Effects of mechanical stress and deficiency of dihydrotestosterone or 17ß-estradiol on Temporomandibular Joint Osteoarthritis in mice.

OBJECTIVE: To observe and analyze the interaction between excessive mechanical stress (MS) and decreased sex hormones on Temporomandibular Joint Osteoarthritis (TMJ-OA), and to discover TMJ-OA disease susceptibility genes by molecular biological analysis to elucidate part of the mechanism of TMJ-OA onset. DESIGN: For experimental groups, orchiectomy (ORX) or ovariectomy (OVX) was performed on sexually mature 8-week-old mice. A metal plate was attached to the posterior surface of the maxillary incisors to apply excessive MS on mandibular condyles. Male mice were divided into control, ORX, MS, and ORX + MS groups, while female mice were divided into control, OVX, MS, and OVX + MS groups. Mandibular condyles were evaluated by histology and molecular biology. RESULTS: Histomorphometric analysis of the TMJ in ORX + MS and OVX + MS groups revealed the thinnest chondrocyte layers, highest modified Mankin scores, and significant increases in the number of osteoclasts. Gene expression analysis indicated upregulation of Angptl7 and Car1 genes in the mandibular condyles of mice subjected to the combined effects of excessive MS and reduced sex hormones. In vitro analysis suggested that cartilage-like cells overexpressing Angptl7 enhanced calcification, and osteoblast-like cells overexpression Car1 suppressed cell proliferation and calcification. CONCLUSIONS: A severe TMJ-OA mouse model was successfully developed by applying excessive MS on the mandibular condyle of male and female mice with reduced sex hormones. Disease-susceptibility genes Angptl7 and Car1 were newly discovered in the experimental groups, suggesting their involvement in the onset mechanism of TMJ-OA.

Semi-allogeneic dendritic cells injected via the intratumoural injection route show efficient antitumour effects in cooperation with host-derived professional antigen-presenting cells.

Dendritic cells (DC)-based immunotherapy is a potent anticancer modality. In DC-based immunotherapy, allogeneic DC may be an alternative source, but the usefulness of allogeneic DC in DC-based immunotherapy is still controversial. When used for immunotherapy, three factors may affect the efficiency of an allogeneic DC-driven antitumour response: (1) survival time, which is affected by T-cell alloresponses; (2) major histocompatibility complex incompatibility with the host cells in the context of antigen presentation; and (3) the role of host-derived professional antigen-presenting cells (pAPC). In addition, it is unclear which injection route is preferable when using allogeneic DC. In this study, we demonstrate that semi-allogeneic DC, which share half of the genes of the recipient, are more effective when used via the intratumoural (i.t.) injection route, rather than the subcutaneous (s.c.) injection route, for the induction of efficient antitumour effects and the generation of a significant tumour-specific CD8(+) T-cell response. The i.t. route has the advantage of not requiring ex vivo pulsation with tumour lysates or tumour antigens, because the i.t.-injected DC can engulf tumour antigens in situ. Allogeneic bone marrow transplantation (BMT) models, which permit us to separately assess the three factors described previously, show that while all three factors are important for efficient antitumour effects, the control of the alloresponse to injected DC is the most crucial for host-derived pAPC to function well when DC are administered intratumourally. This information may be useful for DC-based cancer immunotherapy under circumstances that do not allow for the use of autologous DC.

Complete elimination of established neuroblastoma by synergistic action of gamma-irradiation and DCs treated with rSeV expressing interferon-beta gene.

Dendritic cell (DC)-based immunotherapy has been investigated as a new therapeutic approach to intractable neuroblastomas; however, only limited clinical effect has been reported. To overcome the relatively low sensitivity of neuroblastomas against immunotherapy, we undertook a preclinical efficacy study to examine murine models to assess the combined effects of gamma-irradiation pretreatment and recombinant Sendai virus (ts-rSeV/dF)-mediated murine interferon-beta (mIFN-beta) gene transfer to DCs using established c1300 neuroblastomas. Similar to intractable neuroblastomas in the clinic, established c1300 tumors were highly resistant to monotherapy with either gamma-irradiation or DCs activated by ts-rSeV/dF without transgene (ts-rSeV/dF-null) that has been shown to be effective against other murine tumors, including B16F10 melanoma. In contrast, immunotherapy using DCs expressing mIFN-beta through ts-rSeV/dF (ts-rSeV/dF-mIFNbeta-DCs) effectively reduced tumor size, and its combination with gamma-irradiation pretreatment dramatically enhanced its antitumor effect, resulting frequently in the complete elimination of established c1300 tumors 7-9 mm in diameter, in a high survival rate among mice, and in the development of protective immunity in the mice against rechallenge by the tumor cells. These results indicate that the combination of ts-rSeV/dF-mIFNbeta-DCs with gamma-irradiation is a hopeful strategy for the treatment of intractable neuroblastomas, warranting further investigation in the clinical setting.

