA, USA) was applied to artificially porous hydroxyapatite (HAP) implants. Their fixing ability was assessed with pull-out tests and a preliminary study was done to determine the most suitable drill hole diameter and depth. Effectiveness of the combined use of drill-free titanium screws (Martin GmbH & Co. Ltd., Tuttlingen, Germany) and the mini ROC fastener system was also evaluated. The pull-out strength of the system differed according to the hole diameter and depth of insertion. The strongest physical strength was obtained with holes 2.10 mm in diameter and 6 mm in depth. If inserted under optimum conditions, the mini ROC fastener system was deemed to have adequate fixing ability for clinical applications pull-out strength more than 7 kgf. In addition, the combined use of a drill-free screw for microplate with the mini ROC fastener system had a pull-out strength of around 10 kgf which was superior to the mini ROC fastener system alone. The mini ROC fastener system is best suited to fix HAP implants to the soft tissues such as muscle, tendon or periosteal tissue as well as for plate fixation when used with drill-free screws. The fact that effective fixing was achieved should result in a further increase in the clinical use of artificial HAP implants.

Porous hydroxyapatite ceramics and their ability to be fixed by commercially available screws.

We developed porous hydroxyapatite ceramics (HAP) which are screw fixable and evaluated the fixing abilities of commercially available screws using pull-out tests with the HAP implants. The fixing abilities were higher in the following order: Leibinger micro PLUS titanium screw > Osteomed M3 titanium screw > Leibinger standard mini screw > Martin-drill free screw. In preliminary examinations, the fixing ability of each of the screws differed according to the hole diameter and depth of insertion but if inserted under optimum conditions, all were deemed to have adequate fixing ability for clinical applications. Therefore, screw systems are superior for fixing HAP implants to surrounding bone than the use of thread or wire. Specifically, Leibinger's micro PLUS titanium screws system seems to be a good option currently. The fact that effective fixation was achieved should result in a further increase in the clinical use of synthetic porous HAP implants whose porosity and pore size are completely controlled for those purposes.

Effects of collagen matrix containing transforming growth factor (TGF)-beta(1) on wound contraction.

We evaluated the effectiveness of transforming growth factor (TGF)-beta(1) on wound contraction, both alone and in combination with collagen matrix, using an in vivo delayed wound healing type model. To clarify the mechanisms involved in the effectiveness of TGF-beta(1), we also used a fibroblast-populated collagen gel contraction in vitro model. Although we found that TGF-beta(1) significantly accelerated contraction of the fibroblast-populated collagen gel in vitro, we demonstrated that both collagen matrix alone and 1.0 microg of TGF-beta(1) alone significantly inhibited wound contraction in the in vivo model. In addition, the combination of TGF-beta(1) and collagen matrix was much more effective than TGF-beta(1) alone, a finding which was supported by histopathological examination. Wounds treated with collagen matrix containing TGF-beta(1) showed horizontal rearrangement of collagen fibers in the dermal part as well as evidence of active fibroblast proliferation, which was not observed in the scar regions of controls. These results show that the application of TGF-beta(1) treated collagen matrix is effective for preventing contraction producing so called "neodermis" in treating a delayed healing type model and may be highly beneficial for treating chronic wounds.

Effects of cilostazol lotion on blood flow in rabbit skin.

Cilostazol (Cls) is a inhibitor of phosphodiesterase and increases cyclic AMP (cAMP) in platelets and also raises the vascular smooth muscle cell cAMP level causing vasodilation. Therefore, it was expected to increase local blood flow in the skin. Topical application of Cls may improve local blood flow without systemic effects in clinical situations. In this paper the effect of Cls lotion on skin blood flow was assessed in animal experiments. Application of this lotion allowed skin blood flow to remain at increased levels for about 60-90 min. Tissue assay of the Cls content revealed that Cls is absorbed percutaneously and retained, even in the inner tissue layer, for at least 180 min.

Effects of a collagen matrix containing prostaglandin E(1) on wound contraction.