Important role of tissue angiotensin-converting enzyme activity in the pathogenesis of coronary vascular and myocardial structural changes induced by long-term blockade of nitric oxide synthesis in rats.

The long-term administration of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, produces coronary vascular remodeling and myocardial hypertrophy in animals. This study used a rat model to investigate the role of angiotensin I converting enzyme (ACE) in the pathogenesis of such changes. We studied the following groups, all of which received drug treatment in their drinking water: untreated controls, and those administered L-NAME, L-NAME, and an ACE inhibitor (ACEI), and L-NAME and hydralazine. Cardiovascular structural changes and tissue ACE activities were evaluated after the first, fourth, and eighth week of treatment. In rats treated with L-NAME alone, vascular remodeling was evident at the fourth and eighth week, and myocardial hypertrophy was present at the eighth week of treatment. The vascular and myocardial remodeling were characterized by increased tissue ACE activities and immunodetectable ACE in those tissues. These changes were markedly reduced by ACEI, but not by hydralazine treatment. Increased local ACE expression may thus be important in the pathogenesis of cardiovascular remodeling in this model.

Actions of ZD0947, a novel ATP-sensitive K+ channel opener, on membrane currents in human detrusor myocytes.

BACKGROUND AND PURPOSE: ATP-sensitive K+ channels (K(ATP) channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)-induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated. EXPERIMENTAL APPROACH: Tension measurements and patch-clamp techniques were utilized to study the effects of ZD0947 in segments of human urinary bladder. Immunohistochemistry was also performed to detect the expression of the sulphonylurea receptor 1 (SUR1) and the SUR2B antigens in human detrusor muscle. KEY RESULTS: ZD0947 (> or = 0.1 microM) caused a concentration-dependent relaxation of the CCh-induced contraction of human detrusor, which was reversed by glibenclamide. The rank order of the potency to relax the CCh-induced contraction was pinacidil > ZD0947 > diazoxide. In conventional whole-cell configuration, ZD0947 (> or = 1 microM) caused a concentration-dependent inward K+ current which was suppressed by glibenclamide at -60 mV. When 1 mM ATP was included in the pipette solution, application of pinacidil or ZD0947 caused no inward K+ current at -60 mV. Gliclazide (< or =1 microM), a selective SUR1 blocker, inhibited the ZD0947-induced currents (Ki = 4.0 microM) and the diazoxide-induced currents (high-affinity site, Ki1 = 42.4 nM; low-affinity site, Ki2 = 84.5 microM) at -60 mV. Immunohistochemical studies indicated the presence of SUR1 and SUR2B proteins, which are constituents of KATP channels, in the bundles of human detrusor smooth muscle. CONCLUSIONS AND IMPLICATIONS: These results suggest that ZD0947 caused a glibenclamide-sensitive detrusor relaxation through activation of glibenclamide-sensitive KATP channels in human urinary bladder.

Sp1 decoy transfected to carcinoma cells suppresses the expression of vascular endothelial growth factor, transforming growth factor beta1, and tissue factor and also cell growth and invasion activities.

Vasculature development is thought to be an important aspect in the growth and metastasis of solid tumors. Among the angiogenic factors produced by tumor cells, vascular endothelial growth factor is considered to be the most potent and pathologically important. The synthesis of this growth factor has been shown to be modulated through Sp1 function following stimulation by tumor necrosis factor alpha (TNF-alpha). Oligodeoxynucleotides (ODNs) were synthesized with either the consensus sequence for Sp1 binding (Sp1 decoy ODNs) or a mutated form of this sequence (mt-Sp1 decoy ODNs). Using the hemagglutinating virus of Japan (HVJ)-liposome method, we transferred these ODNs into cultured cancer cells (A549 and U251 cells). The TNF-alpha-mediated expression of both VEGF and transforming growth factor beta1 and tissue factor (TF) by the cancer cells could be simultaneously suppressed to less than 30% by transfection of Sp1 decoy ODNs but not by mt-Sp1 decoy ODNs. In addition, in vitro invasiveness, synthesis of mRNA for urokinase-type plasminogen activator, and cell proliferation of both cell lines were also inhibited to 40% by the transfection of only Sp1 decoy ODNs. These results suggested that the Sp1 decoy strategy would be effective for regulating tumor growth by simultaneously reducing cancer cell (a) angiogenic growth factor expression, (b) proliferation, and (c) invasiveness.

Isolation of a major apolipoprotein of canine and murine pulmonary surfactant. Biochemical and immunochemical characteristics.

We studied some of the biochemical and immunochemical properties of a major apolipoprotein in isolated pulmonary surfactant from dog and rat lungs. These apolipoproteins were purified by DEAE-cellulose chromatography in buffers containing Triton X-100. Purity of the apolipoproteins was assessed by both fused rocket and crossed immunoelectrophoreses. In addition, the apolipoproteins showed one band with an apparent molecular weight of 72 000-73 000 on SDS-polyacrylamide gel electrophoresis. These proteins are composed of two polypeptide chains of 36 000 daltons. When subjected to isoelectric focusing, the major component of the apolipoprotein had an isoelectric point of about 4.4, with very minor components near 4.6. Even though the apolipoproteins of both species had very similar amino acid compositions, including a relatively high glycine content, no immunologic cross-reactivity was observed. Rocket immunoelectrophoretic analysis of several preparations of dog and rat surfactant using the respective purified apolipoproteins as standards indicated that the apolipoprotein constituted 56.9% +/- 4.6. (S.D., n = 3) and 42.1% +/- 2.1 (S.D., n = 2) of the total protein in dog and rat surfactant, respectively.

Purification and characterization of human kidney plasminogen activator dissimilar to urokinase.

The tissue activator was extracted with 2 M ammonium thiocyanate and purified by L-arginine methyl ester, concanavalin A and ion-exchange chromatographies, and Sephacryl S-200 gel filtration in buffers containing Triton X-100 and/or ammonium thiocyanate. The final preparations had specific activities of 25 000-40 000 IU/mg protein and were shown to be a single band with an apparent molecular weight of 54 00 by SDS-polyacrylamide gel electrophoresis with or without reducing agent. When subjected to isoelectric focusing, its major component had an isoelectric point of approx. 8.2 with minor components. (7.8-8.6). The purified tissue activator was a serine protease, dissimilar to urokinase in some respects including antigenicity, strong affinity to insoluble fibrin monomer and hydrolytic activities for synthetic substrates. The crude extract contained another plasminogen activator with antigen identity to urokinase, which constituted approx. 15% of the total activity in crude extract. These findings indicated that human kidney would produce at least two plasminogen activators, namely, the tissue activator as a major plasminogen activator and urokinase.

Role of calcium ions the structure and function of pulmonary surfactant.

Pulmonary surfactant isolated by centrifugation in buffers containing ions contains at least three different morphologic structures. The presence of one of these, tubular myelin, is dependent on calcium ions, since chelation of the calcium ions causes disruption of this structure. Addition of EDTA also decreases the ability of the surfactant to absorb rapidly to air-water interfaces and lower surface tension. Titration with calcium ions (2.5 or 5 mM) restores rapid surface adsorption and restores the tubular myelin structural forms. Magnesium ions cannot substitute for calcium ions in these processes. The reversibility of structure and function induced by calcium ions and EDTA is also accompanied by reversible isopycnic density shifts probably related to aggregation and disaggregation of the lipid-protein complex with calcium ions and EDTA, respectively.

Tumor necrosis factor alpha-induced alteration of glycosaminoglycans in cultured vascular smooth-muscle cells.