In this study, we evaluated the effectiveness of prostaglandin (PG) E(1) in inhibiting wound contraction, both alone and in combination with collagen matrix, using a in vivo full thickness skin defect model. To clarify the mechanisms involved in this inhibition we also used a fibroblast-populated collagen gel contraction in vitro model. We demonstrated that collagen matrix alone significantly inhibited wound contraction PG E(1) alone did not. Interestingly, their combination was much more effective than either collagen matrix or PG E(1) alone, a finding which was also supported by histopathological examination. Wounds treated with collagen matrix, but not control wounds, showed horizontal rearrangement of collagen fibers in the dermal part as well as evidence of active fibroblast proliferation which was not observed in scar regions surrounded by normal dermis. With the fibroblast-populated collagen gel contraction in vitro model, we found that PG E(1) significantly inhibited contraction at a high dose. It was concluded that collagen matrix combined with PG E(1) is effective for preventing contracture producing so called neodermis than collagen matrix alone, which remains one of the most challenging problems in treating full thickness type wounds.

Effects of prostaglandin E1 on cultured dermal fibroblasts from normal and hypertrophic scarred skin.

To investigate the role of prostaglandin (PG) E1 in preventing scar formation as well as that of the related cytokines, we culture fibroblasts from hypertrophic scar tissue (SDF) and normal dermis (NDF) collected from patients with scar contracture. We have compared the type I collagen synthesis, type I collagenase activity, and the production of interleukin (IL)-6, IL-8 and transforming growth factor (TGF)-beta(1) in two types of cultured fibroblasts before and after addition of PGE1. Our results demonstrated that levels of type I collagen and TGF-beta(1) production were higher and that type I collagenase activity and IL-8 production were significantly lower in the culture supernatants of SDF. There was no significance difference in IL-6 production between SDF and NDF culture supernatants. On the other hand, PGE1 significantly increased type I collagenase activity and IL-8 production in the SDF culture supernatants and it increased IL-6 and TGF-beta(1) production in both types of fibroblasts. However, there was no effect on synthesis of type I collagen in either group. To further investigate the role of TGF-beta(1) in NDF and SDF, exogenous recombinant human (rh) TGF-beta(1) was added. In NDF group, rhTGF-beta(1) induced a decrease in the type I collagenase/type I collagen ratio, while rhTGF-beta(1) had no effect on the same ratio in the SDF group. These results suggest that PGE1 may have a role in the prevention of hypertrophic scar by increasing the activity of type I collagenase.

Regulatory effects of 1,25-dihydroxyvitamin D3 and a novel vitamin D3 analogue MC903 on secretion of interleukin-1 alpha (IL-1 alpha) and IL-8 by normal human keratinocytes and a human squamous cell carcinoma cell line (HSC-1).

Pro-inflammatory cytokines mediate their biological functions after they are secreted or released from intracellular to extracellular milieu. Keratinocytes have proven to be able to produce various cytokines including IL-1 and IL-8. Dysregulations of IL-1 and IL-8 were found in psoriatic lesions. Recently, vitamin D3 (VD3) was found to be an effective and safe therapy for psoriasis. In the present study, we investigated the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its analogue MC903 on IL-1 alpha and IL-8 secretion by human keratinocytes in vitro. Cultured normal human keratinocytes (NHKs) produced considerable amounts of IL-1 alpha but secreted less. In contrast, they produced less IL-8 and almost all molecules were secreted to the culture supernatants. Treatment of unstimulated NHKs with 1,25(OH)2D3 or MC903 showed little effects on IL-1 alpha production and secretion though they slightly enhanced IL-8. When NHKs were stimulated with tumour necrosis factor-alpha (TNF alpha), both IL-1 alpha and IL-8 secretions were enhanced and these enhancements were inhibited by 1,25(OH)2D3 or MC903. Stimulation of NHKs with phorbol 12-myristate 13-acetate(PMA) and lipopolysaccharide(LPS) resulted in an increase of IL-8 and decrease of IL-1 alpha in the culture supernatants. Addition of 1,25(OH)2D3 or MC903 inhibited the increased secretion of IL-8 but restored decreased secretion of IL-1 alpha from stimulated NHKs dose dependently. Hydrocortisone and cyclosporin A showed similar inhibitory effects on PMA/LPS-increased IL-8 secretion from NHKs but had little effect of restoring IL-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on cytokines related to wound healing in donor site wound fluid.