We studied alteration of glycosaminoglycans (GAGs) induced by recombinant human tumor necrosis factor alpha (rhTNF alpha) in vascular smooth-muscle cells from bovine aorta in a culture system. It was found that rhTNF alpha at 10 ng/ml and below significantly increased the incorporation of [35S]sulfate (35S) but conversely decreased that of [3H]glucosamine (3H) into GAGs in the trypsinate fraction of the cell layer after a 24-h incubation. These results suggested that rhTNF alpha reduced the formation and/or the anchorage of sugar chains in the cell layer but enhanced their sulfation in whole GAG synthesis by the cells. In results, the ratio of 35S to 3H in the GAGs was markedly increased. This increase occurred after 24 h and longer when the cells were treated with 1.0 ng/ml rhTNF alpha. The TNF alpha-induced alteration of the incorporation of both 35S and 3H was completely blocked by anti-rhTNF alpha antibody. Other cytokines including recombinant human interleukin-1 beta and -6, and platelet-derived growth factor failed to alter the ratio of 35S to 3H in the GAGs of the trypsinate fraction of the cell layer. In cultured vascular endothelial cells from bovine aorta, however, rhTNF alpha at 1.0 ng/ml significantly decreased the incorporation of both 35S and 3H into GAGs of both the trypsinate fraction and the medium; the ratio of 35S to 3H was not changed. Characterization of GAGs in vascular smooth muscle cell trypsinate fraction revealed that rhTNF alpha at 10 ng/ml induced (i) no change of the incorporation of 3H in the hyaluronate fraction, (ii) a marked increase in the incorporation of 35S and no change of that of 3H in chondroitin sulfates (A plus C) fraction, (iii) a significant decrease in the incorporation of both 35S and 3H in the heparan sulfate fraction, and (iv) no change of the incorporation of 35S and a marked decrease in that of 3H in the dermatan sulfate fraction. In the medium, rhTNF alpha also induced various changes of GAGs. It was therefore concluded that TNF alpha may have a capacity of inducing a qualitative change of vascular smooth-muscle cell GAGs, which may be involved in the vascular pathology such as atherosclerosis.

Secretion of surfactant by primary cultures of alveolar type II cells isolated from rats.

Pulmonary surfactant conventionally is prepared from material obtained by endobronchial lavage. Although it has been assumed that the components of surfactant are secreted by alveolar type II cells, direct proof of this assumption has not been available. Furthermore, it is possible that the final material obtained by lavage has been modified after secretion or altered during the isolation procedure. It has been shown previously that type II cells, after 1 day in primary culture, secrete saturated phosphatidylcholine, one of the lipid components of surfactant. Because saturated phosphatidylcholine is not unique to surfactant and because type II cells in culture lose differentiated characteristics over the first several days in culture, it has not previously been established how closely the secretory products of cultures of type II cells resemble surfactant as obtained by endobronchial lavage. We therefore studied the morphologic, physical and chemical characteristics of the material that type II cells secrete under basal conditions and after stimulation with terbutaline or 12-O-tetradecanoyl-13-phorbol acetate. The secreted material resembled surfactant obtained by lavage; it was similar morphologically to the lamellar material and tubular myelin seen in the fluid-filled alveoli of fetal rats, it lowered surface tension to 5 mN per meter, and it contained the 72000 dalton apolipoprotein of surfactant (as measured by the 'rocket' immunoelectrophoresis technique). When cells were incubated for 22 h with [1-(14)C]acetate, the distribution of radioactivity in the secreted material was very similar to the phospholipid composition of rat surfactant. We conclude that the material secreted by alveolar type II cells after 1 day in primary culture is similar to surfactant obtained by endobronchial lavage.

Increased activity of nuclear factor-kappaB participates in cardiovascular remodeling induced by chronic inhibition of nitric oxide synthesis in rats.

BACKGROUND: Chronic inhibition of endothelial nitric oxide (NO) synthesis by the administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) to rats induces early vascular inflammatory changes [monocyte infiltration into coronary vessels, nuclear factor-kappaB (NF-kappaB) activation, and monocyte chemoattractant protein-1 expression] as well as subsequent arteriosclerosis (medial thickening and perivascular fibrosis) and cardiac fibrosis. However, no direct evidence for the importance of NF-kappaB in this process is known. METHODS AND RESULTS: We examined the effect of a cis element decoy strategy to address the functional importance of NF-kappaB in the pathogenesis of cardiovascular remodeling. We found here that in vivo transfection of cis element decoy oligodeoxynucleotides against NF-kappaB to hearts prevented the L-NAME-induced early inflammation and subsequent coronary vascular medial thickening. In contrast, NF-kappaB decoy oligodeoxynucleotide transfection did not decrease the development of fibrosis, the expression of transforming growth factor-beta(1) mRNA, or systolic pressure overload induced by L-NAME administration. CONCLUSIONS: The NF-kappaB system participates importantly in the development of early vascular inflammation and subsequent medial thickening but not in fibrogenesis in this model. The present study may provide a new aspect of how endothelium-derived NO contributes to anti-inflammatory and/or antiarteriosclerotic properties of the vascular endothelium in vivo.

Clinicopathological and molecular evidence indicating the independence of bronchioloalveolar components from other subtypes of human peripheral lung adenocarcinoma.

Although human lung adenocarcinoma has diverse histological subtypes, the correlation between histological subtypes and occurrence of the p53 gene mutation has been given less attention. We investigated 145 surgically resected lung adenocarcinomas to search for the incidence of p53 mutations and for record data on survival in each histological subtype, according to the new WHO criteria (1999). The frequency of p53 mutation in bronchioloalveolar carcinoma (BAC; 0% in 17 cases) and BAC with invasive growth component (BAC-invasive; 11% in 27 cases), which is conventionally categorized as the mixed subtype in WHO typing, were apparently significantly lower than in other types (non-BAC including acinar, papillary, solid, or mixed histology with these subtypes; 48% in 101 cases; P < 0.01). Multivariate analysis revealed that the histological subtype including BAC-invasive was a strong, independent, and significant prognostic factor (P < 0.03), as were tumor size and pathological stage (P < 0.001 and 0.002, respectively) for overall survival. However, the occurrence of p53 mutation itself was seen to be significant only in case of the univariate analysis. Therefore, histological subtyping may be a better prognostic indicator than is p53 mutation. These findings suggest that the WHO classification with the BAC and BAC-invasive from other histological subtypes may prove useful to predict the outcome for surgically treated patients with lung adenocarcinoma.

The role of fibroblast growth factor-2 in the vascular effects of interleukin-1 beta in porcine coronary arteries in vivo.

OBJECTIVE: We recently demonstrated that chronic treatment with interleukin-1 beta (IL-1 beta), a major inflammatory cytokine found in atherosclerotic lesions, induces coronary arteriosclerotic changes and vasospastic responses to serotonin and histamine in pigs in vivo and that those responses are partially mediated by platelet-derived growth factor (PDGF). This study was designed to examine, first, whether the effects of IL-1 beta are also partially mediated by fibroblast growth factor-2 (FGF-2), which is another important growth factor in atherosclerotic lesions, and, secondly, whether chronic treatment with FGF-2 per se also induces histological and functional changes in porcine coronary arteries in vivo. METHODS: Porcine coronary arteries were aseptically wrapped with cotton mesh absorbing IL-1 beta with or without neutralizing antibody to FGF-2. In a separate series of experiments porcine coronary arteries were chronically treated with FGF-2 itself in the same manner. Coronary vascular responses in vivo and histological changes were examined 2 weeks after the operation. RESULTS: Coronary vasospastic responses to serotonin and histamine and neointimal formation were induced at the site of the coronary artery where IL-1 beta was chronically and locally applied. These responses were significantly suppressed by co-treatment with a neutralizing antibody to FGF-2 but not by that with non-immune IgG. Immunostaining revealed the presence of FGF-2 in the endothelial cells, the thickened intima and the media at the IL-1 beta-treated site. Furthermore, chronic treatment with FGF-2 also induced coronary vasospastic responses to serotonin and histamine and neointimal formation. CONCLUSIONS: These results suggest that the vascular effects of IL-1 beta may also be mediated by FGF-2 in our swine model and that chronic treatment with FGF-2 also causes coronary arteriosclerotic changes and vasospastic responses in vivo.

Target gene transfer of tissue plasminogen activator to cornea by electric pulse inhibits intracameral fibrin formation and corneal cloudiness.