This study was performed to evaluate cytokines in donor-site wound fluids and to determine their effect on wound healing. A film dressing was applied to the donor-site wound of 24 patients immediately after a split-thickness skin graft was taken. On the 5th day after treatment, 2-3 ml of the fluid retained under the film dressing was collected by means of puncture with a syringe. Growth factors and cytokines considered to accelerate wound healing were present in relatively large amounts in the exudate. Very low concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were detected by a commercially-available enzyme-linked immunosorbant assay (ELISA) kit. However, the presence of both growth factors in wound fluid could not be confirmed because of the possible cross-reactivity of the antibodies to other EGF and FGF family growth factors. In contrast, platelet derived growth factor (PDGF), interleukin-6 (IL-6), transforming growth factor-alpha (TGF-alpha) and TGF-beta were present in relatively large amounts. The finding that certain cytokines coexist in a balanced state under the film dressing suggests that epithelization can proceed, since an adequate balance would insure proper regulation by the cytokine network. Our present study increases the likelihood that film or hydrocolloid dressings will be used more frequently in the future for treatment of burn wounds, ulcers or donor-site wounds since these dressings were shown to be more capable than ointments of retaining cytokines, particularly intrinsic growth factors secreted at the wound site.

Analysis of microsatellite instability and loss of heterozygosity in uterine endometrial adenocarcinoma.

Microsatellite instability (MSI) and loss of heterozygosity (LOH) were examined in 60 cases of uterine endometrial adenocarcinoma, using 13 microsatellite markers. In non-Smad-related regions, MSI and LOH were noted in 13 of 60 (21.7%) and in 20 of 60 (33.3%) cases, respectively. Genetic alternation of TGF-beta RII was noted in 1 of 60 cases (1.7%). The frequency of MSI and LOH was highest in Stages III and IV, respectively. Cases with G2 carcinoma showed the highest frequency, but LOH frequency did not differ among G1, G2, and G3 carcinoma cases. In Smad-related microsatellite regions, MSI and LOH were noted in 10 of 60 (16.7%) and in 12 of 60 (20.0%) cases, respectively. The frequency of MSI and LOH was highest in Stages III and IV, respectively. LOH was seen only in the Smad2 gene but not in the Smad4 gene. Our results suggest that the alterations in MSI and LOH were associated with middle and late stages of carcinogenesis of endometrial carcinoma. Both MSI and LOH tended to show an association with moderate to severe atypia of carcinoma. Our results also suggest that genetic alteration of the Smad2 gene is more responsible for endometrial carcinogenesis than that of the Smad4 gene. However, the TGF-beta type II receptor gene was considered a minor target of genetic instability in endometrial carcinogenesis.

Non-traumatic gas gangrene in the abdomen: report of six autopsy cases.

Six autopsy cases of non-traumatic gas gangrene in the abdomen are reported. Five of the six were caused by clostridia, as identified by culture or histology. There were associated underlying diseases, such as alcoholism, liver cirrhosis, diabetes mellitus, and malignant disease. Three of the six patients had gas gangrene in the liver. Bacterial proliferation and gas accumulation were found in the sinusoids of the liver, and congestion and edema with extensive gas embolism were found in the lungs. Pulmonary gas embolism was considered to be the direct cause of death in these three patients. The other three patients had intestinal clostridial gas gangrene, with alcoholism as an underlying condition. None of the six patients was clinically diagnosed as having gas gangrene. We suggest that gas gangrene should be considered in any patient with abdominal infection. A review of 19 autopsy cases of gas gangrene in the abdomen reported in the Japanese literature is also presented.

Determination of ascorbic acid in food by column liquid chromatography with electrochemical detection using eluent for pre-run sample stabilization.