Intracameral fibrin formation, a complication of ocular inflammation and intraocular operations, sometimes results in glaucoma and/or corneal damage leading to permanent visual loss. We transferred a therapeutic gene to the corneal endothelium in order to use it as a therapeutic organ. A plasmid encoding tissue plasminogen activator (tPA) was injected into the anterior chamber of rats and electric pulses (EPs) were given subsequently, which transferred a plasmid gene to a highly selected area of corneal endothelium with no inflammation. The biologically active tPA was clearly present for 4 days after treatment. Fibrin formation induced by YAG laser-generated bleeding in the anterior chamber decreased significantly more in treated eyes than in control eyes. Corneal opacity was significantly lower in treated eyes than in control eyes and histological damage was not apparent in the treated eyes. This genetic modification allows us to use the corneal endothelium to treat various ocular diseases and could be a new and effective type of pharmacologic gene therapy.

Chronic inhibition of nitric oxide synthesis causes coronary microvascular remodeling in rats.

The aim of the present study was to investigate the effects of long-term blockade of nitric oxide synthesis with the L-arginine analogue N omega-nitro-L-arginine methyl ester (L-NAME) for 8 weeks on coronary vascular and myocardial structural changes. Four groups of Wistar-Kyoto rats were studied: those with no treatment, those treated with L-NAME 1 g/L (3.7 mmol/L in drinking water), those treated with L-NAME 0.1 g/L (0.37 mmol/L in drinking water), and those treated with L-NAME 1.0 g/L and hydralazine 120 mg/L (0.6 mmol/L in drinking water). After 8 weeks, the heart was excised, and the degrees of structural changes in coronary arteries (wall-to-lumen ratio and perivascular fibrosis), myocardial fibrosis, and myocyte size were quantified by an image analyzer. Chronic inhibition of nitric oxide synthesis increased arterial pressure compared with control animals. Chronic inhibition of nitric oxide synthesis caused significant microvascular remodeling (increased wall-to-lumen ratio and perivascular fibrosis). Cardiac hypertrophy was also observed after chronic inhibition of nitric oxide synthesis. Coadministration of hydralazine prevented arterial hypertension but did not affect microvascular remodeling and cardiac hypertrophy induced by the chronic inhibition of nitric oxide synthesis. In addition, chronic inhibition of nitric oxide synthesis caused scattered lesions of myocardial fibrosis, which was significantly attenuated by cotreatment with hydralazine. These results suggest that long-term blockade of nitric oxide synthesis caused coronary microvascular remodeling and cardiac hypertrophy in rats in vivo by a mechanism other than arterial hypertension. In contrast, arterial hypertension contributed to the development of myocardial fibrosis induced by long-term blockade of nitric oxide synthesis.

Chronic angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade: effects on cardiovascular remodeling in rats induced by the long-term blockade of nitric oxide synthesis.

We have shown previously that angiotensin-converting enzyme (ACE) inhibitors prevent coronary vascular remodeling (medial thickening and perivascular fibrosis) and myocardial remodeling (fibrosis and hypertrophy) in rats induced by long-term inhibition of nitric oxide (NO) synthesis with oral administration of N omega-nitro-L-arginine methyl ester (L-NAME). ACE inhibitors inhibit both the formation of angiotensin II and the catabolism of bradykinin. In this study, we aimed to determine the relative contribution of the latter two mechanisms to the beneficial effects of an ACE inhibitor on structural remodeling. First, we examined the effects of the ACE inhibitor temocapril and the angiotensin II AT1 subtype receptor antagonist CS-866 on the structural remodeling induced by administering L-NAME for 8 weeks. Temocapril and CS-866 were equally effective in preventing remodeling. Second, we examined whether the effect of temocapril on the remodeling induced by L-NAME was reduced by the bradykinin receptor antagonist HOE140. The latter drug did not alter the beneficial effect of temocapril on remodeling. In conclusion, although species differences must be considered to apply our conclusion to clinical conditions, the present results suggest that the inhibition of angiotensin II activity, mediated via the AT1 receptors, is responsible for the beneficial effects of an ACE inhibitor in our animal model of coronary vascular and myocardial remodeling induced by the long-term inhibition of NO synthesis.

Corticotropin-releasing hormone- and adrenocorticotropin-producing pituitary carcinoma with metastases to the liver and lung in a patient with Cushing's disease.

A 53-yr-old man with Cushing's disease was found to have a pituitary carcinoma with metastases to the liver and lung which produced both CRH and ACTH simultaneously. Despite removal of the pituitary tumor, his Cushing's disease worsened. Endocrinological examination then demonstrated elevated plasma CRH and markedly elevated plasma ACTH, beta-lipotropin, and cortisol concentrations, increased urinary 17-hydroxycorticosteroid and 17-ketosteroid excretion, and no suppression of serum cortisol after low or high dose dexamethasone administration. Urinary 17-hydroxycorticosteroid excretion increased in response to metyrapone, and lysine vasopressin elicited a striking increase in plasma ACTH. A computed tomographic scan of abdomen revealed multiple hypodense areas in the liver and bilateral adrenal hyperplasia. Postmortem histological examination revealed a necrotic hemorrhagic pituitary carcinoma with metastases to the liver, lung, and olfactory bulb. Immunohistochemical staining, gel filtration, and Northern blot analysis of liver and lung metastases revealed evidence of the production of both CRH and ACTH in these metastases. We concluded that the patient's pituitary carcinoma produced both CRH and ACTH.

An in vitro invasion model for human renal cell carcinoma cell lines mimicking their metastatic abilities.

We developed a modified in vitro invasion assay system using monolayers of vascular endothelial cells. A type I collagen gel was formed in plastic dishes, and overlaid with type IV collagen. Calf pulmonary arterial endothelial (CPAE) cells were seeded onto these plates, and incubated until they reached confluence. Five human renal cell carcinoma cell lines with various metastatic potentials in vivo were then seeded on the monolayer CPAE cells, and their colony formation and invasion activities were examined for 9 days. At day 4, the highly metastatic cell lines increased the number of colony foci on monolayer CPAE cells several fold higher than their poorly metastatic counterpart. The horizontal spreading patterns were also different between poorly and highly metastatic cell lines. On day 9, the number of carcinoma foci that penetrated the monolayer of CPAE cells and type IV collagen sheets into type I collagen gels in highly metastatic cell lines greatly increased as compared with that of poorly metastatic cell lines. Our in vitro invasion assay using monolayer CPAE cells would be useful to evaluate protease activities and colony formation during invasion.

Osteonecrosis induced by a single administration of low-dose lipopolysaccharide in rabbits.

We succeeded in developing a novel rabbit model of nonsteroid and nontraumatic osteonecrosis (ON) by use of a single- and low-dose lipopolysaccharide (LPS) injection. This model is simple and highly reproducible for the frequent development of multifocal and widespread ON lesions. Male adult Japanese white rabbits intravenously injected with a single injection of 10 microg/kg body weight of LPS were histopathologically examined in the early phase (3 [n = 3], 5 [n = 3], and 24 h [n = 3]) and at 4 weeks (n = 22). Seventy-seven percent of the rabbits developed multifocal ON 4 weeks after LPS injection. ON was also observed in the femoral and humeral condyle. The average percentage of necrotic area/total area examined was 86.7 +/- 29.1% and 78.8 +/- 16.7% in the proximal one third of both the femoral and humeral bones, respectively. Organized thrombi in the intraosseous small-sized arteries and arterioles were frequently seen in and around the necrotic tissues. In the early phase, LPS treatment prominently induced thrombocytopenia, hyperlipidemia, and increased plasma levels of plasminogen activator inhibitor-1 (PAI-1). The plasma level of PAI-1 was significantly higher in the rabbits with ON than in those without ON (p < 0.01). The immunohistochemical expression of tissue factor was exaggerated in monocytes/macrophages and adipocytes in both the femoral and humeral bones of the LPS-treated rabbits. Histologically, marrow necrosis and fibrin thrombi could be observed at 24 h. In addition, pretreatment with an anticoagulant, warfarin potassium, significantly decreased the incidence of LPS-induced ON (33%, n = 9, p < 0.05) associated with elongation of prothrombin time. The results of our study show that a single administration of low-dose lipopolysaccharide induces multifocal and widespread ON characterized by the pathophysiological participation of hypercoagulability in ON development. Therefore, this model would be useful for elucidating the pathogenesis of nonsteroid ON in humans especially inflammatory hypercoagulability-induced as well as for developing preventive and therapeutic strategies.