Determination of ascorbic acid (AA) in food was performed by column liquid chromatography with electrochemical detection using an eluent (100 mM KH2PO4 (pH 3) with 1 mM ethylenediaminetetraacetic acid disodium dihydrate) for pre-run sample stabilization. The applied potential was set at 400 mV vs. an Ag/AgCl reference electrode. The proposed method was simple, rapid (analysis time: ca. 8 min), sensitive (detection limit: ca. 0.5 ng per injection (20 microliters) at a signal-to-noise ratio of 3), highly selective and reproducible [relative standard deviation: ca. 1.8% (n = 5)]. The calibration graph for AA was linear in the range 0.1-16 ng per injection (20 microliters). Recovery of AA was over 90% by the standard addition method.

Role of transforming growth factor-beta 1 in fibroblasts derived from normal and hypertrophic scarred skin.

In order to elucidate the effect of transforming growth factor beta 1 (TGF-beta 1) on normal dermal fibroblasts (NDF) and on fibroblasts derived from hypertrophic scar (HSF) tissue, we compared proliferation, the levels of TGF-beta 1 protein and mRNA, the activity of type-I collagen synthesis and collagenase, and the response to recombinant human (rh) TGF-beta 1 in cultures of both types of cells which had been simultaneously collected from the same patients. We also studied the effects of anti-TGF-beta 1 antibody on the proliferation of these two types of fibroblasts in culture. In spite of the fact that the growth rate of HSF was higher than that of NDF, NDF proliferation was more sensitive to the concentration of rhTGF-beta 1. With respect to rates of synthesis, the results obtained in both groups revealed that the production of type-I collagen was higher and collagenase activity was lower in culture supernatants of HSF. However, the addition of rhTGF-beta 1 resulted in a decrease in the collagenase/collagen ratio in NDF, but failed to induce any change in this ratio in HSF. In addition, the production of TGF-beta 1 and the expression of TGF-beta 1 mRNA in HSF were greater than in NDF. Furthermore, anti-TGF-beta 1 antibody reduced the rate of growth of HSF. These results suggest that HSF are able to produce TGF-beta 1, resulting in enhanced proliferation of these cells as well as in a rapid synthesis of type-I collagen through an autocrine mechanism which may lead to hypertrophic scarring.

Prostaglandin I1 analogues, SM-10902 and SM-10906, affect human keratinocytes and fibroblasts in vitro in a manner similar to PGE1: therapeutic potential for wound healing.

The newly synthesized prostaglandin (PG) I1 analogues, SM-10902 and SM-10906, were compared with PGE1 in terms of their biological effects on cultured normal human keratinocytes (NHKs) and human dermal fibroblasts (HDFs) in order to evaluate their therapeutic potential for cutaneous wound healing. The PGI1 analogues had a direct effect on cell proliferation of HDFs as did PGE1, but inhibited cell growth of NHKs in contrast to the stimulatory effect observed with PGE1. In contrast to NHKs stimulated with PGI1 analogues, which exhibited low levels of adenosine 3,5-cyclic monophosphate (cAMP). HDFs stimulated with these analogues responded in a dose-dependent manner with extremely high levels of cAMP. Conditioned media (CM) derived from media in which HDFs had been incubated with both the PGI1 analogues promoted NHK proliferation. HDF production of interleukin (IL)-6 increased in response to the PGI1 analogues. Since IL-6 was shown to promote cell growth of NHKs, enhancement of NHK proliferation by CM was thought to be due to IL-6 derived from HDFs stimulated with the PGI1 analogues.

In situ hybridization and immunohistochemical study of human papillomavirus infection in adult laryngeal papillomas.

Routinely processed paraffin sections from 20 patients with adult laryngeal papillomas were examined for the presence of human papillomavirus type 11 (HPV-11) DNA and its specific mRNA by in situ hybridization methods using 35S-labeled RNA probes. Immunohistochemical techniques were also used to identify papillomavirus genus-specific common antigen (pgs-antigen). HPV-11 DNA signals and/or papillomavirus genus-specific common antigen were detected in all eight samples of multiple laryngeal papilloma. On the other hand, in 12 samples of single laryngeal papilloma, neither papillomavirus genus-specific common antigen nor HPV-11 DNA were detected. Four patients were positive for both HPV-11 DNA and pgs-antigen. In three of these four patients, HPV-11 mRNA signals were also detected. These results provided direct evidence of the association of HPV and adult multiple laryngeal papilloma.